Mobility and recognition in cell biology: Proceedings of a FEBS Lecture Course held at the University of Konstanz, West Germany, September 6–10, 1982 9783111533407, 9783110095364

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Mobility and Recognition in Cell Biology FEBS Lecture Course No. 82/09

Mobility and Recognition in Cell Biology Proceedings of a FEBS Lecture Course held at the University of Konstanz, West Germany, September 6-10,1982 Editors Horst Sund • Cees Veeger

W DE G Walter de Gruyter • Berlin • New York 1983

Editors Horst Sund, Dr. rer. nat. Professor of Biochemistry Fakultät für Biologie Universität Konstanz D-7750 Konstanz, West Germany Cees Veeger, Ph.D. Chemistry Professor of Biochemistry Faculty of Agricultural Sciences Agricultural University Wageningen, The Netherlands

CIP-Kurztitelaufnahme

der Deutschen

Bibliothek

Mobility and recognition in cell biology : proceedings of a FEBS lecture course held at the Univ. of Konstanz, West Germany, September 6-10,1982/ ed. Horst Sund ; Cees Veeger. - Berlin ; New York : de Gruyter, 1983. ISBN 3-11-009536-X NE : Sund, Horst [Hrsg.] ; Federation of European Biochemical Societies

Library of Congress Cataloging in Publication Data Mobility and recognition in cell biology Bibliography: p. Includes index. 1. Cytochemistry-Congresses. 2. Binding sites (Biochemistry)Congresses. 3. Cell receptors-Congresses. 4. Biological transport, Active-Congresses. I. Sund, Horst, 1926- II. Veeger, Cees, 1929[DNLM : 1. Biopolymers. 2. Macromolecular systems-Congresses. 3. Proteins-Physiology-Congresses. 4. Nucleic acids-PhysiologyCongresses. 5. Chemotaxis-Congresses. QU 55 M6871982] QH611 .M55 1983 574.87'6042 83-2000 ISBN 3-11-009536-X

Copyright © 1983 by Walter de Gruyter & Co., Berlin 30. All rights reserved, including those of translation into foreign languages. No part of this book may be reproduced in any form - by photoprint, microfilm or any other means nor transmitted nortranslated into a machine language without written permission from the publisher. Printing: Kupijai & Prochnow, Berlin. - Binding: Dieter Mikolai, Berlin. Printed in Germany.

PETER HEMMERICH

In August 1981, w h e n w e came together to discuss the details of this meeting, our mutual

friend and colleague Peter Hemmerich, professor of bioche-

mistry at the University of Konstanz, was fatally

ill. Being aware of the

short time given to him to live, we could have decided to organize a symposium in one of Peter's fields of research. W e decided, however, to organize the present lecture course in view of the fact that Peter Hemmerich, chemist, was deeply

involved

the

in biology.

In his work on flavins and f1avoproteins and on the role of metals

in en-

zymes, Peter Hemmerich demonstrated and advocated, as early as I960, the need for the molecular understanding of enzymological systems. His participation

and other

biochemical

in previous Konstanzer Symposia sometimes em-

barrassed participants by asking direct questions on the molecular level or by criticism of the vague "biological" approach. His involvement mental

in environ-

protection and his love for nature made him a true biologist.

A survey of his many contributions to the understanding of

structure-func-

tion relations of flavoproteins, a contribution to his lecture course by his long-lasting friend and collaborator Vince Massey, guest professor at the University of Konstanz, shows the refined insight to which this molecular approach might lead to in terms of recognition and specificity. Up to the last moments of his life he was occupied with his beloved science and nature presented

in many aspects. At the Agricultural in September 1980, shortly before his

about the involvement of metal clusters hydrogenase. During his

illness, new original

ideas

in enzymes like nitrogenase and

illness, he organized

"Single Electron Transfer

University of Wageningen he

in March 1981 a Symposium

in Biology" to honor the many contributions of

his friend and colleague Helmut Beinert, guest professor at the University of Konstanz. Peter Hemmerich finished his memoirs during the last weeks of his life in August 1981 . At the end he wrote:

Peter Hemmerich

VIII

" And so today I am sitting with my tumor on the balcony, and wondering whether it is more important to investigate further iron-sulfur clusters with Helmut Beinert and better to understand the mechanism of nitrogenase with Cees Veeger, or of saving the Bodensee from the 400 million liter of leaking Swiss oil reserves that are to be pumped by diligent entrepreneurs of the canton Graubunden into unsealed caverns of the Alps in order to protect the Swiss Confederation for evermore from a possible lack of oil. Meanwhile I have anyhow come to doubt the sincerity of the Helvetic efforts for the continued existence of humanity, notwithstanding the fact that I owe Switzerland half my development. But the other half I owe the USA, with corresponding doubts regarding the sincerity of Reagan's efforts in that direction. And when I think of oil caverns and neutron bombs, I find it more bearable to live with my tumor despite the fact that I would dearly love to show the light of day to many things starting with molybdopteridines and finishing with an autobahn-free Bodensee, a landscape offering rest and recuperation to hikers and bicyclists and altogether a generous reorganization of all the inherited cultural building blocks with which the Bodensee country abounds and which we, in our lack of restraint and our arrogance and greed, have neglected for twenty years. But we have reached a turning point. " Helmut Beinert and Vince Massey ended their obituary in "Trends in Biochemical Sciences" of February 1982 with a sentence which we should like to use to express ourselves: " For those who were close to Hemmerich, life will have become more calm; but it may also have lost some of its more vivid colors. "

Cees Veeger Horst Sund

PREFACE

The p r e s e n t volume c o n t a i n s "Mobility

and R e c o g n i t i o n

t h e p r o c e e d i n g s o f a FEBS l e c t u r e c o u r s e on

in C e l l

B i o l o g y " h e l d a t the U n i v e r s i t y o f

Kon-

s t a n z , West Germany, on September 6 - 1 0 ,

1982.

In \S7h,

I n t e r a c t i o n s " was o r g a n i z e d a t

a Symposium on " P r o t e i n - L i g a n d

university.

The p r e s e n t

Biochemists

have d i r e c t e d

more o r

less

availability

integral

l e c t u r e c o u r s e m i g h t be c o n s i d e r e d a s a f o l l o w themselves

biological

o f new b i o p h y s i c a l ,

Rapidly deepening

insight

in the l a s t

biochemical

has been a c h i e v e d

Interaction of Proteins with Proteins,

n i s m s and

in N u c l e i c A c i d s ,

fields,

and i m m u n o l o g i c a l

into M o b i l i t y of

either

the o r g a n i z e r s

to assemble s p e c i a l i s t s

r e s u l t s and

ideas

this

Mechanisms

i d e a s between s c i e n t i s t s w o r k i n g lems from a s p e c i f i c small meetings

like

viewpoint.

Mecha-

oriented,

or s p e c i a l i s a t i o n ,

fields

of

and l o o k i n g a t

probthat

r e a s o n , we have not s e t any

b e f o r e the l e c t u r e c o u r s e

the p r o g r e s s o f the p r o c e e d i n g s and d i s c u s s i o n s .

distime

in o r d e r The

were

to

discussions

r e c o r d e d . The p a r t i c i p a n t s were r e q u e s t e d t o p r o v i d e a

w r i t t e n r e p o r t o f what they c o n s i d e r e d w o r t h information

In o t h e r

P h o t o p r i n t s of the submitted t y p e s c r i p t s

c i r c u l a t e d among t h e p a r t i c i p a n t s facilitate

interactions.

not

but

i n t e r a c t i o n and s t i m u l a t i o n

i s the b e l i e f o f t h e o r g a n i z e r s

i d e a s . For t h i s

to the d i s c u s s i o n .

were not d i r e c t l y

to d i s c u s s

t h i s one p r o v i d e a p r o p e r p l a t f o r m f o r e x t e n s i v e

c u s s i o n and e x c h a n g e o f limitation

different

in t h e i r f i e l d s

in d i f f e r e n t It

in the

or b i o l o g i c a l l y

to promote t h e i n t e r d i s c i p l i n a r y

As u s u a l

in

Biopolymers,

N u c l e i c A c i d s and L i p i d s ,

in a d d i t i o n t o e n c o u r a g e d i s c u s s i o n and h o p e f u l l y

This

techniques.

Receptor-Ligand Binding, Transport

physico-chemica1ly

the newest

words,

of

due to the

Chemotaxis.

I t was the aim o f

only

to the s t u d y level

in the t o p i c s c o v e r e d

l e c t u r e c o u r s e w h i c h can be r o u g h l y d e v i d e d

of Recognition

few y e a r s

s y s t e m s on the m o l e c u l a r

this up.

is presented

for symposia,

in t h i s

including

in the

publication.

volume.

t h e r e was no r e f e r e e i n g o f the p a p e r s s u b m i t t e d ,

contents of which are s o l e l y

the a u t h o r s '

responsibility.

the

X

Preface

Unfortunately

f o r the o r g a n i z e r s o f

standing s c i e n t i s t s

this

i n the d i f f e r e n t

l e c t u r e c o u r s e , a number o f

fields

c o u l d not be i n v i t e d ; some o f

them b e i n g

i n v i t e d were u n a b l e to a c c e p t . The o r g a n i z e r s a r e aware

additional

t o p i c s c o u l d have been added; u n f o r t u n a t e l y , we had t o

the number o f p a r t i c i p a n t s

and f i e l d s .

Our a p p r e c i a t i o n

p a r t i c i p a n t s who have c o o p e r a t e d s o e f f e c t i v e l y symposium and p u b l i c a t i o n o f by t h e p o s i t i v e

strive

that restrict

i s extended

in o r d e r

to a l l

to promote

i t s p r o c e e d i n g s . The o r g a n i z e r s were

r e s p o n s e from many p a r t i c i p a n t s ;

t i c i p a t i o n was more than the s c i e n t i s t s '

out-

this

pleased

i t made us aware t h a t f o r m o b i l i t y and

par-

recogni-

tion. Everyone o r g a n i z i n g the d i f f i c u l t i e s

a m e e t i n g t h e s e d a y s o f economic

of f i n d i n g

o f European B i o c h e m i c a l

Societies

l e c t u r e c o u r s e . We g r a t e f u l l y s i t y of Konstanz;

f o r the generous

acknowledge

the A g r i c u l t u r a l

l a n d s ; by the " G e s e l l s c h a f t

i s aware o f

t o the

support for

this

der U n i v e r s i t ä t

und G e s e l l s c h a f t

an der

Kon-

Universi-

Konstanz".

W a l t e r de G r u y t e r , B e r l i n . We a r e a l s o v e r y g r a t e f u l voted a s s i s t a n c e

in a l l

matters concerning

this

interest of

(bio)-physicists,(bio)-chemists

pidly developing

f i e l d of molecular

expect t h a t m e e t i n g s o f a s i m i l a r be as s t i m u l a t i n g

and a p p r o a c h e s

Konstanz,

kind w i l l

as the p r e s e n t one

in the n a t u r a l

November 1,

interaction

lecture

1982

de-

course.

and b i o l o g i s t s in b i o l o g y . The

be h e l d

Verlag

for Mrs. A l l e n ' s

The e d i t o r s hope t h a t the c o n t r i b u t i o n t o t h e s e p r o c e e d i n g s w i l l

will

FEBS Univer-

U n i v e r s i t y o f W a g e n i n g e n , The N e t h e r -

We would l i k e t o a c k n o w l e d g e t h e c o o p e r a t i o n o f t h e s t a f f o f the

the

Federation

the s u p p o r t g i v e n by the

der Freunde und F ö r d e r e r

s t a n z " and by der " S t i f t u n g W i s s e n s c h a f t tät

recession

s u p p o r t . We a r e v e r y g r a t e f u l

stimulate in the

organizers

i n the near f u t u r e

in the development o f new

sciences.

H o r s t Sund Cees V e e g e r

ra-

and

ideas

CONTENTS

SECTION

I.

MOBILITY OF BIOPOLYMERS

S p e c i f i c i t y o f C a t a l y s i s by F l a v o p r o t e i n s by V. Massey Discussion

3 18

Functional S i g n i f i c a n c e of F l e x i b i l i t y W.S. Bennett, Jr Discussion

21 46

in P r o t e i n s by R. Huber and

S t r u c t u r a l Dynamics o f P r o t e i n s as R e f l e c t e d by I s o t o p e K i n e t i c s by R.B. Gregory and A. Rosenberg Discussion

Exchange

S e l f - O r g a n i z a t i o n o f O l i g o m e r i c P r o t e i n s . F o l d i n g and R e c o g n i t i o n o f P o l y p e p t i d e C h a i n s upon Quaternary S t r u c t u r e Formation by R. Jaenlcke Discussion

49 63

67 78

I n t e r c o n v e r s i o n s between [ ' i F e - ' t S ] and [ 3 F e - 3 S ] C e n t r e s in Desulfovibrio gigas F e r r e d o x i n s by I. Moura, J.J.G. Moura and A.V. Xavier Discussion

83 100

Dynamics a t the P r o t e i n S u r f a c e by R.G. Bryant Di s c u s s ion

103 116

R o t a t i o n a l Dynamics o f C e l l S u r f a c e P r o t e i n s by T i m e - R e s o l v e d P h o s p h o r e s c e n c e A n i s o t r o p y by E.D. Matayoshi, A.F. Corin, R. Zidovetzki, W.H. Sawyer and T.M. Jovin Di s c u s s ion

119 13 3

SECTION I I .

INTERACTION OF PROTEINS WITH PROTEINS, ACIDS AND L I P I D S

NUCLEIC

Dynamic A s p e c t s o f P r o t e i n - P r o t e i n A s s o c i a t i o n Revealed by A n i s o t r o p y Decay Measurements by A.J.W.G. Visser, N.H.G. Penners, and F. Müller Discussion

137 15 2

Dynamic A s p e c t s o f A n t i b o d y F u n c t i o n by I. Pecht Discussion

155 170

F l e x i b i l i t y in Chromosomal Discussion

173 193

P r o t e i n s by E.M. Bradbury

Contents

XII H i s t o n e - H i s t o n e and H i s t o n e - D N A I n t e r a c t i o n s i n Chromatin J. Ausio, Y. Haik, D. Seger and H. Eisenberg Discussion

by 195 209

Q u a n t i t a t i v e Approaches to the Autogenous R e g u l a t i o n o f Gene E x p r e s s i o n by P.H. von Hippel and F.R. Fairfield Discussion

213 234

Dynamics o f Gene R e g u l a t i o n in the OR/0|_ C o n t r o l System o f B a c t e r i o p h a g e Lambda by M.A. Shea and G.K. Ackers Discussion

237 251

S p e c i f i c i t y and L i p i d B i n d i n g S i t e o f B o v i n e P h o s p h a t i d y l c h o l i n e T r a n s f e r P r o t e i n by K.W.A. Wirtz, R. Akeroyd, J. Westerman and L.L.M. van Deenen Discussion

253 263

SECTION

III.

MECHANISMS OF RECOGNITION IN NUCLEIC ACIDS

A I l o s t e r i c DNA by F.M. Discussion

267 278

Pohl

Left-Handed Z DNA in P o l y t e n e Chromosomes by M. D.J. Arndt-Jovin, D.A. Zarling and T.M. Jovin

Robert-Nicoud, 281

L o c a t i o n and R e c o g n i t i o n o f S p e c i f i c DNA B i n d i n g S i t e s by P r o t e i n s t h a t R e g u l a t e Gene E x p r e s s i o n by P.H. von Hippel and D.G. Bear . . Discussion

291 316

H i e r a r c h i e s o f Promoter R e c o g n i t i o n D i s p l a y e d by Escherichia RNA P o l y m e r a s e by W.R. McClure and D.K. Hawley Discussion

317 332

N i t r o g e n F i x a t i o n : I n t e r a c t i o n s among nif P r o d u c t s by C. Kennedy and R.L. Robson Discussion

Genes and

M o l e c u l a r A s p e c t s o f R e c o g n i t i o n in the S y m b i o s i s by J.W. Kijne and I.A.M. van Discussion

Rhizobium-Legume der Schaal

coli

their 335 356

C a l m o d u l i n and the I n t r a c e l l u l a r C a l c i u m S i g n a l : S t r u c t u r a l and F u n c t i o n a l I m p l i c a t i o n s o f C a l m o d u l i n mRNA by R.P. Munjaal and J.R. Dedman Discussion

357 372

375 389

Contents

XIII

SECTION I V .

RECEPTOR-LIGAND BINDING AND TRANSPORT MECHANISMS

The A c e t y l c h o l i n e Receptor and i t s L.

Lauffer

Discussion

and

F.

Hucho

Ion Channel by P.

Muhn,

393

406

Modulation of Calcium Ions Fluxes as S i g n a l s for Mast C e l l s and B a s o p h i l s D e g r a n u l a t i o n by I . Pecht, R. Sagi-Eisenberg and 1V. Mazuiek

409

Discussion

426

R e c o g n i t i o n and S p e c i f i c i t y In the I n t r a c e l l u l a r M i t o c h o n d r i a l P r o t e i n s by W. Neupert Discussion

Transfer of

•

The E l e c t r o c h e m i c a l Proton Gradient Induces Di t h i o l - D i s u l f i d e Interchange in T r a n s p o r t P r o t e i n s a t Both S i d e s o f the Membrane by

G.T.

Robillard

and

Di scuss ion

R.G.

Lageveen,

B.

Poolman

S e c r e t i o n and M a t u r a t i o n o f a L y t i c P e p t i d e : to U.

P r o d u c t by L. Haiml

the Final Vilas and

Discussion

SECTION V.

G.

Kreil,

C.

Mollay,

and

W.N.

Konings

From P r e p r o m e l i t t i n A.

Hutticher,

471

480

Rotary Motor by H.C.

Berg

and S.

B a c t e r i a l M o t i l i t y : E n e r g i z a t i o n and S w i t c h i n g of the Motor by R.M. Macnab Discussion

Khan

Flagellar

The Phosphoenolpyruvate-Dependent C a r b o h y d r a t e : P h o s p h o t r a n s f e r a s e System Enzymes I I , a New Class of Chemosensors in B a c t e r i a l C h e m o t a x i s by

A.

Discussion

Pecher,

I.

Renner

and

J.W.

Lengeler

Discussion

by

M.R.

Kehry,

F.W.

Vahlquist

and

M.W.

Bond

A Family of Homologous Genes Encoding Sensory Transducers A.

Boyd,

Discussion

A.

Krikos,

N.

Mutoh

and

M.

Simon

485 496 499 515

517

529

Chemotaxis: The Chemical P r o p e r t i e s o f the cheß-Dependent

Modification

by

449

469

CHEMOTAXIS

A Model f o r the F l a g e l l a r Discussion

Bacterial

.

429 445

533

548 in

E.coli

551

562

XIV

Contents

Genetics of Transmembrane Signaling Proteins in E. coli by J.S. Parkinson, M.K. Slocum, A.M. Callahan, D. Sherris and S.E. Houts Discussion

563 576

Index of Contributors

579

Subject Index

581

LIST

OF

CONTRIBUTORS

G . K . A c k e r s , Department o f B i o l o g y and McCol1 u m - P r a t t H o p k i n s U n i v e r s i t y , B a l t i m o r e , M a r y l a n d , USA G. Adam, F a k u l t ä t

f ü r B i o l o g i e der U n i v e r s i t ä t ,

R. A k e r o y d , L a b o r a t o r y o f B i o c h e m i s t r y , Netherlands

Institute,

The John

K o n s t a n z , West Germany

State U n i v e r s i t y of Utrecht,

The

D . J . A r n d t - J o v i n , A b t e i l u n g M o l e k u l a r e B i o l o g i e , Max P l a n c k - I n s t i t u t B i o p h y s i k a l i s c h e Chemie, G ö t t i n g e n , West Germany

für

J . A u s i ö , Department o f Polymer R e s e a r c h , The Weizmann Rehovot, I s r a e l E . Bade, F a k u l t ä t

f ü r B i o l o g i e der U n i v e r s i t ä t ,

I n s t i t u t e of

K o n s t a n z , West Germany

D.G. B e a r , I n s t i t u t e o f M o l e c u l a r B i o l o g y and Department o f U n i v e r s i t y o f O r e g o n , E u g e n e , O r e g o n , USA W.S. B e n n e t t , J r . , M a x - P l a n c k - I n s t i t u t c h e n , West Germany

W. B o o s , F a k u l t ä t

California

Fakultät

Institute of

bei

Mün-

Technology,

I n s t i t u t e of Technology,

f ü r B i o l o g i e der U n i v e r s i t ä t ,

R. B ö s i n g - S c h n e i d e r , Germany

Chemistry,

für Biochemie, M a r t i n s r i e d

H.C. Berg, D i v i s i o n of B i o l o g y 216-76, C a l i f o r n i a P a s a d e n a , C a l i f o r n i a , USA M.W. Bond, D i v i s i o n o f B i o l o g y , d e n a , C a l i f o r n i a , USA

Science,

Pasa-

K o n s t a n z , West Germany

f ü r B i o l o g i e der U n i v e r s i t ä t ,

H . J . Bosma, Department o f B i o c h e m i s t r y , A g r i c u l t u r a l The N e t h e r l a n d s

Konstanz,

University,

A . B o y d , Department o f B i o l o g y B - 0 2 2 , U n i v e r s i t y o f C a l i f o r n i a , La J o i l a , C a l i f o r n i a , USA

Wageningen, San

Diego,

E.M. B r a d b u r y , Department o f B i o l o g i c a l C h e m i s t r y , U n i v e r s i t y o f C a l i f o r n i a , D a v i s , C a l i f o r n i a , USA

School

J.M. B r a s s ,

K o n s t a n z , West Germany

Fakultät

f ü r B i o l o g i e der U n i v e r s i t ä t ,

R.G. B r y a n t , C h e m i s t r y M i n n e s o t a , USA

of

West

Department, U n i v e r s i t y o f M i n n e s o t a ,

Medicine,

Minneapolis,

A . M . C a l l a h a n , B i o l o g y D e p a r t m e n t , U n i v e r s i t y o f U t a h , S a l t Lake U t a h , USA

City,

C.M. Cheney, Department o f B i o l o g y and M c C o l 1 u m - P r a t t H o p k i n s U n i v e r s i t y , B a l t i m o r e , M a r y l a n d , USA

The John

Institute,

A.F. Corin, Abteilung Molekulare Biologie, Max-Pianck-Institut s i k a l i s c h e Chemie, G ö t t i n g e n , West Germany F.W. D a h l q u i s t , I n s t i t u t e o f M o l e c u l a r B i o l o g y , Eugene, O r e g o n , USA

U n i v e r s i t y of

fur

Biophy-

Oregon,

List of Contributors

XVI

J.R. Dedman, Departments of Internal Medicine and Physiology and Cell logy, University of Texas Medical School, Houston, Texas, USA

Bio-

L.L.M. van Deenen, Laboratory of Biochemistry, State University of Utrecht, The Netherlands H. Eisenberg, Department of Polymer Research, The Weizmann Science, Rehovot, Israel

Institute of

D. Engelhardt-Altendorf, Fakultät für Biologie der Universität, West Germany

Konstanz,

F.R. Fairfield, Institute of Molecular Biology and Department of Chemistry, University of Oregon, Eugene, Oregon, USA S. Ghisla, Fakultät für Biologie der Universität, Konstanz, W e s t Germany R.B. Gregory, The Department of Laboratory Medicine and Pathology, University of Minnesota, Minneapolis, Minnesota, USA Y. Haik, Department of Polymer Research, The Weizmann Rehovot, Israel

Institute of Science,

L. Haiml, Institute of Molecular Biology, Austrian Academy of Sciences, Salzburg, Austria D.K. Hawley, Department of Biological Pittsburgh, Pennsylvania, USA

Sciences, Carnegie-Mellon

University,

P.H. von Hippel, Institute of Molecular Biology and Department of Chemistry, University of Oregon, Eugene, Oregon, USA S.E. Houts, Biology Department, University of Utah, Salt Lake City, Utah, USA R. Huber, Max-Pianck-Institut für Biochemie, Martinsried bei München, W e s t Germany F. Hucho,

Institut für Biochemie der Freien Universität, Berlin, Germany

A. Hutticher, Institute of Molecular Biology, Austrian Academy of Sciences, Salzburg, Austria R. Jaenicke, Institut für Biophysik und Physikalische Biochemie der Universität, Regensburg, West Germany T.M. Jovin, Abteilung Molekulare Biologie, Max-Planck-Institut für Biophysikalische Chemie, Göttingen, West Germany M.R. Kehry, Institute of Molecular Biology, University of Oregon, Eugene, Oregon, USA C. Kennedy, Agricultural Research Council, Unit of Nitrogen University of Sussex, Brighton, England S. Khan, Division of Biology 216-76, California Pasadena, California, USA

Fixation,

Institute of Technology,

J.W. Kijne, Botanisch Laboratorium, Rijksuniversiteit Leiden, The Netherlands R. Knippers, Fakultät für Biologie der Universität, Konstanz, W e s t Germany A. Koltmann, Fakultät für Biologie der Universität, Konstanz, West Germany

L i s t of

XVII

Contributors

W.N. K o n i n g s , Department o f M i c r o b i o l o g y , U n i v e r s i t y o f Groningen, Haren, The Netherlands G. K r e i l , Salzburg,

I n s t i t u t e o f M o l e c u l a r B i o l o g y , A u s t r i a n Academy o f Austria

Sciences,

A. K r i k o s , Department o f B i o l o g y , U n i v e r s i t y o f C a l i f o r n i a , San Diego, La J o l l a , C a l i f o r n i a , USA P. Kroneck, F a k u l t ä t f ü r B i o l o g i e der U n i v e r s i t ä t , Konstanz, West Germany R.G. Lageveen, Department o f P h y s i c a l C h e m i s t r y , U n i v e r s i t y o f Groningen, The Netherlands L. L a u f f e r ,

I n s t i t u t f ü r Biochemie der F r e i e n U n i v e r s i t ä t , B e r l i n , Germany

J.W. L e n g e i e r , I n s t i t u t f ü r Biochemie, Genetik und M i k r o b i o l o a i e der U n i v e r s i t ä t , Regensburg, West Germany R.M. Macnab, Department o f M o l e c u l a r B i o p h y s i c s and B i o c h e m i s t r y , U n i v e r s i t y , New Haven, C o n n e c t i c u t , USA

Yale

M. Manson, F a k u l t ä t f ü r B i o l o g i e . d e r U n i v e r s i t ä t , Konstanz, West Germany V. Massey, Department o f B i o l o g i c a l Ann A r b o r , M i c h i g a n , USA

Chemistry, The U n i v e r s i t y o f M i c h i g a n ,

E.D. M a t a y o s h i , A b t e i l u n g M o l e k u l a r e B i o l o g i e , Max P l a n c k - I n s t i t u t B i o p h y s i k a l i s c h e Chemie, G ö t t i n g e n , West Germany N. Mazurek, Department o f Chemical S c i e n c e , Rehovot, I s r a e l

für

Immunology, The Weizmann I n s t i t u t e o f

W.R. McClure, Department of B i o l o g i c a l P i t t s b u r g h , P e n n s y l v a n i a , USA

Sciences, Carnegie-Mellon

University,

C. M o l l a y , I n s t i t u t e o f M o l e c u l a r B i o l o g y , A u s t r i a n Academy o f S c i e n c e s , Salzburg, Austria I . Moura, Gray Freshwater B i o l o g i c a l I n s t i t u t e , M i n n e s o t a , USA and Centro de Quimica E s t r u t u r a l , Complexo I , I . S . T . , L i s b o n , P o r t u g a l J . J . G . Moura, Gray Freshwater B i o l o g i c a l I n s t i t u t e , M i n n e s o t a , USA and Centro de Quimica E s t r u t u r a l , Complexo I , I . S . T . , L i s b o n , Portugal P. Muhn, I n s t i t u t f ü r Biochemie der F r e i e n U n i v e r s i t ä t , B e r l i n , Germany F. M ü l l e r , Department o f B i o c h e m i s t r y , A g r i c u l t u r a l The Netherlands

U n i v e r s i t y , Wageningen,

R . P . M u n j a a l , Departments o f I n t e r n a l Medicine and P h y s i o l o g y and C e l l l o g y , U n i v e r s i t y o f Texas Medical S c h o o l , Houston, Texas, USA

Bio-

N. Mutoh, Department o f B i o l o g y , U n i v e r s i t y o f C a l i f o r n i a , San Diego, La J o l l a , C a l i f o r n i a , USA W. Neupert, I n s t i t u t f ü r Biochemie, Abt. Biochemie I I der G ö t t i n g e n , West Germany

Universität,

J . S . P a r k i n s o n , B i o l o g y Department, U n i v e r s i t y o f Utah, S a l t Lake C i t y , Utah, USA A. Pecher, I n s t i t u t f ü r Biochemie, Genetik und M i k r o b i o l o g i e der t ä t , Regensburg, West Germany

Universi-

XVIII

L i s t of

I . P e c h t , Department o f Chemical Science, Rehovot, I s r a e l

Immunology, The Weizmann

N.H.G. P e n n e r s , Department o f B i o c h e m i s t r y , A g r i c u l t u r a l W a g e n i n g e n , The N e t h e r l a n d s F.M. P o h l ,

Fakultät

f ü r B i o l o g i e der U n i v e r s i t ä t ,

B . Poolman, Department o f M i c r o b i o l o g y , The N e t h e r l a n d s

Contributors

Institute University,

K o n s t a n z , West Germany

U n i v e r s i t y of Groningen,

Haren,

I . R e n n e r , I n s t i t u t f ü r B i o c h e m i e , G e n e t i k und M i k r o b i o l o g i e der t ä t , R e g e n s b u r g , West Germany M. R o b e r t - N i c o u d , L e h r s t u h l G ö t t i n g e n , West Germany

für Entwicklungsphysiologie

G.T. R o b i l l a r d , Department o f P h y s i c a l The N e t h e r l a n d s

Chemistry,

R.L. Robson, A g r i c u l t u r a l Research C o u n c i l , U n i v e r s i t y of Sussex, B r i g h t o n , England

der

W.H. S a w y e r , Department o f B i o c h e m i s t r y ,

University of

Unit of Nitrogen

Groningen,

Fixation,

Fakultät

University

of Melbourne,

Rijksuniversiteit

f ü r B i o l o g i e der U n i v e r s i t ä t ,

M.A. S h e a , Department o f B i o l o g y and M c C o l 1 u m - P r a t t H o p k i n s U n i v e r s i t y , B a l t i m o r e , M a r y l a n d , USA

M.K. Slocum, B i o l o g y Department, U n i v e r s i t y o f Utah, S a l t USA

Lake C i t y ,

U. V i l a s , Salzburg,

I n s t i t u t e of Molecular Austria

San

Science,

Agricultural

Utah,

Germany

University,

A . J . W . G . V i s s e r , Department o f B i o c h e m i s t r y , A g r i c u l t u r a l W a g e n i n g e n , The N e t h e r l a n d s

Utah,

Lake C i t y ,

B i o l o g y , A u s t r i a n Academy o f

G. Voordouw, Department o f B i o c h e m i s t r y , n i n g e n , The N e t h e r l a n d s

Germany

Diego,

K o n s t a n z , West

C. V e e g e r , Department o f B i o c h e m i s t r y , A g r i c u l t u r a l The N e t h e r l a n d s

Leiden.

I n s t i t u t e , The John

M. S i m o n , Department o f B i o l o g y , U n i v e r s i t y o f C a l i f o r n i a , La J o l l a , C a l i f o r n i a , USA

f ü r B i o l o g i e der U n i v e r s i t ä t ,

Australia

I n s t i t u t e of

B i o l o g y Department, U n i v e r s i t y of Utah, S a l t

H. Sund, F a k u l t ä t

Insti-

K o n s t a n z , West

D. S e g e r , Department o f Polymer R e s e a r c h , The Weizmann Rehovot, I s r a e l

D. S h e r r i s , USA

University

Immunology, The Weizmann

I . A . M . van der S c h a a l , B o t a n i s c h L a b o r a t o r i u m , The N e t h e r l a n d s H. S c h w e i z e r ,

Universi-

Universität,

A . R o s e n b e r g , Department o f L a b o r a t o r y M e d i c i n e and P a t h o l o g y , o f M i n n e s o t a , M i n n e a p o l i s , M i n n e s o t a , USA R. S a g i - E i s e n b e r g , Department o f Chemical tute of Science, Rehovot, I s r a e l

of

Wageningen,

Sciences,

University,

University,

Wage-

L i s t of

XIX

Contributors

J . Westerman, L a b o r a t o r y o f B i o c h e m i s t r y , The N e t h e r l a n d s

State U n i v e r s i t y of

Utrecht,

K.W.A. W i r t z , L a b o r a t o r y o f B i o c h e m i s t r y , The N e t h e r l a n d s

State U n i v e r s i t y of

Utrecht,

A . V . X a v i e r , Gray F r e s h w a t e r B i o l o g i c a J I n s t i t u t e , M i n n e s o t a , USA and Ce C e n t r o de Q u i m i c a E s t r u t u r a l , Complexo I , I . S . T . , L i s b o n , P o r t u g a l D.A. Z a r l i n g , A b t e i l u n g Molekulare B i o l o g i e , M a x - P 1 a n c k - I n s t i t u t B i o p h y s i k a 1 i s c h e Chemie, G o t t i n g e n , West Germany R. Z i d o v e t z k i , C h e m i s t r y D i v i s i o n , P a s a d e n a , C a l i f o r n i a , USA

California

I n s t i t u t e of

fur

Technology,

SECTION I MOBILITY OF BIOPOLYMERS Chairmen:

H.

Eisenberg

and

F.M.

Pohl

SPECIFICITY OF CATALYSIS BY FLAVOPROTEINS

Vincent Massey Department of Biological Chemistry, University of Michigan Ann Arbor, Michigan 48109, U.S.A.

Anybody who has worked with a flavoprotein must have been impressed by the often enormous differences in properties of the enzyme-bound flavin and the same coenzyme free in solution.

Similarly, anybody who has worked with

more than one such enzyme also becomes impressed by the differences between different flavoproteins. For example, in the oxidized state, free flavins have an intense yellow-green fluorescence, which is sometimes enhanced, but mostly quenched, partially or completely, on binding to a particular protein.

In the reduced state, free flavins have a very weak fluorescence

visible only at sub-zero temperatures, whereas many flavoproteins are quite strongly fluorescent in the reduced state (1). Free reduced flavins also react readily with oxygen, in a complex series of reactions involving flavin radical and superoxide radical, yielding finally oxidized flavin and H 2 02 (2-4).

Some flavoproteins retain the ability to react rapidly with 0 2 ,

although the pathway and the fate of 0 2 in the reaction may differ; on the other hand many other flavoproteins have lost almost completely the reactivity with 0 2 .

Free flavins also show a limited thermodynamic

stabilization of the radical state, with the radical having a pK 8.5: F1

ox

+ F1

F1H- ^

red H 2v

~ s

2

F1H

1

'

Fi~ + H+

> 2)

With free flavins the very rapid equilibrium of equation 1) lies heavily to the left, so that an equimolar mixture of oxidized and reduced flavin yields at neutral pH approximately 5% radical (5). flavoproteins, a wide variety of responses is found.

With

Owing presumably to

steric and accessibility constraints, both the forward and reverse rates of equation 1 are generally slowed dramatically, so that it is sometimes difficult to determine if a given flavoprotein radical is stabilized thermodynamically or kinetically.

However, with good anaerobiosis and

Mobility and Recognition in Cell Biology © 1983 by Walter de Gruyter & Co., Berlin • New York

V. Massey

4

patience this question can be answered by mixing equimolar concentrations of oxidized and reduced flavoproteins; in the case of thermodynamic stabilization of the radical state the formation of flavoprotein radical is observed, and often the equilibrium lieS heavily in favor of radical formation.

If no radical is produced under these conditions over a period

of several days there is presumably no thermodynamic stabilization of the radical state.

In such cases considerable amounts of radical may be found

on one-electron reduction of the flavoprotein, due to kinetic stabilization, but then one can observe the dismutation to yield (often very slowly) a mixture of oxidized and reduced enzyme.

In practically all cases where a

flavoprotein radical is observed, the pK is greatly perturbed, so that over the whole pH-range of stability of the particular protein either the blue neutral semiquinone or the red anion semiquinone is stabilized (6). From this brief description it is obvious that the particular environment in which the flavin is located in a given flavoprotein must determine to a large extent the properties and reactivity of the bound coenzyme.

The idea that within a particular class of flavoproteins there

might be common directive influences, differing from those in another class, arose with some findings which we made about 12 years ago.

Thus it was

found that most enzymes of the oxidase class, i.e., those which react rapidly with 0 2 , were found to stabilize the red anionic flavin radical, to stabilize the adduct of sulfite at the flavin N(5)-position, and on reaction of the reduced form with 0 2 to produce oxidized flavoenzyme and H 2 0 2 without any detectable intermediates.

On the

other hand, many enzymes of the "dehydrogenase" class, i.e., those which had lost the ability to use 0 2 efficiently as an acceptor in catalysis, were found to stabilize the blue neutral flavin radical, failed to react with sulfite, even at high concentrations, and on reaction of the reduced form with 0 2 gave rise to the neutral flavin radical and 0 2 as intermediates.

It was also found that in general there was a reciprocal

relationship between the ability to react rapidly with 0 2 or with one electron acceptors such as ferricyanide or cytochrome c; the "oxidases" reacting very slowly with one-electron acceptors and the "dehydrogenases" reacting very quickly (7,8). These correlations excited not only us, but also aroused considerable

S p e c i f i c i t y o f C a t a l y s i s by

5

F1avoproteins

interest in Peter Henmerich, who was always on the lookout for order in nature.

Thus it came about that he took up an abiding interest in

classification of flavoproteins, a subject which occupied his attention right up to his death.

This has led to a more logical and accurate

classification, with five main classes (9»10) as follows: Class 1 Transhydrogenases, carrying out dehydrogenation of one substrate (2e~-oxidation) and "rehydrogenation" of another (2e~reduction).

This group can be subdivided further depending on whether the

centers involved in the hydrogen transfer are carbon, nitrogen or sulfur atoms. Class Z Dehydrogenase-oxidase.

These enzymes carry out dehydrogenation of

one substrate, with the 2e~-reduced flavin of the enzyme being reoxidized by 0 2 , with the production of H 2 0 2 .

These.are the

classical oxidases, and it is convenient to retain the simple term "oxidase." Class 3. Dehydrogenase-oxygenase.

Again, the primary reaction is

dehydrogenation of one substrate (2e~-oxidation) and utilization of 0 2 as another substrate.

Now, however, the oxygen molecule is split,

one half being incorporated into the first (or yet another) substrate, the other half being reduced to H 2 0.

There are two main subdivisions of

this class, the "internal monooxygenases". where the substrate to be oxygenated also supplies the reducing equivalents to form the obligatory 2e"-reduced flavin intermediate, and the "external monooxygenases", where an external source of reducing equivalents (NADH or NADPH) is required, and where the oxygen atom is incorporated into a third substrate. The internal monooxygenases appear in fact appear to belong better to Class 2, the oxygenase function being due to the H 2 0 2 product being slow to leave the reoxidized enzyme, and reacting with the dehydrogenated product while both are still enzyme-bound. Class 4 Dehydrogenase-electron transferase enzymes, again involving a 2e~-dehydrogenation of one substrate and reoxidation of the reduced flavin in 1e~ steps with obligatory 1e~-acceptors, such as ironsulfur proteins and cytochromes.

This class also includes enzymes which

work in the opposite direction, such as ferredoxin-NADP reductase, where the flavin is reduced by single electron steps to the fully reduced form, which

6

V. Massey

is then reoxidized in a 2e"-equivalent step, converting NADP to NADPH. Class 5. Pure electron transferases, where both reduction and oxidation of the flavin appear to be accomplished in 1e~-transfers. The flavodoxins are examples of this class; Our initial correlations of properties and catalytic function may now be reexamined in light of this more detailed and logical classification. Despite a few exceptions, which may have interesting explanations and implications, the correlations have stood the test of time very well, with the qualification that for the original "dehydrogenases" we now read the electron transferases of Classes 4 and 5, and that the oxygenases of Class 3 have very different properties from the oxidases of Class 2.

(In general

they do not stabilize any radical form, do not form sulfite adducts, and as will be discussed later, react with

in a fashion which is

observedly different from other flavoproteins.)

Unfortunately the

transhydrogenases of Class 1 are the least well-characterized as a group. They form either neutral or anion flavin radicals on one-electron reduction, but in general these appear to be stabilized kinetically rather than thermodynamically. As a group they fail to form flavin N(5)-sulfite adducts, and they vary in their capacity to form

versus HgOg.

While it was postulated early that these correlations may be due to specific interactions between protein and the flavin N(5)- or N(1)- C(2a)positions (11,12), it is only in the last few years that strong experimental support has been forthcoming. On the basis of the similarity in spectral properties between the blue flavoprotein radicals and the stable N(5)substituted radicals, it was proposed that protein-stabilization of the neutral radical involved hydrogen bonding interaction between the flavin N(5)-H and some protein residue:

protein B

S p e c i f i c i t y of C a t a l y s i s by

7

Flavoproteins

This postulate received very nice confirmation with the determination of the three dimensional structure of the electron transferase, flavodoxin, where it was found for the blue semiquinoid state that the flavin N(5) atom was in hydrogen bonding distance from a carbonyl oxygen of the polypeptide backbone (13). Further corroboration comes from studies with artificial flavins which serve as "indicators" of the protein environment of the flavin.

The

principle was first ennunciate by Ghisla and colleagues for the naturally occurring 6-hydroxy- and 8-hydroxyflavins, which in their oxidized anionic states exist mainly in the benzoquinoid forms, with the negative charge localized in the N(1)- C(2a)-region of the flavin (11-16):

?

R

©

The pK values for ionization are 7-1 for 6-hydroxyflavin (14) and 1.8 for 8-hydroxyflavin (15), and in both cases there are marked changes in visible spectrum on ionization.

In both cases, a positively charged protein

residue in the region of the flavin N(1)-C(2a) locus would be expected to stabilize the benzoquinoid form and lower the pK of the enzyme-bound flavin. The existence of a protein negatively-charged residue in the vicinity of the flavin 6- or 8- positions respectively would of course be expected to favor the benzoquinoid structure also, but now the pK would be raised rather than lowered. An even more sensitive indicator has become available recently in the form of 8-mercaptoflavins (17).

In this case the pK is very low, pH 3.8,

and in free solution the dominant from of the anion is the 8-thiolate form C, rather than the benzoquinoid form D.

Thus, with rare exceptions with

proteins, the anionic form of the oxidized flavin will be encountered, and the only ways in which the benzoquinoid form would be stabilized would be through interaction with a positively charged protein residue in the

8

V. Massey

vicinity of the flavin N(1)- C(2ct)-locus, or through charge repulsion by a negatively-charged protein residue in the neighborhood of the flavin 8position. I

H

As indicated from model studies the two forms C and D are dramatically different in color, C being bright red and D blue-purple.

Both forms are

found in various proteins, as illustrated in Figure 1, where 8mercaptoflavodoxin is typical of the 8-thiolate (form C) and 8-mercapto lactate oxidase typical of the benzoquinoid form D.

400

500

600

700

Wavelength (nm) Fig. 1. Spectra typical of 8-mercaptoflavoproteins.

Specificity

of

Catalysis

by

9

F1avoproteins

It was very gratifying to observe that all the oxidases of Class 2 stabilize the benzoquinoid form of 8-mercaptoflavin, whereas with electron transferases of classes 4 and 5 the 8-thiolate form is encountered (as this is the predominant form of 8-mercaptoflavin in free solution, finding it in a flavoprotein does not imply any stabilization by the protein).

The

oxygenases of Class 3 do not show any marked stabilization of the benzoquinoid form, although the spectrum does appear to be shifted more toward long wavelengths than with the electron transferases.

As usual, the

transhydrogenases (Class 1) yield somewhat variable results, although the benzoquinoid form does appear to be favored (10,17). These results, and corroborating ones with enzymes substituted with 6hydroxy- and 8-hydroxyflavins (18), suggest that oxidases all share the common structural feature of having a protein positively charged group in the vicinity of the flavin N(1)- C(2a)-locus.

This positive charge would

not be expected to have any pronounced effect with the oxidized native coenzyme, except perhaps to lower somewhat the pK of ionization of the flavin N(3)H.

With the 6- and 8-substituted flavins discussed above, it

would stabilize the benzoquinoid form, as observed.

With the semiquinoid

and fully reduced forms of native flavin, it would stabilize the anion state, lowering the pK so that over the usual pH range of stability of proteins, only the anion form would be observed: -fB-protein I H

R

.0

+

HB-protein R

0

0 The existence of such a positively charged group would also have an inductive effect on the nucleophilic attack of sulfite at the flavin N(5)position, and stabilize this adduct once formed (12).

The mechanistic

significance of this interaction is that in the same way it would favor attack of a transient carbanion form of the normal substrate at the flavin N(5)-position, and stabilize this adduct to such an extent that it could be

V. Massey

10

observed, as has been done with lactate oxidase and where considerable evidence for a carbanion mechanism exists.

Thus it is possible that the

real correlation of sulfite adduct formation lies with a dehydrogenation mechanism involving a substrate carbanion (19,20) rather than with oxygen reactivity. ©B-protein

Another interesting correlation which has recently been made is that all flavoproteins which contain the flavin covalently linked to the protein, yield on partial reduction the red semiquinone anion, irrespective of whether the enzyme reacts rapidly with 0 2 , or not (21).

This may be

connected with the route of biosynthesis of the covalent linkage, which is always at the flavin 8-" Q

A S E C T I O N OF T H E AND T H E REFINED AROUND GLY 1 9 AT

F I N A L F O U R I E R MAP M O D E L OF T R Y P S I N O G E N 1/3 K

A S E C T I O N OF T H E F I N A L F O U R I E R M A P A N D T H E R E F I N E D M O D E L OF T R Y P S I N O G E N AROUND G L Y 1 9 AT 1 0 3 K

Figure 6. Comparison of the Fourier maps of room temperature (a), 170° K (b), and 100° vicinity of the N-terminus (9).

trypginogen at K (c) in the

R. Huber and W.S. B e n n e t t ,

30

disulfide bond to form the group -Cys 191-''"^lnHg-Cys 220used in these experiments. Fig. 7

compares

the

trypsinogen

ternary

complex

with

that

of

the

trypsinogen, PTI, and Ile-Val. complex, indicating

the

anisotropy

intramolecular

In

vanishes

and

no

dynamics

in

in

of

11

this

to

trypsin-like state. Motion in this time

of

ternary

trypsinogen

motion

of

the

in

the

free

range

in

the

nsec

time

was

between

the

free

reorientational

^-emitter with a correlation time molecule

contrast

spectrum

Jr.

range

is

consistent

with the Interconversion of different conformers. The manifestation of disorder in crystalline proteins is strongly influenced by lattice packing. The intramolecular crystal forces may stabilize a particular conformation out of the ensemble of conformers prevailing in solution and simulate the picture of a rigid single conformation. There are several well documented examples of chemically but not crystallographically identical molecules which differ strongly in certain regions with respect to segmental disorder. Quite generally tight packing increases order. The influence of crystal packing on order will be illustrated with the Fc fragment later in this article. Other examples are ovomucoid (7,11) and kallikrein (12,46). Stabilization of a certain conformation may explain why chymotrypsinogen (13) and trypsinogen crystallized under conditions different from those of the structure described above (14) show less disorder in the crystalline state. An order-disorder transition similar to trypsinogen

has

oberved in tobacco mosaic virus (TMV) protein, where a

been stable

fold of the RNA binding segment exists only in the presence of RNA

(15,16).

A

similar

phenomenon

pancreatic prophospholipase (17) and disordered regions of the tyrosyl (18), although the

distinction

appears might

tRNA between

to

occur

in

for

the

account

synthetase static

and

structure dynamic

disorder cannot be made in either of the latter examples.

Functional S i g n i f i c a n c e of F l e x i b i l i t y

«9m

w^hoTO 293 K 10

10

fllP -5

O

31

in P r o t e i n s

0

-5

20

10

TIME lns*c)

0

20

TIME InMc)

Figure 7. Plots of ^-correlation anisotropy versus time mercury trypsinogen (HgTg) and HgTg-PTX-Ile-Val (10).

for

The structural features of segmental flexibility are reflected to some extent in the

amino

acid

domains in trypsinogen and TMV

sequence:

lack

the

aromatic

transition (hinge) region is rich in glycine

disordered

residues. residues.

The These

observations are also valid for the hinge segment in molecules with domain motion discussed below.

Immunoglobulins We now focus on a different

type

of

flexibility,

independent rigid domains move relative to

one

in

another

which with

considerable freedom. Very little conformational change in the connecting segment occurs in such motions. This seems to be quite common phenomenon but has been studied in the

example

of

the

scheme based on repeated

immunoglobulins. structural

thoroughly An

domains

proposed for immunoglobulins on the basis of sequence and is depicted in fig. 8 for the

a

only

organizational was the

IgG

originally amino

class.

acid There

32

R. Huber and W.S. B e n n e t t ,

are two heavy chains and two light chains of molecular 50,000 and 25,000 and subdivided

in

respectively. These domains

linked

are

polypeptide strands. A .wealth of

four

and by

crystal

available (for recent reviews,

see

weight

two

domains,

short,

extended

structure

references

Comparative studies of the same or closely

Jr.

data

19

and

related

is 20).

molecules

are of particular interest in the context of flexibility. The first direct observation of domain flexibility in

protein

crystals came from a study of the IgG Kol crystals (21,22). In these crystals the Fab parts are well ordered, but the Fc part is disordered and does not contribute to the electron

density

(23). The crystal packing is solely determined by the Fab arms with the Fc parts occupying a central channel (fig. 9). The (Fab)j fragment of the Kol molecule, a pepsin

degradation

product cleaved C-terminal to the hinge segment, isomorphously

to

the

intact

molecule.

crystallizes

Analysis

of

the

diffraction intensities of these crystals and comparison

with

those of the intact crystals allowed a quantitative

study

the effect of the Fc portion on the diffraction pattern

of

(23).

If the Fc region were vibrating harmonically around

a

equilibrium position in the crystals of the

molecule,

we would expect to see substantial

intensity

least in the innermost region of the hand,

if

the

Fc

segment

were

intact

differences

pattern.

On

distributed

the

among

different sites, the intensity differences would be case.

As

with

the

activation

domain

in

at

other several

small

all resolutions. The analysis clearly shows the latter the

single

to

at be

trypsinogen

crystals, static disorder prevails for the Fc portion

in

Kol

crystals. The polypeptide segment

linking

conformation consisting of a short

Fab

and

Fc

has

polyproline

a

double

unique helix

with disulfide crosslinks flanked on the N-terminal side by an open turn of helix (23), Flexibility in

Kol

crystals

starts

Functional

S i g n i f i c a n c e of F l e x i b i l i t y

in P r o t e i n s

33

Figure 8. Schematic diagram of an IgG molecule. The heavy chain consists of four domains, the light chain of two. The N-terminal tip of the Fab arms is the antigen binding site. The domains aggregate in the indicated fashion; CH2, to which carbohydrate is bound, is an exceptional single domain.

Figure 9. Crystal packing of Kol IgG projected down the 3 t axis. The three solid pairs of recangles are the (Fab)t components; the Fc components are disordered in the space around the central 3* axis. The open rectangles are related to the solid ones by unit-cell translations (21).

34

L-chain Figure 10. The h i n g e segment in Kol IgG. Filled bonds, light chains; open bonds, h e a v y chain. The amino acid sequence of the h e a v y chain segment is Cys 527-Pro 528-Pro 529-Cys 530. Residues 522 to 526 form an open turn of helix (23). abruptly at the C-terminus o f the polyproline double helix i.e., after the last P o f the - C - P - P - C - P - segment The open turn of helix stabilizing movement

formed by residues

intramolecular contacts

in

solution.

ultracentrifuge

Indeed

522

and

may

electron

(fig.

to

10).

526

allow

lacks

Fab

microscopic

studies o f the complexes b e t w e e n IgG

arm and

antibody

and bivalent h a p t e n s show a large range o f different a n g l e s o f the Fab

arms

differences

around

for these

order-disorder

the

hinge

anugular

and

suggest

positions

energy

The

abrupt

(24).

transition seen in Kol is comparable

seen the previous examples of trypsinogen b e g i n s within a single residue,

small

peptide,

which

and is

TMV.

often

suggesting m o t i o n around a well-defined

to

those

Disorder a

hinge.

glycine

35

Figure 11.. The Fab parts of Kol IgG, Kol Fab and McPC 603 Fab looking along an axis through the switch peptides. The Kol molecules are characterized by an open elbow angle, while McPC 603 forms a closed structure (22). This aspect of flexibility is particularly clear in the motion of the Fab segment. In

fig.

conformations in intact Kol crystals,

11

we

Kol

compare Fab

elbow

the

Fab

fragment

and

McPc 603 Fab fragment (23,25). They differ in elbow

angle

by

R. Huber and W.S. B e n n e t t ,

36 up to 60°.

A closed elbow,

as in McPc 603,

Jr.

is also found in

the other Fab or Fab-like molecules whose structures are known (26,27). Fig. 12 compares hinge

conformations

of

Kol

(open

elbow, ref. 23) and New (closed elbow, ref. 26) in detail. The difference in

conformation

is ' very

small

and

essentially

confined to residues Val 107 to Gly 109 of the light chain and Val 416 to Ser 418 of the heavy chain, which define the hinge. A similar but much smaller conformational difference is observed in Fc with respect to the relative arrangement of the two chemically identical but crystallographically independent CH3 and CH2 domains. An angular difference of about 7° was observed (28). This difference in CH2-CH3 arrangement must be a consequence of crystal packing. In addition CH2 shows asymmetry in the degree of disorder of some segments at one end of the domain. These segments are ordered where they are involved in crystal-lattice contacts but disordered if they face the solvent. Electron microscopy has indicated much larger flexibility in Fc, although the effects of sample preparation are difficult to evaluate in this case. The dynamic aspects of immunoglobulin flexibility were studied as early as in 1970 by

nanosecond

fluorescence

spectroscopy

(29). Although intramolecular motion was detected, the

nature

of this motion was (and is) debated. It now seems likely the larger of the two correlation times observed in the

that decay

of anisotropy in the bound hapten's fluorescence is due to Fab arm motion. The

faster

correlation

time

stems

from

elbow

motion within the Fab arm (29-32). The time constants observed are

consistent

with

the

proposal

that

domain

motion

in

immunoglobulins is controlled by rotational diffusion (33). In the crystals certain conformations are evidently frozen by lattice forces. The extremely open elbow conformation in Kol may also be related to the antigen-antibody-like packing and reflect a functional state (23). In some favourable cases,

Functional

Significance of F l e x i b i l i t y

in

Proteins

Figure 12. Comparison of the switch peptides of the heavy chains of intact Kol and Fab New (23). Filled bonds, Kol; open bonds, New.

such as the crystals of intact IgG Kol, allows multiple conformations

to

occur

the in

crystal the

packing

crystalline

state and thus allows flexibility to be "observed" in the form of disorder. Flexibility in the immunoglobulin molecule clearly allows the molecule to adapt to the variable disposition of antigenic sites on cell surfaces. In addition the extreme arrangements of closed and open elbow in Fab, corresponding to the existence or absence of contacts between variable and constant domains, may reflect different functional states of the immunoglobulin molecule, i.e., unliganded and liganded states (23). However, the fluorescence depolarization data discussed above clearly demonstrate flexible domain arrangement in hapten-antibody complexes; whatever the exact nature of the motion, this observation, would seem to indicate that the energy barriers between different domain arrangements in immunoglobulins are small.

38

R. Huber and W.S. B e n n e t t ,

Jr.

Citrate Synthetase The relatively unhindered domain motion in immunoglobulins may be contrasted with the domain motion observed in some

enzymes

which we now wish to consider as our final example of

protein

flexibility. In these enzymes, the domain motion substrate binding during the catalytic cycle. has

been

observed

and

established

by

analyses of the different forms in yeast liver alcohol dehydrogenase and

appears

also

dehydrogenase Jansonius,

to

(47)

private

(35) and

occur

and

in

in

occurs

upon

The

phenomenon

crystal

structure

hexokinase

citrate

(41,34),

synthase

(36),

glyceraldehyde-3-phosphate

aspartate

communication).

aminotransferase

The

(H.

observation

that

kinases generally have a bilobal structure roughly similar that found in

hexokinase

has

led

to

the

suggestion

to that

analogous domain motions are a common feature of this class of enzymes

(37).

proposal

has

hexokinase,

Structural been

evidence

presented

phosphoglycerate

consistent

for

two

kinase

kinases

(42,43)

with

this

other

than

and

adenylate

kinase (44), while the proposal appears to

be

phosphofructokinase

small-angle

(45).

scattering experiments

Evidence

also

from

suggests

motion in arabinose binding protein

incorrect

ligand-induced

for X-ray

domain

(38).

We now concentrate on citrate synthase, the condensing

enzyme

which catalyses the reaction

A

between

oxaloacetate to form citrate.

acetyl-coehzyme

The molecule is a dimer

of two

identical subunits with 437 amino acid residues each (39). is a very

large

globular

molecule

that

is

formed

entirely of «^-helices (36). Fig. 13 is a schematic

and It

almost

representa-

tion of the helix arrangement in one subunit. The two subunits pack tightly via eight helices in an antiparallel

arrangement.

Each subunit folds into two domains, a large one mediating dimer aggregation and a small domain comprising helices N, 0, P, Q, and R.

of

about

110

the

residues

Functional

S i g n i f i c a n c e of F l e x i b i l i t y

in

39

Proteins

Figure 13. Schematic diagramme of the monomer of citrate synthase viewed from a point on the diad relating the two monomers. The insert indicates the residues within the helical regions A to T (36).

Citrate synthase has been crystallographically analysed in two forms which differ by the relative arrangement small domains as shown filling

in

representations.

fig. The

14

in

and

change

described to a good approximation as

rotation

domain by 18° around an

to

close

large

skeletal

structural

axis

of

space

can

of

residue

and be

the

small

274,

which

represents the hinge (fig. 15). However small deviations a rigid rotation of the structural change between summarizes the

CA

two

domains

open

deviations

of

(P4:open form, C2: closed form), form analysed

recently

(G.

and

are

also

closed large

including

Wiegand

and

involved

forms.

and a S.

Fig.

from in 16

small

domains

third

crystal

J.

Remington,

unpublished). The latter is also a closed crystal form has a dimer

in

the

subunits can also be compared; the

form.

asymmetric

As

the

unit,

the

differences C4/2).

two

between

reflect the relatively small influence of lattice the structure of the two domains (C4/1,

new them

packing It

is

on

clear

that the small domain has a much less rigid structure than the

C ° MODELS OF SUBUNIT, MONOCLINIC ( = ) TETRAGONAL FORMS (

AND

) OVERLAID.

jgg^

Figure 15. The large domains of the open and closed forms citrate synthetase are superimposed. The relative rotation the small domain is obvious (36).

large domain; it appears to respond to the domain

of of

arrangement

and to functional states of the enzyme by changes in

tertiary

structure. What is the functional significance of these molecular The open form (P4) crystallizes these crystals crack in

the

from

presence

closed form (C2) crystallizes when the

phosphate of

or

forms? citrate;

oxaloacetate.

products

The

citrate

and

CoA-SH or an analog of citroyl-CoA is present. The third

form

(C4) crystallizes with oxaloacetate. Neither the P4 nor the C2 crystals are enzymatically active, while the C4 crystals

have

not yet been tested. Citrate in C2 is bound in a cleft between the large and small domains and is completely enveloped

in

a

highly polar pocket by the domain closure. CoA-SH is bound

to

the small domain, and the cysteamine part comes very close

to

42

R. Huber and W.S. B e n n e t t ,

the bound citrate. Only in the closed form is the CoA

Jr.

binding

site completely formed, as contributions from the large domain of

the

other

subunit

are

required.

Domain

closure

thus

demonstrates

that

provides a better binding site for the cofactor. The binary complex with oxaloacetate (C4), oxaloacetate binds to the enzyme exactly as

citrate

the C2 crystals. Oxaloacetate binding apparently

does

in

suffices

to

induce domain closure. It is interesting that the oxaloacetate binding site involves residues that form the hinge between the two domains; these interactions quite likely contribute to the process by change.

which

In

oxaloacetate

addition,

oxaloacetate

is

the

triggers

binding

surrounded

and

shaped

residues and three charged

arginine

this binding site

closed

in

the

energetically unfavourable in

the

the

site

conformational

for by

citrate four

residues.

histidine

Formation

conformation absence

and

of

of

is

probably

the

counter-

charges provided by the negatively charged substrates. Kinetic studies

(40)

show

that

citrate

synthase

has

an

ordered

mechanism with oxaloacetate binding first and show very strong cooperativity between oxaloacetate and acetyl coenzyme A. This observation can be

understood

in

terms

of

the

structural

Catalytic action, condensation of oxaloacetate and

acetyl-CoA

features just discussed.

to form citryl-CoA and hydrolysis of citryl-CoA to citrate and CoA,

proceed

in

the

closed

form.

There

may

be

differences in structure between the form of the enzyme

subtle which

acts as a ligase and that which acts as a hydrolase. The small differences in the tertiary

structure

of

the

small

observed between the C2 and C4 crystal forms might be

domain related

to functional differences of this sort. The open form (P4) probably the

substrate

binding

and

product

release

Domain motion must occur in each catalytic cycle, i.e.

a

thousand times per second

It

under

optimal

conditions.

unclear however whether this is the rate limiting step in enzymatic reaction.

is

form. few is the

Functional

Significance of F l e x i b i l i t y

I C2

Sma 11 Domain C4/!

C4/2

P4

43

Proteins

I C4/I

I C4/2 I

0.72

0.56

0.54

2299

2336

2320

1.35

0.77

0.75

748

2341

2338

C2

P4

I

in

1.00

1.87

0.29

697

662

2360

0.99

1.86

0.32

697

662

666

Large Domain

Figure 16. A comparison of four different subunits in three crystal structures of citrate synthase (C2: the closed form with citrate and CoA bound; P4: the open form in phosphate; C4/1, C4/2: the two halves of the binary complex with oxaloacetate). Each box shows the r.m.s. distance between equivalent atoms, after atoms with d > 2 v were rejected. The number of equivalent atoms is also given (36).

As we have mentioned above, the occurrence

of

domain

motion

has been well established in a few examples. It is probably

a

widespread phenomenon, as it offers a unique way to

a

binding

site

well

shielded

from

the

create

surroundings

where

catalysis can proceed without the interference from water.

References 1.

Huber, R., Bode, W.: 114-122 (1978).

Accounts of Chemical

2.

Bode, W.: J. Mol. Biol. 127, 357-374

3.

Nolte, H. J., Neumann, E.: (1979).

4.

Perkins, S. J., Wuethrich, K.: J. Mol. Biol. (1980).

Biophys.

Research

11,

(1979). Chem.

10, 138,

253-260 43-64

44

R. Huber and W.S. Bennett, Jr.

5.

Bode, W., Huber, R.: FEBS Lett. 68, 231-236 (1976).

6.

Bode, W., Schwager, P., Huber, R.: 99-112 (1978).

7.

Weber, E., Papamokos, E., Bode, W., Huber, R., Kato, I., Laskowski, M., Jr.: J. Mol. Biol. 149, 109-123 (1981).

8.

Bolognesi, M., Gatti, G., Menegatti, E., Guarneri, M., Marquart, M., Papamokos, E., Huber, R.: J. Mol. Biol., submitted (1982).

9.

Walter, J., Steigemann, W., Singh, T. P., Bartunik, H., Bode, W., Huber, R.: Acta Cryst. B 38, in press (1982). Butz, T., Lerf, A., Huber, R.: Phys. Rev. Lett., in press (1982).

10.

J.

Mol.

Biol.

118,

11.

Papamoko s, E., Weber, E•, Bode, W., Huber, R•, Empie, W., Kato, I., Laskowski, M., Jr.: J. Mol. Biol. 158, press (1982).

12.

Bode, W., Chen, Z.: In: Advances in Experimental Medicine and Biology of Kinins, Vol. 3 (Dietze, Fritz, Haberland, and Beck, Eds.), Plenum Press, New York, 1982.

13.

Freer, S. T., Kraut, J., Robertus, J. D., Wright, H. T., Xuong, Ng. H. : Biochemistry 9^, 1997-2009 (1970). Kossiakoff, A. A., Chambers, J. L., Kay, L. M., Stroud, R.H.: Biochemistry 16, 654-664 (1977). Bloomer, A. C., Champness, J. N. , Bricogne, G., Staden, R., Klug, A.: Nature (Lond.) 276, 362-368 (1978).

14. 15. 16.

Stubbs, G., Warren, S., Holmes, K.: Nature 216-221 (1977).

17.

Dijkstra, B. W., Van Nes, Brandenburg, N. P., Hol, W. Cryst. B 38, 793-799 (1982).

18.

Irwin, M. J., Nyborg, J., Reid, B. R., Blow, Mol. Biol. 105, 577-586 (1976).

19.

Amzel, L. M., Poljak, R. J.: Ann. Rev. Biochem. 961-997 (1979). Huber, R.: Klin. Wochensch. 58, 1217-1231 (1980).

20. 21. 22. 23. 24.

G. G.

(Lond.)

M. in

267,

J. H., Kalk, K. H., J., Drenth, J.: Acta D.

M.:

J. 48,

Colman, P. M., Deisenhofer, J., Huber, R., Palm, W.: J. Mol. Biol. 100, 257-282 (1976). Huber, R., Deisenhofer, J., Colman, P. M., Matsushima, M., Palm, W.: Nature 264, 415-420 (1976). Marquart, M., Deisenhofer, J., Huber, R., Palm, W.: J. Mol. Biol. 141, 369-391 (1980). Schumaker, V. N., Seegan, G. W., Smith, C. A., Ma, S. K., Rodwell, J. D., Schumaker, M. F.: Mol. Immunol. 17, 413-423 (1980).

Functional

Significance of F l e x i b i l i t y

in

Proteins

45

25.

Segal, D. M., Padlan, E. A., Cohen, G. H., Rudikoff, S., Potter, M., Davies, D. R.: Proc. Nat. Acad. Sci., U.S.A. 71_, 4298-4302 (1974).

26.

Saul, F. A., Amzel, L. M., Poljak, R. J.: 253, 585-597 (1978).

27.

Ely, K. R., Firca, J. R., Williams, K. J., Aboia, E. Fenton, J. M., Schiffer, M., Panagiotopoulos, N. Edmundson, A.B.: Biochemistry 17, 158-167 (1978).

28.

Deisenhofer, J.: Biochemistry 20, 2361-2370 (1981).

29.

Yguerabide, J., Epstein, M. F., Stryer, L.: J. Mol. Biol. 51, 570-590 (1970).

30.

Hanson, D. C., Yguerabide, J., Biochemistry 20, 6842-6852 (1981).

31.

Pecht, I.: In: The Antigens, Vol. VI (M. Sela, Ed.), Academic Press, New York.San Francisco.London, 1982, p. 26.

32.

Reidler, J., Oi, V. T., Carlsen, W. F., Pecht, I., Herzenberg, L. A., Stryer, L. : Biophys. J. 3_3, 135a (1981).

33. 34.

McCammon, J. A., Karplus, M.: Nature 268, 765-766 (1977). Bennett, W. S., Jr., Steitz, T. A.: J. Mol. Biol. 140, 211-230 (1980).

35.

Eklund, H., Samama, J.-P., Wallen, L., Braenden, C.-I., Akeson, A., Jones, T. A.: J. Mol. Biol. 146, 561-587 (1981) .

36.

Remington, S. J., Wiegand, G., Huber, R.: J. 157, in press (1982).

37.

Anderson, C. M., Zucker, F. H., Steitz, 204, 375-380 (1979).

38.

Newcomer, M. E., Lewis, B. A., Quiocho, F. A.: Chem. 256, 13218-13222 (1981).

39.

Bloxham, D. P., Parmelee, D. C., Kumar, S., Wade, R. Ericsson, L. H., Neurath, H., Walsh, K. A., Titani, Proc. Natl. Acad. Sei. U.S.A. 78, 5381-5385 (1981).

40.

Johansson, C.-J., Pettersson, G.: Biochim. Biophys. 484, 208-215 (1977).

41.

Anderson, C. M., Stenkamp, R. E., McDonald, R. Steitz, T. A.: J. Mol. Biol. 123, 207-219 (1978).

C.,

42.

Banks, R. D., Blake, C. C. F., Evans, P. R., Haser, Rice, D. W., Hardy, G. W., Merrett, M., Phillips, A. Nature (Lond.) 279, 773-777 (1979).

R., W.:

43.

Pickover, C. A., McKay, D. B., Engelman, D. M., T. A.: J. Biol. Chem. 254, 11323-11329 (1979).

J. Biol. Chem.

Schumaker,

T.

V.

Mol. A.:

E., C.,

N.:

Biol. Science

J.

Biol. D., K.: Acta

Steitz,

R. Huber and W.S. Bennett, Jr.

46 44.

Pai, E. F., Sachsenheimer, W., Schirmer, R. G . E . : J. Mol. Biol. 114, 37-45 (1977).

H. ,

45.

Evans, P. R., Farrants, G. W., Hudson, Trans. R. Soc. Lond. B 293, 53-62 (1981).

46.

Bode, W., Chen, Z., Bartels, K., Kutzbach, C., Schmidt-Kastner, G., Bartunik, H.: J. Mol. Biol., submitted (1982).

47.

Dalziel, K., McFerran, N. V. , Wonacott, A. J.: Trans. R. Soc. Lond. B 293, 105-118 (1981).

P.

J.:

Schulz, Phil.

Phil.

Received June 7, 1982 DISCUSSION Von Hippel: Could you say something further about the changes in helix packing rules that your citrate synthetase structural data lead to? Huber: The helix packing rules have been derived for polyalanine models and lead to the concept of ridges into grooves or knobs into holes packing. The helix axes are inclined by seme angle (Chothia et al., Proc.Natl.Acad.Sci. USA (1977) 74, 4130-4134). In citrate synthase the helices are often kinked or bent smoothly over a large angle. The packing is often antiparallel. The reason may be the large number of large amino acids and the dominant interaction of the side chains. (Remington et al., J.Mol.Biol. (1982) 158, 111-152). 2+ Voordouw: In the literature binding of tvro Ca ions to trypsinogen has been described. In your scheme on the trypsinogen •*->- trypsin inhibitor interaction I saw only one Ca ion incorporated. What happened to the second ion? 2+ Huber: Trypsin and trypsinogen have a well established Ca binding site at the loop around residue 80. (Bode and Schwager (1975) J.Mol.Biol. 98, 693717). It influences slightly the thernodynamic parameters of trypsi^+(ogen)inhibitor interaction (Bode (1980) J.Mol.Biol.). The second weak Ca binding site is believed to be to sequence of aspartates at the N-terminal segment of trypsinogen. McClure: You oenmented on the itain chain recognition of the adenine residue CoA in citrate synthetase. Could you sketch the hydrogen bonding pattern seen, and in particular, whether the amino group or N(7) of adenine is involved? Huber: N(7) is involved in an intra-CoA-hydrogen bond with O-H 52 (Remington et al., J.Mol.Biol. (1982) 158, 111-152). The amino group binds to two carbonyl groups of the nain chain, (see figure 7a of the reference above).

Functional

S i g n i f i c a n c e of F l e x i b i l i t y

in

Proteins

47

McClure; Some of my crystallographer friends resist the notion of more than tvo well-defined structural states of proteins {i.e. hemoglobin remains the paradigm). Obviously your data on citrate synthetase provide a good counterexample to this via*. Hew many other proteins have been shown, structurally (i.e. X-ray) to exist in more than two ligand-induced states? Huber: Also citrate synthase at first sight, corresponds to a two state model: open and closed form. However, we have analysed two different closed forms, one with product bound, the other with oxaloacetate and acetonyl-CaA. These differ in the detailed structure of the small danain. The small domain is soft and responds to the functional states of the enzyme. (See reference above). Jaenicke: Is anything known with respect to the structure of the carbohydrate part of IgG? Is it highly flexible, or does it serve as a recognition site, e.g. in connection with complement fixation? Huber: The carbohydrate structure is knewn in atonic detail (Deisenhofer et al., Hoppe Seyler's Z.Physiol.Chem. (1976) 357, 1421-1434; Deisenhofer, J.Biochemistry (1981) 20, 2361-2370; Huber, R. Klinische Wbchenschrift (1980); Marquant N. and Deisenhofer, J. Imnunology Today (1982) 3, 160). It is rigid, except at the termini and interacts extensively with the protein. It is probably not involved directly in interaction with complement. Sundi Is the coenzyme bound to citrate synthase in the open or in the closed form? Huber: The coenzyme is bound only to the closed form (Remington et al., J. Mal.Biol. (1982) 158, 111-152). Sund: The phosphate group of the coenzyme is bound to another polypeptide chain of citrate synthase than the other parts of the coenzyme molecule. This leads to the question whether each active site is constituted by both polypeptide chains or mainly located in one polypeptide chain? Huber: The coenzyme A binding site is made up mainly frcm residues of the large and the small domain. The 3'-phosphate interacts also with an arginine at the other subunit (Remington et al., J.Mai.Biol. (1982) 158, 111-152). Pecht: Do we have any further information concerning the flexibility of the CL 2 domains emerging from other crystal structures of immunoglobulin fragments? Huber: We have information from tMD different crystal structures: human F c and protein A-F (Deisenhofer, Biochemistry (1981) 20, 2361-2370). No other detailed structure is knewn yet, but the crystal structure of rabbit F c is being studied intensively by the Oxford group.

48

R. Huber and W.S. Bennett, Jr.

Neupert: At which site of citrate synthase is ATP bound, which is believed to have a regulatory function? Huber: We have no direct evidence about ATP binding to citrate synthase. Presumably it binds to the CoA binding site in the closed form of the enzyme. Robillard: How similar are the structures of the trypsin-trypsin inhibitor complex and the trypsinogen trypsin inhibitor complex after the exogenous peptide is added back? Huber: They are identical in structure and thermal parameters except at the point of covalent linkage of the Ile-Val dipeptide in the trypsin-PTI complex (Huber and Bode Acc.Chem.Res. (1978) 11, 114-122). Robillard: How large was the exogenous peptide? Huber: A dipeptide. Addition of more residues does not increase binding. (Bode (1979) J.Mol.Biol.).

STRUCTURAL DYNAMICS OF PROTEINS AS REFLECTED BY ISOTOPE EXCHANGE KINETICS

Roger B. Gregory and Andreas Rosenberg The Department of Laboratory Medicine and Pathology University of Minnesota Minneapolis, Minnesota 55 455 Publication No. 207 from the Laboratory for Biophysical Chemistry

Fluctuations in Macromolecules Hydrogen isotope exchange kinetics has been of major importance in establishing the reality of structural fluctuations in macromolecules. FIuctuational behavior of a system of particles is a natural consequence of the random collision processes that provide the mechanisms for the transfer of energy at the microscopic level. The concepts of energy and density fluctuations for a system are well defined by statistical mechanics. In general, for large, non-cooperative systems fluctuations in the ensemble-averaged thermodynamic properties are extremely small so that the average state is well represented by the most probable state. Similarly, the dynamic properties of such systems are often well described by single relaxation times. The situation is rather different for a macromolecule such as a protein. Thermodynamically, a single protein molecule may be considered as a small cooperative system. The small size of the system in itself is sufficient to increase the importance of fluctuations, however, it is the cooperativity of the protein system which deserves emphasis. The restrictions imposed by bonding and secondary structural interactions between atoms cause the atomic displacements associated with fluctuations in energy and volume to couple

Mobility and Recognition in Cell Biology © 1983 by Walter de Gruyter & Co., Berlin • N e w York

50

R . B . G r e g o r y and A .

Rosenberg

with one another in such a way as to maintain the protein at an essentially constant free energy. The sum of fluctuations in energy and volume from 0 K to the temperature, T that enters the expression for the enthalpy is given by the heat capacity integral Cp(T')dT' which is compensated exactly by a corresponding term in the entropy (1, 2). In other words, the heat can never contribute to the free energy. Large fluctuations in energy and volume can occur at nearly constant protein free energy, so that microstates of widely different energy and volume occur with relatively high probability. As a result the distributions of energy and volume for a protein system are very different from those that characterize simple non-cooperative systems. The description of these distributions is contained within the Guggenheim cannonical partition function which is defined for a system at constant T and P as: A = LHL

n

. . ij

• • ( E -' v •) e x Pc - E. A l Ji

x

Jl

l

W

exp - PV . / r J

r k T

.

All the higher moments of the energy and volume probability density functions are obtained by successive differentiation with respect to temperature or pressure.

The variance in the

enthalpy, for example, is simply related to the heat capacity: 2

2

dH/dT = Cp = ajj /RT . In principle, therefore, a thermodynamic description of the accessible states of the protein system is possible in terms of the normalized partition function if we can obtain estimates of a sufficient number of higher moments for E and V. Just as the partition function is the goal of the thermodynamic description of the protein, the relaxation time spectrum for the fluctuations between states is the goal of dynamics studies. Unlike simple non-cooperative systems the time scales for motions in proteins cover a large range. Thus the correlation time of methyl group rotation is as short as 5 ps while more concerted motions involving side chain or main chain displacements or move cooperative domain displacements and "hinge-bending" motions extend the time scale to milli-

Structural

Dynamics o f

Proteins

seconds or longer (3). The occurrence of conformational fluctuations in proteins is now well established and provides a dynamic basis for function. Basic mechanisms by which enzymes lower activation energy barriers and subdivide the reaction pathway into discrete steps are well described (4, 5). However, the details of free energy transduction, the possibilities for time correlation of relevant conformational transitions and the types of coupling between the protein interior and the solvent are currently the subject of much discussion (6). We shall, in the following, discuss what kind of information per taining to conformational fluctations is available from studies of isotope exchange.

The Hydrogen Isotope Exchange Method The rate of the hydrogen isotope exchange reaction: RCONH*R1 + HOH + RCONHR^ + HOH* proves to be exceedingly sensitive to the environment of the amide exchange site. The reaction is specifically catalysed by acid and base (7) together with an important contribution from direct exchange with water (8). The behavior of the che mical exchange reaction and the effects of nearest neighbor substituents, electrostatic and discrete change effects and the influence of ionic strength and medium polarity on the reaction rates have been reviewed in detail elsewhere (7). Regardless of the method employed, we can recognize two basic approaches to such studies; those that provide exchange infor mation for specific single residues within the protein and those that yield the total exchange kinetics of the protein. For each site in a protein the number of hydrogens remaining unexchanged at time t is simply: H(t) = exp - kt. For a pro

R . B . Gregory and A. Rosenberg

52 tein

containing n exchangable sites the expression for total

exchange is: n H(t) = I exp - kit i=l

(1)

The deuterium-hydrogen exchange of single protons in proteins can be studied by nmr methods (9, 10) which have provided detailed information on the pD and temperature dependence of exchange rates for some single protons in BPTI (9, 10). However, despite the analytical simplicity that is introduced, the study of single proton exchange reactions provides only a fragmentary view of the dynamics of the whole protein. In order to understand the correlated fluctuations of the protein machine as a whole it is necessary to examine total hydrogen exchange kinetics. There is, however, a price to be paid for such a general view. The analysis of the simultaneous exchange of many hydrogens that is described by equation (1) is notoriously difficult. Indeed, it is only in recent years that advances in such an analysis have been made.

Analysis of Total Protein Hydrogen Exchange Kinetics Early attempts to describe the hydrogen exchange kinetics of proteins often employed a simplification in which the sum in equation (1) was replaced by a sum of a few exchange classes. However, the parameters derived from this approach generally have little or no physical meaning.

Recently, Knox and

Rosenberg (11) described a more physically meaningful analysis, adapted from the method of Austin et al (12).

When the

value of n is large in equation (1), as is generally the case for proteins, then the sum in equation (1) may be taken over as an integral to give: H(t)

exp - ktdk = L{f(k)}

(2)

Structural

Dynamics of

53

Proteins

The experimental observable, H(t), is simply the Laplace transformation of f(k), the exchange rate probability function

(pdf).

density

In principle, the function f(k) can be reco-

vered as the inverse Laplace transform of H(t).

The hydrogen

exchange data for lysozyme examined by Knox and Rosenberg

(11)

could be fitted quite well by a power law of the form: H(t) = b(1 + a t ) _ n e x p - ct

(3)

which on closed form Laplace inversion gives a simple gamma pdf modified to account for a second slow exchange process which is believed to involve cooperative thermal

unfolding.

Other examples of this approach applied to exchange data for hemoglobin and myoglobin have been reviewed by Barksdale and Rosenberg

(7) who also discuss other distribution

functions

that have been applied to protein hydrogen exchange.

There

is, however, a major problem associated wth the Laplace sion of noisy experimental data.

inver-

There exist an enormous num-

ber of solutions that fit the data to within the noise Furthermore, the errors are unbounded so that the may be quite diverse in character

level.

solutions

(13). Fortunately, the

recent development by Provencher of a computer program for the general solution of integral equations

(13) makes it possible

to perform a model-free, numerical inversion of hydrogenexchange kinetic data.

An example of the application of

Provencher's program to exchange data for lysozyme

(14) is

shown in Figure 1.

6 ÌI 4

2 0

Figure 1.

-8

-6

-4

-2

LOG K

0

2

4

Exchange rate pdf for lysozyme at 5°C pH 7.5. Units of k are m i n - 1 '

R.B.

54

G r e g o r y and A .

Rosenberg

An important concept in the analysis of hydrogen exchange data is the rank order of exchange. For many proteins, the rank order of exchange is conserved with changes in experimental conditions such as temperature, solvent composition or the presence of specific ligands (7). When this is the case, then properties of the exchange reaction determined at a particular value of H(t) will refer to the same exchange site or group of sites.

This idea is exploited in the determination of the * * average values of the activation parameters AH and AS for lysozyme hydrogen exchange (15) which reveal an interesting pattern of enthalpy-entropy compensation: (16).

AH* = a(T) + TcAS*

Its appearance provides a method for obtaining the

probability density functions for AH

and AS .

The derivation

of the activation enthalpy, g(AH*), from f(k) proceeds from appropriate variable transforms suggested by the compensation behavior (15).

Given the relationships k = k B T K* and AG* =

RTlnK* we have from equation (2): H(t) = ^B^ I f(k)exp - ktdK* = _1 / kf(k)exp - ktdAG* h RT o o The AH* - AS* compensation behavior provides the variable

J

J

(4)

transforms: dAS* = dAG*/(Tc-T) and dAH* = TcdAS* from which we obtain:

CO

/ °

kf(k)exp-ktdAS* =

oo (Tc

~ T C) RTT C

/ kf(k)exp-ktdAH* (5) J o

The analysis described above leads to two pdf's, one for free energies of activation and one for the enthalpies of activation. The activation entropy pdf is of course determined by the two functions. If the contributions due to the chemical exchange process could be subtracted, we can describe the attenuation of rates by two pdf's and we also arrive at a major interpretational problem: How can such pdf's be related to the moments of the partition function and the relaxation time distribution functions discussed in the first section of this paper. In order to make any progress in this direction

Structural

Dynamics o f

Proteins

55

we have to define some reasonable models for the process of isotope exchange.

Interpretation of Exchange Data Despite the impressive accumulation of hydrogen exchange data in the literature, the mechanisms of exchange in proteins and the types of internal motion responsible for catalyst accessibility to the peptide exchange sites have been discussed mostly in terms of rather picturesque but general concepts such as breathing, local unfolding, global fluctuations and solvent penetration. The ensuing debate about the validity of such ill-defined models tends to obfuscate an understanding of protein dynamics, which is the goal of hydrogen exchange studies, by becoming an end in themselves. These models have recently been reviewed elsewhere (7, 17, 18) and will not be discussed in any detail here. Instead, we shall attempt to present a more rigorous and unified description of the exchange process, one based on well defined concepts such as hydrogen bond configurations. This allows us to utilize a stochastic approach in the analysis of experimental data. The common feature of all the models for protein hydrogen exchange is their acknowledgement in one way or another of the possibilities for hydrogen bond rearrangements within the protein. They differ principally in the degree of cooperativity of such rearrangements. At one extreme, the local unfolding model (18, 7) involves highly cooperative hydrogen bond breakage and relatively large amplitude motions of a locally unfolded unit that expose the normally protected interior peptide sites to bulk solvent where exchange takes place. At the other extreme, the penetration model (7, 17) proposes that the exchange catalyst penetrates the protein matrix via smallamplitude motions which may collect to form holes or pathways for the migration of catalyst to the buried peptide sites.

R.B.

56

G r e g o r y and A .

Rosenberg

There is little doubt that this whole spectrum of conformational fluctuations occur in proteins. The thermal energy of the protein is certainly sufficient to lead to short-lived locally unfolded species, while the basis for free volume rearrangements or "mobile defect«," has been described by Lumry and Rosenberg (19). For the purposes of a discussion of hydrogen exchange kinetics, we can consider the protein as an irregular network of hydrogen bonds. Each position in the network may be either bonded or nonbonded and a configuration of the protein is defined by the bonding state at each network position (see Figure 2). Rearrangements of the bonds within the protein give rise to a large number of configurations. The range of structures that may be adopted and in particular the distribution of the total number of bonds, Nq, in each configuration will be constrained in a manner consistent with the stability of the protein. LOCAL UNFOLDING

o o

Nb) centre ferredoxin. The Mossbauer study on these ferredoxins was very thorough and as you know all the iron is observed by this technique. Kroneck: The structures presented by you for the [4Fe-4S] or the [3Fe-xS] cluster indicate chemical equivalence among the individual subsites. Frcm your interconversion experiments do you have any indication that there might be iron centers which are preferentially exchanged? Xavier: First let me stress again that the [3Fe-xS] structure drawn in a chair conformation is a speculative one using the Fe-Fe and Fe-S distances obtained frcm EXAFS studies (M.R. Antonio, B.A. Averill, I. Itoura, J.J.G. Moura, W.H. Orme-Johnson, B.-K. Teo, and A.V. Xavier, J.Biol.Chan. (1982) 257, 6646-6649). The iron incorporated by the 3Fe cluster, upon incubation, originates only one of the two quadrupole doublets of the Mossbauer spectrum (see Figure 3). This shows that the added iron occupies only one subsite or at most two structurally equivalent sites.

DYNAMICS AT THE PROTEIN SURFACE

Robert G. Bryant Chemistry Department, University of Minnesota Minneapolis, Minnesota 55455, U.S.A.

Introduction The time scale of interest when considering motions at the surface of a catalytically active macromolecule spans the range from seconds or longer to fractions of a picosecond or shorter. The longer time constants may be studied by the classical techniques including, for example, chemical kinetics where the rate determining' step may report a slow conformation change or a rare collision.

The shorter time constants may be character-

ized by spectroscopy where the motional effects are coupled to the observation by a theory relating the slow time response of the spectroscopic instrument to very rapid events such as molecular rotation, vibration, diffusion, etc.

Nuclear magnetic

resonance and the associated nuclear magnetic relaxation rates provide a powerful method for studying the intermediate time scale from milliseconds to hundreds of psec.

There is a vari-

ety of approaches to local dynamic information including line shape analysis of spin 1 nuclear resonances

(1), analysis of

the averaging of chemical shift tensors in the solid state analysis of multiple quantum spectra

(2),

(3), and interpretation

of nuclear magnetic relaxation rates (4).

The focus of this

presentation will be on the information available from nuclear magnetic relaxation and what information these measurements provide about solvent motion at the protein surface.

Mobility a n d Recognition in C e l l Biology © 1 9 8 3 by W a l t e r d e Gruyter & Co., Berlin • N e w York

R.G.

104

The solute-solvent interaction is very complex.

Bryant

The present

effort makes no claim for completeness, as indeed, several dynamical aspects of the water-protein interface remain unclear.

Nevertheless, we would like to understand the temporal

specifics of the water molecule interacting with a macromolecule surface under a variety of conditions.

The complexity of

this situation is shown by considering the schematic view of a protein solution shown in Figure 1.

This view of the water-

protein surface constrains the water motion by one hydrogen bond considered to be primary; thus, there are two time constants only characterizing the local water motion: the fast reorientation about the primary hydrogen bond, and a slower reorientation of this fast spinning axis indicated as Tg.

The

fast rotation may, of course, be hindered by transient interactions with other potential hydrogen bonds on the water molecule.

Even if we make the usual approximation that the protein

may be characterized as a rotating sphere and represent this component of the total motion by an average rotational

0

1

H

Figure 1. A schematic representation of a protein solution indicating three correlation times describing protein rotation, two time constants characterizing local water motion at the surface, a protein surface-bulk water exchange time, t e x , and a surface diffusion time, i n .

l u

W a t e r S u r f a c e Dynamics

c o r r e l a t i o n time, f r o t ,

We

may

a n t i c i p a t e a d d i t i o n a l d i f f i c u l t i e s b e c a u s e of the c h e m i c a l

s e v e r a l time c o n s t a n t s r e m a i n .

and

s t r u c t u r a l h e t e r o g e n e i t y of the m a c r o m o l e c u l e

surface.

E a c h of

these time c o n s t a n t s , a l r e a d y a s t a t i s t i c a l c h a r a c t e r i z a t i o n the d y n a m i c s at best, m a y be d i f f e r e n t at d i f f e r e n t o n the p r o t e i n s u r f a c e .

A distribution

positions

in e a c h of t h e m is p o s -

sible a n d this l i m i t s the p r e c i s i o n a n d a c c u r a c y of a n y tative dynamical characterization. d e p i c t e d in F i g u r e

The k i n d s of

quanti-

motion

1 affect many different measurements

The p r o b l e m , h o w e v e r ,

of

is s e l e c t i v i t y or the a b i l i t y to

(5, 6). focus

the m e a s u r e m e n t to i n v e s t i g a t e a p a r t i c u l a r m o l e c u l e or p a r t of a molecule.

Though nuclear magnetic resonance

to be i n s e n s i t i v e

p r o v i d e a h i g h d e g r e e of

Magnetic

is o f t e n

in c o m p a r i s o n w i t h o t h e r t e c h n i q u e s ,

thought it c a n

selectivity.

Relaxation

The s t r a t e g y for m a k i n g d y n a m i c a l d e d u c t i o n s f r o m n u c l e a r n e t i c r e l a x a t i o n rates is s u m m a r i z e d

in E q u a t i o n 1, w h i c h

r e l a t e s the time c o n s t a n t for the n u c l e a r equilibrium

mag-

s p i n s y s t e m to

in a d i r e c t i o n p a r a l l e l w i t h the a p p l i e d

reach

magnetic

field, T j , to the c o r r e l a t i o n time, T c , for r e o r i e n t a t i o n of pair of i n t e r a c t i n g n u c l e a r m a g n e t i c d i p o l e m o m e n t s by a d i s t a n c e ,

r.

n „ 4.2 - L = JL iJL_ { T, 10 6 . 1

r

T

g

+ 2 2 ,

1 + W T

4T

c , , 2 2

}

(1)

1 + 4 W T

c

c

w h e r e y is the p r o t o n m a g n e t o g y r i c r a t i o , fi P l a n c k ' s

constant

d i v i d e d by twice pi, and w the L a r m o r p r e c e s s i o n f r e q u e n c y the n u c l e a r m o m e n t s w h i c h field strength,

B Q ( W = Y B Q ) .

of

is p r o p o r t i o n a l to the d c m a g n e t i c This often applied

a s s u m e s that the i n t e r a c t i n g d i p o l e s m o v e c o n s t a n t d i s t a n c e and d o e s n o t , t h e r e f o r e , molecular

a

separated

equation

i s o t r o p i c a l l y at a include any

c o n t r i b u t i o n s to the r e l a x a t i o n rate.

inter-

Nevertheless,

it is clear how a m e a s u r e m e n t of the r e l a t i v e l y long

relaxation

106

R.G. Bryant

time, T^, which may be on the order of a second, leads to information about a very short time constant or very high frequency behavior through the dependence on the precession or resonance frequency, typically on the order of tens to hundreds of MHz for protons. The most direct application of Equation 1 is to measure the relaxation rate as a function of frequency, w.

This measure-

ment avoids the problems of temperature dependent changes in the structure of the protein and differing activation energies for the different dynamical constants in the model.

Extensive

measurements have been made on protein solutions using sophisticated techniques to cycle magnetic fields and maintain reasonable signal to noise ratios for measurements at very low field strengths.

A significant frequency dependence is found

that has been directly related to the rotational motion of the protein

(7, 8).

However, a detailed understanding of the

relaxation coupling between the protein rotation and the water molecule spin relaxation is not presently available various suggestions have been made

(9).

(4), though

Part of the difficulty

is apparent in the number of time constants shown in Figure 1 that may all enter the relaxation problem.

A second limita-

tion, however, is with the lack of magnets that produce fields corresponding to resonance frequencies significantly above several hundred MHz for protons.

The alternative is to measure

the relaxation rate as a function of temperature.

The diffi-

culties in this case are imposed by the temperature dependence of the conformation in the sample which may include decomposition at high temperature so that the overlap of information obtainable from the frequency dependence and from the temperature dependence is less extensive than desirable.

Neverthe-

less, to obtain a reasonable picture of the water-protein surface dynamics, both types of experiment are required.

Recent work in our laboratory is based on an attempt to simplify the complex dynamical situation outlined in Figure 1 by:

107

Water Surface Dynamics

1) immobilizing the protein and thus eliminating the bulk macromolecule rotations from the problem; and, 2) eliminating the bulk solvent phase, and therefore the surface to bulk solution exchange rate.

Though the remaining system is far from

simple, the dynamical characterization is more tractable and the information gained should provide useful limits for interpretation and modelling of data from more complex systems.

Water-Lysozyme Data A dramatic demonstration of the important effects of water on the properties of a protein are shown in Figure 2 where the

0.61-

o o

•

o °

- 0.4|-

„ ° O

0

*

H

o

n(HR')
(HR') n

complex

, and/or

2) a cooperative aggregation. n(HR)
(HR)

with K = f (n) . eq

u

Current e f f o r t s are directed toward determining whether the be

preclustered

if

exposed

to

37° C p r i o r

defining the possible e f f e c t s of agents reactions.

In

to

involved

receptors

hormone binding and to in

the

phosphorylation

a d d i t i o n , the formation of c l u s t e r e d receptors in c e l l u l a r

membranes i s being assessed by a completely d i f f e r e n t technique to

the

static

distribution

and

proximity

of

membrane

Fluorescence energy measurements can be performed on a in

a

flow

sorter/analyser

(37).

Using

and

redistribution

events

responsive components.

cell-by-cell

basis

p r o t e i n s labeled with s u i t a b l e

fluorescence donors and acceptors, s e n s i t i v e determinations aggregation,

may

can

of

proximity,

be made, most recently with

absolute evaluation of the energy t r a n s f e r and thus s u r f a c e

densities

for

each individual c e l l (L. Tron, S. Damjanovich, J . Sz'ollosi, D. Arndt-Jovin, and T. Jovin, in p r e p a r a t i o n ) .

E.D. Matayoshi et a I.

132

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Austin, R. H., Chan, S. S., Jovin, T. M.: Proc. Natl. Acad. Sei. USA 76, 5650-5654 (1979).

2.

Jovin, T. M., Bartholdi, M., Vaz, W. L. C., Austin, R. H.: Ann. N. Y. Acad. Sei. 366, 176-196 (1981).

3.

Zidovetzki, R,, Yarden, Y . , Schlessinger, J . , Jovin, T. M.: Proc. Natl. Acad. Sei. USA 78 , 6981-6985 (1981).

4.

Bartholdi, M., Barrantes, F. J . , Jovin, T. M.: Eur. J. Biochem. 389-397 (1981) .

5.

Cherry, R. J . : Biochim. Biophys. Acta 559, 289-327 (1979).

6.

Lipari, G., Szabo, A . : Biophys. J.

7.

Kinosita, K., Ikegami, A., Kawato, S.: Biophys. J.

8.

Jähnig, F.: Proc. Natl. Acad. Sei.

9.

Heyn, M. P . : FEBS L e t t . 108, 359-364 (1979).

120,

30, 489-506 (1980). 37, 461-464 (1982).

USA 76, 6361-6365 (1979).

10. Skou, J. C., Esmann, M.: Biochim. Biophys. Acta 601, 386-402 (1980). 11. Kawato, S., Kinosita, K.: Biophys. J.

36, 277-296 (1981).

12. Striker, G.: Analysis of Convoluted Exponential Data, in Proceedings of Deconvolution and Reconvolution of Analytic Signals, Nancy, July 1982, in press. 13. Lassen, U. V., Ussing, H. H., Wieth, J. 0 . , eds.: Membrane Transport in Erythrocytes, Proceedings of the Alfred Benzon Symposium 14 (1980). 14. Sheetz, M. P . , Schindler, M., Koppel, D. E.: Nature 285, 510-512 (1980). 15. Golan, D. E., Veatch, W.: Proc. Natl. Acad. Sei. USA 77, 2537-2541 (1980) . 16. Koppel, D. E., Sheetz, M. P . , Schindler, M.: Proc. Natl. Acad. USA 78, 3576-3580 (1981).

Sei.

17. Cherry, R. J . , Bürkli, A . , Busslinger, M., Schneider, G., Parish, G. R.: Nature 263, 389-393 (1976). 18. Nigg, E. A . , Cherry, R. J . : Biochemistry 18, 357-365 (1979). 19. Nigg, E. A . , Cherry, R. J . : Proc. Natl. Acad. Sei. (1980).

USA 77, 4702-4706

20. Johnson, P . , Garland, P. B.: FEBS Lett 132, 252-256 (1981). 21. Salhany, J. M., Gaines, K. C.: Trends Biochem. Sei. 6, 13-15 (1981). 22. G i l l i e s , R. J . : Trends Biochem. Sei. 1_, 41-42 (1982). 23. Yu, J . , Steck, T. L.: J. B i o l . Chem.

250, 9176-9184 (1975).

24. Kliman, H. J . , Steck, T. L . : J. B i o l . Chem.

255, 6314-6321 (1980).

25. Strapazon, E., Steck, T. L.: Biochemistry 16, 2966-2971 (1980). 26. Murthy, S. N. P. , Liu, T . , Kaul, R. K., Kohler, H., Steck, T. L.s

Rotational

D y n a m i c s o f Cell

Surface

133

Proteins

J. Biol. Chem. 256, 11203-11208 (1981). 27. Shaklai, N., Yguerabide, J., Ranney, H. M.: Biochemistry 16, 5593-5597 (1977). 28. Salhany, J. M., Cordes, K. A., Gaines, E. D.: Biochemistry 19, 1447-1454 (1980). 29. Carpenter, G., Cohen, S.: Annu. Rev. Biochem. 48, 193-216 (1979). 30. Cohen, S., Ushiro, H., Stoscheck, C., Chinkers, M. : J. Biol. Chem. 257, 1523-1531 (1982). 31. Maiag, T.: Trends Biochem. Sei. 7, 197-198 (1982). 32. Schlessinger, J., Geiger, B.: Exp. Cell Res. 134, 273-279 (1981). 33. Schlessinger, J.: Trends Biochem. S c i . ^ , 210-214 (1980). 34. Saffmann, P. G., Delbrück, M.: Proc. Natl. Acad. Sei. 3111-3113 (1975).

USA 72,

35. Vaz, W. L. C., Criado, M., Madeira, V. M. C., Schoellmann, G., Jovin, T. M.: Biochemistry, in press (1982). 36. Orly, J., Schramm, M. : Proc. Natl. Acad. Sei. (1975).

USA 72, 3433-3437

37. Jovin, T. M., Arndt-Jovin, D. J.: in Trends in Photobiology (Eds. C. Hélène, M. Charlier, Th. Montenay-Garestier, G. Laustriat), Plenum Press, New York, pp. 51-66 (1982).

Received August 31, 1982 DISCUSSION Robillard: You claimed in one instance that an increased correlation time was evidence for cluster formation. Subsequently you observed decreased correlation times and maintained that you still had clusters but that the clusters were smaller. What was the evidence that you still had clusters? Jovin: The observed correlation times were still longer than those initially measured for the dispersed receptors, (see Zidovetzki et al., ref. 2 in manuscript). Pecht: The induction/delay phase in the dependence of the rotational correlation time of EGF is rather long for a protein conformational change. Could you corrmsnt on that? Jovin: The tine-course might in vivo be slightly dependent upon factors not well controlled in the membrane preparation: manbrane potential, electrochemical gradients, energy, state of phosphorylation. An alternative explanation for the lag phase (oo-aperative aggregation) was also proposed. The time-course of this process is hard to predict.

134

E.D. Matayoshi

et

al.

Pecht: Did you conpare the behavior of native BGF with that of the cyanogen cleaved derivative? Jovin: Not yet. Hucho: To what extent do photochemical reactions take place (e.g. crosslinking)? Jovin: We have no evidence for photochemical processes, probably due to the fact that phosphorescence (and other triplet state) measurements are performed in an 0_-free environment. Macnab: What is the reason for increased translational diffusion rates of band 3 under conditions where microclusters are forming? Jovin: Hie observed phenomena referred to E6-F and not Band 3• The dependence of translational diffusion on size (diameter) is logarithmic, as opposed to the approximately 2nd pcwer (in 2 dimensions; see Selfman and Delbrick, Proc.Natl.Acad.Sci. USA, ref. 34 in manuscript) or 3rd power (in 3 dimensions) dependence in the case of rotational diffusion. Thus, the effect of temperature on membrane microviscosity dominates the translational diffusion but the increase in size is the dominant factor in rotational diffusion (see discussion in Zidovetzki et al., ref. 3 in manuscript). Von Hippel: In terms of your hormone receptor studies, hew do yoi define a 'cluster' of receptors? Hew close together do you think the receptors have to be before they behave as a cluster, in that there is enough interaction between than to be reflected in a changed fluorescence polarization (especially if the main motion should be rotation about the long axis i.e. , perpendicular to the membrane surface)? Jovin: A cluster can be defined as any self-associated form of the hormonereceptor complex, i.e. (HR) with n >2. The rotational correlation times will be approximately proportional to n, if the irolecular assembly is rigid during the time ranging the anisotropy decay (^1 msec).

SECTION II INTERACTION OF PROTEINS WITH PROTEINS, NUCLEIC ACIDS AND LIPIDS Chairmen:

I.

Pecht

and

W.

Neupert

DYNAMIC ASPECTS OF PROTEIN-PROTEIN ASSOCIATION REVEALED BY ANISOTROPY DECAY MEASUREMENTS A.J.W.G. Visser, N.H.G. Penners and F. Miiller Department of Biochemistry, Agricultural University, De Dreijen 11, 6703 BC Wageningen, The Netherlands

Introduction Proteins often possess quaternary structure. Many proteins are composed of (mostly) noncovalent assemblies of subunits. The composition can vary from a single symmetric dimer of two identical subunits to a multienzyme complex built up from a large number of several types of subunits with fixed total size and stoechiometry. The interaction between different proteins are also of vital importance for interenzyme catalysis, like e.g. the coupled reactions involving electron transfer proteins. It can very well be imagined, that the strength of the interaction has a regulatory function. Several questions related to protein-protein interaction can be summarized: How are these complexes stabilized and what is the spatial configuration? The answer is not simple, since each individual system requires knowledge at several structural levels. For instance, in order to estimate the free energy of binding between proteins semiquantitatively, details about primary, secundary and tertiary structures of the proteins involved should be known to determine the surface area accessible for interprotein contact [1]. It is generally accepted, that protein complexes are stabilized by hydrophobic interaction. However, electrostatic interactions are important as well; they are required for proper orientation of protein partners to form catalytically productive complexes. The net charge of each individual protein and especially the charges near the active center provide therefore important information. Another factor

Mobility a n d Recognition in C e l l Biology © 1983 by Walter d e Gruyter & Co., Berlin • N e w York

138

A.J.W.G. V i s s e r ,

N.H.G. Penners and F.

Müller

to consider is that many proteins contain flexible domains of different size, as became apparent from molecular dynamics simulation of protein movement (ps time scale) [2] and from experimental approaches like e.g. fluorescence relaxation techniques (ns timé scale) [3-5]. These dynamic aspects are of basic interest in characterizing the behaviour of protein complexes in solution. In this paper an experimental approach is described to investigate protein-protein interaction. The method is based on time-dependent fluorescence depolarization of proteins containing intrinsic or extrinsic fluorescent molecules. Thanks to the pioneering work of Gregorio Weber [6] the principles of fluorescence (de)polarization and its applications to proteins have already been outlined. Also, the potentials of anisotropy decay methods have been detailed [7,8]. One of the major advantages of the technique is the sensitivity, implicating the study of protein-protein association under thermodynamically ideal conditions, i.e. with low protein concentrations in the (sub)micromolar range. The method, in which polarized fluorescence is created by a short pulse of polarized light, is a relaxation method. It measures the return to randomization of a previously formed, anisotropic distribution of fluorescent molecules. The time constant of this process (the rotational correlation time 0) is proportional to the size of the fluorescent particle. The simplest case, of course, is the assumption of spherically shaped proteins. For such proteins with fluorescent groups rigidly bound a simple, empirical formula can be used, which relates 0 to the molecular weight, M r , partial specific volume, v, viscosity of the solution, n, and degree of hydration, h, of the protein: 0 = Mrn(v + h)/RT. The anisotropy, r, is in this case an exponential function: r(t) = r Q exp(-t/0), r(t) = (I/7(t) - I^(t) )/(l (t) + 2l±(t)) = 2p (t) / (3-p (t)) , I/7(t) and I^(t) are the polarized emission components as function of time, p is the degree of polarization. Deviations from spherical shape will be discussed later. If two proteins form a complex,

139

Anisotropy Decay of Protein Complexes

Pj^ + P 2 J

p

ip2

and

P

1P2

are

fluorescent

) » 0 should increase,

since the molecular weight of the complex increases. In an equilibrium mixture conditions might be such, that the complex is partly dissociated. The total anisotropy decay should then obey a two-exponential functions r(t)/r Q = a.^ exp(-t/01) + a 2 exp(-t/02), with 01 and 02 the correlation times of P.^ and P^Pj. If the fluorescent properties are not altered upon complexation the preexponential factors a^^ and a 2 measure the amounts of free and complexed protein, respectively. Analysis of anisotropy decay can thus be used to describe protein-protein association equilibria. One example will be presented to demonstrate the method of procedure. The example deals with an oligomeric protein: phydroxybenzoate hydroxylase from Pseudomonas

fluoresaens.

The

enzyme is a flavoprotein, catalyzing the NADPH-dependent hydroxylation of p-hydroxybenzoic acid (PHB), with noncovalently bound FAD functioning as the fluorescent reporter group. At ViM concentration the enzyme mainly exists as a dimer, M^ = 82 000, with two identical subunits [9]. At submicromolar concentration dissociation into monomers occurs, which could be followed by monitoring the fluorescence anisotropy of bound FAD. Range of Times and Interactions Natural fluorophores have fluorescence lifetimes ranging from around 0.5 ns (NADH in solution, [10]) to 14 ns (lumazine protein, [11]). As a rule of thumb correlation times up to five times the fluorescence lifetime can be precisely determined from anisotropy decay. Very short correlation times can be extracted with deconvolution methods, since the anisotropy decay will be distorted owing to the finite excitation pulse. Referring to the empirical equation given in the Introduction, anisotropy plots can be generated assuming standard conditions and spherical particles with fluorophores having a lifetime of about 5 ns (Fig. 1). It is directly apparent from Fig. 1, that

140

A.J.W.G. Visser, N.H.G. Penners and F. Müller

Time 200 000 cannot be exactly determined, since the slope of the decay curve approaches zero. On the other hand, the difference between a protein with M = 20 000 and one with M r = 100 000 is clearly visible. Fig. 2 shows a few biexponential decays. One example is for a fluorophore undergoing a rapid independent motion (0 = 1 ns) superimposed on the rotation of the whole protein (0 = 23 ns). Lipari and Szabo have developed a formalism to estimate the amplitude of this motion relative to the rotation of the protein as a whole [12]. The concept is that the fluorescent molecule has an unique symmetry axis (with the emission transition moment pointing along it), which 'wobbles' in a cone of semiangle 9Q independently from the macromolecular rotation. Fig. 2 also shows the difference in biexponential decay, when the amplitudes are varied, while the correlation times remain constant. This example is based on the association equilibrium of lumazine protein with luciferase as detailed in [13]. Idealized behaviour, as illustrated in Figs. 1 and 2, is not encountered in normal practice. Noise propagates in the decay and increases along the decay. This might be an important stumbling block in retrieval of parameters. If a biexponential decay model holds true, it often occurs that an unconstrained analysis, i.e. allowing all four parameters to assume values 2

that minimize x / yields uncorrelated parameters: they can adopt a wide range of values. In order to retrieve meaningful results a constrained analysis, in which only the amplitudes are treated as free parameters, might be preferable. The correlation times of free and bound proteins should then be exactly known. Details are given in [13], If the fluorescence parameters like lifetime and quantum yield do not vary upon protein-protein association, the amplitudes in the decay are direct measures of the species concentration in the equilibrium mixture. Analysis of the decay thus provides the binding constant in a single experiment. Dissociation constants in the range 0.1 pM1 mM can easily be determined.

142

A.J.W.G. V i s s e r ,

N.H.G. Penners and F.

Müller

Deviation from Spherical Shape This section will discuss deviations from spherical shape and its effect on fluorescence depolarization of fluorophores rigidly bound to proteins. Francis Perrin, already in the early thirties, treated this problem for model compounds [14], whilst Tao derived the time dependence of fluorescence depolarization of ellipsoids of revolution (three symmetry axes, two of them are equal) [7]. In general, there exist five correlation times for an irregularly shaped body [7,15]. Ellipsoids of revolution contain three correlation times, 0^, 0 2 and 0^, which can be related to the diffusion coefficients around the three axes of revolution (one is parallel to the symmetry axis z, the two others are perpendicular to z and are degenerate). The correlation times and amplitudes of the triexponential function are listed below: 0. = 1/(6D.) 0 2 = 1/(5D± + D^)

a. = (1.5cos20-O.5)2 2 2 a 2 = 3cos Gsin 9

0 3 = 1/(2D± + 4D^)

a 3 = 0.75sin4Q

where 0 is the angle between the (single) transition moment of the fluorescent molecule and the symmetry axis z. Transition moments of excitation and emission are the same. The diffusion constants of prolate (rod-shaped, rotation about long semiaxis) or oblate (disk-shaped, rotation about short semiaxis) ellipsoids can be expressed in the isotropic diffusion constant D of a sphere of equivalent volume V (D = 1/60 = kT/(6nV)) and in the axial ratio of the ellipsoid p. If we restrict ourselves to prolate ellipsoids, the ratio of the three correlation times and that of an equivalent sphere can be calculated straightforwardly:

An¡sotropy

Decay o f P r o t e i n

Complexes

143

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221

Autogenous Regulation of Protein Synthesis

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-O ai c -rH Mg >_ Ca > K = Na independent of ionic strength (20). It is in this order that these ions bind to the phosphodiester group of phosphatidylcholine (21). This suggests that the recognition site contains basic amino acid residue (s) involved in the interaction. In order to obtain evidence for the possible involvement of Arg residues PC-TP has been treated with the a-carbonyl reagents 2,3-butanedione and phenylglyoxal (22). Both reagents completely inactivate the transfer protein by specific rapid modification of three of the ten Arg residues. In the presence of negatively charged vesicles butanedione (30 mM) rapidly modifies two Arg residues without a significant loss of activity (Fig. 3). This strongly suggests that the phospholipid interface protects one Arg residue essential for transfer activity. So far we have not succeeded in the identification of this Arg residue. The results, however, convincingly show that Arg residues may play an important role in phospholipid-protein interactions.

The Lipid Binding Site At present it is not known how the recognition site spatially relates to the actual lipid binding site on PC-TP. This site has been investigated by use of photoactivable analogues of

K.W.A. Wi rtz et al

258

1009=

0

10

20 30 40 TIME (min)

50

60

Fig. 3. Inactivation of phosphatidylcholine transfer protein by 2,3-butanedione (30 mM) in the presence (•) and absence (o) of vesicles consisting of phosphatidylcholine and phosphatidic acid (80/20, mol/ mol) and the corresponding number of arginine residues modified (•,•).

O

h 2 c-o-c-(ch 2 )-ch 3 HCEl » I (CH3)3-r N - C H 2 - C H 2 - 0 - P - 0 - C H 2 ÒB

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Fig. 4. Chemical structure of l-palmitoyl-2-[û>- (m-diazirinophenoxy) [1- C]acyl]sn-glycero-3-phosphocholine. PC-I (x=5) contains a sn-2-[1-^C] hexanoyl chain and PC-II (x=10) a sn-2-[l-^C]undecanoyl chain.

Phosphatidylcholine Transfer

Protein

259

phosphatidylcholine which carry a diazirinophenoxy group linked 14 to thew-carbon of either the sn-2-[l- C]hexanoyl (PC-I) or 14 sn-2-[1- C]undecanoyl chain (PC-II) (Ref. 22). These carbenegenerating analogues (see Fig. 4) were synthesized as described in (23) and incorporated into PC-TP by incubation with vesicles prepared of these phospholipids (24). Photolysis of the isolated PC-I (II)-PC-TP complex followed by sodium dodecylsulfatepolyacrylamide gel electrophoresis indicated that 30-40% of the incorporated PC-I and II were covalently coupled to the protein. The remaining 60-70% of the photogenerated carbene moieties have probably reacted with water molecules which may have been present in the binding site. Degradation of the photolabeled PC-TP with protease from Staphyloeoeeus aureus, trypsin and cyanogen bromide rendered 14 specific C-labeled peptides which were sequenced by automated Edman degradation. A major site of coupling shown by release of radioactivity was identified as the peptide segment Val 1 177 Phe-Met-Tyr-Tyr-Phe-Asp . Distinct differences in the pattern of coupling were observed depending on whether PC-I or PC-II were used. Thus, coupling occurred preferentially to 173 175 177 171 173 Met , Tyr and Asp1 with PC-I while Val 7 1 and Met were labeled preferentially with PC-II. This shift in coupling is compatible with an increase of 6 X for the sn-2 fatty acyl chains of PC-I and II assuming that the peptide V a l 1 7 1 - A s p 1 7 7 has adopted the strongly predicted B-strand configuration (see Fig. 2) . Fig. 5 shows a tentative model of the binding site being part 170 of two strongly predicted anti-parallel B-strands (i.e. Lys P h e 1 7 6 and G l n l 8 2 - T r p 1 9 0 ) and g-turn (i.e. A s n 1 7 8 - G l y 1 8 1 , see Fig. 2). The alternate sites of coupling demonstrate that the sn-2 acyl chain of the bound phosphatidylcholine molecule is aligned along one site of the B-strand Lys 170 -Asp 177 . No coup1fty T 9o ling was found to occur on the apposed B-strand Gin -Trp

260

K.W.A. W!rtz et al .

Fig. 5. Tentative model of the peptide region in phosphatidylcholine transfer protein involved in the binding of phosphatidylcholine. I and II represent the carbene-generated derivatives of PC-I and II.

suggesting that the sn-2 acyl chain has a restricted freedom of motion in the binding site. Moreover, the pattern of labeling points the phosphorylcholine head group towards the 6178 181 turn Asn -Gly . In conjunction with g-turns being generally found at the surface of proteins one may presuppose that this polar moiety is similarly positioned. Finally it is worth noting that the bound phosphatidylcholine molecule is accommodated in the most hydrophobic segment of the protein which has 172 a very high content of aromatic amino acid residues (i.e. Phe , 174 175 176 186 190 174 Tyr , Tyr , Phe , Trp , Trp ). The residues Tyr 17 and Tyr "* were not subjected to enzymatic iodination (Akeroyd, R., Thesis 1981). Additional evidence for the binding site being shielded from the medium derives from the observation that nitroxide groups on various positions along the sn~2

Phosphatidylcholine

Transfer

Protein

261

acyl chain of bound phosphatidylcholine is not accessible to ascorbate (25).

Conclusion The PC-TP-mediated transfer of phosphatidylcholine between membranes involves both ionic and hydrophobic interactions. The ionic interactions form part of the specific recognition of phosphatidylcholine by PC-TP. Moreover, one should realize that this interaction is strengthened by the relatively low dielectric constant at the interface. We presume that the site of recognition is located near the hydrophobic binding site. Possible conformational changes may expose hydrophobic peptide segments at the interface giving rise to local drastic changes in water structure, e.g. expulsion of water molecules. This would lead to a spontaneous redistribution of phosphatidylcholine between interface and PC-TP.

References 1. Wirtz, K.W.A., Zilversmit, D.B.: J. Biol. Chem. 243, 35963602 (1963) 2. McMurray, W.C., Dawson, R.M.C.: Biochem. J. K2, 91-108 (1969) 3. Kader, J.C.: Dynamic Aspects of Cell Surface Organization (G. Poste, G.L. Nicolson, Eds.), Elsevier/North-Holland, Amsterdam, pp. 127-204 (1977) 4. Wirtz, K.W.A.: Lipid-Protein Interactions (P.C. Jost, O.H. Griffith, Eds.) Wiley-Interscience, New York, pp. 151-233 (1982) 5. Helmkamp, G.M., Harvey, M.S., Wirtz, K.W.A., van Deenen, L.L.M.: J. Biol. Chem. 249, 6382-6389 (1974) 6. DiCorleto, P.E., Warach, J.B., Zilversmit, D.B.: J. Biol. Chem. 2S4, 7795-7802 (1979) 7. van Golde, L.M.G., Oldenborg, V., Post, M.,.Batenburg, J.J., Poorthuis, B.J.H.M., Wirtz, K.W.A.: J. Biol. Chem. 255, 6011-6013 (1980)

262

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8. Dyatlovitskaya, E.V., Timofeeva, N.G., Yakimenko, E.F., Barsukov, L.I., Muzya, G.I., Bergelson, L.D.: Eur. J. Biochem. 123, 311-315 (1982) 9. Erickson, S.K., Meyer, D.J., Gould, R.G.: J. Biol. Chem. 253, 1817-1826 (1978) 10. Metz, R.J., Radin, N.S.: J. Biol. Chem. 255, 4463-4467 (1980) 11. Conzelmann,E., Burg, J., Stephan, G., Sandhoff, K.: Eur J. Biochem. 123, 455-464 (1982) 12. Bloj, B., Zilversmit, D.B.: J. Biol. Chem. 252, 1613-1619 (1977) 13. Poorthuis, B.J.H.M., Glatz, J.F.C., Akeroyd, R. , Wirtz, K.W.A.: Biochim. Biophys. Acta 665, 256-261 (1981) 14. Demel, R.A., Wirtz, K.W.A., Kamp, H.H., Geurts van Kessel, W.S.M., van Deenen, L.L.M.: Nature New Biol. 246, 102-105 (1973) 15. Kamp, H.H., Wirtz, K.W.A., van Deenen, L.L.M.: Biochim. Biophys. Acta 398, 401-414 (1975) 16. Akeroyd, R., Moonen, P., Westerman, J., Puyk, W.C., Wirtz, K.W.A.: Eur. J. Biochem. 114, 385-391 (1981) 17. Wirtz, K.W.A., Moonen, P.: Eur. J. Biochem. 1_7, 437-443 (1977) 18. Akeroyd, R. , Lenstra, J.A., Westerman, J. , Vriend, G. , Wirtz, K.W.A., van Deenen, L.L.M.: Eur. J. Biochem. 121, 391-394 (1982) 19. Kamp, H.H., Wirtz, K.W.A., Baer, P.R., Slotboom, A.J. , Rosenthal, A.F., Paltauf, F., van Deenen, L.L.M.: Biochemistry 1^6, 1310-1316 (1977) 20. Wirtz, K.W.A., Vriend, G., Westerman, J.: Eur. J. Biochem. 94, 215-221 (1979) 21. Hauser, H., Hinckley, C.C., Krebs, J., Levine, B.A., Phillips, M.C., Williams, R.J.P.: Biochim. Biophys. Acta 468, 364-377 (1977) 22. Westerman, J., Wirtz, K.W.A., Berkhout, T., van Deenen, L.L.M., Radhakrishnan, R., Khorana, H.G.: Biochemistry, submitted (1982) 23. Radhakrishnan, R., Robson, R.J., Takagaki, Y., Khorana, H.G.: Methods Enzymol. 72D, 408-433 (1981) 24. Wirtz, K.W.A., Moonen, P., van Deenen, L.L.M., Radhakrishnan, R. , Khorana, H.G.: Ann. N.Y. Acad. Sei. 348, 244-255 (1980) 25. Devaux, P.F., Moonen, P., Bienvenue, A., Wirtz, K.W.A.: Proc. Natl. Acad. Sei. USA 74, 1807-1810 (1977) Received June 5, 1982

Phosphatidylcholine Transfer

Protein

263

DISCUSSIONI CorIn; You have indicated that ionic interactions are involved in the recognition of the bilayer by the protein. Cation inhibition of Pc-Tp mediated transfer iirplies that the protein 'recognition site' of the bilayer surface consists of basic amino acids. Perhaps one could conduct experiments to label lysine residues in the presence and absence of bilayer interface with subsequent protease digestion and peptide analysis. This should identify the lysine residues that are probably involved in the electrostatic protein/ bilayer interaction. Wirtz; We certainly have considered the experiment you just suggested. We also have thought of modifying lysine residues with reagents exclusively present in the interface. This may give us information on what segment of the protein is actually interacting with the membrane interface. Veegar: Could you visualize that the protein acts as a channel between two vesicles, in view of the big hydrophobic structures at the C.terminus? Wirtz: Your 'channel'-suggestion asks for the protein to act by forming a ternary complex with two different membrane interfaces. Both our kinetic studies and our studies with Pc-monolayers argue against the formation of such channels. We presume that the strongly hydrophobic segments are there to remove a Pc-molecule frcm the interface and to accommodate this molecule in the protein. Huchoi Procaryotic cells do not have transfer proteins, apparently cytoplasmic membranes can live without them. What evidence do you have, that the physiological role is truly exchange with membranes? Could they be lipid carriers in metabolism or lipid stores etc.? Wirtz; We go by the hypothesis that eukaryotic cells have a need for phospholipid transfer proteins because of the fact that the biosynthesis of the bulk of the phospholipid is concentrated in the endoplasmic reticulum. These proteins would be required to transfer phospholipids to those sites in the cell in need of phospholipids, e.g. membrane biogenesis, maintenance of merrbrane phospholipid composition. On the other hand it is well possible that these proteins are involved in processes of vrfiich, as yet, ws do not knew. However, our recent studies, using a radioinmmo-assay for rat Pctransfer protein, clearly shew that liver and intestinal mucosa have by far the highest level of this protein. These tissues are the most active ones in Pc metabolism and transport, suggesting that a relationship does exist with the level of Pc-transfer proteins. Jovin: What happens to the transfer rate as one passes through the lipid phase transition of the donor vesicle? Wirtz; Experiments by Helmkamp in the U.S.A. and Tinker in Canada have indicated that the transfer protein is active belcw and above the transition

264

K.W.A. Wirtz et al.

temperature (T ) of the phosphotidylcholine (Pc) involved. As to be expected the activation energy for the transfer process belcw T is distinctly higher than above T . Moreover, kinetic analysis has provided evidence that under both conditions the rate limiting step is the off-reaction by which the protein pulls a Pc-molecule out of the interface. The lipidphase transition per se does not appear to have an effect on the transfer rate. Sund; Frctn your modification experiments with 2,3-butanedione you suggested that the phospholipid interface protects one arginine residue out of ten which is essential for activity, neglecting the fact that the difference of the modification in the presence and absence of vesicles is about 0.5 only, the question arises whether the same arginine residues are modified in the presence and in the absence of vesicles. Wirtz: As you may have noticed frctn the slide even in the presence of vesicles a slew inactivation occurs. You vrould, therefore, not expect to find a difference of one arginine (Arg) residue when you perform the modification reaction in the presence of vesicles but a lewer value. As for your actual question we cannot be sure that the Arg residues modified in the absence and presence of vesicles are the same. On the other hand it has been observed for many enzymes that in general only a very limited number of Arg residues can react with the modifying reagent. This may have to do with lewer than normal pKa values for the reactive Arg residues. As for new, we tentatively conclude that 1 Arg residue is essential for activity but chemical analysis certainly has to be performed to support this conclusion. Robillard; Is the lipid molecule which is found on the enzyme at the end of the purification procedure, the same molecule vAiich is transferred? Wirtz; The answer is yes. We have clearly shewn that the Pc-molecule bound to the protein exchanges freely with a Pc-molecule present in a matibrane interface.

SECTION III MECHANISMS OF RECOGNITION IN NUCLEIC ACIDS Chairmen:

F.

Hucho

and

H.C.

Berg

ALLOSTERIC DNA

Fritz M. Pohl Fakultät für Biologie, Universität Konstanz, D-7750 Konstanz, Germany

Introduction The evidence that DNA may adopt different structures depending, for example, on the base sequence is now rapidly accumulating; this is due mainly to the results from x-ray diffraction studies of single crystals of oligonucleotides [e.g. 1,2]. It has been shown some time ago that sequence specific changes between different double helical structures can occur in solution and the most intensively studied example up to now is the salt induced transition between a right-handed B-form and a left-handed Z-form of poly(dG-dC)'poly(dG-dC) [e.g. 3,4]. Experiments with such model polymers of relative simple chemical structure provide a valuable starting point in the study of the structural variability of DNA, since the complexity of 9

natural DNA with up to 10 base pairs per cell makes the detection of sequence specific conformations and their interconversions extremely difficult. A number of recent developments make more detailed investigations possible, e.g. the chemical synthesis of defined oligonucleotides or the ability to insert defined DNA into small plasmid DNA, and studying its properties in topologically constrained covalently closed circular DNA, retaining dji vitro this unique and apparently also biologically important property of DNA. The recent discovery that a particular conformation of DNA the left-handed Z-DNA - induces in animals a strong immune res-

Mobility and Recognition in Cell Biology © 1983 by Walter de Gruyter & Co., Berlin • New York

F.M. Pohl

268

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74

25

NaCl(M) Fig. 1 Transition of poly (dG-dC)-poly(dG-dC) with a chainlength of about 1000 base pairs from the right-handed B-form at low salt to a left-handed Z-form at high salt as followed by the change of absorbance at 295 nm. 5M NaCl solution was added continuously at low speed to a cuvette, stirred at 45 C, containing the polymer. ponse allows the application of immunochemical methods with their high selectivity and sensitivity in the study of DNA structure [5,6].

Such antibodies bind, for example, to the

polytene chromosomes of Drosophila, making it very likely that such structures do occur in the cell [7]. The term "allosteric" is borrowed from the protein field, where it was coined to explain cooperative, regulatory functions of oligomeric proteins [8].

It should mainly indicate that simi-

lar phenomena may occur in DNA as will be shown in the case of some model reactions, involving also the recognition of lefthanded DNA by specific monoclonal antibodies. B-Z transition of poly(dG-dC)"poly(dG-dC) The salt induced transition between a right- and left-handed structure of this DNA has been studies in detail [3,9] and Figure 1 shows as an example the extreme cooperativity of this

A)losteric

269

DNA

interconversion.

By the continuous, slow titration of the

polymer a very high precision is obtained.

Varying the speed

of titration allows to establish those conditions where the equilibrium is closely attained. (This is important because e.g. at low temperatures the transition becomes extremely slow for polymers.) From the steepness of the curve in Fig. 1 the cooperative length N c can be estimated.

The equilibrium constant between

the two forms for polymer with about 1000 base pairs changes with a high power of the salt concentration: Krl

=

[L]/[R] ~

[NaCl] 240

indicating a kind of phase transition within the macromolecules. From the salt dependence of the free energy change per base pair, as obtained from measurements with short oligomers [9] the length of sequences of one conformation in the middle of the transition is estimated to be

N c =400 base pairs.

The

corresponding nucleation parameter for the formation of a lefthanded DNA within a right-handed one is then of the order of -5 10 , with an unfavorable free energy change of about 7.2 kcal/ mole. Recognition of Z-DNA by antibodies The observation that Z-DNA induces a strong immune response [5,6] prompted us to establish several cell lines producing monoclonal antibodies against this form of DNA [R. Thomae et al., in preparation]. Such antibodies provide convenient and reproducible reagents with high specificity and sensitivity. Fig. 2 shows the precipitation of radioactive labelled poly(dGdC)*poly(dG-dC) by such monoclonal antibodies as a function of the salt concentration.

The precipitation curve follows

rather closely the transition between the two forms (Fig. 1) which is one of the indications for their conformational spe-

270

F.M.

Pohl

32 Fig. 2: Precipitation of P-labelled poly(dG-dC)"poly(dG-dC) by monoclonal antibodies to Z-DNA as function of the salt concentration. 0.2 nM poly(dG-dC) at the indicated NaCl concentration was heated 30' at 70°C, cooled to 25°C, incubated with 1:5000 dilutions of ascites fluid for 60', crosslinked with goat antimouse serum for 60', centrifuged and the radioactivity of the washed pellet determined. cificity.

All the results obtained are in agreement with the

idea that these antibodies recognize only left-handed doublehelical DNA of the Z-type. The high sensitivity of such assays is illustrated by the about 10 000 fold lower amount of DNA necessary in these experiments as compared to the one in Fig. 1. The high selectivity is shown by the observation that radioactive Z-DNA is precipitated in the presence of a 10 OOOfold excess of e.g. calf thymus DNA.

Ethidium bromide as a "direct" effector of a DNA structure At high salt the binding of the intercalating dye ethidium bromide (EB) to poly(dG-dC)-poly(dG-dC) becomes an extremely cooperative process [10]. This was explained by its low affinity to the L-form and the high affinity to the R-form, shifting thereby the equilibrium between both. This is also reflected

271

A1losteric DNA

Fig. 3: Binding of ethidiumbromide ( ) to poly(dG-dC)"poly (dG-dC) in 4.4 M NaCl as determined from the absorbance at 284 nm [10]. 0.2 nM 32 P-poly (dG-dC) (-•-•-•-) bound to purified monoclonal antibody Z-D11 in 4 M NaCl, 60 mM sodium phosphate, 30 mM EDTA (pH = 7.5) measured by the precipitation assay described in Fig. 2. by the interaction with antibodies to Z-DNA as shown in Fig. 3. Within a narrow range of the free ethidiumbromide concentration the polymer is not recognized any more and therefore not precipitated.

The high cooperativity observed here as compared to

the case of oligomeric proteins is due to the large number of binding sites in a cooperative unit and the large difference of the affinity of the dye to the two forms of DNA. Such a binding process offers an interesting possibility: the transition between the two forms of the DNA may act as a "buffer" for regulating the free concentration of molecules within a very narrow range.

But it is not known at present, whether

such type of phenomena are of biological significance, for example, in regulating the free ion, water or protein concentration in the nucleus. It rather illustrates the potentials

272

F.M.

Pohl

inherent in such structural variations of DNA. "Z-DNA" in covalently closed circular DNA The experiments shown up to now involved linear duplex DNA and thereby miss an important property of DNA in cells, namely the topological restrictions imposed e.g. by the chemical closure to a double-stranded ring. Our first experiments to see whether a natural DNA being under such topological restrictions is recognized by antibodies to Z-DNA involved form V-DNA [F.M. Pohl et al.,in press, Nature 1982]. This form of DNA is obtained by the association of complementary, circular single-stranded molecules and therefore has a topological linking number of zero. Any right-handed duplex turns in such a molecule have to be compensated by lefthanded structures [11]. Such a DNA, although it lacks alternating (dG-dC) sequences longer than 6 base pairs, nevertheless shows strong interaction with the antibodies at high as well as at low salt. Further experiments revealed that increasing the negative superhelical density above the natural value is enough to promote recognition of the plasmid pBRßG2.17 by the antibody [E. Di Capua et al.,in prep., A. Nordheim et al., pers.comm.]. It has been shown that by cloning alternating oligo(dG-dC) into plasmid DNA a change of the superhelicity occurs at high salt in such recombinant plasmids [12,13], For investigating the interaction of monoclonal antibodies with such a recombinant plasmid, pLP32 was kindly provided by L.J. Peck and J.C. Wang. It was constructed by inserting (dC-dG) into the filled-in Bam H1-site of pBR322 [13]. Fig. 4 gives the results of radioimmuno-assays using

32 P-

labelled poly (dG-dC) in the high salt form and unlabelled co-^ valently closed,circular pLP32-DNA as isolated from E.coli. or poly (dG-dC) as competitor.

The concentration of the plas-

mid DNA necessary for a 50% inhibition is about 200 times

273

AI losteric DNA

[DNA]/i a a P-poly (dG-dC)] F i g . 4: C o m p e t i t i v e r a d i o i m m u n e a s s a y w i t h p u r i f i e d Z-D11 a n t i b o d y (0.2 mM 3 2 P - p o l y ( d G - d C ) in 4M N a C l ) . T h e effect of unlabelled poly(dG-dC) ( — o — ) , covalently closed c i r c u l a r D N A of the p l a s m i d p B R 3 2 2 (-A-) a n d p L P 3 2 (-•-) r e s p e c t i v e l y (DNA c o n c e n t r a t i o n in m o l e n u c l e o t i d e / 1 ) . I n t h e a b s e n c e o f c o m p e t i n g D N A 0.05-0.1 (iM o f the l a b e l l e d p o l y ( d G dC) w a s p r e c i p i t a t e d . higher

(in n u c l e o t i d e s )

t h a n the o n e of the p o l y ( d G - d C ) .

Con-

s i d e r i n g o n l y the c o n c e n t r a t i o n of the a l t e r n a t i n g

(dG-dC)., i n 16 the p l a s m i d DNA, w h i c h c o m p r i s e s 0.72% o f the b a s e p a i r s , the

c o n c e n t r a t i o n for a 50% i n h i b i t i o n is p r a c t i c a l l y the same that of

as

poly(dG-dC).

S i n c e a n e g a t i v e s u p e r h e l i c a l d e n s i t y f a v o r s the f o r m a t i o n o f Z - D N A , e x p e r i m e n t s a t low s a l t w i t h p L P 3 2 o f n a t u r a l helical density were performed.

super-

A s s h o w n in F i g . 5 b y a d i f -

f e r e n t k i n d o f a s s a y , p L P 3 2 - D N A is s l i g h t l y r e t a r d e d d u e

to

the b i n d i n g of the a n t i b o d y , w h i l e no e f f e c t w i t h p B R 3 2 2

is

observed.

(The s m a l l a m o u n t o f o p e n c i r u c l a r D N A s e r v e s as

i n t e r n a l s t a n d a r d i n this type of assay.)

Thus when

f a c i l i t a t e the B - t o Z - t r a n s i t i o n , the n a t u r a l

an

sequences

superhelicity

274

F.M.

Pohl

a) oc ccc 1 2 3 4 5 6 7

1 2 3 4 5 6 7

Fig. 5: Electrophoresis of ccc-DNA molecules (3nM) of plasmid pLP32 (a) and pBR322 (b) after incubation with varying amounts of purified Z-D11 antibody in 0.2 M NaCl, 60 mM Na-phosphate, 30 mM EDTA at room temperature for 60' in a volume of 15 JJI, electrophoresis in 1 % agarose gel and staining the DNA with ethidium bromide. Lanes 1-6: 0.6, 2.3, 9.4, 37, 150, 600 nM antibody, lane 7: no antibody. is enough to allow the switch of such a sequence to a lefthanded conformation to occur at "physiological" conditions.

Ethidium bromide as an "indirect" effector of a DNA structure The intercalation of this dye unwinds superhelix density [14].

DNA and changes the

pLP32-DNA was incubated in the pre-

sence of the antibody at different concentrations of ethidium bromide,using pBR322-DNA as one control (Fig. 6). Within a narrow range of the ethidium bromide concentration:the binding of the antibody is abolished, indicating a strong dependence of the formation of the 3-DNA on the superhelical density. Although the net effect, namely the disappearance of Z-DNA in the presence of the intercalating drug, is the same as shown above for poly(dG-dC) the mechanism is probably somewhat dif-

AIlosteric

275

DNA

1 2

3 4 5 6 7 6 9

10 11

Fig. 6: Influence of ethidium bromide (EB) on the mobility of ccC-DNA of plasmid pLP32 (a) and pBR322 (b). Lanes 1,11: no antibody, no EB; 2-10: 0.8 nM Z-D11 antibody and 0.02, 0.07, 0.2, 0.6, 1.8, 5.5, 16.6, 50, 0.0 uM EB in the incubation mixture described in Fig. 6. Arrow indicates the sudden change in mobility of pLP32. ferent. By the intercalation into topologically constrained DNA the negative superhelix density, which promotes the formation of left-handed DNA, is decreased and the change in free energy transmitted "mechanically" around the DNA to the (dC-dG)^g sequence which then switches to a right-handed conformation at low salt. Such a mechanism is able to produce a cooperative change in the structure of a small part of a topologically constrained DNA even if the binding isotherm of the effector molecule itself is a linear function of the concentration. strates such a model in a schematic way.

Fig. 7 illu-

(A comparable effect

may also be produced by proteins which unwind DNA.)

It empha-

276

F.M. Pohl

Fig. 7; Schematic reaction scheme for an "allosteric mechanism" in topologically constrained DNA. The stress due to negative superhelical turns can be relieved e.g. by switching parts of the DNA to left-handed conformation (indicated in black), which can then be recognized by a specific antibody or protein molecule. Intercalation of ethidium bromide can also relax such DNA, making the formation of L-DNA less likely and abolishing thereby the binding site (thin lines). sizes the unique topological properties of such a DNA.

Conclusion The simple model system of the B-Z transition of poly- and oligo(dG-dC) shows a number of phenomena which illustrate the potentials as inherent in a variability of the DNA structure. A transition between right- and left-handed double helical structures is certainly a rather drastic change and much more subtle effects can be visualized

to play a role e.g. in a

regulated expression of the genetic information, which is stored chemically in the sequence of bases.

277

AIlosteric DNA

The picture of DNA with a beautiful but monotonous and static structure, which has prevailed the thinking in the past, has possibly to be modified and extended in the future.

It may

also change our ideas about recognition mechanisms in DNA.

Acknowledgements I thank R. Thomae for preparing monoclonal antibodies to Z-DNA, L. Peck and J. Wang for generously providing plasmid pLP32, the Fonds der Chemischen Industrie and the Deutsche gemeinschaft

Forschungs-

(Po 15 5/6) for support.

References 1.

Wang, A.H.-J., Quigley, G.J., Kolpak, F.J., Crawford, J.L., von Boom, J.H., van der Marel, G., Rich, A.: Nature 282, 680-686 (1979) .

2.

Dickerson, R.E., Drew, H.R., Conner, B.N., Wing, R.M., Fratini, A.V., Kopka, M.L.: Science 216, 475-485 (1982).

3.

Pohl, F.M., Jovin, T.M.: J. Mol. Biol. 62, 375-396

4.

Pohl, F.M.: Nature 260, 365-366

(1972).

5.

Lafer, E.M., Möller, A., Nordheim, A., Stollar, B.D., Rich, A.: Proc. Natl. Acad. Sei. USA JQ, 3546-3550 (1981).

6.

Malfoy, B., Leng, M.: FEBS Lett. 132, 45-48

7.

Nordheim, A., Pardue, M.L., Lafer, E.M., Möller, A., Stollar, B.D., Rich, A.: Nature 294, 417-422 (1981).

8.

Monod, J., Wyman, J., Changeux, J.-P.: J. Mol. Biol. 12, 88-118 (1965) .

9.

Pohl, F.M.: Cold Spring Harb. Symp. Quant. Biol. 41_ (in press (1982).

(1976).

(1981).

10. Pohl, F.M., Jovin, T.M., Baehr, W., Holbrook, J.J.: Proc. Natl. Acad. Sei. USA 6£, 3805-3809 (1972). 11. Stettier, U.H., Weber, H., Koller, T., Weissmann, C.: J. Mol. Biol. V3±, 21-40 (1979). 12. Klysik, J., Stirdivant, S.M., Larson, J.E., Hart, P.A., Wells, R.D.: Nature 290, 672-677 (1981). 13. Peck, L.J., Nordheim, A., Rich, A., Wang, J'.C.: Proc. Natl. Acad. Sei. USA 79, 4560-4564 (1982).

278

F.M. Poh1

14. Bauer, W., Vinograd, J.: J. Mol. Biol. £7, 419-435 ( 1970).

Received September 3, 1982

DISCUSSION Von Hippel: Sinoe the antibody to Z-DNA binds selectively to one form that is in equilibrium with others, it must perturb the equilibrium between the forms. To see what the free molecule itself is doing, can one scmehow extrapolate the position of the transition to zero antibody concentration? Pohl; In the case of poly(dG-dC), as shown in Fig. 2, the perturbation of the equilibrium is not measurable, but for a quantitative study the use of oligo(dG-dC) will be of advantage. The coupling between the structural transition and the binding of the antibody can then be investigated in detail. For plasmid-DNA there are experimental difficulties, but in principle such an extrapolation should provide the information about the conformation in the absence of the antibody. Corin: Is anything kncwn about which structural aspects of the Z-ENA. are recognized by the antibodies? Pohl: The hybridottes were selected for binding to poly(dG-dC) at 4 M salt. They all shew the strong salt dependence of binding to the high salt form and do not interact with single stranded or linear double stranded DNA. But three out of seven monoclonal antibodies do not interact with poly(dG-msdC) in the Z-form and therefore appear to recognize a different epitope. But a definite answer will only be provided by X-ray diffraction studies. Jaenicke; Have histone or non-histone proteins any effect on the B -» Z transition? Pohl; I do not knew about experiments with non-histone proteins and at present the results on reconstitution of histories with Z-DNA in the labs of Felsenfeld and van der Sande, respectively, are not in ocmplete agreement. Jaenicke; There are now many DNA sequences available; did you check the frequency of poly(dG-dC) sequences occurring in natural DNA? Pohl; I checked my private library of DNA-sequenoes with about 300 000 bp, but the occurrence of alternating G-C or purine-pyrimidine sequences is viiat one expects for a random distribution. Jaenicke; How does the insertion of A and/or T change the B -» Z transition?

Al losteric DNA

279

Pohl; Experiments with d(CQCATGCG) indicate that introducing an A.T basepair involves an unfavourable free energy change of more than 1.2 kcal/mole. Jaenicke; How can you stabilize the Z-form upon inraunizing your rabbits? Pohl: By chemical bromination poly (dG-dC), it adopts a Z-form at lew salt (reference 5 of manuscript) and ccmplexation with methylated serum albumin then provides an efficient immunogen. Bradbury; How does the level of methylation of cytosini affect the B -» Z transition? Pohl: It has been shown by Behe and Felsenfeld (1981) that the complete methylation of poly(dG-dC) at the 5-position of cytosin lowers the transition from about 2.3 M NaCl to 0.7 M NaCl, which corresponds to a free energy change of about -0.35 Kcal/mole base pair (ref. 9 of manuscript). If the free energy change is additive one should be able to calculate the transition for different degrees of methylation. Pecht: Can you conpete with oligonucleotide of G-C content with the antiZ-DNA reacting with the Z-DNA? In other words are there antibodies which react specifically with exposed sets of the CG bases or antibodies which bind to other parts of the polynucleotide, characteristic of the Z-form? Pohl: Double stranded oligo(dG-dC) in the Z-form ccnpetes with the binding of poly (dG-dC) in high salt (ref. 9 of manuscript), but no competition is observed with single stranded DMA., so I do not think that the antibody recognizes for example, the bases only, but is specific for the left handed duplex structure. Neupert: What is the relationship between Z-DNA and auto-antibodies in systemic Lupus Erythermatodes? Pohl: Antibodies to Z-DNA were observed in the serum of SLE-mice (ref. 5 of manuscript). Ralf Thomae has tested many human sera frem SIE-patients, but at present no clear picture emerges. Robillard: When you look at long segments of synthetic DNA and induce a transition from the B- to Z-form, do you retain alternating regions of B + Z or does the entire segment convert to the Z-form? Pohl: For short segments an all-or-none transition is adequate. But as shown in Fig. 1 for polymers above the cooperative length N, which appears to be roughly 400 base pairs, one expects alternating segments of B- and Z-DNA within one molecule in the transition region. This is directly related to the probability of nucleation within the molecule.

LEFT-HANDED Z DNA IN POLYTENE CHROMOSOMES

Michel

Robert-Nicoud

Lehrstuhl für Entwicklungsphysiologie, D-3400 Göttingen, FRG Donna J.

Arndt-Jovin, David A.

Universität

Göttingen,

Zarling, Thomas M.

Abteilung Molekulare Biologie, Max-Planck-Institut Biophysikalische Chemie, D-3400 Göttingen, FRG

Jovin für

Introduction Changing

the

represent factors

conformation

the as

most

well

of

direct

as

DNA

at

mechanism

specific

specific by which

effectors

can

structural and functional state of chromosomes. transition (2,3) such

differ

DNA

has

processes, greatly

properties Inside

may

environmental regulate

the

The reversible

(1) between the right-handed B and the left-handed Z

helical

physiological conditions in

sites

the

left-handed chromosomal

in

been

shown

to

occur

under

near

key

role

(4-9) and thus could play a

particularly their

since the two conformations

physicochemical

and

biochemical

(1,5,6,10-12). cell DNA

nucleus, may

be

proteins.

(e.g. C-methylation,

transitions mediated

between

Furthermore,

9)

and

increase the predisposition

base

topological of

right-

and

by cations, polyamines and

alternating

stress

modification (4,7,13) may

purine-pyrimidine

tracts to undergo the B->Z transition. Since

polynucleotides

immunogens

(6,14-16),

in

the

Z

antibodies

conformation

are

can

as a tool to

be

used

detect and locate Z DNA in cytological preparations

Mobility a n d Recognition in C e l l Biology © 1 9 8 3 by W a l t e r d e Gruyter & Co., Berlin • N e w Y o r k

potent

(6,17,18).

M. Robert-Nicoud et al. .

282

We briefly describe here the results of study

on

the

immunofluorescence

binding of antibodies directed against Z DNA to

salivary gland chromosomes of the thummi.

an

Polytene

chromosomes,

dipteran which

Chironomus

thumml 13 up to a 2 -fold

have

amplification of DNA sequences, exhibit different morphological structures

in

active

and

inactive

regions.

They provide a

unique opportunity to search for possible correlations particular

between

DNA conformations and the structural and functional

states of chromosomal loci.

Results and Discussion Binding of anti-Z DNA antibodies to fixed polytene chromosomes. Polyclonal

and

monoclonal

the left-handed laboratory al.

in

Z

antibodies directed against DNA in

conformation

have

been

prepared

in

our

according to published procedures (6,14, Zarling et preparation),

chromosomes

of

with

staining

with

demonstrates

to

contrast,

H-33342

in

stain

thummi

A comparison

phase a

used

Chironomus

immunofluorescence. obtained

and

fixed

polytene

by

indirect

thummi

(Figure

1)

of

the

immunofluorescence,

stretched

chromosomes

images and

DNA

clearly

strong binding of the antibodies in regions of

high contrast and high DNA density, the bands. The same result has been from

Drosophila

salivary

with an earlier report by fluorescence

obtained

restricted

with

glands, Nordheim to

polytene

chromosomes

an observation at variance et

al. (17)

interband

regions.

showing Results

further experiments providing a possible explanation discrepancy

of this

as well as information on the effects of enzymatic

treatments and DNA-specific drugs on the antibody presented

for

the

in

other

reports (6,18).

the binding to the chromosomal bands of

binding

are

The latter also document an

antibody

directed

Left-Handed Z DNA in Polytene

Chromosomes

283

Figure 1. Antibodies to left-handed Z DNA bind to the bands of fixed polytene chromosomes. Comparison of (A) phase contrast, (B) indirect immunofluorescence, and (C) DNA staining with H-33342 (Hoechst) in a stretched chromosome of Chironomus thummi. Ethanol:acetic acid (3:1) fixation. Rabbit anti-Z DNA IgG fraction Rab 24 (20 Hg/ml). For additional details regarding antibodies and experimental details see refs. 6 and 19). Bar denotes 10 microns.

284

M. Robert-Nicoud et al .

against

5-methylcytosine.

These

observations

possible correlation between the distributions

suggest

of

Z

DNA

a and

ra5C. The characteristic and highly reproducible patterns of immunofluorescence elicited by the binding of anti-Z DNA antibodies to the four salivary gland chromosomes of Chironomus thummi is shown in Figure 2A. The ubiquitous localization of the fluorescence in the regions of high contrast is striking (Figure 2B). Some bands, e.g. in telomeric regions and in the vicinity of the centromeres, display an exceptionally intense fluorescence. Since this differential staining is also observed with direct labelled antibodies (to be published), we conclude that it reflects true variations in the distribution of Z DNA rather than in the accessibility of the binding sites to the antibody.

Binding

of

anti-Z

chromosomes.

DNA

antibodies

to

unfixed

polytene

The physiological significance of left-handed DNA

in polytene chromosomes can preparations.

be

Fortunately,

best in

assessed the

using

case

of

unfixed

Chironomus,

techniques are available for the isolation and manipulation unfixed direct

salivary and

antibodies

gland chromosomes (19,20).

indirect also

bind

immunofluorescence to

isolated

binding is strongly dependent isolation

and

upon

that

anti-Z

unfixed chromosomes. the

ionic

of

We have shown by

conditions

DNA The of

of treatment prior to or during incubation with

Figure 2. Indirect immunofluorescence pattern obtained with anti-Z DNA antibodies bound to fixed polytene chromosomes of Chironomus thummi. (A) Immunofluorescence, (B) phase contrast. Chromosomes are identified by roman numerals. NO: nucleolus organizer region; c: centromeres; r: right hand end of chromosomes; BRc: Balbiani ring c. Fixation and antibody concentration as in Fig. 1.

Left-Handed Z DNA in Polytene Chromosomes

285

286

M. Robert-Nicoud et al.

antibody.

As

fluorescence

an is

example, observed

a

particularly

in

chromosomes

strong after

immuno-

exposure to

350-600 mM NaCl in the presence of divalent cations (Figure 3). These

ionic

conditions

are

known

decondensation of chromosome bands histone

HI

as

well

to

(19,21)

images

demonstrates

and

that

restricted to highly compact regions detailed

report

and

the

loss

of

as some high mobility group proteins.

comparison of the immunofluorescence contrast

induce a differential

of

corresponding

A

phase

the

fluorescence

is

the

chromosomes.

A

on the results obtained with isolated unfixed

chromosomes will be published elsewhere (Robert-Nicoud, et

al.

in preparation). Possible role of Z DNA in chromosomes. the

ubiquitous

localization

the chromosomes and interchromosomal peculiar

its

of Z DNA in condensed regions of

enrichment

associations.

physicochemical

left-handed

Our results demonstrate

and

in

regions

involved

in

Taking into consideration the biochemical

properties

of

polynucleotides in solution (see Introduction), we

may speculate on the potential chromosomes.

The

property

roles of

of

Z

DNA

demonstrated by the aggregated form Z* [e.g. poly[d(G-m^C)]

in

the

in

polytene

self association of Z DNA, as poly[d(G-C)]

and

presence of divalent cations (5,6,10)]

may be responsible for the linkage between adjacent chromomeres and

for

the

interaction

between

closely related repetitive

sequences known to be present in telomeres

(22)

and

at

many

sites scattered through the chromosomes (23).

Figure 3. Antibodies directed against left-handed Z DNA bind to isolated unfixed polytene chromosomes. Comparison of (A) indirect immunofluorescence and (B) phase contrast. Salivary gland chromosome of Chironomus thummi pretreated and stained in 475 mM NaCl in the presence of divalent cations. Arrows point to regions of high contrast and bright fluorescence.

Left-Handed Z DNA in Polytene Chromosomes

287

288

M. Robert-Nicoud et al.

Furthermore, the bidirectional nature of the B-Z transition may serve

as

a mechanism to determine the structural state of the

chromosomes and to regulate the level of gene is

interesting

in

the

state

chromatin

expression.

It

that the ionic conditions which elicit changes of

condensation

(19,21,24-28)

of

the

correspond

to

chromosomes many

of

and

the

of

ionic

requirements for inducing the B->Z and Z->B transitions. Finally, it would appear unlikely that the phenomena here

are

restricted to polytene chromosomes.

described

One can imagine

that changes occuring in the structural and functional state of the

chromatin

cell cycle proteins,

may

during be

enzymes,

development,

mediated etc.)

by

cell differentiation, and

factors

(ions,

polyamines,

acting directly or indirectly (e.g.

through alterations in topological stress) on the conformations of

specific DNA tracts with a potential for the B-Z transition

determined by the primary

sequence

and

the

extent

of

base

modification.

Acknowledgements

The authors acknowledge the Alexander von Humboldt the

Volkswagen

Foundation,

and

the

Max

Foundation,

Planck Society for

fellowship and grant support.

References 1. 2. 3.

Pohl, F.M., Jovin, T.M.: J^ Mol. Biol. 67, 375-396 (1972). Wang, A.H.-J., Quigley, G.J., Kolpak, F.J., van der Marel, G., van Boom, J.H., Rich, A.: Science 211, 171-176 (1981). Dickerson, R.E., Drew, H.R., Conner, B.N., Wing, R.M., Fratini, A.V., Kopka, M.L.: Science 216, 475-485 (1982).

Left-Handed Z DNA in Polytene

Chromosomes

289

4.

Peck, L . J . , N o r d h e i m , A . , R i c h , A., W a n g , J . C . : Natl. Acad. Sei. U S A 79, 4 5 6 0 - 4 5 6 4 (1982).

5.

v a n d e S a n d e , J . H . , J o v i n , T . M . : T h e E M B O J. (1982).

6.

J o v i n , T . M . , v a n de S a n d e , J . H . , Z a r l i n g , D.A., A r n d t - J o v i n , D . J . , E c k s t e i n , F., F ü l d n e r , H . H . , G r e i d e r , C., G r i e g e r , I., H a m o r i , E., K a i i s c h , B., M c i n t o s h , L . P . , Robert-Nicoud, M.: Cold Spring Harbor Symp. Quant. Biol. 47, in p r e s s .

7.

S i n g l e t o n , C . K . , K l y s i k , J., S t i r d i v a n t , S . M . , R . D . : N a t u r e 299, 3 1 2 - 3 1 6 (1982).

8.

M a l f o y , B., R o u s s e a u , N., L e n g , M . : B i o c h e m i s t r y , press.

9.

B e h e , M., F e l s e n f e l d , G . : P r o c . 78, 1 6 1 9 - 1 6 2 3 (1981).

10.

v a n de S a n d e , J . H . , M c i n t o s h , L . P . , J o v i n , T . M . : The J^ 1, 7 7 7 - 7 8 2 (1982).

11.

B e h e , M., Z i m m e r m a n , S., F e l s e n f e l d , G . : N a t u r e 2 3 3 - 2 3 5 (1981).

12.

Pohl, F . M . : C o l d S p r i n g H a r b o r S y m p . in p r e s s .

Natl.

115-120

Wells,

Acad.

Quant.

Proc.

in

Sei.

USA EMBO

293,

Biol.

47,

13.

Pohl, F . M . , T h o m a e , R., D i C a p u a , E . : N a t u r e , in p r e s s .

14.

L a f e r , E . M . , M ö l l e r , A., N o r d h e i m , A . , S t o l l a r , B.D., Rich, A.: Proc. Natl. Acad. Sei. U S A 78, 3 5 4 6 - 3 5 5 0 (1981).

15.

M a l f o y , B . , L e n g , M . : F E B S L e t t e r s 132, 4 5 - 4 7

16.

Pohl, F . M . : t h i s

17.

N o r d h e i m , A . , P a r d u e , M . L . , L a f e r , E . M . , M ö l l e r , A., S t o l l a r , B . D . , R i c h , A . : N a t u r e 294, 4 1 7 - 4 2 2 (1981).

18.

A r n d t - J o v i n , D . J . , R o b e r t - N i c o u d , M., Z a r l i n g , D.A., G r e i d e r , C., J o v i n , T . M . : Proc. Natl. Acad. Sei. USA, submitted.

19.

R o b e r t , M . : C h r o m o s o m a 36, 1 - 3 3

20.

R o b e r t , M : M e t h o d s in C e l l B i o l .

21.

L e z z i , M., R o b e r t , M . : R e s u l t s a n d P r o b l e m s i n C e l l D i f f e r e n t i a t i o n ( B e e r m a n n , W . , R e i n e r t , J., U r s p r u n g , Eds.), Springer, N.Y., vol. 4, 1972, p p . 35-37.

22.

Rubin, G.M.: Cold Spring Harbor Symp. 1041-1046 (1978).

23.

Finnegan, D-J., Rubin, G.M., Young, M.W. and Hogness, D.S.: Cold Spring Harbor Symp. Quant. Biol. 42, 1 0 5 3 - 1 0 6 3 (1978).

(1981).

volume.

(1971). 9, 3 7 7 - 3 9 0

Quant.

(1975).

Biol.

H. 42,

290

M. R o b e r t - N i c o u d et a l .

24.

Robert, M., Mondrianakis, E.N.: J ^ (1976).

25.

Leake, R.E., Trench, M.E., Barry, J.M.: Exptl. 71, 17-26 (1972) .

26.

Olins, D.E., Olins, A.L.: J^ (1972).

27.

Ausio, J., Haik, Y., Seger, D., Eisenberg, H.: this volume.

28.

Bradbury, E.M.: this volume.

Received October 7, 1982

Cell Biol.

Cell Biol.

53,

70, 62a Cell Res. 715-736

LOCATION AND RECOGNITION OF SPECIFIC DNA BINDING SITES PROTEINS THAT REGULATE GENE

EXPRESSION

P e t e r H. v o n H i p p e l a n d D a v i d G. Institute

of M o l e c u l a r

University

of

BY

Bear*

Biology and Department

of

Chemistry,

Oregon

Eugene, Oregon

97403,

U.S.A.

INTRODUCTION To understand gene expression develop a detailed regulatory proteins specific

target

The

forces

interactions)

interactions

the m i l l i o n s of

of p r o t e i n s w i t h D N A

intermolecular that are

interact with other double

involve

the s a m e

involved

a n d the s a m e s i m p l e

in the r e l a t i v e l y

between small molecules

folded

because

into very

(1).

because

and

functional

nonspecific However,

the

inter-

overall

b o t h the p r o t e i n a n d t h e n u c l e i c a c i d

specific

conformations;

functional g r o u p s can form m u l t i s i t e great positional

basic

(hydrogen bonds, charges, dipoles

a c t i o n s of s u c h s i m p l e g r o u p s c a n d e v e l o p e n o r m o u s selectivity

their

genome.

solvent-driven groups

and

must

whereby

in the s u p e r f i c i a l l y m o n o t o n i c

D N A of the

interactions

l e v e l , we

the m e c h a n i s m s

locate, recognize,

sequences among

sequences present helical

at the m o l e c u l a r

k n o w l e d g e of

and directional

this specificity

t h u s the

interacting

interaction domains

specificity.

with

Moreover,

is c o n f o r m a t i o n d e p e n d e n t ,

* Present address: D e p a r t m e n t of C e l l B i o l o g y , U n i v e r s i t y of N e w M e x i c o S c h o o l of M e d i c i n e A l b u q u e r q u e , New Mexico 87131

Mobility and Recognition in Cell Biology © 1983 by Walter de Gruyter & Co., Berlin • New York

are

small

con-

292

P.H.' von Hippel and D.G. Bear

f o r m a t i o n a l c h a n g e s can a l t e r the total s t a b i l i t y of c o m p l e x by o r d e r s of m a g n i t u d e . interacting decrease overall

A minor mispositioning

f u n c t i o n a l g r o u p s c a n thus r e s u l t

seem to a p p l y to the

and s t a b i l i t y of p r o t e i n - n u c l e i c gene regulation: affinity

for DNA

(i)

acid interactions

Nonspecific binding

involving c h a r g e - c h a r g e

i n t e r a c t i o n s of

The

free

phosphates energy

from the e n t r o p y of d i l u t i o n of

the s m a l l c a t i o n i c c o u n t e r i o n s

(K+, Na+, Mg+^)

the D N A w h e n the p r o t e i n b i n d s

(6).

displaced

(iii) S p e c i f i c

r e q u i r e s the i n t e r a c t i o n of a m a t r i x of D N A h y d r o g e n double-helix)

(located

in the g r o o v e s of

site

f a v o r e d at e q u i l i b r i u m ,

(2).

bond

the

(iv) For s p e c i f i c b i n d i n g

the p r o d u c t of K and

c o n s t a n t and

(3). (v)

[D]

binding

nonspecific

G e n e t i c r e g u l a t o r y p r o t e i n s m a y bind

f i c a l l y to D N A sites c o n t a i n i n g

to be

(where K is

[D] is the c o n c e n t r a t i o n of

site) m u s t be g r e a t e r for s p e c i f i c t h a n for binding

from

binding

w i t h the c o m p l e m e n t a r y a c c e p t o r s and d o n o r s of

the p r o t e i n b i n d i n g the b i n d i n g

sites

"electrostatic",

i n t e r a c t i o n s b e t w e e n DNA

this p r o c e s s r e s u l t s

d o n o r s and a c c e p t o r s

speci-

a few "wrong" base p a i r s ;

but

w i t h too m a n y u n f a v o r a b l e m i s p a i r i n g s of p r o t e i n and

nucleic

a c i d h y d r o g e n b o n d i n g g r o u p s the p r o t e i n

into a

nonspecific DNA binding conformation a t t e m p t to a p p l y these p r i n c i p l e s important protein-nucleic

is " p u s h e d "

(2).

to a

In this p a p e r we

physiologically

acid i n t e r a c t i o n ,

to ask how an R N A

p o l y m e r a s e m i g h t find, r e c o g n i z e and b i n d to a " c l o s e d " m o t e r site on a D N A g e n o m e . developed

in

these

relevant DNA target

is l a r g e l y

and the b a s i c r e s i d u e s of the p r o t e i n . driving

specificity involved

Nonspecific binding plays a central

in b o t h e q u i l i b r i u m and k i n e t i c (ii)

the

M a n y r e g u l a t o r y p r o t e i n s show a g e n e r a l

(3,4).

p r o t e i n s w i t h their p h y s i o l o g i c a l l y (5).

of

large

(2).

The f o l l o w i n g g e n e r a l i z a t i o n s

role

in a

in b o t h the s p e c i f i c i t y and the s t r e n g t h of interaction

the

in m o r e d e t a i l

(Some a s p e c t s of these

in ref.

7.)

pro-

ideas

are

Location and Recognition of Specific DNA Binding

-35

-60 antisense sense

-10

TAtaaT ATattA

TTGaca•AACtgt •

5'' 3'

+1

+ 25

G C

3' 5'

5-7 bp

I7(£l)bp

Figure 1: Summary of ters recognized by E. text). Modified from and Siebenlist et al.

293

Sites

consensus features of procaryotic promocoli RNA polymerase (for details see data compiled in Rosenberg and Court (7) (8).

The Bacterial Promoter:

A promoter is generally defined as a

segment of DNA [usually immediately preceding a gene(s)] that contains signals for the proper binding and subsequent activation of RNA polymerase holoenzyme to a form capable of initiating the synthesis of RNA.

Some operons also contain an

additional (recognition) regulatory site in the vicinity of the promoter, for the specific binding of repressor or activator proteins that can modulate the activity of the promoter. Due to the advent of rapid DNA sequencing techniques, a great number of promoter sequences can now be compared; this has revealed certain common features (for reviews see refs. 8 and 9), including a so-called "consensus sequence" (Figure 1).

Surprisingly, there is a relative paucity of common base pair sequences throughout the promoter region.

We infer certain

base pair replacements may not perturb the interaction with amino acids of the polymerase, and that sequence variation in promoters may play an important role in gene regulation by controlling promoter "strength".

Thus, the search for highly

conserved sequences may be of only limited utility. Nevertheless some important regularities do emerge: (i) The promoter sequence is asymmetric.

This reflects the inherent

asymmetry of the prime function of the promoter, which is to initiate mRNA synthesis in the correct direction and on the correct ("sense") template strand.

(ii) There are highly con-

served sequences around the -10 and -35 positions of the promoter.

At -10 the consensus sequence is TAtaaT, and at

294

P.H. von Hippel and D.G. Bear

-35, TTGaca.1

(iii) The d i s t a n c e b e t w e e n the m o s t

p o s i t i o n s of the - 1 0 and the - 3 5 s e q u e n c e s (± 1) base p a i r s , w i t h 17 r e s u l t i n g strength

conserved

is g e n e r a l l y

in m a x i m u m

17

promoter

(10).

The i m p o r t a n c e of the c o n s e r v e d r e g i o n s at - 1 0 and - 3 5 is also manifested

in three o t h e r w a y s .

tions o c c u r

First, most promoter

in, or near these r e g i o n s

(8,9).

muta-

In a d d i t i o n ,

m o s t of the a c t u a l c o n t a c t s b e t w e e n f u n c t i o n a l g r o u p s of promoter

(hydrogen-bonding groups

in the g r o o v e s of the

d o u b l e - h e l i x , and b a c k b o n e p h o s p h a t e s )

and of the

(protein side c h a i n s ) , as d e t e r m i n e d by " c h e m i c a l analysis, also occur

in or near these r e g i o n s

nuclease digestion experiments s e q u e n c e s are i n c l u d e d complex

formation

(9).

into four s e q u e n t i a l

show that the - 3 5 and - 1 0

"closed"

(and p r o b a b l y

steps

(12,13):

promoter-polymerase

complex;

promoter-polymerase

complex

(i) p r o m o t e r

Processes

(iii)

and

location;

"closed" "open"

(iv) m R N A

(i) and (ii) w i l l be c o n s i d e r e d for the l o c a t i o n and

t i o n of DNA t a r g e t s i t e s by g e n e t i c r e g u l a t o r y (iii) and

divided

f o r m a t i o n of the

("melting-in");

in d e v e l o p i n g m e c h a n i s m s

Processes

from

"open")

i n t e r a c t i o n can be

(ii) p r o m o t e r r e c o g n i t i o n and f o r m a t i o n of the

initiation.

Finally,

(11).

The o v e r a l l p r o m o t e r - p o l y m e r a s e

here,

polymerase probe"

in the r e g i o n that is p r o t e c t e d

DNAse by p o l y m e r a s e d u r i n g

the DNA

(iv) w i l l be d i s c u s s e d ,

M c C l u r e and H a w l e y e l s e w h e r e

in this v o l u m e

further

recogni-

proteins.

in p a r t ,

by

(see also ref.

7).

^ P o s i t i o n n u m b e r s w i t h i n the p r o m o t e r s e q u e n c e r e p r e s e n t p o s i t i o n s " u p s t r e a m " (-) or " d o w n s t r e a m " (+) from the f i r s t m R N A i n c o r p o r a t e d , w h i c h is u s u a l l y a p u r i n e a n d is n u m b e r e d +1. S e q u e n c e s listed are o n s e q u e n c e s w i t h i n the m R N A (with U r e p l a c i n g T). R e s i d u e s listed in c a p i t a l l e t t e r s are c o n s e r v e d in a l m o s t all p r o m o t e r s ; those l i s t e d in lower case l e t t e r s are c o n s e r v e d to a lesser e x t e n t .

295

Location and Recognition of Specific DNA Binding Sites PROMOTER The

L O C A T I O N BY R N A

POLYMERASE

l o c a t i o n of a u n i q u e

functional

m o t e r or an o p e r a t o r ) protein

is n o t a s i m p l e

chromosome

carries

distributed

among

r o l e of the m a n y

is,

tion —

was

is p e r m i t t e d

(16).

Following

been subjected

to c o n s i d e r a b l e analysis.

formation may

Location

(R) c a n l o c a t e

to l a c o p e r a t o r

for the

interaction, we will

e s t i m a t e of in M - 1

one-step

of

and

Using

+

0

experisteps

summarize

indicated

kd the D e b y e - S m o l o c h o w s k i

equation:

our

(0)

above,

second

order

repressor

in a

process:

RO

has

complex

ka R

reac-

the

first

with which a

a n d b i n d to an o p e r a t o r

diffusion-controlled,

more

here.

the m a x i m u m

sec-1)

able

sites

(17-21)

is a T w o - S t e p P r o c e s s : A s

(kajcaic?

to be

the l a c s y s t e m

theoretical

of the l a c s y s t e m

we can make a reasonable

fast.

seems

DNA target site m u c h

s e r v e as a n i m p o r t a n t m o d e l

present understanding

rate constant

protein

polymerase

(14,15)

Since operator-repressor

of the p r o m o t e r - p o l y m e r a s e

Operator

evidence

initial observation,

mental

(22,23)

nonspecific

in iji v i t r o m e a s u r e m e n t s

lac repressor

this

dif-

with

in a f u l l y d i f f u s i o n - c o n t r o l l e d

first observed

b i n d i n g of E^. c o l i

Clearly

coupled

in f a c t , v e r y

that a regulatory

t o l o c a t e a n d b i n d to a s p e c i f i c than

sites.

l o c a t i o n by

Y e t the a v a i l a b l e location

promoters

slow three-dimensional

to m a k e p r o m o t e r

that promoter

This p a r a d o x — i . e . ,

pro-

the E^. c o l i

hundred

RNA polymerase m o l e c u l e ,

competitive

a very slow process.

rapidly

Thus,

than several

the r e l a t i v e l y

should combine

suggests

( s u c h as a

genome-regulatory

several million nonspecific

f u s i o n of t h e m a s s i v e the p o t e n t i a l l y

DNA site

undertaking.

no m o r e

t h e c o m b i n a t i o n of

sites,

by a s p e c i f i c

(1)

P.H. von Hippel and D.G. Bear

ka,calc

4

=

f

™

w h e r e k and f e i e c are

elec

b

(dimensionless)

f a c t o r s , b is the i n t e r a c t i o n r a d i u s the free d i f f u s i o n c o n s t a n t s (in c m ^ / s e c ) , and N 0 that k a , c a l c

c a n

be

NQ/1000

(2)

s t e r i c and

electrostatic

(in c m ) , Dr and Dq are

for lac r e p r e s s o r

and

operator

is A v a g a d r o ' s n u m b e r , we have

no

lar

9er

than ~ 1 0 8 M - 1

interaction,

i^f the p r o c e s s

v a l u e s of k a

(eq.l), m e a s u r e d

sec-1

estimated for the

RO

is fully d i f f u s i o n - c o n t r o l l e d and 2 n o n s p e c i f i c b i n d i n g d o e s not interfere (2,21) • In c o n t r a s t , in v i t r o

for the lac

operator

i n t e r a c t i o n , have b e e n s h o w n to e x c e e d

1010 M - 1

sec-1

(16, 22, 23).

by r e p r e s s o r m u s t

C l e a r l y the l o c a t i o n of

operator

involve m e c h a n i s m s by w h i c h e i t h e r

d i m e n s i o n a l i t y or the v o l u m e of the s e a r c h p r o c e s s in o r d e r to f a c i l i t a t e

target

(operator)

that the a c t u a l k i n e t i c m e c h a n i s m m u s t two-step

repressor-

is

location.

involve

the reduced

This

means

(at least)

a

process: kl R

+

D

+

0

—

v

k2 RD

+

0

k-1

~ — R O

+

D

(3)

k_2

The first step c o n s i s t s of the d i f f u s i o n - c o n t r o l l e d of a c o m p l e x b e t w e e n r e p r e s s o r and n o n s p e c i f i c f o l l o w e d by f a c i l i t a t e d

translocation

(via

DNA

formation (D),

intermediate

n o n s p e c i f i c RD c o m p l e x e s ) , p r i o r to final RO c o m p l e x

formation.

2 In an a n a l y s i s of the t w o - s t e p m e c h a n i s m (eq. 3), Berg et al. (21) and W i n t e r et a 1. (23) have s h o w n t h e o r e t i c a l l y and e x p e r i m e n t a l l y that k^ (the initial b i n d i n g rate c o n s t a n t for r e p r e s s o r to a n o n s p e c i f i c DNA site) is ~ 1 0 7 to 10® M--*- sec-''' (per m o l e of b a s e p a i r s ) . Since k a for a o n e - s t e p RO i n t e r a c t i o n (eq. 1) c a n n o t be a p p r e c i a b l y larger than k^ (see ref. 21), this e f f e c t i v e l y sets the m a x i m u m u n f a c i l i t a t e d f o r w a r d rate c o n s t a n t at ~ 1 0 7 to 1 0 8 M - 1 s e c - 1 for the RO i n t e r action process. P o l y m e r a s e is a l a r g e r p r o t e i n than lac r e p r e s s o r ; t h e r e f o r e k a f l n a x for a o n e - s t e p pol'ymerasep r o m o t e r i n t e r a c t i o n m u s t be less than ~ 10' sec .

L o c a t i o n a n d R e c o g n i t i o n o f S p e c i f i c DNA B i n d i n g

The o v e r a l l

(observed)

step m e c h a n i s m be w r i t t e n

the

for s u c h a t w o =

a r r a n g e d so that k_2

= 1

+

D

^ T RD K

D

TKRD k 2 V

+

k

y

(4)

- 1

i n d i v i d u a l rate c o n s t a n t s are as d e f i n e d

in eq.

D T a n d 0 T are the total c o n c e n t r a t i o n s of n o n s p e c i f i c binding

ma

(19,21): k

where

forward rate c o n s t a n t

(experimentally

297

Sites

sites and o p e r a t o r sites, and K R D is the

(3),

DNA

nonspecific

b i n d i n g c o n s t a n t (B Using c a l c u l a t e d v a l u e s of kj (21) that have b e e n experimentally ka

(23), the v a r i o u s p a r a m e t e r s of eq.

mined

for the lac r e p r e s s o r - o p e r a t o r

of salt c o n c e n t r a t i o n s lengths

(4) have b e e n

parameters

strong and v a r i a b l e

DNA

(see Figure

2) are

range

fragment

sufficiently

f u n c t i o n s of b o t h salt c o n c e n t r a t i o n

total DNA f r a g m e n t l e n g t h so that an u n a m b i g u o u s t i o n is p o s s i b l e .

In d i l u t e s o l u t i o n

(Figure

f r a c t i o n of

(3).

Furthermore,

in o r d e r

f o r m a t i o n to be f a c i l i t a t e d , RD c o m p l e x

not be i n h i b i t i n g ; must occur during

operator),

3), the RO i n t e r a c t i o n c l e a r l y

p r o c e e d as d e f i n e d by eq.

in fact, a c t i v e r e p r e s s o r

(see Figure

the

does for RO

formation must

translocation

the n o n s p e c i f i c b i n d i n g p r o c e s s

in o r d e r

i n c r e a s e the rate of target l o c a t i o n to the m e a s u r e d R e p r e s s o r S l i d e s to the O p e r a t o r T a r g e t :

and

interpreta-

lO--^ M

the D N A "domains" o c c u p y o n l y a small

total s o l u t i o n complex

deter-

s y s t e m o v e r a wide

and o p e r a t o r - c o n t a i n i n g

and

(23).

The r e s u l t i n g

where

confirmed

(23) w i t h m e a s u r e d v a l u e s of K R D (24,25)

A n a l y s i s of

value. data

2) shows that these a s s o c i a t i o n r e s u l t s c a n be

fitted quantitatively

if one a s s u m e s that t r a n s l o c a t i o n

r e p r e s s o r to o p e r a t o r

involves a s e r i e s of

intra-domain

of

to

P.H. von Hippel and D.G. Baer

298 log K r d 16

14

12

10

8

6

4

log [KCl]

F i g u r e 2. Plot of log k a v e r s u s log [KCl] for: (a) X p l a c 5 D N A (~50,000 b a s e p a i r s ) ; (b) EcoRI l a c - o p e r a t o r - c o n t a i n i n g DNA f r a g m e n t (~6700 base p a i r s ) ; and (c) H a e l l l l a c - o p e r a t o r c o n t a i n i n g DNA f r a g m e n t (203 base p a i r s ) . Association rates d e t e r m i n e d by filter b i n d i n g at ~ 2 0 ° C . D a t a from W i n t e r et al. (23). T h e o r e t i c a l c u r v e s from Berg et al. (21).

dissociation-association

events,

i n t e r s p e r s e d by

active

" s l i d i n g " of the r e p r e s s o r o v e r the n o n s p e c i f i c DNA w h i l e repressor

is b o u n d as an RD c o m p l e x

(Figure

3).

Figure

s h o w s that the d e p e n d e n c e of log k a o n log

[KCl] c a n be

theoretically

(salt

independent) constant)

(solid lines) using a single v a l u e of D^

(the o n e - d i m e n s i o n a l

for three o p e r a t o r - c o n t a i n i n g

widely varying

length.

the

2 fitted

concentration

diffusion

DNA f r a g m e n t s

The b e s t - f i t v a l u e of D^ is

of

299

Location and Recognition of Specific DNA Binding Sites

F i g u r e 3. S c h e m a t i c v i e w of lac r e p r e s s o r (R) i n t e r a c t i n g w i t h o p e r a t o r - c o n t a i n i n g (0) X p l a c 5 D N A d o m a i n s in d i l u t e solution. (For further d e t a i l s see text, and refs. 21 and 23) . approximately

9 x 10-10

the d a t a of Figure follows:

2 can be e x p l a i n e d

At h i g h salt c o n c e n t r a t i o n s

b, and c) n o n - s p e c i f i c complexes

c m 2 / s e c at ~ 2 0 ° C . 3

is short.

binding

in these terms

location

e n t i r e l y d e p e n d e n t on a t h r e e - d i m e n s i o n a l concentration

walk 3

relatively

slow p r o c e s s .

is g i v e n m o r e

site on n e i g h b o r i n g

(sliding).

RD

the

As the

salt

complexes

time to " s e a r c h "

DNA v i a a o n e - d i m e n s i o n a l

T h i s leads to an increase

a,

is a l m o s t

s e a r c h of

is d e c r e a s e d the l i f e t i m e of RD

i n c r e a s e s and r e p r e s s o r operator

as

(right side of c u r v e s

is weak and the l i f e t i m e of

Thus, operator

D N A by the r e p r e s s o r — a

Qualitatively,

in the

for

the

random

ka-

S o m e w h a t b e t t e r fits to the low salt d a t a for the A p l a c 5 and the H a e - 2 0 3 f r a g m e n t s , as w e l l as to the p l a t e a u of the EcoRI f r a g m e n t d a t a , c a n be o b t a i n e d by using s l i g h t l y d i f f e r e n t v a l u e s of K R D a n d / o r D^ in these e x p e r i m e n t s (see ref. 22). H o w e v e r in Figure 2 we show the fit o b t a i n e d for all three D N A f r a g m e n t s usinQ the best sihqle v a l u e s of D^ a n d K J ^ Q •

P.H. von Hippel and D.G. Bear

300

Everrtually, for the l a r g e s t DNA f r a g m e n t v a l u e of k a is r e a c h e d . sliding

A t this p o i n t the d u r a t i o n of

search process, relative

(non-correlated)

each

to the rate of o c c u r r e n c e

intra-domain dissociation-association

is o p t i m a l l y b a l a n c e d

(this o c c u r s at K R D - 1 / D T ) .

lower salt c o n c e n t r a t i o n s search processes tively extended

(curve a), a m a x i m u m

(curve a) the

(by o n e - d i m e n s i o n a l

sliding)

are

events,

At

individual

still

correlated nonproduc-

(since this s c a n n i n g o p e r a t i o n w i l l

often

involve D N A s e g m e n t s d i s t a n t from the o p e r a t o r ) , u n t i l , v e r y low salt c o n c e n t r a t i o n s ,

k a becomes

i n d e p e n d e n t and the t a r g e t

is l o c a t e d

single n o n s p e c i f i c binding

event.

For s m a l l e r D N A d o m a i n s salt.

be s c a n n e d by sliding (correlated)

during

in w h i c h k a

is

decreases

is s m a l l e r ,

the e x t e n d e d

is e l i m i n a t e d .

fragments

nonproductive

For still

(curve c), w h i c h can

smaller

essentially

be a p p r o x i m a t e d as rigid rods, the s e a r c h p r o c e s s

is not

l i m i t e d by RD c o m p l e x

every

binding event sliding.

(at

l i f e t i m e s since e s s e n t i a l l y

[KC1]

< ~ 0.1 M) leads to t a r g e t l o c a t i o n

W h i l e DNA f r a g m e n t s of this size c a n c o n t r i b u t e

the o v e r a l l rate by d i f f u s i o n , ka

a

Since the o v e r a l l D N A t a r g e t w h i c h m u s t

scanning phase

operator-containing

phase

at

salt-concentration-

(by sliding)

(curve b), a h i g h e r p l a t e a u

r e a c h e d w i t h o u t the intervening with decreasing

the final p l a t e a u value

locate and bind the s m a l l e r DNA is

by

to

of

is l o w e r e d b e c a u s e the rate at w h i c h the r e p r e s s o r c a n

tially

of

ini-

significantly

reduced. Molecular ceding

I n t e r p r e t a t i o n of R e p r e s s o r S l i d i n g :

s e c t i o n we s u m m a r i z e d e v i d e n c e

facilitated

that o p e r a t e s w h i l e the

t e i n is ( n o n s p e c i f i c a l l y )

b o u n d to n o n - o p e r a t o r

m e c h a n i s m has b e e n termed

"sliding".

fusion constant calculated fic DNA

(Di)

DNA.

10~9 cm2/sec, which

pro-

This

The o n e - d i m e n s i o n a l

for r e p r e s s o r t r a v e r s i n g

is a p p r o x i m a t e l y

pre-

for the e x i s t e n c e of a

contour-length correlated, diffusion-driven, t r a n s f e r m e c h a n i s m for r e p r e s s o r

In the

dif-

nonspeci-

corresponds

L o c a t i o n and R e c o g n i t i o n o f S p e c i f i c D N A B i n d i n g

to an i n s t a n t a n e o u s r a n d o m walk ~

10

6

(sliding)

"jumps" between neighboring

per second

U

sites

(base

is (D\t/i2) Vl, c o r r e s p o n d i n g

0.1 sec., e t c . ) .

i.e.,

to the s c a n n i n g

of

down

follows:

(i) Is the a b o v e s l i d i n g rate r e a s o n a b l e

cmvsec)

for a

if the rate is o n l y

limited by hydrodynamic considerations? —• Q O

Clearly

is m u c h s m a l l e r than the o r d i n a r y

three-

d i m e n s i o n a l d i f f u s i o n c o n s t a n t e s t i m a t e d for a p r o t e i n of (~ 150,000 d a l t o n s ; D 3 = 5 x 1 0 " 7 c m 2 / s e c ) .

size

repressor

s l i d e s l i n e a r l y along

frictional

resistance

bulk s o l v e n t , limit

to this p r o c e s s

is c o m p a r a b l e

to that

In fact,

limit for Di if r e p r e s s o r s l i d e s along backbone

to the

in

the Schurr the

the DNA by

sugar-

(i.e., if it s l i d e s

r o t a t i n g a r o u n d the D N A m o l e c u l e once for e v e r y a l o n g the D N A a x i s ) .

the

f u r t h e r by c a l c u l a t i n g

a fixed orientation relative

phosphate double-helical

this

if the

the m e a s u r e d value of Di is c l e a r l y b e l o w

imposed by h y d r o d y n a m i c c o n s i d e r a t i o n s .

hydrodynamic

Thus

the DNA " c y l i n d e r " , and

(26) has c a r r i e d this c o n s i d e r a t i o n maintaining

in

be

T h i s q u e s t i o n m a y be b r o k e n

m o l e c u l e of the size of r e p r e s s o r ,

(- 10"

walk

(and ~ 3.2 x 1 0 2 base p a i r s

How m i g h t such a s l i d i n g p r o c e s s

visualized molecularly?

Di

pairs)

The l e n g t h "scanned" by such a r a n d o m

~ 1 0 3 base p a i r s i n one s e c o n d

f u r t h e r , as

(r^ = D-^/A 2 ) of

rate

is the l e n g t h of one base p a i r ;

3.4 x 1 0 ~ 8 c m ) . process

binding

301

Sites

by

34 A m o v e d

S c h u r r e s t i m a t e d that Di s h o u l d be no

l a r g e r than ~ 5 x 1 0 - ^ c m 2 / s e c

in this m o d e l ; thus e v e n

the o b s e r v e d v a l u e of Dj is below the h y d r o d y n a m i c

here

limit.

(i i) W h a t f o r c e s h o l d r e p r e s s o r to the D N A d u r i n g the s l i d i n g p r o c e s s ?

It has b e e n s h o w n e l s e w h e r e

(24,25)

that

the b i n d i n g of r e p r e s s o r to n o n s p e c i f i c D N A is p u r e l y electrostatic, per repressor

and i n v o l v e s ~ 11 c h a r g e - c h a r g e tetramer.

interactions

This m e a n s that 9 to 10 m o n o v a l e n t

(K + ) c o u n t e r i o n s are d i s p l a c e d from the D N A d o u b l e - h e l i x the

(locally) p o l y c a t i o n i c r e p r e s s o r ,

[see R e c o r d et al.

by

302

P.H. von Hippel and D.G. Bear

(9)], and it is the e n t r o p y g a i n e d by these d i s p l a c e d t e r i o n s that a c c o u n t s

for the f a v o r a b l e b i n d i n g

t h a t h o l d s the r e p r e s s o r to the DNA in the binding mode.

F i g u r e 4 i l l u s t r a t e s this

free

nonspecific

situation

s c h e m a t i c a l l y , and also shows that s l i d i n g of the along

the D N A r e s u l t s

in no net c h a n g e

in ion

repressor

displacement;

the m o n o v a l e n t c a t i o n d i s p l a c e d from "in front" of r e p r e s s o r as it m o v e s DNA "behind"

the r e p r e s s o r . (or r e p l a c e m e n t ) there

r e p r e s s o r s l i d i n g along ion a t m o s p h e r e

to the

In this s e n s e , b e c a u s e no n e t is i n v o l v e d ,

the r e p r e s s o r

be c o n s i d e r e d to be sliding o v e r the DNA on an Therefore,

the

is simply r e p l a c e d by one b i n d i n g

displacement surface".

counenergy

"isopotential

is no t h e r m o d y n a m i c b a r r i e r

to

the DNA, since the r e l a x a t i o n of

is fast r e l a t i v e

and thus c a n be c o n s i d e r e d

to the r e p r e s s o r

the

sliding

to r e m a i n at e q u i l i b r i u m .

c o n t r a s t , d i s s o c i a t i o n of the r e p r e s s o r

ion

can

rate,

(In

from the DNA,

d i r e c t l y or by sliding off the e n d s of DNA f r a g m e n t ,

either requires

net c o u n t e r i o n r e p l a c e m e n t and is thus t h e r m o d y n a m i c a l l y v o r a b l e at low and m o d e r a t e salt c o n c e n t r a t i o n s . )

unfa-

Furthermore

there s h o u l d be little or no a c t i v a t i o n e n e r g y b a r r i e r

to

sliding

site

if the p o s i t i v e c h a r g e s

are e f f e c t i v e l y negative

"delocalized"

(DNA p h o s p h a t e )

counterparts.

since the p o t e n t i a l e n e r g y is not a strong

in the p r o t e i n b i n d i n g

relative

to their

individual

T h i s seems

(E) of c h a r g e - c h a r g e

interactions

f u n c t i o n of i n t e r c h a r g e d i s t a n c e

and there are ~ 11 c h a r g e - c h a r g e

likely,

(E

Œ

1/r),

interactions between DNA

p r o t e i n l o c a t e d w i t h i n ~ 60-80 A along

the DNA. It has

and

also

b e e n s h o w n that m o n o v a l e n t c a t i o n s , at least, bind to DNA in a relatively delocalized

(non-site-bound) manner

s u g g e s t s that the o v e r a l l sliding hydrodynamically-limited

(27). T h i s

rate s h o u l d a p p r o a c h

v a l u e , as a p p e a r s

to be the

(iii) H o w d o e s the r e p r e s s o r r e c o g n i z e the o p e r a t o r site w h e n it s l i d e s "over"

it?

the

case.

(and b i n d

to)

A s Figure

4 also

L o c a t i o n and R e c o g n i t i o n o f

O-Binding

Specific

it

D-Binding

k +

+ *

Ro

Repressor (D-Binding

\

© © © © © © © ©1+ + + +

303

Sites

Form

+ + + +

•+ + + + t t

DNA B i n d i n g

t t t t

Form

tilt + ••• + +

Form)

^

• + + +1

+ + + +

+

+ +

Sliding

/

©©©©©©©©©©©©©©©©©©©©©©©©©©©©©© Figure 4. Schematic models of the operator-binding and nonspecific DNA-binding conformations of the lac repressor (see text; figure from ref. 23).

shows, repressor binds to DNA in two binding modes. One, which we call the non-specific binding mode, is totally electrostatic and involves ~ 11 charge-charge interactions. The other is the (much tighter) specific binding conformation corresponding to RO complex formation; this mode involves only ~7-8 charge-charge interactions, and the binding free energy is only ~ 40% electrostatic at moderate salt concentrations (28). In the latter conformation repressor recognizes (and articulates with) the specific matrix of hydrogen bond donors and acceptors that identifies the operator (see below). In order for repressor to recognize operator as it slides over it, the first order rate constant for the interconversion of the two repressor conformations (e.g., k R o and k R Q ) shown in Figure 4 must be at least 106 per second. Rapid reaction measurements (e.g., see refs. 29-31) suggest that repressor conformations can indeed interconvert at rates of this order. We now attempt to apply this knowledge of the kinetics of the lac repressor-operator interaction to the somewhat more complex problem of how RNA polymerase locates a promoter.

P

304

-H-

von

Hippel and D.G. Bear

Does R N A P o l y m e r a s e L o c a t e P r o m o t e r S i t e s b y S l i d i n g ?

Clearly

RNA polymerase

transfer

mechanisms footnote

2

finds the p r o m o t e r site by f a c i l i t a t e d

involving

).

initial b i n d i n g

W h e t h e r sliding

to n o n s p e c i f i c

DNA

is i n v o l v e d c a n n o t be

as u n a m b i g u o u s l y as for the r e p r e s s o r - o p e r a t o r

interaction.

T h i s f o l l o w s b o t h b e c a u s e the r e l e v a n t p a r a m e t e r s of polymerase-DNA-promoter

(see

answered the

s y s t e m have not all b e e n m e a s u r e d

as

f u n c t i o n s of salt c o n c e n t r a t i o n and p r o m o t e r - c o n t a i n i n g

DNA

f r a g m e n t l e n g t h , and b e c a u s e

been

m a d e are c o m p l i c a t e d , polymerase-promoter

the m e a s u r e m e n t s

in p a r t , by s u b s e q u e n t steps of

interaction to s e p a r a t e

However, several interaction

Such s t e p s are o f t e n

from c l o s e d c o m p l e x

(i) k 3 f 0 b s

for the

with

polymerase-

is m u c h too large for a o n e - s t e p

(ii) N o n s p e c i f i c b i n d i n g of the R N A p o l y m e r a s e m a y be t o t a l l y e l e c t r o s t a t i c

process,

holoenzyme

(32,33), t h o u g h there

is some

d i s a g r e e m e n t at this time o v e r the e x a c t p r o p e r t i e s of nonspecific complexes moter selection

that are a c t u a l

(32-34).

(iii)

intermediates

Sliding of the

type has b e e n i n f e r r e d for c e r t a i n o t h e r

endonucleases(36), Certain anomolies

(35), for c e r t a i n

and for RNA p o l y m e r a s e in p o l y m e r a s e b i n d i n g

m i g h t find m o r e ready e x p l a n a t i o n f u r t h e r e x p e r i m e n t s , p e r h a p s along

[e.g., for

itself

(37).

to c l o s e d

the lines of sliding

certainty.

(iv)

promoters

(see b e l o w ) .

r e q u i r e d b e f o r e the role of p o l y m e r a s e

EcoRI

repair

involve

Clearly

the

repressor-operator-DNA model system outlined above, l o c a t i o n c a n be a s s i g n e d w i t h

in p r o -

repressor

if these p r o c e s s e s

s l i d i n g of the p o l y m e r a s e on the DNA

the

salt-concentration-

dependent proteins binding non-specifically restriction endonuclease

"open" opera-

formation.

lines of e v i d e n c e are c o n s i s t e n t

s u c h an i n t e r p r e t a t i o n : promoter

the

(e.g., f o r m a t i o n of the

complex, mRNA initiation, etc.). tionally difficult

that have

are

in p r o m o t e r

L o c a t i o n and R e c o g n i t i o n of S p e c i f i c DNA B i n d i n g

FORMATION OF THE CLOSED POLYMERASE-PROMOTER H a v i n g c o n s i d e r e d the p r o c e s s of p r o m o t e r ask:

How d o e s R N A p o l y m e r a s e r e c o g n i z e

a specific sequence

305

Sites

COMPLEX

l o c a t i o n , we

now

the p r o m o t e r and

i n t e r a c t i o n w i t h the fully d o u b l e - s t r a n d e d

(commonly r e f e r r e d to as the " c l o s e d

form

promoter

complex")?

A v a r i e t y of k i n e t i c and e q u i l i b r i u m e x p e r i m e n t s

(see

12,38,39)

polymerase

show quite c l e a r l y that h o l o e n z y m e R N A

c a n form a stable c o m p l e x w i t h c l o s e d p r o m o t e r s at

refs

low

t e m p e r a t u r e , and that e v e n at e l e v a t e d

(physiological)

peratures a closed promoter-polymerase

c o m p l e x s e r v e s as an

i n t e r m e d i a t e on the p a t h w a y to a stable o p e n holoenzyme complex. (summarized regions)

promoter-

F r o m the r e s u l t s of m a n y

studies

in refs. 8 and 9) r e c o g n i t i o n by the

of s p e c i f i c base p a i r s e q u e n c e s is i n v o l v e d

(presumably

in this p r o c e s s .

s i m p l e w a y to c o n s i d e r this

tem-

holoenzyme

in the - 1 0 and - 3 5

We p r e s e n t here a

specificity.

R e c o g n i t i o n is the I n t e r a c t i o n of C o m p l e m e n t a r y M a t r i c e s Hydrogen Bond Donors and Acceptors:

The n a t u r e and

relative

p o s i t i o n i n g of the h y d r o g e n d o n o r and a c c e p t o r g r o u p s point

into the m a j o r

(above) and the m i n o r

a n A » T and a G « C base p a i r are shown cular representation

in Figure

5a.

of

that

(below) g r o o v e s

in the c o n v e n t i o n a l In o r d e r to s i m p l i f y

of

molethis

r e p r e s e n t a t i o n , as w e l l as to focus a t t e n t i o n on the m o s t i m p o r t a n t p o i n t s , the same base p a i r s c a n be d i s p l a y e d "stick-figure"

form

(40)

(see Figure

5b).

In this f o r m a t ,

b a s e p a i r is d e p i c t e d as a s t r a i g h t line from w h i c h e x t e n d at r i g h t a n g l e s r e p r e s e n t i n g acceptors parallel

(a) and m e t h y l g r o u p s to a line joining

(me).

(N-H

(d),

The base line is d r a w n

the N1 a t o m of the p y r i m i d i n e

N) h y d r o g e n bond of the base pair. into the m a j o r g r o o v e are

a

bars

hydrogen bond donors

the N9 a t o m of the p u r i n e , w h i c h p a s s e s t h r o u g h the pointing

in a

central

Functional

i n d i c a t e d above

to

the

groups

P.H. von Hippel and D.G. Bear

306

a

a

(a)

(b)

F i g u r e 5. a. M o l e c u l a r m o d e l s of the A » T and G « C base p a i r s , i n d i c a t i n g f u n c t i o n a l g r o u p s w h i c h p r o t r u d e into the m a j o r ( a b o v e ) , and into the m i n o r (below) g r o o v e s of the D N A d o u b l e helix. b. " S t i c k - f i g u r e " m o d e l s of the A » T and G - C base p a i r s , indicating the same f u n c t i o n a l g r o u p s as in F i g u r e 5a (see a l s o text).

307

Location and Recognition of Specific DNA Binding Sites

base-line, line.

those p o i n t i n g

Slight differences

various groups protrude

into the m i n o r g r o o v e are b e l o w in the d i s t a n c e s by w h i c h

into the g r o o v e s are

ignored;

however

the s p a c i n g s of the p r o j e c t i o n s of the f u n c t i o n a l g r o u p s the base line are to Several

i m p o r t a n t f e a t u r e s are e s p e c i a l l y c l e a r .

it d i f f i c u l t

G«C pair

along

scale. For

in the m i n o r g r o o v e the s y m m e t r y of f u n c t i o n a l g r o u p makes

the

the

example, types

to d i s t i n g u i s h an A * T from a T * A p a i r ;

is a l m o s t e q u a l l y d i f f i c u l t to d i s t i n g u i s h

a

from a

O G

f r o m this side, a l t h o u g h e i t h e r an A » T or a T » A pair can be distinguished

from a G » C or a O G

central donor

(d) g r o u p in the latter p a i r s .

from the m a j o r g r o o v e

p a i r by the p r e s e n c e of

is m u c h e a s i e r :

T » A p a i r s c a r r y the a - d - a s e q u e n c e g r o o v e ) , the r e l a t i v e spacing

Discrimination

although both A«T and

(reading a c r o s s

along

a

the m a j o r

the b a s e l i n e d i f f e r s

for

the two p a i r s , and of course the m e t h y l g r o u p of T falls o n the left in the former p a i r and on the right The s e q u e n c e of d o n o r s and a c c e p t o r s in the m a j o r g r o o v e modified

in the

is a - a - d for the G * C p a i r

(and d - a - a for C * G ) ;

in a d d i t i o n the

(glucosylated, methylated, halogenated)

of c y t o s i n e

( r e p r e s e n t e d by x in F i g u r e s

r i g h t for G*C.

In s h o u l d be kept

in the m a j o r g r o o v e

the m i n o r g r o o v e the base p a i r

position

in m i n d that

and on

and those

are on o p p o s i t e

(and of the d o u b l e - h e l i x ) ;

on the

functional

(above the b a s e l i n e )

(below the baseline)

C5

often

5a and b) falls

the left of the o t h e r f u n c t i o n a l g r o u p s for O G , groups

latter.

in

s i d e s of

thus p r o t e i n s

cannot

g e n e r a l l y be in c o n t a c t w i t h f u n c t i o n a l g r o u p s l o c a t e d

in b o t h

the m a j o r and the m i n o r g r o o v e s of the same base pair at the same

time.

E x a m i n a t i o n of a single base pair

in this "stick

form"

repre-

s e n t a t i o n e m p h a s i z e s the r e l a t i v e p l a c e m e n t of h y d r o g e n d o n o r s and a c c e p t o r s , and, by i m p l i c a t i o n ,

the nature

bond

and

p o s i t i o n of the c o m p l e m e n t a r y p r o t e i n h y d r o g e n bond d o n o r s a c c e p t o r s that are r e q u i r e d to r e c o g n i z e

them.

This

and

308

P.H. von Hippel and D.G. Bear

o b s e r v a t i o n , p l u s the p o i n t that at least a p a i r of d o n o r s a c c e p t o r s are r e q u i r e d

for u n a m b i g u o u s

p a i r , has been m a d e p r e v i o u s l y just recognize

(41).

r e c o g n i t i o n of a base H o w e v e r , p r o t e i n s do

i n d i v i d u a l base p a i r s —

s e q u e n c e s of base p a i r s .

they

T h u s , the r e l a t i v e

inter-base

i m p o r t a n t as the i n t r a - b a s e p a i r p o s i t i o n i n g of these t i o n a l g r o u p s and their p r o t e i n c o m p l e m e n t s . p a i r r e p r e s e n t a t i o n c a n be u s e d to p r o v i d e a functional groups

pair

in Figure

sets of base p a i r s

as

func-

The same

base

three-dimensional in

neighboring

base p a i r s in their c o r r e c t r e l a t i o n s h i p s as c l o s e l y p o s s i b l e , by p l a c i n g

not

recognize

p o s i t i o n i n g of h y d r o g e n bond d o n o r s and a c c e p t o r s m a y be

p e r s p e c t i v e , and to p r e s e n t

and

as

in s e q u e n c e as

shown

6.

3'

A

T-

3'

A

5'

Figure 6. " S t i c k - f i g u r e " r e p r e s e n t a t i o n of t h e . f u n c t i o n a l g r o u p s of the six base p a i r s of the - 1 0 (Pribnow box) r e g i o n of the c o n s e n s u s p r o c a r y o t i c p r o m o t e r (see Figure 1).

L o c a t i o n and R e c o g n i t i o n o f S p e c i f i c

DNA B i n d i n g

Sites

309

In Figure 6, for illustration, we show the six base pairs of the Pribnow box of the consensus sequence around position -10 (see Figure 1).

The DNA chains run 5' + 3' reading down on

the left side of the base pairs (and, of course, 5' •»• 3' reading up on the right).

The base pairs are offset (to the

left, reading down) by ~ 20% of the baseline length per base pair, to correspond to the rotational relationships of functional groups which actually apply in a right-handed B-form DNA double-helix.

Thus functional groups that fall over one

another in adjacent base pairs are actually approximately so located in the double-helix as well.

We note that since B-

form DNA has a pitch of ~ 10 base pairs per turn, functional groups in the major groove of the top position lie approximately above functional groups located in the minor groove of base pairs ~ 5 below it in the sequence.

Thus, for example,

a protein which runs straight down the double-helix axis could interact with major groove functional groups of base pairs 1 and 2 (numbering base pairs from the top of the sequence in Figure 6), with minor groove functional groups in base pairs 5, 6 and 7, and again with major groove groups in base pairs 10, 11 and 12, etc.

(Such longer-range interactions are best

brought out in more complex representations.)

Possible Recognition Patterns in The -10 and -35 Sequences: Careful study of the 55 E. coli RNA polymerase binding promoters summarized in refs. 8 and 9, reveals the following features:

(i) the vast majority of these promoters

(42 of 55) show TA in the first two positions of the Pribnow box, and only 5 other base pairs, of the 16 possible combinations, are used at all in these positions; (ii) no wild type or mutant promoters are AT_ _ _T, indicating that sequence features other than simply A«T-richness are involved; (iii) "down" promoter mutations (resulting in decreased promoter strength) in TA

T that result in 4 of the 6 wild

type combinations in positions 1 and 2 have been

P.H.

310 observed;

(iv) p r o m o t e r m u t a t i o n s r e s u l t i n g

unobserved tions

von H i p p e l

(in the w i l d type p r o m o t e r s )

1 and 2 are all "down" m u t a t i o n s ;

mutations observed

in s e v e r a l of

combinations (v) all

in the P r i b o w box are

and 6, w h i l e m u t a t i o n s o b s e r v e d

in p o s i t i o n s

in P l

an(

3

p

Rf

(vi) the s e q u e n c e G A _

GA

strong p r o m o t e r

3, 4 and 5 are

and

from the T A _ _ _ T in d e f i n i n g

(though we note that some fairly weak

p a r t s of the sequence are a l s o

suggesting

other

donor-acceptor position

in c o m m o n , w h i c h the o t h e r o b s e r v e d

couplets

Figure 7, in w h i c h these s e q u e n c e s are c o m p a r e d

the o t h e r w i l d type s e q u e n c e s

that c h a r a c t e r i z e

(a-d/d-a)

these p o s i t i o n s

bridging

sets of h y d r o g e n

in the m a j o r

A s i m i l a r a p p r o a c h can be t a k e n to the h i g h l y "triplet" of base p a i r s consensus promoter stick-figure

(TTG)

(Figure

with

found w h i c h also o c c u r as d o w n

m u t a t i o n s of TA, s u g g e s t s that it m a y be the acceptor-donor/donor-acceptor

the

system).

s y s t e m , do the TA and the GA first and s e c o n d " c o u p l e t s " have

a

promo-

that

i m p o r t a n t , or can o f f s e t

f e a t u r e s of the T A _ _ _ T

W h a t , then, in terms of our h y d r o g e n bond

1).

form, in F i g u r e 8.

This sequence

bonds

groove. conserved

in the - 3 5 r e g i o n of

the

is p r e s e n t e d ,

A feature of the m a t r i x

f u n c t i o n a l g r o u p s that i m m e d i a t e l y sequence

(A in the

T occurs

important

ters also have the T A _ _ _ T s e q u e n c e ,

do n o t ?

1, 2,

T h e s e r e s u l t s s u g g e s t that the s e q u e n c e T A _ _ _ T or

_ T c a r r i e s f e a t u r e s that are

favorable

the

"down"

two of the s t r o n g e s t p r o m o t e r s of p h a g e X,

is not g e n e r a t e d by any "down" m u t a t i o n s sequence.

Bear

in p o s i -

in p o s i t i o n s

all "up" and involve c h a n g i n g a base pair to A » T antisense strand).

and D . G .

s t r i k e s the eye

in

of

in this

is the "ridge" of a c c e p t o r h y d r o g e n b i n d i n g

groups

that runs d o w n the left side of the c e n t e r of the m a j o r g r o o v e , and the p a r a l l e l ridge of d o n o r g r o u p s that runs the r i g h t c e n t e r of this g r o o v e . this f e a t u r e

The p o s s i b l e

down

significance

is s t r e n g t h e n e d w h e n we a g a i n e x a m i n e

a v a i l a b l e s e q u e n c e d p r o m o t e r s ; of the 55 l i s t e d by

of

the Siebenlist

L o c a t i o n and R e c o g n i t i o n o f S p e c i f i c

DNA B i n d i n g

Sites

me

me

• J

L

J

L J

L

me

I

J

L

me

- L r

Figure 7. "Stick-figure" representations of the first two base pairs of the Pribnow box as observed in 55 promoters recognizing E. coli RNA polymerase. The top two "couplets" represent the sequence seen in the strongest promoters; note the a-d/d-a hydrogen donor-acceptor recognition pattern. This pattern is absent in the lower four sequences seen in weaker promoters (see text).

et al. (2), 50 have a recognizable -35 region.

The sequences

either show the consensus TTG sequence, or consist of T and G in various permutations (e.g., TGT,GTG, TTT, etc.); thus in all these sequences the "double ridge" seen in Figure 8 is preserved intact.

Furthermore the promoter mutations detected

in this triplet are largely "down" (see ref. 1), and all down mutations result in "breaking" this "double ridge" sequence by replacing a T or G on the antisense strand by an A or a C. We do not wish to emphasize these observations unduly, since other features of promoter sequence and structure are cer-

P.H. von Hippel and D.G. Bear

312

Figure 8. " S t i c k - f i g u r e " r e p r e s e n t a t i o n of the m o s t h i g h l y c o n s e r v e d (TTG) " t r i p l e t " of base p a i r s of the - 3 5 r e g i o n of the c o n s e n s u s p r o c a r y o t i c p r o m o t e r s e q u e n c e (see Figure 1). The "double ridge" of h y d r o g e n bond a c c e p t o r and d o n o r g r o u p s that runs d o w n the m a j o r g r o o v e is i n d i c a t e d (see t e x t ) .

tainly

i m p o r t a n t as w e l l ^ .

a t t e n t i o n here on s p e c i f i c

R a t h e r our p u r p o s e

is to

f e a t u r e s that m a y p l a y an

focus important

role in the m a t r i x of h y d r o g e n bond d o n o r s and a c c e p t o r s are c e n t r a l

in p r o m o t e r r e c o g n i t i o n , and to e m p h a s i z e

for m o l e c u l a r p u r p o s e s , m u t a t i o n s and changes

chemically-induced

in base p a i r s e q u e n c e are m o s t p r o f i t a b l y v i e w e d

alterations

that

that,

in f u n c t i o n a l g r o u p s , r a t h e r than as c h a n g e s

as in

base pair s e q u e n c e per se.

4 A m o r e e x t e n s i v e c o m p i l a t i o n of p r o m o t e r m u t a t i o n s has just b e e n c o m p l e t e d ( Y o u d e r i a n , P., B o u v i e r , S. and S u s s k i n d , M . ; C e l l , in p r e s s ) . The r e s u l t s are l a r g e l y i n . a c c o r d w i t h the above " r u l e s " , but d e m o n s t r a t e that some a d d i t i o n a l f e a t u r e s in e a c h of these r e g i o n s m a y also need to be c o n s i d e r e d .

Location and Recognition of Specific

DNA B i n d i n g

3

Sites

Summary In this p a p e r we have o u t l i n e d some m o l e c u l a r a s p e c t s of

how

g e n e t i c r e g u l a t o r y p r o t e i n m i g h t locate and r e c o g n i z e a speci fic DNA t a r g e t site, using

information and approaches

o u t i n i t i a l l y w i t h the lac r e p r e s s o r - o p e r a t o r specific protein-nucleic find-ings

acid i n t e r a c t i o n s ,

extra-polations

formation.

s u b j e c t to m o d i f i c a t i o n

the

polymerase-promoter

speculative

in n a t u r e ,

based on future r e s e a r c h .

s e n t e d here w i l l turn out to be u s e f u l

and

However

do b e l i e v e that the p r i n c i p l e s and m e t h o d s of a n a l y s i s v a r i e t y of p r o t e i n - n u c l e i c

thes

polymerase-

As a r e s u l t , some of

and c o n c l u s i o n s a b o u t

i n t e r a c t i o n s m u s t be c o n s i d e r e d

worked

other

and applying

to the initial steps of 12. c o l i R N A

closed promoter complex

and

in the e x a m i n a t i o n

acid i n t e r a c t i o n

we

preof

systems.

Acknowledgments We w i s h to thank James M c S w i g g e n , T e r r y P i a t t , and S c h m i d for c r i t i c a l reading of the m a n u s c r i p t . T h i s was supported

in p a r t by USPHS g r a n t s G M - 1 5 7 9 2 and

(to P H v H ) , and U S P H S P o s t - D o c t o r a l

Molly research GM-29158

Fellowship GM-06676

(to

DGB) .

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v o n H i p p e l , P.H. and M c G h e e , J.D.:

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v o n H i p p e l , P.H.:

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B i o l . 1 0 0 , 459-472 31.

(1976).

F r i e d m a n , B . E . , O l s o n , J.S. and M a t t h e w s , K.S.: J. Mol. B i o l . Ill, 27-39

32.

P. and W u , C . - W . : J. Mol.

(1977).

d e H a s e t h , P . L . , L o h m a n , T . M . , B u r g e s s , R.R. and M . T . , J r . : B i o c h e m i s t r y r7, 1 6 1 2 - 1 6 2 2

33.

Record,

(1978).

R e v z i n , A. and W o y c h i k , R.P.: B i o c h e m i s t r y

20,

250-256

(1981). 34.

K a d e s c h , T . R . , W i l l i a m s , R.C. and C h a m b e r l i n , M.J.: M o l . B i o l . 1 3 6 , 65-78 and 7 9 - 9 3

35.

(1979).

J a c k , W . E . , T e r r y , B.J. and M o d r i c h , P.: P r o c . Natl. Sei. U S A 79, 4 0 1 0 - 4 0 1 4

36.

J.

(1982).

L l o y d , R . S . , H a n a w a l t , P.C. a n d D o d s o n , M.L.s Res. 8,

5113-5126

Acad.

(1980).

Nuc.

Acid.

316

P.H.

von Hippel

and D.G.

Bear

37.

Belintsev, B.N., Zavriev, S.K. and Shemyakin, M.F.: Nuc. Acid Res. 8, 1391-1404 (1980).

38.

Strauss, H.S., Burgess, R.R. and Record, M.T.,Jr.: Biochemistry 3^9, 3496-3515.

39.

Kadesch, T.R., Rosenberg, S., and Chamberlin, M.J.:

40.

Woodbury, C.P. Jr. and von Hippel, P.H.: In Gene

J. Mol. Biol. 155, 1-129 (1982). Amplification and Analysis (J.G. Chirikjian, ed.) Elsevier/North Holland, New York, 1981, Vol. I, pp. 181-207. 41.

Seeman, N.C., Rosenberg, J.M. and Rich, A.: Proc. Natl. Acad. Sci. USA 72, 804-808 (1976).

Received September 3, 1982

DISCUSSION Jovin: How does one evaluate the relative importance of the mechanisms for recognition involving a) specific H-bonding interaction (as you discussed) and/or b) stereochemical adaptation directly to other local features of the conformation (e.g. sequence-specific wedging or bending as discussed by Trifonor)? Von Hippel: I think the specific patterns of hydrogen bond donor and acceptor recognition we discussed have to be the minimal basis for recognizing a base-pair, or a sequence of base-pairs, in double-helical ENA. Local conformational twists and tilts may well reposition these H-bonding groups relative to one another slightly, and thus further 'fine-tune1. The fit, but this seems to me likely to be secondary. Roblllard: You propose frcm your inspection of the patterns of donor and acceptor groups in the promotor region that there are regular arrays. If this is correct then it provides a mechanism for a stepwise increase in the association constant of the polymerase to the prcmotor region as the polymerase passes over the correct sequence. Would you care to oomrent on that aspect? Von Hippel: While that is possible I think it likely to be fortuitous in these rather special cases. In general, I would expect that when a specifically-binding protein is mispositioned (along the double-helix axis) by one base pair relative to an operator or a prcmotor, it is effectively bound nonrspecifically, with the same order of affinity as if it is far frcm the specific target site.

HIERARCHIES OF PROMOTER RECOGNITION DISPLAYED BY ESCHERICHIA COLI RNA POLYMERASE

William R. McClure and Diane K. Hawley Department of Biological Sciences, Carnegie-Mellon University, Pittsburgh, PA 15213

Introduction RNA synthesis in procaryotes is controlled principally at the stage of transcription initiation.

RNA chain initiation frequencies in

Escherichia coli vary over a range of about one thousand-fold (1).

The

interesting biochemical question is, how does RNA polymerase recognize the DNA sequence within the promoter region in a manner that results in 3 a functional hierarchy spanning 10 in reaction rate? The answer to this question is beginning to emerge and has several parts.

First, chemical

and enzymatic protection studies indicate that the RNA polymerasepromoter interaction occurs along one side of the DNA and that functional groups in the major groove of the helix play an important role in recognition (2,3).

Second, sequence homologies among wild-type promoters

(3,4) and the locations of ^50 mutations show that two separate sites within each promoter determine the frequency of initiation (see Fig. 1). The range of promoter strength must in part be a consequence of the base sequence diversity found within these two regions.

A further considera-

tion is that although promoter sequence is the main determinant of initiation frequency for some promoters, others are further modulated by accessory proteins that either activate or repress the rate of initiation. The regulation of transcription initiation by both DNA sequence and ancillary proteins is now amenable to mechanistic studies because it has recently become convenient to quantitate the two functional steps in the initiation reaction that determine initiation frequency (5).

The in vitro

measurements follow from a consideration of a simple model originally proposed by Zillig (6) and extended by Chamberlin (7).

Mobility and Recognition in C e l l Biology © 1983 by Walter d e Gruyter & Co., Berlin • N e w York

This model can

W . R . McClure and D.K. Hawley

318

A. RNAP DNA Template -35

-10

>

URNA start

B. — — T T G ACA

1 7 b p — T A T A AT

- 3 5 region

-

- IO region

Fig. 1. Structures and sequences that control RNA polymerase recognition of E. coli promoters. A. The enzyme is depicted schematically as an arrow covering about 65 base pairs of double-stranded DNA (-v/220 K in length). Specific interactions occur at the RNA start site, and at two regions located at -10 and -35 with respect to the RNA start site. B. The consensus promoter sequence in the -35 and -10 regions is shown. At each position the base shown occurred in 50%-95% of the E. coli promoters that have been sequenced (4).

be represented schematically as follows: KB R + P

k2 RP

c

NTP's RP„ o

>•

...

RNA

(1)

where the free enzyme (R) and promoter (P) combine in a binding step (characterized by Kg) to form an inactive intermediate termed the "closed complex" (RP C ); conversion of the closed complex to the transcriptionally active "open complex" (RP ) proceeds with rate constant, k 2 -

The sub-

sequent triphosphate binding and elongation steps in RNA synthesis are rapid and do not ordinarily limit initiation frequency (8). In the following sections we show that the hierarchy of promoter selectivity depends largely on DNA sequence within the homologous regions of promoters.

We will argue that the hierarchy encoded in the DNA

sequence is important even in cases where ancillary proteins modulate

319

Promoter Recognition

initiation frequency.

We will also suggest that certain characteristics

of overlapping promoters may be involved in control of transcription initiation.

Finally, we will consider complex systems in which all

three of these hierarchies combine to regulate promoter recognition.

I.

DNA Sequences Affect Promoter Recognition

For many promoters the determination of jn_ vitro promoter strength is now relatively straightforward.

The two-step mechanism is Eq. 1 leads

to a simple formalism for analyzing the kinetics of open complex formation (5).

The reaction progress curves can be analyzed to obtain the

rate constant for open complex formation according to the following equation: KR[R]k? k

where Kg and k 2

are

oo b s bs

=

—

(2)

— 1 + Kb[R]

apparent constants defined above and [R] is the RNA

polymerase concentration.

The separate quantitation of Kg and k 2 is

based on the observed enzyme concentration dependence of k ^ .

Several

wild-type and mutant promoters have been analyzed in this fashion.

A

compilation of some of the values determined in our laboratory is presented in the promoter selectivity map (9) shown in Fig. 2. Several important features of promoter function are highlighted in Fig. 2.

The Kg and k 2 values each span two orders of magnitude.

The

strongest promoters have high values for Kg and k 2 ; the weakest promoters have low values for each parameter.

The range of magnitudes of these

values suggest that Kg and k 2 are primarily responsible for the determination of RNA chain initiation frequency.

Second,- a promoter mutation

(the x3 mutation of X P R , the major rightward promoter of bacteriophage x) that decreases initiation frequency in vivo (10) is shown to decrease Kg 20-fold and to reduce k 2 5-fold (11).

Conversely, the u£-l mutation

of x P R M , which increases initiation frequency in vivo (12), was found to increase both Kg and, to a lesser extent, k 2 (13).

Both x3 and ujd-1 are

single base pair changes in the -35 region (4,12).

The UV5 mutation

W.R. McClure and D.K. Hawley

320

\ 10"

h

\

\

\

T7AI

\

UV5 X r

\

2

\

\

\

(sec"1)

\

\

T7D

min

—

r2 h N N 10'

-

0.33-

+

T7A2

\

+

'

1.0--

min.

XPR + 3.0 •

mm

\

X

+ lac

up-1 9.0 —

+x3

min.

_r3 +

*Prm

—

I07

I08 KB( M

10® _l

)

Fig. 2. The in vitro selectivity of promoters results in a hierarchy of calculated in vivo initiation frequency. Each promoter was placed on the promoter selectivity map according to the values determined in vitro for k2 and Kg. (The logarithmic axes were used to accommodate the range of values.) Each dashed curve corresponds to combinations of Kg and k2 that were calculated to result in the same initiation frequency. The average time (l/k 0 b s ) between chain initiation events is shown at the right for each curve. The calculations used the reciprocal of Eq. 2 in the text and an RNA polymerase concentration of 30nM. The calculated boundaries can also be viewed as contour lines on a three-dimensional surface that rises out of the plane of the figure.

occurs in the -10 region of lac P + (the lactose operon promoter); in this case the effect of the mutation is to increase k2 about 20-fold (14). Finally, we believe that the Kg and k 2 values for the promoters shown in Fig. 2 relate in a simple way to in vivo initiation frequency.

McClure

(15) has correlated calculated initiation frequency (Eq. 2) with in vivo gene expression.

In that analysis, the value used for the free concentra-

tion of RNA polymerase was 30nM. it is also imprecise (+ 50%).

(This value was determined indirectly;

Neither limitation affects our argument

Promoter

321

Recognition

in the f o l l o w i n g . )

In other words, promoter strength measured in v i t r o

depended on RNA polymerase concentration a£ vf the in vivo enzyme concentration were 30nM. The functional hierarchy of i n i t i a t i o n frequency shown in F i g . 2 increases from lower l e f t to upper r i g h t .

For example, T7 A1 i s pre-

dicted to i n i t i a t e about 600 times more frequently than

The upward

curvature of the boundary l i n e s in F i g . 2 r e s u l t s from the effect of the f r a c t i o n a l enzyme saturation of the promoters on the l e f t side of the s e l e c t i v i t y map.

This means that two promoters with rather d i f f e r e n t

values for Kg and k 2 might each i n i t i a t e with comparable frequencies in v i v o .

As an example, consider the lac UV5 and xP R promoters.

The Kg

for UV5 i s only 1% of that determined for a P r ; the r e l a t i v e l y low binding a f f i n i t y of UV5 i s compensated by a higher isomerization rate.

We

suggest that t h i s example corresponds to a general d i v e r s i t y in promoter c h a r a c t e r i s t i c s and that the optimal combination of Kg and k 2 for a p a r t i c u l a r promoter w i l l be related to other forms of control beyond the simple determination of i n i t i a t i o n frequency.

In p a r t i c u l a r , the

f r a c t i o n a l s a t u r a t i o n of the closed complex at each promoter might be an additional component of the regulation of t r a n s c r i p t i o n (see Section

III

below). The r e s u l t s obtained with mutant promoters show that in v i t r o measurements of Kg and k 2 are appropriate for a s s e s s i n g the c o n t r i b u t i o n of individual base pairs to overall promoter s e l e c t i v i t y .

At present,

however, two d i f f i c u l t i e s l i m i t our a b i l i t y to tabulate detailed rules f o r recognition.

F i r s t , the set of wild-type promoters characterized

thus f a r d i f f e r in too many p o s i t i o n s for v a l i d comparisons.

Second,

there are simply too few measurements on mutant promoters containing s i n g l e base pair s u b s t i t u t i o n s .

Nevertheless, i t i s clear that the up-

promoter mutations increase Kg and/or k 2 and that down-promoter mutations decrease Kg and/or k 2 -

Moreover, a l l of the promoter mutations char-

acterized i_n vijtro obey the following general r u l e :

down-mutations

decrease homology with the consensus sequence of F i g . I B ; up-mutations increase that homology.

The detailed rules f o r promoter recognition are

expected to r e s u l t from the study of a s i n g l e promoter with many base pair s u b s t i t u t i o n s .

An excellent candidate for such a study i s the

W . R . M c C l u r e and D . K .

322

antirepressor promoter of Salmonella phage P22.

Hawley

Youderian, et cH. (16)

have selected, sequenced, and characterized in vivo 25 d i f f e r e n t mutations that change the DNA sequence within the -35 and -10 regions of P a n t The in v i t r o c h a r a c t e r i z a t i o n of these promoter mutations i s c u r r e n t l y in progress (Hawley and Youderian, unpublished r e s u l t s ) .

II.

Protein Modulators Affect Promoter Recognition The modulation of i n i t i a t i o n frequency by a n c i l l a r y proteins i s a

second level of control found in the i n t e r a c t i o n between RNA polymerase and promoters.

In these cases the DNA binding s i t e s f o r the a c t i v a t o r

and repressor proteins overlap or are adjacent to the promoter region. We have begun to characterize two a c t i v a t o r proteins jji v i t r o .

The DNA

recognition s i t e s f o r these proteins and t h e i r r e l a t i o n s h i p to RNA polymerase binding at each promoter are shown schematically in Fig. 3. The XPR|V| promoter i s p o s i t i v e l y activated by X repressor, the product of the Xcl gene (10,17,18).

The c l protein binds cooperatively

to two s i t e s , 0 R 1 and 0 R 2 ( 1 9 ) , that are close to XP RM ( F i g . 3A).

In so

doing, rightward t r a n s c r i p t i o n from XPR i s repressed and t r a n s c r i p t i o n from xPRm i s activated (12).

Hawley and McClure (13) have shown that

RNA polymerase at P R M i s activated by cl protein in v i t r o by a d i r e c t 11-fold enhancement of by cl protein. xP

RM

mu

the isomerization rate.

Kg was not affected

A s i m i l a r pattern of a c t i v a t i o n was a l s o observed f o r the

tant in v i t r o .

These r e s u l t s i l l u s t r a t e the f a c t that the

a c t i v a t i o n mechanism i s superimposed on the basic determinants of promoter strength encoded in the promoter DNA sequence.

Moreover, the

binding of c l protein to 0 R 1 and 0 R 2 near the promoter i s affected by mutations within those s i t e s (19); the r e s u l t i n g hierarchy of recognition has been shown to play an important role in the a c t i v a t i o n of X P ^ (12). F i n a l l y , f o r both wild-type and mutant XP RM promoters the extent of a c t i v a t i o n observed in v i t r o corresponded q u a n t i t a t i v e l y to the increase in gene expression determined in vivo (13). C y c l i c AMP receptor protein (CRP) from E. c o l i binds to cAMP.

The

r e s u l t i n g conformation change enables the complex (CRP*cAMP) to bind to

Promoter

Recognition

323

0

A.

D N A

start - Fe protein N2.

Pyruvate is implicated because

its addition to toluenized cultures of wild-type K. pneumoniae supported as much nitrogenase activity as did dithionite addition; little activity was restored by formate although in some reports it was as effective in crude extract assays as pyruvate (11).

NifJ protein has physical characteristics like

those of an oxidoreductase and is a candidate for coupling pyruvate oxidation.

NifF protein is more likely than nifJ

product to interact directly with nitrogenase Fe protein for two reasons: 1) nifF protein was reported to co-purify with

n i f Gene

343

Interaction

Fe protein on DEAE-cellulose which may suggest affinity (14) and 2) it is a flavoprotein and in two species of Azotobacter flavodoxin has been reported to provide electrons for nitrogenase activity directly (15).

Also Azotobacter

flavodoxin was shown to couple électron flow from pyruvate or formate to nitrogenase in nifF but not in nifJ extracts and was visibly reduced only in the former (11). Final verification of the path of electron flow to nitrogenase must await not only demonstration of redox couples in vitro but also proof of physiological significance in vivo.

The

latter will be trickier. Unknown activities: 4 or 5 nif genes

Uncertainties relating to nifS product function have been discussed. and nifU.

Other genes of unknown function include nifQ Mutants of the latter type are very leaky and

no protein correlation has been made.

Function is also

unknown for the two genes nifX and Y assigned on the basis of 18,000 and 24,000 dalton MW proteins found to be encoded by otherwise unassigned regions of the nif gene cluster (16). Regulation of nitrogenase synthesis: 2 nif genes and the ntr system Recent advances in understanding nif gene regulation in K. pneumoniae have been underpinned by the developing technologies of molecular genetics to a greater degree than any other aspect of nif genetics.

In particular, genetic

fusions between nif and lacZ genes has allowed expression of individual nif genes to be measured by the convenient 3galactosidase assay (17,18).

Also, studies with individual

regulatory genes cloned onto manipulable plasmids by recombinant DNA techniques have confirmed and extended details

C.

344

Kennedy and R . L .

Robson

of nif regulation suggested by more conventional studies of gene mutations (19,20). It has long been recognized that nitrogenase activity is absent from NH^-sufficient cultures of K. pneumoniae or cultures exposed to more or less vigorous aeration. assumed that

It was

prevented nitrogenase synthesis because its

addition to nitrogen-fixing cultures had no effect on enzyme activity already present.

Since C>2 inactivates nitrogenase,

its effect on synthesis of the enzyme was only recognized more recently by failure to detect, in cultures exposed to O2 (or NH^ ) , newly formed nitrogenase proteins by immunoprecipitation or pulse-labelling and analysis of nitrogenase polypeptides on SDS gels (21,22).

Other sources of fixed N that decrease

levels of nitrogenase enzyme are NO^ (24).

(23) and amino acids

Also incubation at 37° prevents nitrogenase from being

made in K. pneumoniae (25). The current working model to explain these observations is diagrammed in Fig. 2.

The most important feature is that

there are two levels of nif gene control:

The first, termed

external regulation, links nif expression to 2 genes, ntrA, and C, that control the levels of many different enzymes of nitrogen metabolism (for a mini-review see ref. 26).

Internal

regulation is nif specific whereby two nif gene products, L and A, control expression of the other 15.

The link occurs

because ntrABC control expression of the nifLA operon.

The

temperature sensitivity of nif expression is explained by thermolability of one or both of the nifL and A proteins. Evidence for the involvement of the ntr genes will not be reviewed here because it follows a tortuous path. Until recently, it was thought that the enzyme glutamine synthetase (GS) not only assimilated NH^ to make glutamine but also controlled expression of N-related genes such as nif. Instead, the gene product of ntrC (closely linked to glnA

345

ntr gene product(s)

External control

-NHi, (or other signal of N-defIciency)

v nlf

A

LP

-fixed N, 0 2 Internal control

Repressor

Activator

+fixed N, 0 2 /

turns on

* Inactive Repressor

turns off

nlf gene expression nlf QBP (ALP) Fp MP VSUP XNEp YKDHp Jp

Figure 2.

Regulation of nif gene expression in Klebsiella pneumoniae.

that encodes GS) in conjunction with the product of ntrA (unlinked to ntrC) are now thought to be necessary for activating expression at the nifLA promoter.

This activation

only occurs at relatively low levels of fixed N (approx. 1 mM or less) supplied to cultures.

The role of ntrB in nif

regulation is less well established although it may function as a repressor of operons under ntr control in other nonnitrogen fixing enteric organisms. The product of the nifA gene is necessary for transcription of the other nif promoters located as shown in Fig. 1. Evidence for this activity is based primarily on the failure of nifA mutants to produce 1) nif gene products visualised on polyacrylamide gels

(6,27) or 2) B-galactosidase in nif;:lac

fusion strains (17,19).

The nifA product itself is

insensitive to NH.+ or O, since when produced from a

C. Kennedy and R . L .

346

Robson

constitutive promoter on a small multicopy plasmid, it allows +

expression of nitrogenase or 8-galactosidase under NH^ sufficient or aerobic conditions (19).

However, expression

at 37° is reduced suggesting that the activity of nifA protein is heat sensitive.

It is not yet known how the nifA product

activates transcription: whether it binds to promoter regions to facilitate initiation by RNA polymerase or whether it interacts with polymerase or another regulatory protein to allow their binding to nif promoter regions. In apposition to nifA, the nifL gene product prevents expression of other nif operons in response to increasing levels of both fixed N and O^ (1,28, 29).

The evidence is

based, similarly to nifA, on the effect of nifL mutants on expression of other nif operons.

In this case however,

derepression of nif transcriptional units is observed under most conditions that normally prevent their expression. exception is high levels of supplied

The

when nifLA is not

turned on by the ntr system and nifA product is absent.

Lac

fusions within nifLA have confirmed that this operon is, as expected, less sensitive to intermediate levels of NH^

(1.5

mM), amino acids, 0 9 or incubation at 37°, conditions under *

+

which nifL but not nifL strains synthesize other nif gene products. Derepression of nifL mutants as 37° suggests that this protein may also be temperature sensitive but in the sense of being more active or 'locked' in the active mode at high temperature. Alternatively, nifL mutants may need less nifA product for activation and there is sufficient nifA product in these mutants at 37° (19) . How

the nifL product prevents expression of nif genes is not

known.

It has been shown that mRNA hybridizing to nifHDKY

and nifJ regions is reduced in strains carrying a multicopy plasmid that overproduces

nifL product (20).

Also, the

nif::lac fusion alleles used in conjunction with nifL

nif

Gene

Interaction

347

mutations were transcriptional fusions such that g-galactosidase mRNA, if present, should be translated normally in these strains. Nevertheless there is no direct evidence that the nifL gene product is a repressor in the classical sense exemplified by lacl binding to the lac operator to impede RNA polymerase. Most experiments involving nifL have shown that in the absence of functional L product, derepression occurs under conditions where a nifL + strain would not. Repression experiments in which C^ is added to cultures already expressing nif genes have shown that production of nitrogenase subunits is rapidly curtailed in nifL + but not in nifL strains (28). Nif::lac fusion strains are less amenable to repression experiments because small decrements in specific activity of 3-galactosidase are difficult to assess. However, recent publication by Kaluza and Hennecke (30) demonstrates that the decreasing rate of nitrogenase synthesis occurring after exposure of cultures to or C>2 correlates with the net rate of decrease of nif mRNA production. What is apparent from this study and from that of Kahn et al. (31) is that metabolic shifts change the rate of mRNA decay and addition of NH^ + or C>2 results in a dramatic and rapid decrease in the average half-life of not only nif but also bulk mRNAs. Thus repression of nif by NH^+ and O^ can be explained solely through transcriptional control and it is therefore most likely that nifL product prevents nif transcription. Possible mechanisms include not only direct binding of nifL protein to nifDNA but also the inactivation by nifL product of nifA or some other protein involved in nif expression. Elucidation of the specific mechanisms by which the regulatory proteins encoded in ntrABC and nifLA control nitrogen fixation will probably require their purification and assay in defined transcription systems. Parallel

C. Kennedy and R . L .

348

Robson

amassment of DNA sequence information will complement such studies and together will continue to make research on nif regulation an exciting field. Two other interesting aspects of nif regulation that may or may not be related to the nifLA system concern autoregulation of the nitrogenase structural genes (nifHDKY operon) and secondly, the effect of certain his mutations on nitrogen fixation.

This former mode of regulation was discovered

during a course of experiments to study the effect of Mo starvation on nif derepression (17).

Nif-lac fusions in each

of the nif transcriptional units showed that none of the nif 2 operons had a requirement for MoO^ for expression. However maximum expression of nifHDKY required both the presence of 2 -

MoO^ and a functional nifHDKY operon suggesting that a molybdoprotein product of this operon is involved in its control. His mutations that influence nif expression map in his A, B, C or D and reduce the recycling of adenine that normally occurs at step 6 of histidine biosynthesis to replenish the ATP consumed by the first enzyme of the pathway, phosphoribosylphosphotransferase (32). During derepression at levels of histidine sufficient to supply auxotrophic demands, nitrogenase synthesis and activity are normal at early times. At later times, however, ATP rapidly decreases to about 10% of the normal levels, acetylene reducing activity is drastically reduced and synthesis of nitrogenase subunits eventually stops while all the other proteins observed on 1 D SDS gels continue to be made. At 5 fold higher levels of supplied histidine both ATP levels and nitrogenase synthesis are normal. Thus a correlation has been made between intracellular ATP levels and nif gene expression. Little is known about why this occurs nor whether nif transcription is reduced through an effect of decreased ATP levels on nifL or A product activities.

nif G e n e

349

Interaction

Nif genes in organisms other than K. pneumoniae Nitrogen-fixing species are found to be widely distributed among different families of prokaryotes.

Despite their

divergent physiological modes and hence adaptations to diazotrophy, the nitrogenase enzyme complex is remarkably similar. With a relative wealth of knowledge about nif structure and function in K. pneumoniae coupled with recombinant DNA technology that allows analysis of genes in virtually any organism, it will now be possible to see how similar or divergent the nif genes are across a wide taxonomic spectrum. Obvious questions being asked are: 1) Amongst diazotrophs, which nif genes are ubiquitous and which are peculiar to a particular genus or species? 2)

Where similar proteins are specified, how homologous are their amino acid and DNA base sequences?

3)

Does the organization of nif genes vary?

4)

Are nif genes located on chromosomes or plasmids?

5)

How similar are the nif regulatory mechanisms?

Despite divergent physiological states, all nitrogen fixing organisms examined can be repressed by NH^ + and/or C>2 (for review see 33).

Are there systems analogous to ntrABC and

and nifLA to mediate regulation? Some answers are now emerging. In 19 80, Ruvkun & Ausubel (34) reported screening 14 nitrogen-fixing species for DNA sequences that hybridized to K. pneumoniae nifDNA. In every case, homology to nifH (encoding Fe protein) was detected and in many cases to nifD (a subunit of MoFe protein). Comparison of published amino acid sequences and those deduced from DNA sequences shows that the Fe proteins from the divergent organisms Clostridium pasteurianum, Anabaena, A. vinelandii, K. pneumoniae and Rhizobium meliloti are 60 to 70% homologous overall (35-39).

Different regions of the proteins show

350

C.

different degrees of homology;

Kennedy and R . L .

Robson

the five invariant cysteine

residues occur in highly conserved regions.

Interestingly,

although about 70% of amino acid sequences are homologous in the Fe proteins of R. meliloti, Anabaena and K. pneumoniae only 27-35% of the triplet DNA codons are the same (39). This suggests that constraints on protein structure are great indeed if such divergence of triplet codon usage has occurred.

Turning to the nitrogenase FeMo-cofactor, it has been demonstrated that those of K. pneumoniae and A. vinelandii are interchangeable in so far as extracts of cofactor deficient mutants of both species can be activated by cofactor prepared from either (40).

It is likely therefore that the

genes for FeMo cofactor assembly are similar in these two species at least. K. pneumoniae is the only diazotroph known with certainty to carry nif on the chromosome.

Circumstantial evidence obtained

in this laboratory for nif being on the chromosome of Azotobacter species is that 1) A. vinelandii appeared to contain no plasmids after being screened by a variety of methods for their detection and 2) A. chroococcum strain which carries 5 or 6 indigenous plasmids has been cured for all but one with no loss of nitrogen fixation (the remaining one is too small to carry more than about 3 genes).

Nif

genes are located on large plasmids that also carry genes for nodulation in at least some strains of Rhizobia (41-44). In Rhizobium sesbania the sequence of the nifHDK genes is the same as in K. pneumoniae (45) while in Anabaena, nifK is located some 10 kb away from nifHP (46). Although nitrogenase homologies have been found among divergent nitrogen-fixing organisms little is known of the

n i f Gene

351

Interaction

extent to which nif gene regulation is similar. of nitrogenase synthesis by

Repression

and/or 0 2 has been shown

to occur in all nitrogen-fixing organisms examined.

In order

to look for functional analogy between nif regulatory genes we have cloned the nifA gene of K. pneumoniae from pMC71a (19) into a site adjacent to a constitutive promoter of a transmissible wide host range cloning vector pKT230 of the IncQ incompatability group (47). pCKl,

The resulting plasmid,

directs production of nifA constitutively and can be

transferred, using a Tra + helper plasmid, to a number of gramnegative, organisms.

In wild-type (Nif+) strains of

A. vinelandii and A. chroococcum carrying pCKl, +

was no longer repressed by NHjJ .

nitrogenase

Of equal interest was the

finding that in 6 independently isolated Nif

mutants of the,

regulatory type (producing neither nitrogenase component), pCKl

restored a Nif + phenotype and nitrogenase activity was

again found in cells growing with normally repressive concentrations of

.

Thus the nifA gene product can

activate expression of nif genes in what is generally regarded as a fairly distant and unrelated genus of bacteria. It will be of great interest to see how far the nifA analogy will broaden to include other nitrogen fixing organisms. There is evidence that Rhizobia may also contain a nifA product since in certain slow growing cowpea varieties, explanta

nitrogenase activity correlates with the

production of a pink compound in the presence of 6-cyanopurine (48).

This conversion in K. pneumoniae is associated

with a functional nifA gene (49).

Thus it seems likely that

a gene analogous to nifA is ubiquitous among diazotrophs. If the molecular interactions at both the regulatory and nitrogenase levels are similar then it is likely that the nitrogenase modifying functions are as well.

It will then

be of particular interest to examine the electron transfer proteins and their genes in aerobic and anaerobic bacteria

C. Kennedy and R . L .

352

Robson

since the milieu of redox-active molecules is so different. In any event, the scope of interesting molecular interactions associated with nitrogen fixation is vast and their dissection may open avenues to genetic manipulations of consequence to agriculture. We wish to thank Linda Batsleer and Paul Woodley for technical assistance, R.R. Eady and J.R. Postgate for helpful criticism of this manuscript. The transcriptional units of the nif cluster are indicated by arrows in Figure 1.

These conform to published data

cited in references 1 and 2 except for the groupings of nif ^XNE and nif ( VSU which have been indicated by recent data of M. Cannon, J. Beynon and F. Cannon in this Unit.

We

acknowledge with thanks their communication of these unpublished results.

References 1.

Kennedy, C., Cannon, F.C., Cannon, M.C., Dixon, R.A., Hill, S., Jensen, J.S., Kumar, S., McLean, P., Merrick, M.J., Robson, R., Postgate, J.R.: Recent advances in the genetics and regulation of nitrogen fixation, In: Current perspectives in nitrogen fixation (Gibson, A.H. and Newton, W.E., Eds.), Australian Academy of Science, Canberra, 1981, pp. 146-156.

2.

Roberts, G.P., Brill, W.J.: Ann. Rev. Microbiol. 35, 207-235 (1981).

3.

Eady, R.R., Kahn, D., Buchanan-Wollaston, V.: Israel J. Botany 20» (1982) (in press).

4.

Mortenson, L.E., Thorneley, R.N.F.: Ann.Rev. Biochem. 48, 387-418 (1979).

5.

McLean, P.A., Dixon, R.A.: Nature 292, 655-656

(1981).

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Gene

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5a.

MacNeil, T., MacNeil, D., Roberts, G.P., Supiano, M.A., Brill, W.J.: J. Bacterid. 136, 253-266 (1978).

6.

Roberts, G.P., MacNeil, T., MacNeil, D., Brill, W.J.: J. Bacteriol. 13J5, 267-279 (1978).

7.

Carithers, R.P., Yoch, C.D., Arnon, D.J.: J. Bacteriol. 137, 779-789 (1979).

8.

Ludden, P.W., Burris, R.H.: Biochem. J. 175, 251-259 (1978) .

9.

Ludden, P.W., Preston, G.G., Dowling, T.E.: Biochem. J. (1982) (in press) .

10.

Yoch, D.C.: J. Gen. Microbiol. £3, 153-164

11.

Hill, S., Kavanagh, E.: J. Bacteriol. 141, 470-475 (1980) .

12.

St. John, R.T., Johnston, H.M., Seidman, C., Garfinkel, D., Gordon, J.K., Shah, V.K., Brill, W.J.: J. Bacteriol. 121, 759-765 (1975) .

13.

Nieva-Gomez, D., Roberts, G.P., Klevickis, S., Brill, W.J.: Proc. Natl. Acad. Sei. USA TT_, 2555-2558 (1980).

14.

Bogusz, D., Houmard, J., Aubert, J.-P.: Eur. J. Biochem. 120, 421-426 (1981).

15.

Yates, M.G.: Physiological aspects of nitrogen fixation. In: Recent developments in nitrogen fixation (Newton, W.E., Postgate, J.R. and Rodriguez-Barrueco, C., Eds.), Academic Press, London, 1977, pp 219-270.

16.

Piihler, A., Klipp, W. : Fine structure analysis of the gene region for Nj-fixation (nif) of Klebsiella pneumoniae. In: Biological metabolism of inorganic nitrogen and sulphur compounds (Bothe, H. and Trebst, A., Eds.), Springer, 1981, pp. 276-286.

17.

Dixon, R., Eady, R.R., Espin, G., Hill, S., Iaccarino, M., Kahn, D., Merrick, M.: Nature 286, 128-132 (1980).

18.

MacNeil, D., Zhu, J., Brill, W.J.: J. Bacteriol. 145, 348-357 (1981) .

19.

Buchanan-Wollaston, V., Cannon, M.C., Beynon, J.L., Cannon, F.C.: Nature 294, 776-778 (1981).

20.

Buchanan-Wollaston, V., Cannpn, M.C., Cannon, F.C.: Mol. Gen. Genet. 184, 102-106 (1981).

(1974).

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21.

St. John, R.T., Shah, V.K., Brill, W.J.: J. Bacteriol. 119, 266-269 (1974).

22.

Eady, R.R., Issack, R., Kennedy, C., Postgate, J.R., Ratcliffe, H.: J. Gen. Microbiol. 104, 277-285 (1978).

23.

Horn, S.S.M., Hennecke, H., Shanmugam, K.T.: J. Gen. Microbiol. 117, 169-179 (1980).

24.

Shanmugam, K.T., Morandi, C.: Biochim. Biophys. Acta 437, 322-332 (1079).

25.

Hennecke, H., Shanmugam, K.T.: Arch. Microbiol. 123, 259-265 (1979).

26.

Merrick, M.J.: Nature 279, 362-363

27.

Dixon, R., Kennedy, C., Kondorosi, A., Krishnapillai, V. , Merrick, M.: Molec, Gen. Genet. 157, 189-198 (1977).

28.

Hill, S., Kennedy, C., Kavanagh, E., Goldberg, R.B., Hanau, R.: Nature 290, 424-426 (1981).

29.

Merrick, M., Hill, S., Hennecke, H., Hahn, M., Dixon, R., Kennedy, C.: Mol. Gen. Genet. 185, 75-81 (1982).

30.

Kaluza, K., Hennecke, H.: Arch. Microbiol. 130, 38-43 (1981) .

31.

Kahn, D., Hawkins, M., Eady, R.R.: J. Gen. Microbiol. (1982) (in press).

32.

Jensen, J.S., Kennedy, C.: EMBO J. 1, 197-204

33.

Eady, R.: Regulation of nitrogenase activity. In: Current perspectives in nitrogen fixation (Gibson, A.H. and Newton, W.E., Eds.), Australian Academy of Sciences, Canberra, 1981, pp. 172-181.

34.

Ruvkun, G.B., Ausubel, F.M.: Proc. Natl. Acad. Sei. USA 77., 191-195 (1980) .

35.

Mevarech, M., Rice, D., Haselkorn, R.: Proc. Natl. Acad. (1980). Sei. USA 72, 6476-6480

(1982).

(1982).

35a. Hausinger, R.P., Howard, J.B.: Proc. Natl. Acad. Sei. USA 77, 3826-3830 (1980). 36.

Hase, T., Nakano, T., Matsubara, H., Zumft, W.G.: J. Biochem. 9£, 295-298 (1981).

37.

Scott, K.F., Rolfe, B.G., Shine, J.: J. Mol. Appl. Genet. 1, 71-81 (1981).

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Interaction

3

38.

Sundaresan, V. , Ausubel, F.M.: J. Biol. Chem 256, 2808-2812 (1981).

39.

Török, I., Kondorosi, A.: Nuc. Acid Res. 9, 5711-5723 (1981) .

40.

Shah, V.K. Brill, W.J.: Proc. Natl. Acad. Sci. USA 74, 3249-3253 (1977) .

41.

Nuti, M.P., Lepidi, A.A., Prakash, R.K., Schilperoort, R.A., Cannon, F.C.: Nature 282, 533-535 (1979).

42.

Banfalvi, Z., Sakanyan, V. , Koncz, C., Kiss, A., Dusha, I., Kondorosi, A.: Mol. Gen. Genet. 184, 318-325 (1981) .

43.

Hombrecher, G., Brewin, N.J., Johnston, A.W.B.: Mol. Gen. Genet. 182, 133-136 (1981).

44.

Rosenberg, C., Boistard, P., Dénarié, J., Casse-Delbart F.: Mol. Gen. Genet. 184, 326-333 (1981).

45.

Elmerich, C., Dreyfus, B.L., Reysset, G., Aubert, J.-P. EMBO J. 1, (1982) (in press).

46.

Mazur, B.J., Rice, D., Haselkorn, R.: Proc. Natl. Acad. Sci. USA 21, 186-190 (1980)

47.

Bagdasarian, M., Lurz, R., Riickert, B., Franklin, F.C.H Bagdasarian, M.M., Frey, J., Timmis, K.N.: Gene 16, 237-247 (1981).

48.

Kennedy, C., Dreyfus, B., Brockwell, J.: J. Gen. Microbiol. 125, 233-240 (1981).

49.

MacNeil, D., Brill, W.J.: J. Bacteriol. 136, 247-252 (1978) .

50.

Smith, B.E.: J. Less Common Metals 5_4, 465-475

51.

Smith, B.E.: Reactions and Physiocochemical Properties of the Nitrogenase MoFe Proteins. In: Nitrogen Fixation The Chemical/Biochemical/Genetical Interfaces (Müller, A. and Newton, W.E. eds.) Plenum, New York in the press

52.

Smith, B.E., 0'Donne 11, M.J., Lang, G., Spartalian, K.: Biochem. J. 191, 449-455 (1980).

53.

Pienkos, P.T., Shah, V.K., Brill, W.J.: Proc. Natl. Acad. Sci. USA 7j4, 5468-5471 (1977).

Received August 16, 1982

(1977).

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DISCUSSION Jaenicke: Can native Fe protein be reconstituted in vitro after preceding dissociation, and, if so, does the process require the nif M and/or nif S product? What does processing of the nitrogenase ccrrponents mean in molecular terms? Kennedy: As far as I am aware, Fe protein subunits cannot be dissociated in vitro without irreversible loss of activity. Processing as used here refers to the activation or modification of the Fe protein by the nif M and/or nif S gene products (Roberts et al., 1978). We have suggested possible involvement of these products in 4Fe-4S cluster formation or modification by addition of an adenine containing moiety, as occurs with the Fe protein of Rhodospirillum and related organisms. Hcwever, there is no evidence that the Klebsiella protein is or needs to be modified in this way. McClure; In addition to ^ product control, are the nif genes under carbon, phosphate, or any other general metabolic regulation? Kennedy: There is evidence that the cellular energy status affects nif expression. As presented in our paper, sane mutations in the histidine biosynthetic operon interfere with maintenance of ATP levels under certain grcwth conditions. Under these conditions nitrogenase synthesis stops while most or all other proteins are synthesized at only slightly reduced rates. Evidence is lacking for a more specific role for carbon metabolism in nif expression; neither cAMP nor its binding protein appear to be involved. On the other hand, the 'stringent' response may be involved in nif expression since vel A rrutants of K.pneumoniae are nif (Kari et at., Mol.gen.Genet. 1981).

MOLECULAR ASPECTS OF RECOGNITION IN THE RHIZOBIUM - LEGUME SYMBIOSIS. Jan W. Kijne and Ineke A.M. van der Schaal. Botanisch Laboratorium, Rijksuniversiteit Leiden, Nonnensteeg 3, 2311 VJ LEIDEN, The Netherlands.

SUMMARY I n f e c t i o n of legume roots and the induction of N ^ - f i x i n g root n o d u l e s by the soil b a c t e r i u m K h i z o b i u m is c h a r a c t e r i z e d by h o s t s p e c i f i c i t y . A v a i l a b l e data about the m o l e c u l a r b a s i s of r h i z o b i a l h o s t s p e c i f i c i t y are d i s c u s s e d , e s p e c i a l l y in v i e w of the lectin r e c o g n i t i o n h y p o t h e s i s . T h e lack of data about the general R h i z o b i u m - l e g u m e r e c o g n i t i o n p h e n o m e n a is e m p h a s i z e d .

INTRODUCTION Infection of legumes by the Gram-negative bacterium Rhizobium under low-nitrogen conditions in the soil can lead to the induction and development of nitrogen-fixing root nodules. Symbiotic nitrogen fixation is of considerable importance in the nitrogen cycle of the biosphere. The commonly adopted speciation of the genus Rhizobium is based on the host specificity in the symbiosis (table 1, see also 64). Adequate definition of many other rhizobia, responsible for the nodulation of host plants such as Lotus, Cicer and various tropical legumes, requires more study. table Is Species of Rhizobium. species

preferred host plant

R. trifolii R. leguminosarum

Trifolium Vicia, Pisum, Lathyrus, Lens

R. phaseoli R. meliloti

Phaseolus Medicago, Melilotus

R. lupini

Lupinus, Ornithopus

R. japonicum

Mobility and Recognition in Cell Biology © 1983 by Walter de Gruyter & Co., Berlin • New York

Glycine

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van d e r

Schaal

The site of root infection for many rhizobia is a growing root hair tip, and most root nodules will be found in the primary and secondary root zones with induced (5) and/or developing root hairs at the time of inoculation (for a review see 10). Induction of root hair formatioa or root hair branching (leading to a new root hair area with tip growth) might be stimulated by Rhizobium in concert with host plant factors (6). The host specificity is expressed in an early step of the infection process: the induction of "marked" root hair curling (67) and the formation of an infection thread (38)(fig. 1). Despite of several claims for host-specific phenomena in the rhizosphere (e.g. enrichment of the homologous Rhizobium by host root exudate constituents: 62; specific chemotaxis: 9), rhizosphere effects could not clarify the existence of the classical crossinoculation groups of Rhizobium and its host plants (e.g. 21) . Several observations indicate that attachment of Rhizobium to the root hair surface might be the first specificity determining step in the infection process (13, 56). The unsuccessful attempts to identify a rhizobial root hair curling factor led to the view that a viable Rhizobium-cell or a floe of rhizobia are the curling factor themselves. The molecular basis of host specific attachment is suggested to be a specific binding fig. 1: Early steps in the infection process of Rhizobium.

root hair attachment

root hair curling

infection thread formation

Rh i zob i um-Legume Recognition

359

of certain sugar sequences in/on the rhizobial surface by sugarbinding proteins (lectins) in/on the root hair tip surface (1 (review), 11 (review), 8,12,23). The general applicability of the lectin-recognition hypothesis is a matter of considerable discussion ( e.g. 53). Unfortunately the research into and the discussion about the specificity on the species level have drawn the attention away from the central and most important matter in rhizobial host specificity: the almost exclusive symbiosis of the genus Rhizobium with the family of leguminous plants. Much work in the area of rhizobial host specificity is motivated with a view on the transfer of rhizobial nodulation to non-leguminous plants. In this respect the central unanswered question is: what makes a leguminous root hair infectable by Rhizobium in comparison to the many, many other root hairs present in the soil? THE LECTIN-RECOGNITION HYPOTHESIS The lectin-recognition hypothesis states that attachment of Rhizobium to the roots of its legume host is mediated by specific complementary host lectin - rhizobial (poly)saccharide interactions. Work of Dazzo and co-workers (for a review see 11) has demonstrated that R. trifolii specifically binds to a multivalent clover lectin, trifoliin A, present in clover seeds but also on growing root hair tips and in the root exudate. Haptenic monosaccharides are 2-deoxyglucose and quinovosamine (2-amino2,6-dideoxyglucose). The rhizobial lectin receptors are present in capsular (CPS)(16) and lipopolysaccharide (LPS)- related (28) polysaccharides (fig.2), and their presence or accessibility is transient and dependent on culture conditions. Transformation of Azotobacter vinelandii with R. trifolii-DNA resulted in the recovery of Azotobacter-isolates which specifically adsorbed trifoliin A and bound to clover root hairs (7). The ability of R. trifolii to attach to clover root hairs in a 2deoxyglucose-inhibitable way is plasmid-encoded (71). A so-

J.W. Kijne and I.A.M. van der Schaal

3 60

fig. 2: Rhizobial surface polysaccharides. EPS extracellular polysaccharides; CPS capsular polysaccharides; LPS lipopolysaccharides, inserted in the outer membrane of the bacterial cell wall. called sym-plasmid from R. trifolii, carrying a.o. the nodulation (nod) genes, could be transferred to R. leguminosarum and Agrobacterium tumefaciens, which resulted in successful infection and nodulation of clover plants by the transconjugants (26). A relation between nodulation plasmids and rhizobial clover lectin receptors has to be demonstrated as yet. Russa et al. (54) reported plasmid-dependent changes in R. trifolii-LPS, including quantitative differences in quinovosamine content. It should be noted however that quinovosamine also is a constituent of R. leguminosarum-LPS (48). The data concerning the clover symbiosis are well in accordance with the lectin-recognition hypothesis. Quite different is the situation in the soybean - R. japonicum symbiosis. Nacetylgalactosamine (galNac) and galactose are specific haptenic monosaccharides of the classical soybean seed lectin (SBL or SBA). GalNac-inhibitable binding of R. japonicum to Glycine soja root hairs has been demonstrated but not quantified (56). Data of Bohlool & Schmidt (8) suggested that SBL from soybean seeds could differentiate between nodulating and non-nodulating R. japonicum strains. SBL-binding polysaccharides of R. japoni-

Rhizobiurn-Legume Recognition

361

cum-have been isolated and characterized (41,42,58). SBL-receptors are found in EPS and CPS. The capsules have been shown to contain an acidic polysaccharide with a pentasaccharide repeating unit (41,42). The repeating unit structure in the CPS has a backbone consisting of one residue of mannose, two residues of glucose and one residue of galacturonic acid, and a side chain consisting of one residue of either galactose or 4-0methylgalactose. A decline in SBL-binding activity of R. j aponicum accompanying culture aging was concurrent with a decline in galactose content and a rise in 4-0-methylgalactose residues. Galactose has a lower affinity for SBL when it is 4-0-methylated (24). However, binding of rhizobia to soybean root hairs has been reported to be non-specific (2); capsule-less but nodulating strains of R. japonicum have been isolated (37); there is no relationship between the ability of Rhizobium sp. to nodulate soybean and to bind SBL (50); presence of SBL at the growing soybean root hair tip has been questioned, and, moreover, recent work demonstrates the existence of many (especially noncultivated) soybean plants lacking SBL (49,51,52,63). SBLdeficient soybean plants are infected and nodulated by the usual Rhizobium strains in the usual way (51,52), which suggests rather strongly that SBL is redundant in the soybean root nodule symbiosis. The genetical background of the infectivity of R. japonicum is unknown. Specific adsorption of R. leguminosarum to pea or vetch root hairs has not been demonstrated as yet. On the contrary, attempts to inhibit attachment of R. leguminosarum to pea root hairs with various sugars demonstrated non-specific binding of the bacteria (32). Solheim (55) could not see any difference in binding to Vicia hirsuta-roots by infective and non-infective strains of R. leguminosarum, when inoculated roots were examined in the scanning electron microscope. Haptenic sugars of pea seed lectins are mannose, glucose and derivatives (65). Sugar-specific adsorption of pea seed lectins to various rhizobial species has been demonstrated (55,60). Pea lectin receptors are present in CPS and LPS of R. leguminosarum (30,31,47,59). Presence of

362

J.W. Kijne and A.I.M. van der Schaal

glucans (e.g.£>-1,2-glucans: 68,69) as surface polysaccharides (47,54,68,70) could clarify the observed binding of pea lectins to various rhizobia (60). Presence of pea lectins in/on growing root hair tips has not been demonstrated, although pea isolectin 2 could be extracted from intact pea roots with an acid buffer (32,35,36) . Only very low amounts of root lectin can be washed from the pea root surface with use of a high molarity solvent, even when the solvent contains a haptenic sugar (27). However, recent indications for a superficial mannose-specific agglutinin from R. leguminosarum have enlarged the scope of sugar-specific attachment possibilities considerably (36). Pea lectin receptors in/on the root hair surface could equally function as receptors for rhizobial agglutinin. Nodulation genes of R. leguminosarum have been located on plasmid pRLlJI and successfully transferred to R. phaseoli (3,29). First indications have been obtained for co-transfer of pea nodulation ability with pea lectin-mediated agglutinability to R. phaseoli (60). Influence of the presence of nodulation plasmids on the composition of CPS and LPS of R. leguminosarum is unclear yet; experiments are complicated by the existence of the generally occurring pea lectin receptors in Rhizobium. Data concerning lectins and lectin receptors in relation to host specificity in other cross-inoculation groups are scanty. The most important observation was the demonstration of specific agglutination of R. meliloti by a non-haem-agglutinin from Medicago-seeds (46). The agglutination experiments however were done at pH 4.0, at which the growth of R. meliloti as well as root hair curling and nodulation of Medicago are inhibited (43). Nodulation genes of R. meliloti could be demonstrated on very large plasmids ( > 300 Md) (18,33) . Nod" mutants of R. meliloti could attach to the host roots but were unable to induce root hair curling (18).

Rhizobium-Legume Recognition

363

REGULATORY ASPECTS Legume root hairs are resistant to rhizobial infection when the roots are grown in the presence of critical concentrations of combined nitrogen, e.g. nitrate (14,43,57). This observation provides an experimental system in which the factors essential for successful rhizobial infection can be studied. Dazzo and co-workers found that detectable levels of trifoliin A and the specific binding of R. trifolii to clover root hairs decreased with increasing concentrations of (up to 15 mM) in the rooting medium (14). Also the detectable levels of trifoliin A in clover root exudate, and the accessibility of trifoliin A receptors on purified clover root cell walls are significantly reduced when clover seedlings are grown in the presence of 15 mM NO^ (17). Nitrate (18 mM) inhibits root hair attachment by R. meliloti and subsequent root hair curling in Medicago (43, 57), and also the nodulation of Lens (66) and Pisum (19) by R. leguminosarum. The slime and pectinic fractions of pea root cell walls grown with 20 mM NO^ contain much more uronic acids than cell walls grown in a medium without or with 2 mM NO^ (19). The inhibition of pea root nodulation by R. leguminosarum in presence of 20 mM nitrate might be correlated with masking of lectins and/or lectin receptors by polyuronate. The presence or accesibility of rhizobial lectin receptors is transient and dependent on cultural conditions (4,15,28,60). The regulating factors nor the regulatory mechanisms are known. Moreover, much work will be needed to elucidate the regulation of rhizobial plasmid expression in general and especially under legume rhizosphere conditions. RHIZOBIAL SPECIFICITY ON THE SPECIES LEVEL Well known exceptions to the classical cross-inoculation groups of Rhizobium and its legume host plants have been used to question the lectin-recognition hypothesis (53). For•instance, R. leguminosarum also infects Trifolium subterraneum (25), Phaseo-

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lus (64), and various other leguminous species (Greenwood, cited in 53), although the infection leads to non nitrogen-fixing (ineffective) nodules. It is improbable (although unproven) that all anomalous host plants of R. leguminosarum harbour the "mannose/glucose-type" Vicieae-lectins. Likewise it is improbable that a promiscuous host plant as Macroptilium atropurpureum, infectable by various rhizobia (64), should contain all the corresponding specific lectins. On the other hand, presence and expression of dissimilar nodulation plasmids in Rhizobium is possible (26), and promiscuity of a rhizobial species can be clarified by the presence of several sets of nodulation genes within one bacterium. With the present set of data, and the unacquaintance with the presence and distribution of rhizobial agglutinins and corresponding plant lectin receptors, it is impossible to confirm or invalidate the role of specific sugar-binding proteins in the Rhizobium-legume symbiosis in general. It is quite clear that observations made with respect to one cross-inoculation group revealed gaps in the knowledge about other groups: is trifoliin A a common constituent of all infectable clover plants? does R. japonicum produce a sugar-specific agglutinin? are non-haem-agglutinins a normal constituent of leguminous plants other than alfalfa? etc. etc. RHIZOBIUM - LEGUME RECOGNITION As already stated, the discussions about the lectin hypothesis and the seemingly conflicting data from the different crossinoculation groups have clouded the view on the general mechanism of rhizobial infectivity and legume root hair infectability. Moreover, the lectin-recognition hypothesis does not offer an outlook on the infection phenomena after root hair attachment, unless one assumes that lectins and/or lectin receptors are involved in plant cell wall metabolism (35). Induction of marked

Rhizobium-Legume Recognition

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root hair curling and initiation of an infection thread demands an interference with root hair cell wall metabolism. Virtually nothing is known about the composition of leguminous root hair cell walls and the regulation of their synthesis and degradation, let alone about similarities and differences between leguminous root hairs. An important factor in root hair infection is time. Although many rhizobia attach (specifically or not), only a relatively few root hairs become infected. When the infection-sensitive area of the root hair indeed is only the growing tip, rhizobial interference with cell wall metabolism should take place within a few hours or less after attachment. The fast growth of the root hair (clover: 30 fxm/hr, 53) causes a displacement of the attached Rhizobium to the non-infectable parts of the root hair body in a short time. Bhuvaneswari et al. (5) found that soybean root responses leading to infection and nodulation are initiated in less than two hours after inoculation with R. japonicum. Not only the composition but also the structural organization of the cell wall will be important. Ultrastructural studies in our laboratory (Kijne & Mommaas-Kienhuis, in preparation) have shown that the generally accepted view on the structure of root hair cell walls (an "amorphous" outer o(-layer and a fibrillar inner (b-layer, 1,45) does not hold in pea root hairs. In pea root hairs from the developing root hair zone the fibrillar and/or lamellar organization of the cell wall seems to start at the outside and not at the inside of the cell wall (plate 1). Location of structurally important processes at the outside of the cell wall enables a short-term interference by Rhizobium. The observation accentuates the need of information about the plant side of the specificity problem. The data with regard to Rhizobium emphasize more the differences between the rhizobia than their similarities. An exception is the discovery of the rhizobial p-1,2-glucans, also present as EPS (68). These particular molecules might be rhizobial factors involved in the interference with root hair cell wall metabolism. Glucan metabolism in important in plant cell wall syn-

plate 1: Cell wall of a developing pea root hair. Detail of a cross-section perpendicular to the root hair axis and just below the tip, 41,500 x. cw cell wall; n nucleus; a amyloplast; g Golgi apparatus; m mitochondrion. thesis (e.g. 40), and could be disturbed by rhizobial glucans. In this respect it is interesting that colchicine, known to interfere with microtubules and thereby microtubule-directed cell wall organization (45), increases the percentage of deformed clover root hairs and the number of infection threads considerably (61) . Agrobacterium tumefaciens, closely related to R. meliloti, also produces (b-1,2-glucan (22) and can be transformed into a legume-nodulating bacterium (26) . Complexing factors in the analysis of the general rhizobial infection mechanism are plant-Rhizobium interactions and the changed environmental conditions for Rhizobium after attachment

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Recognition

367

to the root surface. A recently discovered product of the Rhizobium-legume root association is a root hair "branching factor", able to induce new tip growth areas in root hairs (6). The origin of the branching factor is unknown and a possible role in host specificity has to be determined,. Also polygalacturonase has been reported to be a Rhizobium-legume association product (39). The production of polygalacturonase was strongly correlated with host root infection, and a role of the enzyme(complex) in root hair cell wall weakening was suggested. This work has to be repeated with modern methods and with use of the increasing number of defined rhizobial nodulation mutants. The altered growth conditions for Rhizobium after attachment can lead to the induction or repression of hitherto unknown infection factors. Inclusion of Rhizobium in root slime and root hair curls could implicate that metabolism of Rhizobium under relatively low 0^ conditions might be important in this respect. CONCLUDING REMARKS From both partners in the legume root nodule symbiosis the plant is the least investigated with respect to recognition and infection phenomena. The secret of rhizobial host specificity is hidden in the legume root hair cell wall metabolism. Study of the processes leading to root hair synthesis under nitrogen deficient conditions will reveal at which points Rhizobium with its specific polysaccharides and/or proteins can interfere. The role of plasmids in the adherence of pathogenic bacteria to the surface of eukaryotic cells is wellknown (20) . As the ability to nodulate a legume host plant is the key characteristic of the genus Rhizobium, one could say that "it is the plasmid that makes the Rhizobium". Determination of the host range of nodulation plasmids will reveal other interesting aspects of rhizobial host specificity. The lectin-recognition hypothesis, generally applicable or not, has been an important contribution in the field of rhizobial host specificity in that it focussed the attention on

J.W.

368

K i j n e and A . I .M. v a n d e r

Schaal

specific protein-sugar interactions. Sugars are main components of plant cell walls and bacterial capsules. The study of rhizobial multivalent sugar-binding proteins has been started only recently (36) and deserves much attention. Remains the observation that rhizobial infection of anomalous host plants usually results in the development of ineffective

non-fixing nodules. The expression of nitrogenase genes

seems to be under specific host control. Also in this respect it is worthwile to note that during the symbiosis under normal conditions the rhizobial cells remain separated from the host plant cytoplasm by the host plasma membrane or a membrane derived from it. Plant cell wall metabolites and other plant products secreted in the extracellular space might constitute an important part of the rhizobial growth conditions from the beginning to the end of the symbiotic state. Ultrastructural studies provided grounds for the suggestion that rhizobia in root nodules come into contact with ER- and Golgi-derived material and host cell debris (34). The presence of host plant specific factors in the extracellular/peribacterial space essential for nitrogenase induction has to be demonstrated

as

yet. REFERENCES 1. 2.

Bauer, W.D., Ann.Rev.Plant Physiol. 22_, 407 (1981) Bauer, W.D., Plant Physiol. 69 (suppl.), 143 (abstr.)(1982).

3.

Beynon, J.L., Beringer, J.E., Johnston, A.W.B., J.gen. Microbiol. 120, 421 (1980).

4.

Bhuvaneswari, T.V., Pueppke, S.G., Bauer, W.D., Plant Physiol. 6£, 486 (1977).

5.

Bhuvaneswari, T.V., Turgeon, B.G., Bauer, W.D., Plant Physiol. £6, 1027 (1980).

6.

Bhuvaneswari, T.V., Solheim, B., Plant physiol.(in press).

7.

Bishop, P.E., Dazzo, F.B., Applebaum, E.R., Maier, R.J., Brill, W.J., Science 198, 938 (1977).

8.

Bohlool, B.B., Schmidt, E.L., Science 185, 269 (1974).

369

Rh i zob i urn-Legume Recognition

9.

Currier, W.W., Strobel, G.A. , FEMS Microbiol .Lett. .1, 243 (1977) .

10. Dart, P.J., in: Quispel, A. (ed.), The biology of nitrogen fixation, North Holland Publ. Cy., Amsterdam, 381 (1974). 11. Dazzo, F.B., J.Supramol.Struct.Cell.Biochem. 29 (1981). 12. Dazzo, F.B., Hubbell, D.H., Appl.Microbiol. 30, 1017 (1975). 13. Dazzo, F.B., Napoli, C.A., Hubbell, D.H., Appl.Environ. Microbiol. 32., 166 (1976). 14. Dazzo, F.B., Brill, W.J., Plant Physiol. 62,

18 (1978).

15. Dazzo, F.B., Urbano, M.R., Brill, W.J., Curr.Microbiol. 2, 15 (1979) . 16. Dazzo, F.B., Brill, W.J., J.Bacteriol. 137, 1362 (1979). 17. Dazzo, F.B., Hrabak, E.M., J.Supramol.Struct.Cell.Biochem. 133 (1981) . 18. Denarie, J., Rosenberg, C., Boistard, P., Truchet, G., Casse-Delbart, F., in: Gibson, A.H., Newton, W.E. (eds.), Current perspectives in nitrogen fixation, Australian Academy of Science, 280 (1981) . 19. Diaz, C., Kijne, J.W., Quispel, A. , see ref. 18, p. 426. 20. Elwell, L.P., Shipley, P.L., Ann.Rev.Microbiol. 34, 465 (1980) . 21. Gaworzewska, E.T., Carlile, M.J., J.gen.Microbiol. 128, 1179 (1982) . 22. Gorin, P.A.J., Spencer, J.F.T., Westlake, D.W.S., Can.J. Chem. 39 , 1067 (1961) . 23. Hamblin, J., Kent, S.P., Nature New Biol. 245, 28 (1973). 24. Hammarstrfim, S., Murphy, L.A., Goldstein, I.J., Etzler, M.E., Biochem. _16, 2750 (1977) . 25. Hepper, C.M., Lee, L., Plant Soil 5^, 441 (1979). 26. Hooykaas, P.J.J., Brussel, A.A.N, van, Dulk-Ras, H. den, Slogteren, G.M.S. van, Schilperoort, R.A., Nature 291, 351 (1981). 27. Hosselet, M., Van Driessche, E., Van Poucke, M., Kanarek,L., in: B(zSg-Hansen, T.C., Spengler, G. (eds.), Lectins, biology, biochemistry, clinical biochemistry, Vol. 3', W. de Gruyter, Berlin (1982) (in press) .

J.W. Kijne and A.I.M. van der Schaal

370

28. Hrabak, E.M., Urbano, M.R., Dazzo, F.B., J.Bacterid. 148, 697 (1981). 29. Johnston, A.W.B., Beynon, J.L., Buchanan-Wollaston, A.V., Setchell, S.M., Hirsch, P.R., Beringer, J.E., Nature 276, 634 (1978). 30. Kamberger, W. , FEMS Microbiol. Lett. 6, 361 (1979). 31. Kato, G., Maruyama, Y., Nakamura, M., Agric.Biol.Chem.

44,

2843 (1980) . 32. Kato, G., Maruyama, Y., Nakamura, M., Plant Cell Physiol. 22,

759 (1981) .

33. Kondorosi, A. , Banfalvi, Z., Sakanyan, V. , Koncz, C., Dusha, I., Kiss, A., see réf. 18, p. 407. 34. Kijne, J.W., Planqué, K., Physiol. Plant Pathol. 14_, 339 (1979) . 35. Kijne, J.W., Van der Schaal, C.A.M. , De Vries, G.E., Plant Sei.Lett. 1_8, 65 (1980) . 36. Kijne, J.W., Van der Schaal, C.A.M., Dîaz, C.L., Van Iren, F.,see réf. 27, (in press). 37. Law, I.J., Yamamoto, Y-, Mort, A.J., Bauer, W.D., Planta 154, 100 (1982). 38. Li, D., Hubbell, D.H., Can. J .Microbiol. 1_5, 1133 (1969). 39. Ljunggren, H., Fahraeus, G., J.gen.Microbiol. 2j>, 521 (1961). 40. Meier, H., in: Robinson, D.G., Quader, H.(eds.), Cell Walls '81, Wissenschaftliche Verlagsgesellschaft mbH, Stuttgart, 75 (1981). 41. Mort, A.J., Bauer, W.D., Plant Physiol. 66, 158 (1980). 42. Mort, A.J., Bauer, W.D., J.Biol.Chem. 257, 1870 (1981). 43. Munns, D.N., Plant Soil 28, 129 (1968). 44. Munns, D.N., Plant Soil 2£, 33 (1968). 45. Newcomb, E.H., Bonnett, H.T., J.Cell Biol. 27, 575 (1965). 46. Paau, A.S., Leps, W.T., Brill, W.J., Science 213, 1513 (1981). 47. Planqué, K., Kijne, J.W., FEBS Lett. 73, 64 (1977). 48. Planqué, K., Van Nierop, J.J., Burgers, A., Wilkinson, S., J.gen.Microbiol. 110, 151 (1979). 49. Pueppke, S.G., Friedman, H.P., Su, L.-C., Plant Physiol. 68, 905 (1981).

Rh i zob i urn-Legume

Recognition

371

50. -Pueppke, S.G., Freund, T.G., Schulz, B.C., Friedman, H.P., see ref. 18, p. 423. 51. Pueppke, S.G.,see ref. 27, (in press). 52. Pull, S.P., Pueppke, S.G., Hymowitz, T., Orf, J.H., Science 200, 1277 (1981) . 53. Robertson, J.G., Lyttleton, P., Pankhurst, C.E.,see ref. 18, p. 280. 54. Russa, R., Urbanik, T., Kowalczuk, E., Lorkiewicz, Z., FEMS Microbiol. Lett. 12, 161 (1982). 55. Solheim, B., see ref. 27, (in press). 56. Stacey, G., Paau, A., Brill, W.J., Plant Physiol. 66^, 609 (1980) . 57. Truchet, G.L., Dazzo, F.B., Planta 154, 352 (1982). 58. Tsien, H.C., Schmidt, E.L., J.Bacterid. 145, 1063 (1981). 59. Van der Schaal, C.A.M., Kijne, J.W.,see ref. 18, p. 425. 60. Van der Schaal, C.A.M., Kijne, J.W., Diaz, C.L., Van Iren, F., see ref. 27, (in press). 61. Van der Starre-Van der Molen, L.G., Bossink, G.A.H., Quispel, A., Plant Soil 26, 397 (1967). 62. Van Egeraat, A.W.S.M., Plant Soil 42, 381 (1975). 63. Vodkin, L., Plant Physiol. 68, 766 (1981). 64. Vincent, J.M., Nutman, P.S., Skinner, F.A., in: Skinner, F.A., Lovelock, D.W.(eds.), Identification methods for microbiologists, Academic Press, London, New York, 49 (1979). 65. Wauwe, J.P. van, Loontiens, F.G., De Bruyne, C.K., Biochim. Biophys.Acta 329, 456 (1975). 66. Wong, P., Plant Physiol. 66, 78 (1980). 67. Yao, P.Y., Vincent, J.M., Plant Soil 4j>, 1 (1976). 68. York, W.S., McNeil, M., Darvill, A.G., Albersheim, P., J. Bacteriol. 142, 243 (1980). 69. Zevenhuizen, L.P.T.M., Scholten-Koerselman, H.J., Ant.v. Leeuwenhoek 4J5, 165 (1979) . 70. Zevenhuizen, L.P.T.M., Ant.v.Leeuwenhoek 47, 481 (1981). 71. Zurkowski, W., Microbios 27, 27 (1980). Received

July

21,

1982

372

J.W. Kijne and A . I . M . van der

Schaal

DISCUSSION Boama: Can the oontradiction between the lectin binding results and the capability to form root nodules be explained by assuming that the lectins are more involved in the triggering of roothair curling or infection thread formation than cell to cell attachment? Can this be checked experimentally by adding sugars to a grouping plant? Kijne; Unfortunately the function of the lectins in plants is unknown, but I do not believe that leguminous plants have lectins only for catching rhizdbia. On the other hand I should mention again the existence of spybean plants without soybean lectin, and evidently in those plants this lectin is not involved in root hair curling or infection thread formation. To answer your second question I should say that addition of sugars to a growing plant can heavily influence for instance cell wall metabolism thereby causing an artefactual situation and in seme instances even death of the plant root. Jovin: Is continued attachment of the bacterium required for maintenance of the synbiotic state within the established infection thread? Kijne: No, the rhizobia are in fact actively dividing in the tip of the infection thread, a state difficult to achieve if they were continuously attached. However, continuous interaction of the bacteria with plant cell wall metabolism might very well be necessary for the development of the infection process. Bosing-Schneider: There are also lectin-like structures on mairmalian cells. Do you believe that they are important for cellular interactions? Kijne: Yes, and I am not the only one who does so. Kennedy: You msntioned that Dazzo found less binding to root hairs of a non-infective mutant of B . t r i f o l i i . What proportion of non-infective or Nod mutants of R . t r i f o l i i or other Rhizobia fail to bind to root hairs or fail to induce root hair curling? Kijne; As the number of nod -nutants isolated in different laboratories is rapidly increasing, it is impossible for me to answer your question at this ncment. I would like to say that in via/ of the high amount of non-specific binding of various_Rhizdbia to root hairs it will be difficult to properly define a binding -mutant. Veeger: Could you visualize the possibility that the lectins and/or agglutinins are the binding sites for the bacterium at th^^lant root-tips, but that the triggering of the infection is induced by Ca . The cap of bacteria binds Ca rather well and removal seems to lead to changes in the bacterial membrane. Such changes are necessary for interaction between the two genetic systems. Although I do not knew vAiether calmodulin is present

Rh i zob i um-Legume

373

Recognition

in the root-tips, such a role oould be visualized in view of Dr. Dedraan's data shewing involvement in the genetic apparatus. 2+

2+

Kijne: It certainly will turn out that Ca and the Ca regulating mechanisms are of extreme importance in root hair development and root hair infection. Unfortunately the infection-situation is complicated by the fact that bogi the plant cell wall and the bacterial surface polysaccharides are good Ca -exchanges. I think that it vrould be wise to start with a study of the role of Ca in the development of uninfected leguminosarum root hairs. Manson: Is there any convincing evidence for chemotaxis of Khizobium tcward root hairs of their appropriate hosts? Kijne: Rhizobia are attracted by several simple ccmpounds knewn to be present in the rhizosphere of many plants, and there is no evidence that rhizobial chemotaxis clarifies the existence of the cross-inoculation groups. May I refer to Gavrorzewska and Carlile (J.Gen.Microbiol. (1982) 128, 1179) for more detail? Manson: Are there mutants of Rhizobium sp. that bind to the root hairs of their normal horts but fail in later stages of infection? That is, does binding simply trigger subsequent plant responses or are specific bacterial functions required for later stages of infection? Kijne: Such mutants exist. It is not likely that binding 'simply' triggers exclusive plant responses nor that the bacteria is a passive partner.

CALMODULIN AND THE INTRACELLULAR CALCIUM SIGNAL: STRUCTURAL AND FUNCTIONAL IMPLICATIONS OF CALMODULIN mRNA

Ravi P. Munjaal and John R. Dedirfan Departments of Internal Medicine and Physiology and Cell Biology, University of Texas Medical School, Houston, Texas 77025

Summary Calcium is a ubiquitous regulator of cellular events acting as an intracellular second messenger. The mediation of calcium is via specific protein receptors represented by calmodulin, a major calcium mediator. Using conventional techniques we have isolated electric eel calmodulin mRNA and cloned its complementary cDNA. This molecular probe has been utilized to analyze its genomic structure and the transcriptional expression in various cellular systems. The cloned calmodulin structural gene is also being used to trans feet normal cells in culture to produce calmodulin over-production and expression of the neoplastic phenotype. Such studies should provide insight into the complex web of cell regulation.

Introduction During the past century there has been mounting evidence that the ubiquitous divalent metal ion, calcium, is an important regulator of cell functions.

Perhaps the best described exam-

ple is the coordinate regulation of contraction and energy metabolism in fast striated skeletal muscle.

Release of ace-

tylcholine from pre-synaptic vesicles results in sacrolemmal depolarization and transient increases in sacroplasmic calcium levels.

The mediation of this tropic response is via high af-

finity, high specificity sacroplasmic calcium receptors. These calcium "switches" are a pair of homologous proteins, troponin C (TnC) and calmodulin (CaM).

As the calcium levels in-

crease each of the four binding sites become saturated in a

Mobility a n d Recognition in C e l l Biology © 1983 by Walter d e Gruyter & Co., Berlin • N e w York

R.P. Munjaal and J.R. Dedman

376 sequential fashion.

Metal binding induces marked conformation-

al changes which is transmitted to associated proteins.

In the

case of TnC a cascade of reversible conformational changes result in muscle contraction and hydrolysis of ATP.

Calcium bin-

ding to calmodulin results in the activation of phosphorylase kinase (of which calmodulin represents the delta subunit). Phosphorylase kinase activation phosphorylates phosphorylase causing its activation (b to a form) which leads to glycogen catabolism and generation of ATP necessary to support contraction.

Sequestration of free calcium reverses both interdepen-

dent pathways. Calcium is also important in a wide variety of other, noncontractile functions, such as cell proliferation, differentiation, secretion and adhesion presumably mediated through specific intracellular calcium receptors (1).

Since calmodulin

is ubiquitous it represents a strong candidate for non-muscle calcium mediation.

However, recently other calcium mediators

have been described, the calcimedins (2) and the calcium phospholipid dependent protein kinase (3).

The coordinate regula-

tion of these calcium receptors is currently under investigation.

A major obstacle in most systems is the general comple-

xity of the cellular response.

Unlike muscle contraction, an

exaggerated event which can be demonstrated in vitro under defined conditions,

most cellular events such as proliferation

appear to be far more complex with many systems interdependent. In this chapter we discuss the isolation of calmodulin mRNA, synthesis and cloning of its cDNA, and initial analyses of the regulation of the structural gene.

Our long term goal is to

develop specific probes in order to define and dissect, in precise chemical terms, the role of calmodulin and other calcium mediators in complex cell functions such as proliferation. Isolation of the Calmodulin mRNA Most tissues contain calmodulin at a concentration of 0.25 % or less with respect to total protein. However, the electric or-

Structural

and F u n c t i o n a l

Figure 1.

Implication

of

C a l m o d u l i n mRNA

377

Translation analysis of calmodulin mRNA.

Poly A containing RNA from electroplax translated in vitro. Lane A, [35s] labeled translation products were run on 15 % SDS-polyacrylamide slab gel. Lane B, calmodulin mRNA enriched fractions from the sucrose density gradients were used to translate in vitro. Translation products ( ) with and ( ) without exogenous mRNA. Lane C, immunoprecipitable translation products using anti-calmodulin antibodies; and Lane D, 10 ng authentic calmodulin was added to the translationproducts in addition to anti-calmodulin antibodies. gan of the electric eel (Electrophorous electricus) calmodulin represents a major portion of the soluble protein fraction (4). Since calmodulin is structurally conserved it was reasoned that the electric organ would serve as an exceptional source for calmodulin mRNA. Total nucleic acids were extracted from fresh tissue using a phenol:SLS:chloroform solvent system (5). Poly-(A) containing RNA was obtained by oligo (dT) chromatography (twice) as described by Aviv and Leader (6). Poly-(A) RNA (200-300 jig in 0.2 ml) were layered onto 13-25 % (w/v) sucrose gradients (12 ml) containing 2 mM EDTA and 1 % SLS and centrifuged at 28,000 rpm (Beckman SW-40) for 22 hrs at 4°C. Each gradient was then fractionated into twenty tubes and ana-

R.P. Munjaal and J.R. Dedman

378

0 0

I

(

II cw . Xcw—ccw .,

Fig. 3. Response regulator model in which sensory transducers send distinct excitation and adaptation signals to the flagella.

Fig. 4. Response regulator model in which the signal for switching from CCW -+> CW is distinct from that for switching from CW CCW.

Fig. 5. Response regulator model in which distinct types of signal ( X ^ X 2 ) are emitted by different transducers (Tl, T2).

Fig. 6 Response regulator model in which signal X from transducers to flagella is subject to feed-back modulation by signal ~ from flagella to transducers.

(iv) The signal for enhanced CCW rotation may not be the same as that for diminished CW rotation (Fig. 4).

In other words, it may be pos-

sible to increase one probability (say, CCW

CW) without affecting

the other, or it may be possible to increase both. (v) The signals from different sensory transducers may not be the same

(Fig. 5).

For example, binding of serine to methyl-accepting

Chemotaxis protein I (MCP I), of aspartate to MCP II, or of fructose to its specific Enzyme II, might all generate different signals. (vi) The flow of signals may be unidirectional, or may involve feedback to the transducers (Fig. 6).

R.M. Macnab

506 It is convenient they

are

part

flagella (T

of,

only F).

At

(Fl)

that

not.

directly protein

the

that

(F2),

impinge

or

small

9b),

on,

whether

flagella are

Among

indirectly ligand

(Fig.

latter, (F3),

be

transducer communicate

8), we may

only

(T), or

between

distinguish

components,

and

be proteins

that

not by

(Fig.

properties

at

all

(F4).

a number 9a), such

The

of means

covalent as

bind

various 9)

modification

factors such as pH or ionic strength

F3 F2

those

(Fig.

protonmotive

9d), and thermal fluctuations in internal structure

two

between

there may

or

the

the

structural

altered

binding

trans-membrane

(Fig. 9c), environmental

the

they

permanent

the

components might

including (Fig.

or

(F),

proteins are

(Fig. 7) to classify components according to whether

force (Fig.

(Fig. 9e).

n Fl

( F4

Fig. 7. Classification of components of sensory transduction chain as operating at the transducers only (T), at the flagella only (F), or between the two (T F).

H TT (a)

Fig. 8. Classification of protein components of the flagellar switching mechanism (see text).

ÙII (C)

XL PH

"F (d)

Fig. 9. Possible mechanisms for causing flagellar switching or altering switching probabilities: (a) Ligand binding, (b) Covalent modification. (c) Transmembrane strain, (d) Physicochemical environment (e.g., pH). (e) Thermal fluctuations in internal structure.

E n e r g i z a t i o n and S w i t c h i n g o f

the F l a g e l l a r

Motor

507

Proteins Involved in Switching There are no protein components, known to be involved in switching, which are also known to be within the permanent structure of the motor

(i.e. in category Fl of Fig. 8).

However, this statement

refers to the motor defined as an isolated, purified structure (22, 23).

There are two proteins, the cheC and cheV gene products, that

affect switching and also are needed for flagellar assembly (1, 243B).

In the case of cheV. gene defects can cause impairment of motor

rotation (31), and the properties of one temperature-sensitive mutant demonstrate that the defect does not involve genetic regulation, since cells can be converted from fully motile to paralyzed within 0.5 s (Dean & Macnab, in preparation).

From the above evidence, it

seems likely that the cheV and cheC proteins are an integral part of the motor, functioning in switching and cheV) in energy transduction.

(at least in the case of

They could well be central enough to

be needed for flagellar assembly, yet still be labile with respect to experimental procedures for isolation. There are other che proteins (X,

H) for which there is genetic

evidence for direct physical interaction with the motor (1, 28), but the fact that deletion mutants are flagellated suggests the proteins are not a permanent part of the motor (i.e. they would be in category F2 of Fig. 8). The methyltransferase (filifiE protein) and methylesterase (cheB protein) appear to communicate between the receptor/sensory transduction system and the motors (1,32) (i.e. be in category T F of Fig. 6). It is interesting that cheC and cheV mutants can be of either the CCW- or CW-biased type (1, 26, 27, 33; Dean & Macnab, in preparation) whereas mutants in the other genes are of only one type, CCW-biased (fihsE, I, A, H) or CW-biased (chefir Z) (26, 34). This is consistent with separate CW- and CCW-generating pathways, with the cheC and cheV proteins being recipients of both types of signals.

R.M. Macnab

508

The Stochastic Process —

Global or Local?

The behavior of motile cells, alternate swimming and tumbling, suggests that there may be a "tumble generator", a device that sends out, at random intervals, a CCW —* CW command to all of the flagella. This would require the generation of "noise" or fluctuation in some parameter, and simultaneous transmission of the information to various points in the cell

(Fig. 2a or 2c).

Note that the two aspects

are to some degree contradictory, since the first (noise generation) is usually a small-number phenomenon, while the second transmission) is a large-number phenomenon.

(simultaneous

For example, suppose the

tumble generator is a sequestering device for a molecule that binds to flagellar motors and causes switching events.

Then if the free

pool is small, proportional fluctuations will be large, but it will not be possible for all flagella to receive equivalent information. Conversely, if the pool is large, flagella will receive equivalent information, but the proportional

fluctuations will be small.

amplification process is therefore indicated:

An

A hypothetical example

would be the spontaneous opening or closing of a small number of ion gates, resulting in quite large ion fluxes.

Regardless of the actual

mechanism, the "tumble generator" concept implies coordinated switching of the flagella on a cell.

It is difficult experimentally to

determine whether such coordination exists. is possible

under

special

circumstances,

However, the experiment and

then we

112 evidence whatsoever lot CQQgdjnated switching preparation).

have

found

(Macnab & Han, in

Flagella on the same cell were commonly seen in the

opposite sense of rotation and, when the recorded data were subjected to statistical analysis, it was found that the state (CCW vs CW) of a given flagellum at any instant was independent of the states of the other flagella. tion

ratio

For example, in pairwise comparisons, the time frac-

fccw(others

CCW)/fccw(others

CW)

for

each

flagellum

was close to unity (mean value 1.04, standard deviation of the mean 0.07).

Thus the stochastic process must be operating locally, at the

level of the individual flagella (Fig. 2b or 2d). tions ized

If these observa-

(made on partially de-energized cheC mutants) can be generalto

fully

energized wild-type

cells,

we

are

faced with

the

Energization

and

Switching

of

the F l a g e l l a r

Motor

509

paradox of apparently coordinated behavior, consisting of swimming punctuated by tumbles, in the absence of a coordinating switch signal. Arguments have been presented (20? Macnab & Han, in preparation) for how this might come about, with the time overlap for flagella in CCW, rotation being quite high, and possibly further enhanced by mechanical over-ride of potential CCW — • CW switching events of flagella in the bundle. A tumble then becomes any event where sufficient flagella happen to be in CW rotation for sufficient time to appreciably disrupt swimming. Once again the bacterium has managed to utilize a simpler, more passive, strategy than we supposed at first.

The Chemotactic Response Signal What are the characteristics of the response regulator (X in Figs. 2-6), i.e. the signal that determines switching probabilities and is modulated by tactic stimuli? Is it distinct from the locally fluctuating signal that actually causes switching events? Tactic signals are of global origin, but still they might have stochastic impact on individual motors (Fig. 2b). However, we have found that flagella on the same cell not only switch independently, but have different longterm CCW/CW. biases (Macnab & Han, in preparation). This suggests that some component of X is distinct from the rapidly fluctuating signal Y in Fig. 2d. It also suggests that that component is distinct from the rapid signal Xexc in Fig. 3. There is no evidence that X^ and X , (Fig. 3) are distinct sigeXC aQapt nals from the transducers to the motor. It has been suggested (1) that there may be feed-back from the motor to the transducers (Fig. 2+ 6), with an initial fast signal to the motor (Ca ? c-GMP?) resulting in an imbalance between free cheY and cheZ proteins and hence of cheR and cheB proteins available to the transducers. Then X would still be a single forward signal with -a CW switching and CW — » CCW switching

(Fig. 4) ?

There certainly are processes

which favor CCW rotation (e.g. those mediated by the cheB. 2. proteins) and others that favor CW rotation (e.g. those mediated by the cheR. Y, h, if proteins), but the concept of "favoring CCW rotation" (meaning increasing the proportion of time spent in CCW rotation) does not distinguish between increasing C W — > CCW switching probabilAs it happens, the two

ities and decreasing C C W — * CW probabilities.

processes are intimately linked, since no means has yet been found of increasing (20).

one

switching probability without

decreasing

the other

Thus the cheB protein, for example, acts to enhance CCW bias

by decreasing the mean duration of CW. intervals as well as by increasing the mean duration of CCW intervals (33). Do different transducers generate different signals (Fig. 5)?

Does

the same transducer generate a different signal when acted on by different effectors? the answers are no.

Among the MCP transducers, it seems likely that They are acted on by the same enzymes, and dif-

ferent stimuli for a given MCP have algebraically summing effects on methylation.

[Incidentally, proton concentration, both external and

internal, is among the stimuli detected by MCP I (35-37).]

It would

seem to be an unnecessary complexity in evolution if the various MCPs generated distinct signals to the motor.

However, if we compare

stimuli that are mediated by MCPs, and those that are not, such as C>2 (38, 39) or the phosphotransferase system sugars (39-42), there are likely to be major differences (X^ distinct from Xj in Fig. 5).

In

the case of 02, there is probably only one output, the protonmotive force that is generated by electron transport (38) (another example

Energization and Switching of the Flagellar Motor

of the participation of protons) yet adaptation occurs.

It seems

quite unlikely that the electron transport chain transmits a second signal

(X a dapt in Fig. 3), and there is no feed-back modulation of

the first signal, since protonmotive force remains altered.

Thus if

the transducer is not responsible, the motor must have some intrinsic capacity for adaptation.

There is increasing evidence that the pro-

cess of adaptation is not uniquely linked to changes in MCP methylation level (39, 43).

What Controls Switching? There are a number of factors known (or believed) to affect switching probabilities. A, H with cheC.

In addition to protein/protein interactions icheY. , there is also the steady-state value of proton-

motive force (20) (Fig. 9c), and low pH (Fig. 9d) acting directly on the motor

(37) as well as via an MCP I generated signal.

Ca2+ (44)

and cyclic GMP (45) have been implicated and might bind to the motor (Fig. 9a).

There is evidence for changes in membrane potential or

surface potential associated with chemotactic stimulation (46-48; M. Eisenbach,

personal

communication),

they constitute signals.

although

it is not clear

that

There is no evidence to date for a covalent

modification of the motor (Fig. 9b); it is tempting to speculate that methylation reactions might extend from transducers all the way to the flagella. It is not known what causes the actual switching event, but the observation (20) that switching probabilities are reciprocally related is intriguing.

Why, for example, are there no mutants with abnormal-

ly long CCW. intervals but normal CW intervals?

Why are there none

where both CCW. and CW intervals are abnormally long?

The absence of

such mutants, or of physiological circumstances producing the same effects, argues against any mechanism in which the forward and reverse reactions could be subject to independent (or parallel) modulation.

For example, it argues against simple ligand binding, since it

should be possible to alter the QQ rate (by altering ligand concent-

R.M. Macnab

512

ration) without altering the off rate; it also argues against a covalent modification such as methylation/demethylation, if mediated by two distinct, non-interacting enzymes operating far from equilibrium. Displacement binding of two species (each with high affinity, so that the site is always occupied) is a possibility, because increasing the concentration

or

binding

affinity

of

one,

say

the

cheY

protein,

would increase its time occupancy, and decrease that of the other, say the cheZ protein

(1).

[Note that if binding of a protein to

the motor truly mandates the rotation sense, a deletion mutation in the

corresponding

opposite state.

gene

should place

the motor

permanently

in the

So far, it has proved to be much more difficult to

generate mutants with an extreme CW, bias than an extreme CCW bias.] A covalent modification could exhibit the observed reciprocal switching probabilities if a single enzyme, operating at equilibrium, were involved —

for example, it might shuttle a methyl group between two

sites on the motor.

The problem with this type of mechanism is that

mis-sense mutants in the relevant gene should have reduced switching probabilities

in

both

directions,

but

no

such mutants

have

been

found. The above models for switching all involve fluctuating interactions between the motor and other molecules, but it should not be forgotten that fluctuations

can also occur within the structure of a given

molecule or complex

(Fig. 9e).

An elegantly simple mechanism for

switching would be a thermally-induced "flip-flop" or isomerization between CCW and CW. states (17, 20), with the energy levels of the two states subject to modulation by environmental signals.

I would like to acknowledge the research contributions of my colleagues G. Dean, D. Han, S. Khan, M. Kihara, and J. Slonczewski. This work has been supported by United States Public Health Services Grant AI 12202.

Energization

and S w i t c h i n g

of

the F l a g e l l a r

Motor

513

References

1.

Parkinson, J.S.: Genetics of Bacterial Chemotaxis. In: Genetics as a Tool in Microbiology (S.W. Glover & D.A. Hopwood, Eds.), Cambridge University Press, Cambridge, 1981, pp. 265-290.

2.

Macnab, R.M.: Sensory Reception in Bacteria. In: Prokaryotic and Eukaryotic Flagella (W.B. Amos & J.G. Duckett, Eds.) Cambridge University Press, Cambridge, 1982, pp. 77-104.

3.

Silverman, M., Simon, M.: Nature (London) 2àl, 73-74 (1974).

4.

Larsen, S.H., Reader, R.W., Kort, E.N., Tso, W.-W., Adler, J.: Nature (London) 249f 74-77 (1974).

5.

Berg, H.C.: Nature (London) 242/ 77-79 (1974).

6.

Macnab, R.M. : Proc. Natl. Acad. Sci. USA 24f 221-225 (1977).

7.

Macnab, R.M., Ornston, M.K.: J. Mol. Biol. 112. 1-30 (1977).

8.

Berg, H.C., Brown, D.A.: Nature (London) 239. 500-504 (1972).

9.

Macnab, R.M., Koshland, D.E., Jr.: Proc. Natl. Acad. Sci. USA 62., 2509-2512 (1972).

10.

Manson, M.D., Tedesco, P., Berg, H.C., Harold, F.M., van«der Drift, C.: Proc. Natl. Acad. Sci. U.S.A. 2A, 3060-3064 (1977).

11.

Glagolev, A.N., Skulachev, V.P.: Nature (London) 272. 280-282 (1978).

12.

Matsuura, S., Shioi, J.-I., Imae, Y., Iida, S. : J. Bacteriol. 1 M , 28-36 (1979).

13.

Manson, M.D., Tedesco, P.N., Berg, H.C. : J. Mol. Biol. 541-561 (1980).

14.

Khan, S., Macnab, R.M. : J. Mol. Biol. 1 M , 599-614 (1980).

15.

Shioi, J.-I., Matsuura, S., Imae, Y.: J. Bacteriol. 144. 891-897 (1980).

16.

Oosawa, F., Masai, J.: J. Phys. Soc. Japan 31, 631-641 (1982).

17.

Macnab, R.M.: An Entropy-driven Engine — The Bacterial Flagellar Motor. In: Biological Structure and Coupled Flews (A. Oplatka, Ed.), Balaban International Science Services, Israel (in press, Oct. 1982).

18.

Berg, H.C., Khan, S.: A Model for the Flagellar Rotary Motor. In: Symposium on Mobility and Recognition in Cell Biology (H. Sund, Ed.), Walter de Gruyter, Berlin. (This volume.)

19.

Hilmen, M., Simon, M.: Motility and the Structure of Bacterial Flagella. In: Cell Motility (R. Goldman, T. Pollard & J. Rosenbaum, Eds.), Cold Spring Harbor, New York, 1976, pp. 35-45.

20.

Khan, S., Macnab, R.M.: J. Mol. Biol. 138. 563-587 (1980).

21.

Berg, H.C., Tedesco, P.M.: Proc. Natl. Acad. Sci. U.S.A. 22, 3235-3239 (1975).

R.M. Macnab

514 22.

DePamphilis, M.L., Adler, J.: J. Bacteriol. 105. 384-395 (1971).

23.

Komeda, Y., Silverman, M., Matsumura, P., Simon, M.: J. Bacteriol. 1 M , 655-667 (1978).

24.

Yamaguchi, S., lino, T., Horiguchi, T., Ohta, K.: J. Gen. Microbiol. Ill, 59-75 (1972).

25.

Silverman, M., Simon, M.: J. Bacteriol. 116. 114-122 (1973).

26.

Warrick, H.M., Taylor, B.L., Koshland, D.E., Jr.: J. Bacteriol. 1 M , 223-231 (1977).

27.

Rubik, B.A., Koshland, D.E., Jr.: Proc. Natl. Acad. Sci. USA 7.5, 2820-2824 (1978).

28.

Parkinson, J.S., Parker, S.R. s Proc. Natl. Acad. Sci. USA 2£.f 2390-2394 (1979).

29.

Kutsukake, K., lino, T., Komeda, Y., Yamaguchi, S.: Molec. Gen. Genet. 12£, 59-67 (1980).

30.

DeFranco, A. L., Koshland, D.E., Jr.: J. Bacteriol. 15£Lr 1297-1301 (1982).

31.

Enomoto, M.: Genetics 54, 715-726 (1966).

32.

DeFranco, A.L., Parkinson, J.S., Koshland, D.E., Jr.: J. Bacteriol. 139. 107-111 (1979).

33.

Khan, S., Macnab, R.M., De Franco, A.L., Koshland, D.E. Jr.: Proc. Natl. Acad. Sci. USA 25, 4150-4154 (1978).

34.

Parkinson, J.S.: J. Bacteriol. 135. 45-53 (1978).

35.

Repaske, D.R., Adler, J.: J. Bacteriol. 145. 1196-1208 (1981).

36.

Kihara, M., Macnab, R.M.: J. Bacteriol. 145, 1209-1221 (1981).

37.

Slonczewski, J.L., Macnab, R.M., Alger, J.R., Castle, A.: J. Bacteriol. 152f (in press, Oct. 1982).

38.

Laszlo, D.J., Taylor,B.L.: J. Bacteriol. 145 f 990-1001 (1981).

39.

Niwano, M., Taylor, B.L.: Proc. Natl. Acad. Sci. U.S.A. 79. 11-15 (1982).

40.

Adler, J., Epstein, W.: Proc. Natl. Acad. Sci. USA 71. 2895-2899 (1974).

41.

Lengeler, J.: J. Bacteriol. 124. 39-47 (1975).

42.

Lengeler, J., Auburger, A.-M., Mayer, R., Pecher, A.: Molec. Gen. Genet. Ifii, 163-170 (1981).

43.

Stock, J.B., Maderis, A.M., Koshland, D.E., Jr.: Cell 22, 37-44 (1981).

44.

Ordal, G.W.: Nature (London) 270. 66-67 (1977).

45.

Black, R.A., Hobson, A.C., Adler, J.: Proc. Natl. Acad. Sci. USA 77, 3879-3883 (1980).

E n e r g i z a t i o n and S w i t c h i n g o f the F l a g e l l a r

Motor

46.

Szmelcman, S., Adler, J.: Proc. Natl. Acad. Sci. USA 73. 4387-4391 (1976).

47.

Armitage, J.P., Evans, M.C.W.: FEBS Lett. 126. 98-102 (1981).

48.

Goulbourne, E.A., Greenberg, E.P.: J. Bacteriol. 143. 1450-1457 (1980).

Received August 6, 1982

DISCUSSI^ Manson: Spudich and Koshland found that individual thetered cells of Salmonella typhimuriwn had persistently different ratios of OCW/CW rotation intervals and that cells with a greater OCW bias had longer adaptation times upon addition of attractants. They postulate that this variability might be of adaptive significance in a population of cells. You find that individual flagella on a single cell have different OCW/CW ratios. Can you add chemoattractants to cells and observe the adaption behavior of individual flagella? If the individual flagella adapt at rates reflecting their CCW/CW ratio then the arguments about the evolutionary significance of individual cell variation cannot be correct. Macnab: The experiment is adding attractants to de-energized cells under conditions where the individual flagella can be monitored. If it could be carried out, I would predict that a flagellum with a high CCW bias would give a longer adaptation time. I am uncertain of the possibly evolutionary significance of the variability. Dedman: Can you identify, though the various mutants, the protein ccmponents of the motile apparatus? Macnabi Perhaps, but the approach is not a straightforward one, because synthesis and assaribly of the apparatus is such a highly coordinated process. Deletion mutants (in a single gene) generally result in total absence of the organelle. The use of conditional mutants may be helpful. Berg: Is it possible that your flagella have different mean biases because they are interacting mechanically in different ways with other flagella on the same cell? I ask because we also have data indicating that the switching of different flagella motors on the same cell is not correlated; data that suggest that the mean CW/CCW bias is the same for different motors (Ishihara, Segall, Berg, in preparation). Our measurements were made under conditions in which the flagella were not able to interact mechanically. Macnab; I doubt that this is the explanation, since flagella pointing in essentially opposite directions scrretimes possess different mean biases. It could be that the use of partially de-energetized cells reveals a differential susceptibility to reduced proton notive force. Recall that seme flagella on a cell were stationary while others were rotating.

516

R.M. Macnab

Voordouw: You started your lecture by saying that bacteria move basically by a random walk of alternating tumbles and runs. Now a random walk is a_ well-defined concept in physical chemistry relating mean displacement (AX), number of steps (N) and length per step (1) as (AX) 2 = Nl 2 . Is this equation (or a more ccrnplex one for a restricted walk) of more than qualitative use in describing bacterial notility? Are there definite effects of attractants, repellents, or proton motive force on either 1 or N (expressed as the number of tumbles per unit time)? Macnab: Yes, there are definitie effects on 1 as a function of the temporal gradient sensed as a result of the component of swittrning velocity in the direction of a spatial gradient of attractant or repellent. The dominant effect is an increase of 1 in favourable gradient directions. I am not aware of any tracking measurstents of cells in gradients of a proton motive force-related stimulus such as oxygen. In temperal gradient experiments, the effect is conventional (increase of oxygen leading to transient suppression of tumbles). There is also the phenomenon I discussed, of steadystate suppression of tumbles under conditions where proton motive force is lew. This might be related to the adaptation nechanism of aerotaxis, and might be of survival value (by increasing 1 regardless of direction of swinming, and hence increasing h) under conditions of energy deprivation. Berg; A cannent: The motion of E.aoli is a true random walk. A cell swims steadily at a constant speed along a nearly straight path, tumbles with a probability that is constant per unit time, and then swims in a new direction chosen at randem (but frcm a distribution with a slight forward bias). When the notion occurs in a spatial gradient of a chemical attractant, the random walk is biased in the following way: If the cell happens to swim up the gradient, the concentration of the chemical increases with time, and this causes the cell to reduce the probability that it will tumble. Thus, segments of the random walk that carry the cell in a favourable direction are lengthened. Other features of the random walk remain essentially constant.

THE PHOSPHOENOLPYRUVATE-DEPENDENT CARBOHYDRATE : PHOSPHOTRANSFERASE SYSTEM ENZYMES II, A NEW CLASS OF CHEMOSENSORS IN BACTERIAL CHEMOTAXIS

Anita Pecher, Irene Renner, Joseph W. Lengeier Institut für Biochemie, Genetik und Mikrobiologie der Universität Regensburg, D-8400 Regensburg, Germany

Summary The phosphoenolpyruvate-dependent carbohydrate:phosphotransferase system enzymes II constitute a new class of chemosensors in bacterial chemotaxis. Enzyme II-mediated transport and chemotactical stimulation as well as signal integration and phosphorylation processes are tightly coupled, while methylation/demethylation reactions and "Methyl-Accepting ChemotaxisProteins" (MCP's) seem not directly involved.

Introduction Chemotaxis, the orientated movement of freely motile organisms toward attractants or away from repellants, is a behavioural response of primary importance for the survival of most bacteria in a rapidly changing environment. Bacteria sense chemotactical stimuli by means of a series of chemosensors, multifunctional and membrane-bound protein-complexes. In Escherichia coli K12 and in Salmonella typhimurium, for many chemicals three transmembrane "Methyl-Accepting Chemotaxis Proteins" (MCP I to III; products of the genes tsr, tar or trg respectively) are such chemosensors [1-3]. Chemosensors, first of all, detect the stimulus, either directly or indirectly through a periplasmic binding-protein (called chemoreceptor and also involved in the transport of chemotactical substrates). Binding and dissociation of a substrate or

Mobility and Recognition in C e l l Biology © 1983 by W a l t e r d e Gruyter 4 Co., Berlin • N e w York

518

A.

Pecher,

I.

R e n n e r a n d J.W.

Lengeier

substrate/binding-protein complex to and of an MCP usually constitutes the decisive stimulus, which eventually triggers a positive or a negative behavioural response. Transport or metabolism of the substrate are not a prerequisite to stimulate chemotaxis, although under physiological conditions they are its final object. Chemosensors, furthermore, convert a stimulus into a signal, which they transduce to the tumble-regulator, the central part of the chemotactical machinery. Hence their designation as signal-transducer or signaler. Finally, they seem to play an important role in signal-integration and in the phenomenon of adaptation [References in 4-11]. A different class of transmembrane chemosensors in bacteria is represented by the phosphoenolpyruvate-dependent carbohydrate: phosphotransferase system (PTS) enzymes II (EII) [11-15]. Two types of Ell's can be distinguished, depending on whether an additional protein, called enzyme III (EIII), is present or not (Fig. 1). MEDIUM

MEMBRANE

PLASMA

sr® S,

Ejj (1-10) £)-HPr

-ePyr

HPr

Pyr

S2 s2

Fl

9- 1 • Schematic representation of the PTS (16). The PTS consists of two general proteins enzyme I (EI) and HPr, and of a series of substrate-specific and transmembrane enzymes II (EII 1-10), two of which (EII 11, 12) require an additional phosphocarner protein enzyme III (EIII). Pyr pyruvate; P-^ePyr phosphoenolpyruvate; S substrate; S-P substrate-phosphate.

Enzymes

II, a New Class of

Chemosensors

519

Most, e.g. the three Ell's specific for the hexitols D-mannitol, D-glucitol (formerly sorbitol), and galactitol (formerly dulcitol), contain only one membrane-bound protein (13-17). Among the twelve Ell's analyzed thus far in E.coli and related Enterobacteria, only the major D-glucose- and a plasmid-encoded sucrose-transport system have been shown conclusively to require EIII (15-19). In the presence of phosphoenolpyruvate (P^ePyr) and two general, plasmatic proteins enzyme I (EI) and HPr, all Ell's catalyze with a high affinity the vectorial phosphorylation of the substrates together with their translocation through the membrane into the cell. In the absence of P^ePyr, EI or HPr, no phosphorylation by Ell's is possible, while they still catalyze an energy-dependent vectorial transphosphorylation or the facilitated diffusion of substrates [references in 20]. In the present communication, additional data on the role of PTS Ell's in bacterial chemotaxis will be given. Together with previously published data [11-15, 22, 23], they indicate a different role of Ell's in chemo-sensing and signal-integration compared to MCP's.

Materials and Methods To minimize problems caused by the presence of unknown transport systems or chemosensors and by the varying motility of strains, all mutations were introduced into E.coli K12, strain L141 or its derivatives [13]. A complete description of the construction of strains, of their properties, of the culture media and of the growth conditions has been given before [13, 15]. The capillary tube assay for chemotaxis according to Adler was performed in a slightly modified form [15]. The number of bacteria accumulating in 1 h at 30°C inside a 1 ul micropipette

520

A.

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(Drummond Sci., Colorado, USA) was determined by plating out its content on tryptone plates. The numbers given in the figures were normalized to the value of an L-aspartate (1 mM) containing control tube and a tube containing wash-medium, to correct for differences in day to day motility and strain differences . Transport and enzyme tests, also described before [13, 15] are expressed in nmoles • min ^ • mg of protein.

Results and Discussion PTS Ell's differ from MCP-chemosensors in many respects. MCP's are not involved in the transport of their stimulating substrates. For all PTS Ell's, by contrast, a strict correlation between the processes of transport/phosphorylation and chemoreception has always been observed. Thus, uninduced wild-type cells or mutants, unable to take up or to phosphorylate a substrate by Ell's showed a low chemotactical response to this substrate, whereas fully induced or constitutive strains reacted strongly. Furthermore, substrate specificities or affinities measured by transport and chemotaxis tests were equal, even in mutants which, due to a mutated EII, showed altered affinities [12-15]. Such a similarity is expected, if transport/phosphorylation and chemoreception are catalyzed by the same transmembrane protein EII. From the analysis of a series of behavioural mutants carrying known mutations in the general PTS proteins EI/HPr or in the substrate-specific proteins EII/EIII it has been concluded [15], that for Ell-mediated chemotaxis the decisive stimulus was not simply binding and dissociation of the substrate, but more likely the reversible alternation of this enzyme between a phosphorylated and a dephosphorylated configuration. To become active in transport/phosphorylation and in chemoreception, Ell's must be phosphorylated by EI, HPr and eventually by EIII

Enzymes II, a New Class of Chemosensors

521

(Fig. 1). After binding to an active EII, a substrate is translocated through the membrane and phosphorylated, leaving a dephosphorylated EII. Another cycle must be started by rephosphorylation of the EII. Since non-phosphorylated Ell's still were able to bind the substrates (though with a low affinity and in energized cells only; J. Lengeler, unpublished data) during transphosphorylation and facilitated diffusion, but did not trigger a chemotactical reaction, binding alone could not be the stimulus. Metabolism of an attractant beyond the phosphorylation step, on the other hand, was not a prerequisite to cause chemotaxis either. Despite an intensive search by several groups [5-10], no major fourth MCP has been found for the large group of PTS Ell's, while none of the known MCP's seemed directly involved in EIImediated chemotaxis. Thus, tsr-tar double-mutants lacking MCP I and II still responded to Ell-mediated stimulation, although adaptation was significantly delayed. For tsr-tar-trg triplemutants this delay was so strong that the capillary tube assay or the tethered-cells test failed, while trg single-mutants still reacted to Ell-mediated stimuli [6]. Since in the original strains HB234 (trg::Tn5) and HB2 35 (trg::Tn10) (kindly donated by G.L. Hazelbauer) induction and transport activity of several PTS-substrates and the corresponding chemotactical reactions appeared abnormally low (data not shown), the trg-null mutation caused by insertion of transposon Tn10 into trg in strain HB235 was transduced by bacteriophage P1 into our standard strain L182 with a constitutive expression of the D-glucitol specific EII and a well defined genetic background. As shown in Fig. 2, the chemotactical response of strain L182 and its trg::Tn10 derivative L329 to D-glucitol (and other PTSsubstrates, data not shown) was nearly identical, although the latter, after normal growth on D-ribose, barely responded to this pentose and its MCP Ill-mediated stimulation. This MCP, therefore, seemed not involved in Ell-mediated chemotaxis either, a conclusion in accordance with previous data of other groups [4-7, 21].

A. P e c h e r ,

522

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Fig. 2. Chemotaxis of strains L182 and L329 (trg:;Tn1Q) to D-glucitol or D-ribose after pregrowth on the corresponding substrate. LI 82 LI 82 L329 L329

0

0001

00»

0.100

1000

1000

to to to to

D-glucitol D-ribose D-glucitol D-ribose

(o) (•) (A) (A)

mM

Substrate

Meantime, by using the "blue-light" chemotaxis test [3], the positive reaction of tsr-tar-trg triple-mutants to PTS-substrates has been shown directly [22], further substantiating the conclusion that MCP1 s are not directly involved in EIImediated chemotaxis. Finally, mutants of E.coli K12 and S.typhimurium lacking either the methyltransferase coded for by the gene cheR, the demethylase coded for by the gene cheB, or both, were also tested. Such mutants too, though greatly impaired in the frequency of run/tumble changes and unable to react to MCP-mediated stimuli, still reacted to Ell-mediated stimuli [5, 9, 10, 22], Coupling of new Ell's to the chemotaxis machinery. According to the results of a "conformational suppression" study of J.S. Parkinson [personal communication], the products of genes cheY and cheZ must be in direct physical contact to the flagella proteins flaA and flaB, while other general chemotaxis proteins seem linked through biochemical reactions only. To test whether for Ell's and further signaler-proteins similar conclusions could be obtained, a new EII specific for and regulated by sucrose was transferred into E.coli K12, where it normally is not found. As shown in Fig. 3 and previously by

Enzymes

I I , a New C l a s s o f

523

Chemosensors

Fig. 3. Chemotaxis of derivatives of E.coli K12 to sucrose. Strains, after growth on glycerol (0.2%) [and D-fructose (0.2%) for strain L336] were tested as described [15]. L182 Scr + DS409 (pUR404 scrR A + B + )Scr L331 err (pUR404) Scr" L332 glcA (pUR404) Scr + L333 glcA (pUR404) Scr+ L334 pts::Mu (pUR404) Scr" L336 glcA fruA manA (pUR406 scrR+ A+B Scr

(•) (A) (•) (•) (O) (•)

(A)

(For explanation of the genetic symbols see text and [18])

0001

0010

0.100

1000

10.00 TTM

Sucrose

J. Adler and coworkers [23], wildtype cells of E.coli K12 (strain LI 82) did not react to purified sucrose. Sucrose-positive (Scr+) derivatives were isolated by infection with plasmid pUR404 [18, 24]. They took up and phosphorylated sucrose Scr by an EHI-dependent EII (product of the plasmid-gene scrA) and a hydrolase (product of gene scrB) hydrolyzed sucrose-6phosphate to D-glucose-6-phosphate and D-fructose, the inducer of the scr-operon and its repressor (gene scrR). All derivaScr tives with a functional EII reacted positively to sucrose in the capillary tube assay too (Fig. 3). The low reaction of strain DS409, not isogenic to the other strains, was due to its low motility reinforced by a strong catabolite repression caused by sucrose on the flagella synthesis. The positive reaction was not due to chemotaxis toward intermediates since the hydrolase-negative scrB mutant L336 which in addition lacks all known chemosensors for D-glucose and Dfructose (i.e. the Ell's coded for by the genes glcA, manA and fruA) still reacted to the purified disaccharide. Mutants, by contrast, which were unable to phosphorylate EII Scr (e.g.

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strain L331 lacking EIII or L334 lacking EI/HPr) though containing the EII in their membrane, did not react. A possible explanation for this apparent lack of specificity Scr in the coupling of EII to the signal-transducing machinery of E.coli K12 is that this coupling is not direct, but through the mediation of EIII and/or the general PTS-proteins EI/HPr. Further support for this hypothesis came from the mutational analysis of strain L184 with constitutive expression of the three hexitol-specific Ell's [15]. By serial transfer on hexitol-containing swarm-plates and by the use of preformed attractant gradients, a series of mutants were selected which did no longer react to any hexitol, while still reacting normally to MCP-mediated stimuli. All the mutants isolated thus far lacked one or both of the general PTS-proteins EI and HPr, and consequently were also unable to transport/phosphorylate or to grow on PTS-substrates (data not shown). Other apparently uncoupled mutants described before synthesized Ell's with altered affinities for their substrates or took up the substrates through different Ell's [15]. Stimulus-/signal-integration at the Ell's. The central role of Ell's and the PTS in chemotaxis is underlined further by the fact, that both are used to integrate signals emanating from different Ell's. To this end, the cells make use of the highly evolved regulatory mechanisms also involved in the control of transport system synthesis and activity (i.e. induction, catabolite repression, transient repression and several catabolite inhibitions [25]). From a series of elegant competition experiments, Adler and coworkers [23] established groups of substrates which competed in capillary tube assays either at the level of chemoreception or of signal-integration. In such tests, an attractant in the capillary tube is competed by a second substrate included in the pond. Several PTS-substrates clearly taken up through different Ell's (e.g. D-glucose and the hexitols) showed such a

Enzymes II, a New Class of Chemosensors

525

competition. Since, however, the inhibition of the chemotactical response was always parallel to the inhibition of the transport activity (data not shown), interference at the level of the PTS seemed possible. Known interference are [15, 20, 2 5-28]: competition for P^HPr, inhibition by intracellular hexose-phosphates at the Ell-level, unexplained mechanisms involving the "proton-motive-force", the respiratory state of the cell and the unphosphorylated EIII. To differentiate between competition effects at the level of PTS or EII and effects at the level of later signal-transduction steps D-glucose-6-phosphate (Glc-6-P) can be used. This phosphate has been shown to inhibit the Ell-mediated transport of D-glucitol in vivo or phosphorylation in vitro [28] and to lack attracting properties in chemotaxis tests [23]. Thus Glc6-P was added to cells of strain IR10 with a constitutive expression of the uhp-coded hexose-phosphate transport system (Fig. 4). Their chemotactical response to D-glucitol (1 mM) in the capillary tube decreased rapidly with increasing amounts of the competitor in the pond. This inhibition paralleled the inhibition of D-glucitol transport activity. At lower concentrations ( < 1 0 yM) of the inhibitor a characteristic increase of the chemotactical response toward the hexitol was seen. This increase might be explained by the decreased affinity of the EII toward its substrate in the presence of Glc-6-P [28] Fig. 4. Competition experiment between D-glucitol and D-glucose-6-phosphate (Glc-6-P). To cells of strain IR10, pregrown on D-glucitol, were added increasing amounts of Glc-6-P. The inhibition (expressed in % of the value without inhibitor) of D-glucitol uptake (25 uM) (A) and of chemotaxis toward this hexitol (1 mM in the capillary) (o), as well as the chemotaxis toward Glc-6-P (•) are shown. C

0001 0.010 0.100 1.000 D-Glucose - 6 - P

«00

mM

526

A.

Pecher,

I.

Renner

and J . W .

Lengeler

and the consequent "blinding" of the cells against the hexitol diffused around the mouth of the capillary tube into the pond. Since below 1 mM, a concentration of Glc — 6 — P at which transport and chemotaxis inhibition were already maximal, this substrate was chemotactically inert, the inhibition could not be at the level of signal-transduction, but must have affected the EII and/or PTS directly. When D-glucose was tested in a similar way as a competitor for D-glucitol-chemotaxis in mutants taking up this hexose through its major or one of its minor transport systems [15], the chemotaxis inhibition could again be shown to resemble its inhibitory effect on D-glucitol transport, and not its effect as a chemotactical attractant (data not shown). Here again, stimulus interference must be more pronounced at the chemosensor level then at later signal-transduction steps. Conclusions. The available data on the role of PTS Ell's in bacterial chemotaxis thus indicate that, compared to MCP's, they constitute a new class of chemosensors. According to these data, Ell-mediated chemoreception might proceed in the following way: In an energized cell, the transmembrane Ell's are phosphorylated by P^HPr or PM5III, the phosphate-group bound in an ester or thio-ester bond. Specific substrates can bind to such activated Ell's with a high affinity, will be phosphorylated in an exchange reaction and translocated by an unknown process through the membrane to yield intracellular substrate-phosphate and a dephosphorylated EII (Fig. 5). The phosphorylated substrate normally will be metabolized further, or, if it accumulates to high internal concentrations, may exchange with free substrate in the medium. This transphosphorylation seems to require energy, but not P^HPr, P^EIII or P^ePyr [20; J. Lengeler, unpublished data]. Under physiological conditions, however, the cycle is restarted by another phosphorylation of the EII. Apparently, the alternations of an EII between the phosphorylated and the dephosphorylated configuration is a chemotactical stimulus. Since all (twelve) Ell's are fi-

Enzymes II, a New Class of Chemosensors MEMBRANE

S-OH :

WM

\ . >En-R-X-®Q (

®-His-HPr — •

M , Hex - ®

V--

Fig. 5. Schematic representation of the phosphorylation/dephosphorylation steps at an EII. Different (hypothetical) steps involved in vectorial phosphorylation, transphosphorylation, translocation and chemoreception are indicated.

HPr

En-R-XH

527

• -HPr

El• R - x - ® 0 7 - — ®-His - HPr

S-OH substrate; R-X hypothetical amino acid to which the phosphate-group is reversibly bound (ester or thioester-bond); dark arrows prefered direction of the reactions. Further abbreviations see Fig. 1.

nally phosphorylated by EI/HPr, these two general PTS proteins are predestined and seem to be used to integrate the stimuli by regulating the binding and phosphorylating capacity of Ell's. We have speculated [11] that for MCP- and Ell-chemosensors stimulation is followed by a rapid signal, communicated to the tumble regulator by the products of genes cheZ and cheY in an unknown process. We have speculated further, that this rapid signal is followed by a slower biochemical alteration of the inner parts of the transmembrane chemosensors which eventually annulates the stimulus and leads to adaptation. For MCP's, this biochemical alterations would be methylation/demethylation, for Ell's and/or the PTS it could be phosphorylation/dephosphorylation. As indicated by the tight linkage between Ell-mediated transport/phosphorylation and chemoreception, both processes must have evolved together and in close connection to the evolution of the glycolytic enzymes. This coevolution is still reflected in the positioning of the genes coding for PTS, EII and glycolytic enzymes on the genome of the Enterobacteria [25, 26]. A similar coevolution between metabolic pathways and a chemoreception system is also indicated for aerotaxis and the respiratory chain or several taxes and the enzymes controlling the

A.

528

Pecher,

I.

Renner and J.W.

Lengeier

membrane potential of bacteria [11, 22], By contrast, no homology seems to exist between the genes for these chemosensors and the genes for the three MCP's [5].

References 1.

Adler, J.: Ann. Rev. Biochem. 44, 341-356 (1975).

2.

Macnab, R.M.: CRC Critical Rev. Biochem. 5, 291-341

3. 4.

Koshland, D.E. Jr.: Physiol. Rev. 59, 812-862 (1979). Hedblom, M.L., Adler, J.: J. Bacteriol. 144, 1048-1060 (1980).

5. 6.

Boyd, A., Krikos, A., Simon, M.: Cell £6, 333-343 (1981). Hazelbauer, G.L., Engström, P.: J. Bacteriol. 145, 35-42 and 43-49 (1981).

7.

Minoshima, S., Ohba, M., Hayashi, H.: J. Biochem. £9, 411— 420 (1981).

8. 9.

Rollins, Ch., Dahlquist, F.W. : Cell 2J5, 333-340 (1 981 ). Sherris, D., Parkinson, J.S.: Proc. Natl. Acad. Sei. USA 78, 6051-6055 (1981).

(1978).

10. Stock, J.B., Maderis, A.M. Koshland, D.E. Jr.: Cell 2J7, 37-44 (1981). 11. Lengeier, J.: The biochemistry of chemoreception, signaltransduction and adaptation in bacterial Chemotaxis. In: Plasmalemma and Tonoplast: Their functions in the plant cell (D. Marme, E. Marre, R. Hertel, Eds.), Elsevier Biomedical Press B.V., Amsterdam, 1982, pp. 337-344. 12. Adler, J., Epstein, W. : Proc. Natl. Acad. Sei. USA T\_, 2895-2899 (1974). 13. Lengeler, J.: J. Bacteriol. 124, 26-38 and 39-47 (1975). 14. Melton, T., Hartman, P.E., Stratis, J.P., Lee, T.L., Davis, A.T.: J. Bacteriol. 133, 708-716 (1978). 15. Lengeler, J., Auburger, A.-M., Mayer, R., Pecher, A.: Mol. Gen. Genet. 183, 163-170 (1981). 16. Postma, P.W., Roseman, S.: Biochim. Biophys. Acta 475, 213-257 (1976). 17. Jacobson, G.R., Lee, C.A., Saier, M.H. Jr.: J. Biol. Chem. 254, 249-252 (1979). 18. Lengeler, J.W., Mayer, R.J., Schmid, K.: J. Bacteriol. 151, July (1982).

Enzymes I I, a New Class of Chemosensors

529

19. S c h ö l t e , B . J . , Schuitema, A . R . , Postma, P.W.: J. o l . 14J3, 257-264 (1981 ) .

Bacteri-

20. D i l l s , S . S . , Apperson, A . , Schmidt, M.R., S a i e r , M.H. M i c r o b i o l . Rev. 44, 385-418 (1980). 21. Kondoh, H., B a l l , C . B . , A d l e r , J . : P r o c . N a t l . Acad. USA 76, 260-264 (1979). 22. Niwano, M., T a y l o r , 11-15 (1982).

Sei.

B . L . : P r o c . N a t l . Acad. S e i . USA 79, —

23. A d l e r , J . , Hazelbauer, G . L . , 115, 824-847 (1973).

Dahl, M.M.: J.

Bacteriol.

24. Schmid, K . , Schupfner, M., Schmitt, R . : J. B a c t e r i o l . July (1982). 25. L e n g e l e r , J . :

Jr.:

Forum M i k r o b i o l . 2/ 359-365

151,

(1980).

26. R i l e y , M., A n i l i o n i s , A . : Ann. Rev. M i c r o b i o l . (1978).

32, —

519-560

27. L e n g e l e r , J . , 169 (1978).

Steinberger,

H.: Mol. Gen. Genet.

164,

163-

28. L e n g e l e r , J . , (1978).

Steinberger,

H . : Mol. Gen. Genet.

167, 75-82

Received June 18, 1982

DISCUSSION Dahlquist: Are there any non-phosphorylorable structural analogues of hexoses which act as attractants or repellents. This might allcw one to distinguish protein phosphorylation from substrate phosphorylation. Lengeler: The D-gluoose analogue 6-deoxy-D-glucose, which cannot be phosphorylated has been tested by J. Adler and was found to be negative. A l l other analogues tested thus far either are phosphory lated and trigger Chemotaxis or are negative in both reactions. Rpbillard: Could not your signalling event be the appearance of sugar phosphate on the inside of the c e l l instead of a change in the phosphorylation state of EI;]; or HPr? Lengeler: This i s unlikely since feeding of e.g. D-glucose-6-phosphate d i rectly to the c e l l through the uhp-coded hexose-phosphate transport system does not cause chemotaxis (see Fig. 4). Sund: Since your system resembles in some respect that of muscle Phosphorylase, I would like to ask the following questions:

530

A.

Pecher,

I.

Renner

and J . W .

Lengeier

Did you investigate the physico-chemical properties like molecular weight and quaternary structure? Do you assume a conformational change connected with phosphorylation and dephosphorylation? Lengeler: Unfortunately we do not have isolated the molecule in larger amounts to test its physico-chemical properties except for the molecular weight (~58 kD). According to G. Robillard, phosphorylation-dephosphorylation is accompanied by a strong conformational change. McClure: How do you know that the effects you observe with glucose-6-phosphate are not due indirectly to a reduction of CRP*cAMP in the cell because of catabolite repression by G-6-P? I ask this because Milton Sawer has reported direct effects of CRP*cAMP on the phosphorylation of E for lactose by P-HPr. Lengeler: The experiment described in Fig. 4 was done with cells pregrown on D-glucitol, thus fully induced for the corresponding enzyme II u , such that they do not need cflMP anymore when D-glucose-6-phosphate has beeg^added. The other effect you mention is by non-phosphorylated enzyme III on the lactose transport system, which is not of the enzyme II type but an active transport system. Enzymes II are regulated by cAMP directly at the level of their synthesis and indirectly by regulation of the synthesis of HPr and enzyme I of the PTS and these are slow effects, while D-glucose-6-phosphate after its conversion to D-fructose-6-phosphate inhibits directly several enzymes II, even in vitro. Macnab: Is it possible to monitor kinetics of membrane phosphorylation and its relation to tactic behaviour? Lengeler; In theory yes, since chromatographical systems to detect phosphorylated membrane proteins have been set up recently by G. Arresz and toluenized cells are still able to respond to chemotactical stimuli mediated by enzymes II as shown in our group. Unfortunately, we have not done the complete experiment yet. Boos; Do different ohe mutations affect EII mediated chemotaxis in a different way than MCP mediated chemotaxis? Lengeler; As far as I know, no. The majority of mutations, however, has never been tested and such a test is difficult, due to the normally already altered swimming behavior of such mutants. Brass: You think HPr is involved in signal transfer. Is it the state of phosphorylation of this protein or a change in a different dctnain of this protein which carries the information? To use the concentration of HPr~P as a signal would not be wise for the cell, since it changes only at high carbohydrate concentrations and not at low concentrations, which are relevant for chemotaxis.

Enzymes

II, a New Class of

Chemosensors

531

Lengeler: The pool of P-HPr normally does not seem to be very large due to a very sophisticated regulation of its synthesis and activity by mechanisms which feedback on the F~ePro and on the hexose-phosphate pool. The pool is low enough that competition of enzyme III and different enzymes II is measurable and in fact is one of the regulatory mechanisms used by the cells to control enzyme II activity.

BACTERIAL

CHEMOTAXIS:

DEPENDENT

M.R.

THE

Kehry

and

F.W.

PROPERTIES

Biology, University

of

THE

CheB-

Oregon,

Eugene,

Bond*

D i v i s i o n of B i o l o g y , Pasadena, California

California 91125

Institute

The

chemotaxis

proteins

in

transduction

methyl-accepting

play

important

(1).

The

roles

proteins

mation

concerning

across

the

events

inside

inner cell

the

the

the

cell.

themselves

receptor

occupancy rotation

sense

of

which

the

cells

to

*Present Cellular

MCPs

are

address: Biology,

in

(3, is

of

4,

then the

to

are

in

in

route is

these by

two

appear In

present

infor-

seem

used

to

and

during

the of

some the

to

be

cases

the

concerning

control the

the

to

in

information

flagella

modified

with

interior

ways.

and

bacteria

which

biochemical

MCPs

they

coli

transmitted

the

The

The

bacterial

Escherichia

associated

cases

6).

Technology,

control

proteins

processed

covalently

the

(2).

other 5,

of

separates

space

receptor

environmental

California

which

while

receptor

MCPs

information

with

of

environment

membrane

The

membrane

space

provide

outside

periplasmic

interact

periplasmic

to

inner

environmental

MCPs

sensory

appear

bacterial

bacterial from

gather the

OF

Dahlquist

I n s t i t u t e of M o l e c u l a r Oregon, 97403 U.S.A.

M.W.

CHEMICAL

MODIFICATION

the

extent

adaptation

of

stimuli.

DNAX Inc.,

Research 1450

Institute

Page

Mill

94304

Mobility and Recognition in C e l l Biology © 1983 by Walter de Gruyter & Co., Berlin • N e w York

of

Road,

Molecular Palo

Alto,

and

to

M.R.

534 There

are

class

of

four MCP

MCPs

tain

subset

gene

product,

of

such tar

aspartate

repellents responses product MCPII,

to

its

physiological

(11,

reactions

methyl

and

methyl

mechanism during

The a

net

ester

the

be

The

a

cer-

the

serine

8).

tsr and

Similarly

to

the

attrac-

ion controls

fourth

methylated normal

content,

MCP,

the

similarly

to

conditions

adaptation by

the

in

methyl

repellents

cheR

donor

residue

attractants in

the

hydrolyzed

clearly

regulates

in

methyl

glutamyl

Likewise,

is

by

take

place

gene

product

(13,

the

14).

cheB

The

gene

and

producing

the

environment

ester

content

produce

implicating

a

a

net

metha-

of

the

decrease

precise

methylation-demethylation

on

control

reactions

adaptation.

to

the

to

another

lyzed

by

(16,

17).

the

cheB

We

modification. result

in

methylation

the

as

of

increase

MCP.

that

the

subject

more

(7,

Bond

Each

to

MCPI,

divalent

under

catalyzed

can

presence

addition

effect

role

Thus

product,

be

date.

cell

responses

(10).

to

to

the

attractant

acids

gene

involved

are

esters

regenerating

stimulates

of

and M.W.

12).

residues

(15).

are

appears

S-adenosylmethionine

appropriate

to

gene

the

certain

trg

galactose

but

product

In

and

to

weak

and

tap

resulting

in

ribose

identified

mediates

the

the

glutamate

nol

maltose

Dahlquist

repellents.

and

product,

MCPIII,

methylation

using

leucine

and

(9).

and

responses

of

unclear

The

as gene

been

F.W.

responsiveness

attractants

repellents

tants

have

the

mediates

MCPII,

the

which

mediates

Kehry,

of

cheB

have The

reversible

gene

product

designated

function.

gene

In

product.

groups

to

of

and this

CheB be

is

the

modification

sense

other

which

apparently

reaction

tumbling

this the

reactions

modification,

CheB-dependent

suppression

demethy1 ation,

methyl

methylation

covalent

it

is

into

MCPs

to

associated the

the

appears

lacking

similar

of

MCPs cata-

irreversible

of

bacteria

modification

incorporated

is

CheB-dependent

in

activity

the

MCPs

MCP,

the

the with allows

while

not

Chemical

Properties

actually the

creating

CheB

all

that

additional

methylation, chemicals

thereby

to which

a result

of

methylation,

low Some can

effects

E^. c o l i

of

into

are

gene

product

duct

or that

is

of

are

host the

Figure

illustrates in

for

their

production

the

cheRB

background.

is

2)

are

These

referred

distinct

sites

synthesized

in

coresponding forms

bands

two b a n d s

that

to

in

1 is

of

require

absence

with

closely

X-E^

col i

addition labeled

hybrid

of

As can cells

host

against

strains

forms strain

the

all

The

phage

a very

of

any

in

1C).

of

and

synthesized

strain

of

the

MCPI CheB in

(Figure

ID)

migrating of

the

Figure

form),

in

The

CheR

migating

only

pro-

form of

bands MCPs

from CheB m o d i f i c a t i o n of

the

gene

produced

(Figure

forms

MCPs

when MCP

a p o l a c r y 1 amide

A single

faster

the

lacking

of MCPI

species

be s e e n

to

of

presence

slower

consists

correspond

spe-

radioactive

forms

single

result

be

ultraviolet

generated

species

cheR - d e l e t e d

of

related

MCPs can

of m u l t i p l e

band 4 ( u n m o d i f i e d

do not

and

the

as CheB m o d i f i e d

(18).

gels.

backgrounds.

I n the

seem t o cheB

of

an a u t o r a d i o g r a m

from the

of

proteins.

mutant

of M C P I .

to

range

m e t h y l t r a n s f e r a s e ) , t h e cheB

MCPI

a complete

(methylated)

phage

MCPs

to

an a p p r o p r i a t e

Following

t h e cheRB - d e l e t e d of

subject

adapt.

a family

the m u l t i p l e

bands

are

concentration

lysogen

genetic

remaining

there

as

functions

(the

different

produced

the

efficiently

or

expressed

both.

of

may

infecting

by e x a m i n a t i o n

cheR

cells

by

(19).

t h e MCPs

insight

conformation

of CheB m o d i f i c a t i o n

(Xind")

an MCP gene

be g a i n e d

appears

on . S D S - p o l y a c r y l a m i d e

molecules

gel

bacteria

each MCP m i g r a t e s

background

it

the

radiolabeled

acid,

Thus

enlarging

forms

amino

the

(18).

residues

cifically irradiated

sites

may a l t e r

molecular

carrying

these

glutamate

the

the

535

Modification

of

CheB m o d i f i c a t i o n

such

As

of

IB,

(16). at

two

MCPI

3 bands, and two

( 1 and

one

methylated

wild-type

bands

536

M.R.

Kehry,

F.W.

Dahlquist

and M.W.

Bond

RA

A

w.t. B~ R6

F i g u r e 1. Forms o f MCPI S y n t h e s i z e d i n D i f f e r e n t G e n e t i c Backgrounds. D i f f e r e n t s t r a i n s of c o l i ( X i n d " ) were programmed t o s y n t h e s i z e MCPI i n t h e p r e s e n c e o f 3 5 S - m e t h i o n i n e ( 1 9 ) . Only t h e MCP r e g i o n o f t h e a u t o r a d i o g r a m i s s h o w n . The b a n d s have been numbered 1 - 8 i n c o r r e s p o n d e n c e w i t h p r e v i o u s work ( 1 6 ) . ( l a n e A) w i l d - t y p e c e l l s ( 1 5 9 Xi nd~ i n f e c t e d w i t h X f 11 a 9 1 ) ; s t i m u l a t e d w i t h 4 mM L - s e r i n e ; ( l a n e C) cheRB c e l l s ( R P 2 8 5 9 X i n d " i n f e c t e d w i t h X f l a 9 1 ) ( 1 a n e D) cheR c e l l s (RP2859 X i n d ~ i n f e c t e d w i t h x f l a 9 1 and x c h e 2 2 A l 6 f 1 a 3 A 2 8 ) l a b e l e d i n t h e p r e s e n c e o f 5 mM L - s e r i n e t o p r e v e n t c o m p l e t e CheB m o d i f i c a t i o n o f band 4. The band i n l a n e D t h a t c o r r e s p o n d s t o band 6 i s a p r o t e i n s y n t h e s i z e d in i n f e c t i o n s u s i n g only the c o l i h y b r i d phage c o n t a i n i n g cheB ( X c h e 2 2 & 1 6 f l a 3 & 2 8 ) and i s not a form of M C P I . RBA i n d i c a t e s the s t r a i n d e l e t e d in cheRB. B " i n d i c a t e s t h e cheB s t r a i n ( f r o m r e f e r e n c e 1 8 ) .

(compare both

Banding show 21).

Figure

1,

CheB m o d i f i e d patterns

a similar In

multiple classes

forms

have

the

control

shown

of

these

for

CheR

suggests

characterization III

and m u l t i p l e

MCP m o l e c u l e s

specificity,

A and B ) .

complexity

addition, of

lanes

and m e t h y l a t e d

Thus in

forms MCPs

MCPs

appear

wild-type

on t w o - d i m e n s i o n a l I,

II,

III

and

and C h e B - d e p e n d e n c e

that are

the

covalent

homologous

and f u n c t i o n s the m o d i f i e d peptides

to

to

E_. c o l i

in

IV

of

regard

in

of

all

to Moreover,

from MCPs

be h o m o l o g o u s

20,

these

chemotaxis.

peptides

gels (18,

modifications

with

be cells.

I,

II

chroma-

and

Chemical

P r o p e r t i e s o f CheB

tographic we have

behavior

MCP c l a s s e s tical

in

role

with

shown t h a t arginine methyl

resolution

being

residue

are

tryptic

times. find

based

below). in

Thus,

different

necessarily

a common

iden-

functional

peptides

peptide

3H - l y s i n e

have

CheB m o d i f i e d of

MCPII

in

the is

MCP)

the

from MCPI

(data

modification methionineproperties

also

similar

and

II.

with

increased

of

results

in

the

existence

peptide

in

retention

a peptide

35

by

comparing

strains

times not

or

peptide Peptide

is maps

shift

peptide peptide

CheB-dependent elution

position

with

of a

chromatographic

CheB m o d i f i e d

peptides

from

two f o r m s

of

this

peptide

produced

in

cheR

cells

are

complete.

(21).

are

These

S-methionine

peptide

two s i t e s

from M C P I I I

which

MCP).

CheB m o d i f i e d

MCPIII,

the

results

conditions.

(18,20).

an a l t e r e d of

longer.

same C h e B - d e p e n d e n t

this

MCPIII,

is of

of

S-methionine-1abeled

of In

glutamate

be r e t a i n e d

in

lysine the

35

an

to those

CheB m o d i f i c a t i o n

suggests

contains

shown).

However,

a

longer

to

a methionine-containing

and l y s i n e - c o n t a i n i n g

MCPI

to

liquid

peptides,

having

(CheB m o d i f i e d with

high

peptides.

the

identified

labeled

counterpart

not

of

these

synthesized

show e x a c t l y

position

MCPII

were

and cheR

of MCPI

and a l s o

under

con-

performance these

group

peptide

one

methionine,

used the

hydrophobic

a methyl

time

peptides

from MCPs

molecules

high

polarity

the

and

analyze

CheB m o d i f i c a t i o n

shown t h a t

elution

of

retention

maps

phase

on the

peptides,

lysine We have

to

or more

causes

that

in

(20).

pH 2 . 2

Addition

(unmodified

this

not

have

tryptic

reverse

at

larger

The C h e B - m o d i f i a b l e

in

of

on a p e p t i d e

an i n c r e a s e

which

i n MCPI

(HPLC)

either

We l i k e w i s e

that

while

two d i f f e r e n t

technique

retention

HPLC

that,

(see

modifications

and one c o n t a i n i n g

separations

cheRB

covalent

idea

esters

chromatography

in

content

chemotaxis.

We have

The

acid

the m o d i f i c a t i o n s

taining

those

the

the

number,

in

accept

and amino

investigated

537

Modification

which

This are

in

strongly CheB-modifiable

538

M.R.

W-t. MCPI bands 4-8

T4

wt MCPII bands 5-8

vçL_

24

120 140

Ï6Ô

Dahlquist

and

M.W.

Bond

F i g u r e 2. C o m p a r i s o n of M e t h y l - L a b e l e d T r y p t i c P e p t i d e s f r o m M C P I , M C P I I and M C P I I I P r o d u c e d in W i l d - T y p e co1i. M e t h y l g r o u p s in MCPI w e r e l a b e l e d w i t h [ m e t h y l - 3 H ] m e t h i o n i n e , and t r y p t i c p e p t i d e s of b a n d s w e r e s e p a r a t e d by H P L C . .Only the r e g i o n of the e l u t i o n p r o f i l e c o n t a i n i n g the p e p t i d e s is s h o w n . B a c k g r o u n d c o u n t s w e r e f o u n d in all o t h e r fractions. (A) m e t h y l - 3 H - 1 a b e l e d t r y p t i c p e p t i d e s from MCPI (bands 4-8 c o m b i n e d ) p r o d u c e d in M S 5 2 3 5 (tar) , a s t r a i n c h e m o t a c t i c a l l y w i l d - t y p e w i t h r e s p e c t to MCPI. T r y p t i c p e p t i d e s are l a b e l e d T2 ( f i r s t e l u t e d ) to T 6 ; (B) m e t h y l - J H labeled tryptic peptides from MCPII (bands 5 - 8 c o m b i n e d ) p r o d u c e d in M S 5 2 3 4 ( t s r ) , a strain chemotactical1y wild-type with r e s p e c t to M C P I I . A l t h o u g h the o r d e r of e l u t i o n of the two m e t h y l a t e d p e p t i d e s in M C P I I w a s r e v e r s e d f r o m t h a t of the c o r r e s p o n d i n g t w o m e t h y l a t e d p e p t i d e s in M C P I , the p e p t i d e s w i t h p r o p e r t i e s a n a l o g o u s to T1 and T4 h a v e b e e n a s s i g n e d the s a m e n u m b e r s to f a c i l i t a t e c o m p a r i s i o n s ; (C) m e t h y l - 3 H - 1 a b e l e d t r y p t i c p e p t i d e s f r o m M C P I I I ( b a n d s 2 - 5 c o m b i n e d ) p r o d u c e d in H B 2 4 3 (tar t ^ r ) ( 3 0 ) .

WPL

o

F.W.

A

T3

20

Kehry,

180 200

FRACTION NUMBER

In M C P I ,

II,

containing accepting

and

I I I , the

peptide arginine

is

also

Figure

trypsin

of

digestion of the

L-[methyl Many

MCPs

each

are

peptide,

2 shows

the

protein

peptides

in the are

the

for

of each

methyl -

is

maps

by

ester

using

protein MCP.

not derived

methyl

radiolabeled

absence

seen

The

however,

peptide

after

specifically

methionine

radioactive

methionine-lysine

methyl-accepting .

containing

CheB-modifiable. groups

CheB-modifiable

synthesis. These

pep-

Chemical

tides

P r o p e r t i e s of CheB

are

generated

methionine-lysine methylated peptide. tides

form As

exhibit

further

by m u l t i p l y

peptide

of

can

the

be

that

three

MCPs

is

of

unmethylated

by

HPLC

sugesting

This

was

identical

a great based

do

not

large

fingerprint

between

pep-

conserved. to

at

and

of

be

or

away

from

had

MCPII

observed

these

homologous

sites we

identical

peptides

The

(containing

work,

MCPI

of

of

one

properties,

appear

structure

number

analyses

methylated

MCPs

In e a r l i e r

similarity

on the

the

methyl-accepting

strongly

or a r g i n i n e )

in

least

at

methyl-accepting

these

region.

of

of

the

of

a divergence

forms

elution

the

(are)

methionine-containing

dimensional

maps,

peptides

methyl-accepting

suggested

in t h e s e

idea

lysine

presence

chromatographic

remaining

the

methylated

the

the

region(s)

methionine,

the

and

arginine-containing

seen

similar

supporting

539

Modification

(31). nearly

in

two-

proteins.

It

is

TABLE I Modifications

MC P

N u m b e r s of cheB

CheB

2

2

2

n.d.a

3

II

MCPs

N u m b e r s of Modifications

3

I 111

Methyls

of

N u m b e r s of M e t h y l s Wi1d-Type 6 4b >

5C

a

MCPIII

b

N u m b e r s of m e t h y l s gels only.

c

N u m b e r s of m e t h y l s in M C P I I I d e t e r m i n e d f r o m b a s e - c a t a l y z e d d e m e t h y 1 a t i o n of m e t h y l a t e d p e p t i d e s . Some higher m e t h y l a t e d f o r m s w e r e not a n a l y z e d .

now

clear

variously

was

not

that

analyzed

many

chemically

of

these

peptide

and

in t h e

background.

determined

peptides

modified

methyl-accepting is c o n s e r v e d

in a c h e B

in M C P I I

which

forms

simply

of t h e

contains

different

from

MCP

two-dimensional

represent

the

CheB-modifiable ,

methionine

species.

and

lysine

540

M.R.

While

the

tion

MCPs

appear

and m e t h y l a t i o n ,

tions

vary.

Table

methylation determined tions

and

numbers

summarizes II

II,

and

which

occurs

a maximum of

increases

Similarly,

MCPII four

is

three

the

are

number

MCPIII

in

cheB

appear

to

be a v a i l a b l e

of

bacteria

in

six

times

twice

in

in

sites

but

at

maximum have

in

protein

the

extent be

background cells

of

and

cells.

while

cells.

for

five

three

can

wild-type

cheB

available

modifica-

modified

MCPI

a cheB

in

been

two CheB

wild-type

least

this

modifica-

of

Thus

times

found

covalent

to c o n t r o l

MCPs.

Bond

modifica-

which

are

a n d M.W.

CheB

may be CheB

appears

methylated

sites

There

the

to -a maximum of

maximum of

examined

in

these

of

numbers

events

MCPIII

Dahlquist

sites

of

the

III.

while

F.W.

similar

The CheB m o d i f i c a t i o n

methylated this

the

I

in MCPI,

i n MCPI

times.

have

and CheB m o d i f i c a t i o n

methylation

not

to

Kehry,

a

We have

methylation

of

sites

of

methylation

produced

in

wild-type

cells.

The m e t h y l - a c c e p t i n g

and C h e B - m o d i f i a b l e

peptide

Kl,

from MCPI

further

characterized

isolated

determinations. of

The

5

the ^ S - c o u n t s

tion

of

peptide

is

found

as

the

at of

residue in

methyl-1abeled

lowermost

peptide were

panel

of

have

also

peptide,

Rl,

A clear

of

peak

of

This

radioactive

Figure

suggests

forms

is

These by

3 shows that

the

positions

radioactivity is

subjected

observed

at

(T3

incorporation

The

profile

arginine-containing

MCPI,

peak

methionine

are m e t h y l - a c c e p t i n g . the

plot

degrada-

been c h a r a c t e r i z e d

Kl

3 shows

only

establishes

form by HPLC.

panel

a

radiochemically

The m e t h y l a t e d

methyl-^H-labeled

analysis.

Edman

the

by ^ H - m e t h y l

pure

peptide

Figure

of in

been

sequence 3 shows

The

Kl.

labeled

observed.

17 of

when t h e

step

has

Figure

isolated

15 w h i c h

The m i d d l e

profile

methyl-accepting sequence

cycle

peptides

10 and p e r h a p s

observed

each

was

strain

protein of

f o r m by HPLC.

in Kl

at

it

panel

radiochemically

analysis.

radioactivity 3,

pure

peptide

and i s o l a t e d sequence

after

sequenator

15th

and T 5 )

radiolabeled

uppermost

released

Kl

(35S-methionine)

by

methionine-lysine

from a cheRB

to

cycle

11,

Chemical

Properties

2 0. 0

of

CheB

MET

541

Modification

peptide Kl

1 V)

METHYL

Figure

peptides T 3 + T 5 (KD

R a d i o l a b e l e d Sequence A n a l y s i s o f the M e t h y l A c c e p t i n g P e p t i d e s from MCPI

300

200 2

0.

o I X

3

100 0

METHYL

peptide T 4

(Rl)

200 100 2

6

10

14

18

SEQUENATOR I Kl Rl

-

Me -Glu -

10

5 - -

-

Me Glu -

22

CYCLE

-

Me - Gfu -

15 Me Met - (Gfu) ... Lys • Arg

Peptides Kl and Rl labeled with [methyl-^HJmethionine or 35s-methionine were i s o l a t e d preparati vely by HPLC. The methyl-labeled peptides were i s o l a t e d from MCPI synthesized in wild-type c e l l s (MS5235, t a r ) , and 35s- m ethioninelabeled peptide Kl was i s o l a t e d from MCPI produced in cheRB c e l l s (RP2859 Xind" infected with A f l a 9 1 ) . The sequence of 35s-methiomne-labeled peptide Kl was determined with a s i n g l e cleavage program as described p r e v i o u s l y (28,29). To minimize demethylation during sequence determination of the methyl-labeled peptides, the sequenator was modified to bypass the convers i o n f l a s k ; the a n i l i n o t h i a z o l i n o n e d e r i v a t i v e s were extracted from the cup with 1-chlorobutane, 0.1% d i t h i o t h r e i t o l and collected d i r e c t l y . For each sequenator c y c l e , the derivatized-amino acid-containing f r a c t i o n was dried under argon and d i s s o l v e d in 200 pi a c e t o n i t r i l e . The r a d i o a c t i v i t y in the entire f r a c t i o n was determined d i r e c t l y by l i q u i d s c i n t i l l a t i o n counting. A l l samples were loaded on the sequenator in 0.15% SDS (BioRad, twice r e c r y s t a l l i z e d ) , extracted with 1-chlorobutane 0.1% d i t h i o t h r e i t o l (4 ml), and double coupled with an extended f i r s t cleavage time. Top panel, r a d i o a c t i v i t y in f r a c t i o n s of a sequenator run on ^ s - m e t h i o n i n e - l a b e l e d peptide K l ; center panel, r a d i o a c t i v i t y in f r a c t i o n s of a sequenator run on methyl-^Hlabeled peptides T3 and T5 ( K l ) combined; bottom panel, r a d i o a c t i v i t y in f r a c t i o n s of a sequenator run on methyl-^H-labeled peptide T4 ( R l ) . The sequence information on peptides Kl and Rl i s summarized below the graphs and includes the COOH-terminal Lys and Arg residues determined by biosynthetic incorporation of these ^H-labeled amino acids into the peptides.

542

M.R. Kehry, F.W. Dahlquist and M.W. Bond

F i g u r e 4. E l u t i o n O r d e r of C h e B M o d i f i e d P e p t i d e K1 R e l a t i v e t o U n m o d i f i e d P e p t i d e Kl C h a n g e s w i t h p H . M C P I I I w a s l a b e l e d w i t h ^ ^ S - m e t h i o n i n e in c h e R B or c h e R coli mini cells (21). The bands (band 4 for c h e R B , band 1 f o r c h e R ) w e r e p e p t i d e m a p p e d ( s e e l e g e n d t o F i g u r e 2; r e f s . 2 0 , 2 1 ) at p H 2 . 2 in 35 m M s o d i u m p h o s p h a t e b u f f e r ( p a n e l A). ( + ) E l u t i o n p o s i t i o n of u n m o d i f i e d p e p t i d e K l ; t h i s p e p t i d e is a b s e n t in t h e b o t t o m e l u t i o n p r o f i l e s in ( A ) a n d ( B ) . ( ? ) I n d i c a t e s t h e p o s i t i o n of t h e t w i c e C h e B m o d i f i e d f o r m of p e p t i d e Kl ( 2 1 ) . T h e u n m o d i f i e d a n d C h e B m o d i f i e d f o r m s of p e p t i d e Kl w e r e i s o l a t e d p r e p a r a t i v e l y f r o m m a p s s i m i l a r to t h o s e s h o w n in (A) a n d r e c h r o m a t o g r a p h e d u s i n g an i d e n t i c a l a c e t o n i t r i l e g r a d i e n t w i t h 10 m M a m m o n i u m a c e t a t e (pH 5 . 8 ) (panel B). O n l y t h e r e l e v a n t p o r t i o n of t h e p e p t i d e m a p s is s h o w n ; r e m a i n i n g f r a c t i o n s in ( A ) w e r e i d e n t i c a l a n d t h o s e in (B) w e r e b a c k g r o u n d c o u n t s .

Chemical

P r o p e r t i e s of CheB

suggesting

a methyl

543

Modification

glutamate

residue

at

position

11 i n

this

pepti de. Since the

CheB m o d i f i c a t i o n

MCPs

(16),

some c l u e s

CheB-dependent

in

These

later

than

less

position

the

time

lower

the

peptide

the

CheB m o d i f i e d

unmodified

form when the

5.8.

strongly

an

hydrophobic and l e s s

and i s

The most

at

group

in

peptide

chromatographic

derivatives shown

in

of

than

tive

elutes

The

protein

at

from t h e

would

with to

to

at

Figure at

the

is

This of

acid at

recent

and Simon

peptide

Kl

by e x a m i n a t i o n

DNA s e q u e n c e . a methionine

PTH-glu the

than

(22).

pH

is

more

deprotonated unmodified

behavior

is

of

car-

a new

supported

the

of

and g l u t a m i n e has

by

shown

MCPI

below.

Only

one

acid

sequence

15 r e s i d u e s

tsr

one

from a

can

sequence sequence

sequence

in

here

(MCPI)

acid This

deriva-

derivative.

presented of

The amino

amino

retention

acid

glutamine

the

(PTH-gln)

a longer

glutamic

of

residue

at

phenylthiohydantoin

DNA s e q u e n c e

is

out

to t h e this

conclusion

pH 5 . 8 ,

elution

the

but

partially

(PTH-glu)

time

5.8.

modification

generation

the

its

form.

protonated

for

pH

than

carried

CheB

compared

determinations

the

are

elu-

in

a longer

earlier

pH

elute the

and at

in

unmodified

least

the

4 shows

pH 2 . 2

the

to

from M C P I I I

results

explanation

pH 2 . 2 ,

while

be e x p e c t e d

isolated

which

Kl.

an e a r l i e r

by Boyd

corresponding identified

At

sequence

be c o r r e l a t e d

contained

5.

PTH-gln

determined

chromatography

involves

glutamic

Figure

time

different

that

behavior

the

phase

pH 5 . 8 as

likely

CheB m o d i f i c a t i o n

boxyl the

group

pH 2 . 2

hydrophobic

peptide. that

ionizable

the

K1 at

separations

at

of

by e x a m i n i n g

reverse

form e l u t e s

suggests

nature

of

peptide

forms

relative

charge

by

pH, CheB m o d i f i c a t i o n

the

chemical

forms.

and u n m o d i f i e d

negative

the

are

K1 p e p t i d e

for

produces

of

net

be g a i n e d

forms

However,

This

can

hydrophobic

of

CheB m o d i f i e d At

properties separations

w h i c h more h y d r o p h o b i c

tion

the

as t o t h e

modification

chromatographic values.

increases

was

deduced

reading trypsin

frame

544

M.R. Kehry, F.W. Dahlquist and M.W. Bond

F i g u r e 5. E l u t i o n O r d e r of P T H - g l u t a m i c A c i d R e l a t i v e to P T H - G 1 u t a m i ne C h a n g e s w i t h pH. P h e n y l t h i o h y d a n t o i n (PTH ) - g l u t a m i c a c i d and P T H - g l u t a m i n e were s e p a r a t e d by r e v e r s e p h a s e HPLC as f o r t r y p t i c p e p t i d e s ( s e e F i g u r e s 2 and 4 ; r e f e r e n c e 20) and d e t e c t e d by a b s o r b a n c e at 254 nm. ( A ) S e p a r a t i o n i n 35 mM s o d i u m p h o s p h a t e (pH 2 . 2 ) shows t h a t P T H - g l u t a m i c a c i d has a l o n g e r r e t e n t i o n t i m e t h a n P T H - g l u t a m i n e ; ( B ) s e p a r a t i o n i n 10 mM ammonium a c e t a t e (pH 5 . 8 ) r e s u l t s in a very s h o r t r e t e n t i o n time f o r P T H - g l u t a m i c a c i d r e l a t i v e to the P T H - g l u t a m i n e . ( ) i n d i c a t e s the g r a d i e n t of a c e t o n i t r i l e u s e d to e l u t e t h e P T H - a m i n o a c i d s . Under the c h r o m a t o g r a p h i c c o n d i t i o n s i n ( B ) , P T H - g l u t a m i c a c i d a l w a y s e l u t e d as a d o u b l e p e a k , one p a r t e l u t i n g w i t h t h e f l o w through. T h i s i s most l i k e l y due t o t h e h i g h i n i t i a l starting c o n c e n t r a t i o n of a c e t o n i t r i l e . G i n , g l u t a m i ne ; G l u , g l u t a m i c acid.

Chemical

Properties

of

site.

The

cleavage lysine,

in

of

incorporation. in

with

peptide Further,

the

545

Modification

peptide

agreement

COOH-terminus conserved

CheB

is

23 ami no a c i d s

our

determination

K1 by

radioactive

this

amino

DNA s e q u e n c e s

1

acid

long

of

for

10

in

the

amino

acid

sequence

determined

ending

is

tar

highly

and

tap.

20

T E Q Q A A S L E E T A A S M E Q L T A T V K Proposed The p e p t i d e with

the 17,

contains

position

although and

amino

the

of methyl

DNA s e q u e n c e

these

cells.

are m o d i f i e d

suggests

We have

of

This

that

the

and

also

that

peptide Yet

it

K1 of

suggesting

but when one

site

has

from M C P I I I that

the

longer

product lyze

in

data has

for

proteases true, toward

have

however. amides.

of t h e

For

that

are

example

this Most For

3

incorporation the

in

glutamine

wild-type

cells

and

may be a

is

that

can

subject

both

are

the

positions

at

second the

either site

it

appears

that

and an a m i d a s e primarily (23).

esterases example,

is

analogous

3

site, no peptide

suggesting

CheB m o d i f i c a t i o n

may no

have

t h e cheB

activity.

amidases

chymotrypsin

property

only

CheB-modifiable.

occur

that

to

(21).

here,

an e s t e r a s e

enzymes

esters.

positions

t o two CheB m o d i f i c a t i o n s ,

peptide

presented

both

interesting

nature

this

MCPI

both

been m o d i f i e d ,

subject

either/or

hold

From t h e common

is

agreeing

modification

appears

CheB m o d i f i c a t i o n is

at

that in

10

Interestingly,

glutamine

acid

K1

glutamate.

that

It

position

implies

implies

reactive.

peptide

show m e t h y l

glutamic

to

17 can be m e t h y l a t e d

longer

at

CheB-dependent

one C h e B - m o d i f i c a t i o n . This

of

incorporation.

strongly to

glutamine

determined

acid

predicts

two p o s i t i o n s

positions conversion

sequence

glutamic

wild-type again

acid

The

and t h e

to

converse

essentially

is no

acetylcholinesterase

It

also

other

gene is

hydroserine

rarely activity

which

is

3 4 0

M.R.

also

a serine

substrate greater during

active

stability

ester.

This

product

may have

of

became

adaptation

systems.

Recent

(methyltransferase) This

perhaps allow

able

least

have

and c e r t a i n suggest

for

providing

Dr.

A.

Boyd

results

in

that

is

role

Drs.

communicating and D r .

and

ship

from t h e

Damon R u n y a n - W a l t e r

American

supported Cancer

M.R.K.

by a F a c u l t y

et

methylation

on

semisolid

adaptation, sufficient

Finally that

the

cheR

regain

to

Niwano

and

chemotaxis

to

but

oxyis

These

CheB-dependent

Parkinson X-E^

Hood f o r

facilities.

is

bacterial and S t o c k

coli

and d i s c u s s i n g

sequence

sense, primitive

of m e t h y l a t i o n

the

its

in

is

gene

and

modifi-

compounds.

J.S.

L.

cheB

by cheB m u t a t i o n s .

for

strains

this

form of

evidence

energy

the

of

amide

the

(24)

swarms

chemotaxis.

to t h e s e

We t h a n k

progress,

al .

independent

a primary

In

was

the

and M. hybrid

Winchell

Simon phage,

sequencing use

supported

Research

of by

Cancer Award

the a

pro-

fellow-

Fund.

from

the

Society.

Refe r e n c e s 1.

Springer, (1977).

2.

Ridgway, 1 3 2 , 657

M.S.,

Goy,

M.F.,

H.F., Silverman, (1977).

Adler,

J.:

M. , S i m o n ,

Nature M.:

Bond

corresponding

a more

stimuli

modification,

tein

F.W.D.

later.

some o t h e r

of

an a m i d a s e

do not

eliminated

bacterial

for

evolution as

revertants

which

toward

state

its

et

a n d M.W.

because

to t h e

represent

pseudo

presented or

activity

act

form c h e m o t a c t i c

chemotaxis

Acknowledgments.

to

may

mutants

sugars

altered

during

during

that

Dahlquist

transition

Parkinson

rudimentary

(26,27)

studies

of

low

compared

important

CheB-dependent

drastically cation

to

suggests

the

at

Taylor gen

are

as

to e n v i r o n m e n t a l

wo'rk

demonstrates

function

higher

modification

f o r m of

agar.

that

very

F.W.

be j u s t i f i e d

been d e s i g n e d

activity

(25)

has can

an amide

suggests

CheB-dependent

al.

This

and hence

hydrolysis

esterase

enzyme

analogues.

Kehry,

J.

289,

279

Bacterid.

Chemical

P r o p e r t i e s of CheB

3. C l a r k e , (1979).

S.,

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Koshland,

D.E.,

Jr.:

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4. W a n g , E . A . , K o s h l a n d , 77, 7157 (1980).

D.E.,

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5. H a z e l b a u e r , G . L . , P a r k i n s o n , J . S . : In M i c r o b i a l I n t e r a c t i o n s , R e c e p t o r s and R e c o g n i t i o n , S e r i e s B, 3. (J. R e i s s i g , E d . ) , C h a p m a n and H a l l , L o n d o n , 1 9 7 7 . 6. K o i w a i ,

0.,

7. Hedbl o o m , 8.

Kihara,

Hayushi,

M.L.,

Adler,

M. , M a c n a b ,

9. R e p a s k e ,

D.R.,

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R.M.:

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H a z e l b a u e r , .L., I 45 , 43 (1 981 ).

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Springer, M.S., S c i . US 7 4 , 533

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DeFranco, A.L., Parkinson, J.S., J . B a c t e r i o l . 1 3 9 , 107 ( 1 9 7 9 ) .

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Toews,

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C.,

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F.W.:

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Silverman,

Kehry, M.R., (1 9 8 2 ) .

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Kehry, M.R., Dahlquist, G . L . : In p r e p a r a t i o n .

22.

Boyd, A., Krikos, proceedi ngs .

23.

In E n z y m a t i c R e a c t i o n M e c h a n i s m s , C h a p t e r 3 (C. W a l s h , Ed.), W.H. Freeman and C o . , San F r a n c i s c o , 1979.

24.

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N i w a n o , M. , T a y l o r , II ( 1 9 8 2 ) .

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DISCUSSION Von Hippel: If the ahe-B modification is essentially irreversible, what purpose does it serve in chenotaxis. Dahlquist: There are at least three alternatives which are consistent with the data we have to date. One is that the ohe B modification is a 'oneshot' adaptation itechanism. This would be quite inefficient unless protein turnover were rapid but would be better than no ability to adapt at all. A second possibility is that the modification is essentially a processing step which allows activation of the protein after it has been properly inserted in the membrane. Perhaps the additional negative charges observed after ohe B modification do not allow insertion into the membrane. Thus the protein must be inserted in the 'de novo' form and then the negative charges are introduced generating a functional enzyme. A third alternative is that ohe B modification is a reflection of some evolutionary event but has been maintained because it is of seme advantage, perhaps by enhanced chemotaxis capability. McClure: With respect to your suggestion that ohe B dependent deamidation of MCP represents an 'irreversible adaptation' I wonder whether MCP's might turnover in the cell more rapidly than a generation time? Dahlquist: Evidence in normal cells suggests that turnover is very slow, at least in the absence of protein synthesis. We do see an exceedingly rapid cleavage of this protein by an endogenous protease after solubilization by detergent. Such cleavage of other proteins is not seen in the bulk. Manson: It was not clear to me hew the number of ohe B dependent nodifications is correlated with the number of new sites exposed for msthylation after ohe B modification. Can you explain again hew ohe B modification and methylation relate to one another? Dahlquist: It is clear that one ohe B modification allows an additional three methyl groups to be added to the lysine-methionine peptide of tsv. In the case of tar, the single ohe B modification allows two additional sites

Chemical

P r o p e r t i e s o f CheB

Modification

549

to be methylated. Thus it appears that pre-existing glutamate residues are made susceptible to methylation. The single site of ohe B modification appears to generate one of these sites directly by the glutamine to glutamate conversion. We are less able to state the role of the second ohe B modification which occurs on the arginine peptide but does not change its apparent access to the methyl transferase.

A FAMILY OF HOMOLOGOUS GENES ENCODING SENSORY TRANSDUCERS IN E. COLI

Alan Boyd, Alexandra Krikos, Norihiro Mutoh and Melvin Simon Department of Biology, University of California, San Diego, La Jolla, California 92093 USA

Introduction Chemotactic behavior in Escherichia coli consists of an ability to migrate through concentration gradients of chemicals. E_. coli exhibits both attractant responses, e.g. to serine, aspartate, maltose, ribose, and galactose; and repellent responses, e.g. to leucine, weak acids, Co and Ni ions (1,2). Swimming bacteria detect changes in concentrations of these chemoeffectors as a function of time (3,4).

This temporal

sensing strategy enables them to compare concentrations over distances much greater than their own length, and thus to detect very small gradients.

The means by which this sensory

information is converted into directed migration has been reviewed extensively (5,6,7). We have found it useful to define a number of functions that would be required for temporal sensing.

We envisage a

regulatory device composed of the following elements: (a) a measure of the current environment; (b) a measure of the past environment, continuously updated; (c) a comparator which gauges the relative values of past and present; and (d) a signal from the comparator which influences flagellar activity and ultimately the direction of migration.

Mobility a n d Recognition in C e l l Biology © 1 9 8 3 by W a l t e r d e Gruyter & Co., Berlin • N e w Y o r k

552

A. Boyd et al.

A group of transmembrane proteins termed transducers, signalling proteins or methyl-accepting chemotaxis proteins (MCPs) is apparently responsible for comparator function. These proteins are the products of four genes: tsr, tar, tap and trg (8,9,10,11,12). Each transducer species integrates information pertaining to one or a very few chemoeffectors: tsr, serine; tar, aspartate, and maltose; trg, ribose and galactose; tap, unknown. Two properties of a transducer seem to embody the cell's information about present and prst environments: (a) The degree of occupancy of a transducer as chemoreceptor measures the cell's current surroundings. The tsr and tar products act as chemoreceptors for the amino acid attractants, serine and aspartate respectively (13,14). Sugar responses are mediated via periplasmic proteins which bind a sugar molecule and then interact with the corresponding transducer (15). (b) The level of methylation of a transducer population appears to reflect the cell's environment in the recent past (16). Transducers are subject to two types of post-translational covalent modification, which are intimately related. The proteins are reversibly methylated and demethylated at the carboxyl groups of multiple glu residues through the activities of the CheR methyltransferase and the CheB methylesterase (17,18,19,20,21,22,23,24). The CheB protein also performs a second covalent modification which is irreversible and which exposes sites of methylation, but not necessarily on a one-to-one basis (25,26). Thus, transducers may be made up of three functional domains; an extracellular domain responsible for chemoreception, and two intracellular domains one of which gets methylated and the other of which signals to the flagellar motors. Comparator function would then reside in the regulation of the signalling domain by the other two. For example, it is possible that a

553

Sensory Transducers in E.coli

transducer signals if and only if its chemoreceptor domain is occupied, but that its level of signalling is determined by its state of methylation. Thus, the proportion of a transducer population actively signalling at a given instant would be determined by the external concentration of chemoeffector, whereas the total signal output of the population would be a function of the mean level of methylation in the population, the latter parameter being varied during sensory adaptation.

Transducers Are Encoded by Homologous Genes In order to lay a foundation for fine-structure genetic analysis of the sensory transducers, we used the transposon Tn5 to generate insertion mutations throughout cloned fragments of bacterial DNA known to carry the tsr, and tar genes.

In

this way, by combining genetic complementation analysis with restriction endonuclease mapping of DNA, the genetic and physical maps of these two loci have been aligned (12). By the use of DNA-DNA hybridization analysis, performed under conditions of reduced stringency (20-3 0% mismatch tolerated) it was found that the tsr and tar genes possess considerable sequence homology (12).

However, this homology is restricted

to the promoter-distal segments of the two genes, comprising approximately half of each gene.

These findings suggest a

bipartite structure for the genes encoding transducers, in which the promoter-proximal and promoter-distal segments correspond to separate domains of structure and function in the transducer proteins. Thus, the closely related promoter-distal segments might encode cytoplasmic domains concerned with methylation and signalling to the flagellar motors, since these functions involve interactions of all transducers with the same components of the central information-processing system.

Conversely,

promoter-proximal segments might encode extracytoplasmic domains cocerned with chemoreception.

These latter gene segments,

554

A. Boyd et al .

being not detectably homologous, may have simply diverged considerably, or may perhaps have arisen from unrelated sequences. The tap gene A combination of Tn5 mapping and hybridization analysis led to the discovery of a gene, tap which lies between tar and the cheR-cheB-cheY-cheZ genes. transcribed.

These six genes are co-

The tap gene exhibits strong homology to tsr

and tar (12), and upon further analysis the tap gene product was found to form multiple bands in SDS-PAGE, a characteristic property of the transducer proteins which is caused by multiple methylation (21).

This evidence indicates that the

tap product is a sensory transducer.

Attempts to uncover a

chemoreceptor function for the tap product have so far proved unsuccessful. The trg gene; other transducers No nucleotide sequence homology is detected between trg and the tsr-tar-tap family under those hybridization conditions which first uncovered the familial relationship (12).

This

does not rule out the possiblity that the trg sequence has simply diverged beyond the limits of detection imposed by the hybridization conditions which were used.

In genomic

blots a tar + tap probe shows cross hybridization with tsr only, indicating that no other closely-related genes are present in E. coli.

It would be of interest to determine

whether a trg probe cross-hybridizes with any other sequences. Certainly, the failure to observe trg hybridization was surprising and suggests that trg may have diverged from the tsr-tar-tap lineage very early.

Sensory Transducers

in

555

E.coli

Primary Structures of Transducer Proteins The finding of bipartite gene structure in tsr and tar led us to the idea that these transmembrane proteins are constructed from structural domains which perform separate functions. We have therefore undertaken to determine the nucleotide sequences of these genes in order to analyze in detail the nature of the homologous and non-homologous gene segments with the expectation that sequence comparisons will allow us to assign specific functions to different portions of these molecules. The primary structure of the Tsr transducer We have determined the complete nucleotide sequence of the tsr gene (Boyd et_ al., in preparation).

A preliminary

analysis of the derived amino acid sequence of the tsr product has revealed several notable features which extend out understanding of Tsr structure and function.

The primary gene

product of tsr is composed of 535 amino acids (Figure la). The first 3 0-35 residues bear a striking resemblance to the signal sequences found in secreted proteins in E. coli (27).

Such a

signal sequence would be expected to direct the export of subsequent sequences to the outside of the bacterial membrane. We do not know if the tsr product is subject to posttranslational proteolytic processing to remove this putative signal sequence. Further downstream in the primary sequence, around amino acid position 200, is a sequence of 2 9 uncharged, predominantly hydrophobic residues. This sequence might be expected to act as a stop-transfer signal, anchoring the protein in the membrane. The finding of this amino-terminal domain of Tsr flanked by a putative signal sequence and a potential membranespanning sequence is consistent with the notion that this stretch of sequence comprises a domain which assembles at the extracytoplasmic face of the membrane where it acts as a

A. Boyd et al

556

chemoreceptor. To probe the structure of Tsr iri vivo we have treated spheroplasts of E. coli with trypsin. Preliminary data indicate that in such experiments the limit digestion product retains the multiple band pattern of intact Tsr, indicating that it retains the sites of methylation. This is further evidence consistent with the broadly sketched topography of Tsr deduced from the primary sequence. The remaining carboxy-terminal sequence of some 3 00 amino acids includes two stretches of sequence which we have tentatively identified as the methylated regions of Tsr. To make this identification, we analyzed the predicted tryptic cleavage products of Tsr and found that only two peptides (K and R of Figure 1) matched the information about the methylated peptides which has emerged from recent studies (28,29). The sequences of these two peptides, derived from the tsr nucleotide sequence, are presented in Figure lb; asterisks denote the sites of methylation determined by Kehry et a_l. (30) ; the bracketed asterisk denotes a further site proposed by us on the basis of homology with the other sites. The K and R peptides are separated by approximately 200 residues in the primary sequence. They are drawn in Figure lb to emphasize homologies between them. This homology does not extend at all beyond the sequences shown. Two of the proposed sites of carboxymethylation of glu residues correspond to gin residues in the primary sequence. This makes it almost certain that the CheB-modification (25,26) is the conversion of certain gin residues to glu residues in a deamidation performed by the CheB methylesterase.

The other notable feature of the sites of methylation

is that they all occur at the second position in a pair of glu residues.

Sensory Transducers

in

557

E.co)i

Figure 1

100

200 i

I

m

300 i

500 i

i

V/A

VA

K

R

(a) Primary structure of the Tsr transducer. Filled regions; stretches of uncharged predominantly hydrophobic residues. Domains K and R; methylated peptides. Cross-hatched regions; the two major blocks of Tsr-Tar identity, 48 and 26 residues long.

TAP K:

A Q

TAR K: TSR K:

.

ii S A

i i

*

*

TSR R:

T E Q Q A A S L E E T A 1 t 1 1 1 1 1 1 1 * V T Q Q N A A L V E E S A

TAR R:

i

i« S

TAP R:

Q

ii S

Q

•

i i

i

G

i i

i

i

*

A S M E Q L T A T V K 1 1 U) A A A A A L E E Q A S •

i •

i

A A V A T E Q . A N .

•

D

(b) Methylated regions of transducer proteins. Sequences are in standard one-letter code and are derived from nucleotide sequences. Asterisks in Tsr sequences denote sites of carboxymethyl-glu residues observed by Kehry et al. (30); the bracketed asterisk denotes a further site proposed on the basis of sequence homology. The K-R nomenclature is that used in Ref. 30. Note that in Tap neither K nor R is actually a tryptic peptide.

558

A. Boyd et al

Tsr-Tar sequence homology

At the time of writing our determination of the nucleotide sequence of tar is approximately 8 0% complete; this sequence includes the part of the gene which encodes the carboxyterminal two-thirds of the protein. This region of Tar is very similar to the corresponding Tsr sequence. The two protein sequences can be aligned precisely over a stretch of more than 3 00 residues without the.need to introduce any gaps and exhibit almost 80% identity over this region. Furthermore, a potential membrane-spanning sequence occurs at the same position in both proteins suggesting a conservation of transmembrane structure. The carboxy-terminal sequence homology can be divided into two broad types: (1) regions of conserved and diverged residues, interspersed, and (2) more or less uninterrupted stretches of identical sequence. The two most striking examples of the latter class are indicated in Figure la and consist of blocks of 48 and 26 identical residues. The sequences at the methylated regions also fall into the block homology class (See Figure lb). Since the nucleotide sequences encoding these blocks of homology are saturated with base substitutions at silent sites we may conclude that these amino acid sequence identities reflect powerful selective forces acting to maintain identical amino acid sequence. This could well reflect the need for both Tsr and Tar to interact with a single set of cytoplasmic enzymes, namely the CheR-CheB system.

Nature and function of the tap product. Lesions in tap cause no mutant phenotype that has been detected as yet (31). The suggestion that the tap product can act as surrogate for Tar (3 2) has not found support in similar studies by Parkinson et al. (31). We would argue that tap does encode

E.coli

559

a functional transducer.

Sequence analysis of tap has revealed

Sensory Transducers

in

some evidence in support of this idea. Firstly, we have noted above the existence of a major block of 48 amino acids which is identical in Tsr and Tar. In the equivalent sequence of Tap, 47/48 of these residues are conserved. This finding indicates that the tap gene is evolving under selective constraint, not merely drifting as would be expected for a gene encoding a non-functional protein. Secondly, the sequence in Tap which aligns with the R peptides of Tsr and Tar has diverged considerably from the R sequences, but has come to more closely resemble the K peptides of Tsr and Tar with respect to the spacing of gluglx sequences. Again, this finding may best be understood in terms of a functional tap product.

Conclusions The finding that the major sensory transducer proteins of E_. coli are encoded by homologous genes has led to the development of a model of transducer structure and function in which separate structural domains are responsible for the functions of extracellular chemoreception and of intracellular signalling and temporal integration of information.

Deter-

mination of the nucleotide sequences of the genes which encode the transducers is providing a framework for thinking about details of the structure and function of these molecules. Scrutiny of the primary amino acid sequences of the transducers allows the formulation of testable hypotheses concerning, on the one hand, the assembly and membrane topography of the proteins, and on the other hand, the nature and identity of domains concerned with chemoreception, and methylation.

CheR-CheB binding,

The sequence information also provides a firm

foundation for testing these ideas by a combination of genetic and biochemical approaches.

A. Boyd et al .

560 Acknowledgements

Research from this laboratory was supported by a grant from the Office of Naval Research.

We are grateful to M.R. Kehry for

communicating peptide sequence data prior to publication.

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Brown, D.A., Berg, H.C.: Proc. Natl. Acad. Sei. USA 71^ 1388-1392 (1974). Macnab, R.W., Koshland, D.E.: Proc. Natl. Acad. Sei. USA 69, 2509-2512 (1972).

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Emr, S.D., Hall, M.N. and Silhavy, T.J.: J. Cell Biol. 86, 701-711 (1980).

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A. Boyd et al

DISCUSSION

Macnab: There are 3 pronounced regions of homology between the tar and tsr gene products. You have related 2 to the regions which are methylated. Could the other be related to a common signalling domain? Boyd: The other region of homology is in fact, the most highly conserved sequence of amino acids in the two proteins. This domain may be related to the signalling function of the transducers, or alternatively it could represent a site at which the methyl transferase and methyl esterase bind in order to reach their substrate sites. Certainly it would be of interest to knc*/ what mutant phenotypes arise when this danain is altered. Boos; Is there any negative dominance with rrulticopy tsr or tar mutations over wildtype tsr or tar on the dyamosome? Boyd: The notion that transducer multimers are somehow involved in the mechanism of sensery transduction predicts that the kind of effects you suggest in your question should be observed. Indeed it is interesting to speculate that the ohe D alletes of the tsr gene, which create a dominant ahe defect might somehow be related to a multimerisation of transducers, perhaps in mixed multimos. One of the attractions of this nultimerisation idea is that it is much easier to test than a transmembrane propagation of a conformational change. Engelhardt-Altendorf: Do your data about the sequence of tsr and tar give you any hint if there are one or tsro binding sites for aspartate and maltose binding protein? Boyd: The sequence information we have, gives no clue as to the nature of any of the ligand binding sites. The mapping of specific mutations onto the sequence should help to localize these sites. Manson: A cannent: My colleague Claudia Wulff has isolated an E.ooli mutant that has no detectable chemotactic response to maltose but retains an essentially normal aspartate response. The mutation causing this phenotype appears to be located in the tar gene since maltose taxis is restored by reintroduction of a good copy of the tar gene. Our expectation is that the binding site on MCP II for maltose binding protein is altered in this mutant. We plan to isolate more strains lacking only maltose or only aspartate receptor function. By mapping the mutations response, vie hope to determine whether there are domains of receptor recognition.

G E N E T I C S OF T R A N S M E M B R A N E S I G N A L I N G P R O T E I N S

J o h n S. P a r k i n s o n , M a r y K. S l o c u m , Ann M. D a v i d S h e r r i s and S u s a n E. Houts B i o l o g y D e p a r t m e n t , U n i v e r s i t y of S a l t Lake C i t y , U t a h , USA 84112

IN E. COLI

Callahan,

Utah

Introduction Chemotactic sensory

behavior

transduction

genetic stimuli,

episodes

about

CCW

stimuli

or

are

adaptation

in in

response in

phase

that

temporal a

produces

pattern

membrane

chemotaxis

proteins",

excitation

and

[8].

The

MCP's

proteins.

They

signaling with

occupied

membrane, trigger

and changes

flagellar state

binding then

that

in

result

in

produced

movements

perceived

by are

of

changes

[3] .

fashion

rapid

CW in

Chemotactic

[4, 5]

and

excitation and

elicit

phase

a

that

subsequent

slow

return

to

proteins

known

as

or

adaptation are

proteins a

MCP's,

with

on

the

signal

the

pre-

flagellar

rotation. undergo

sensory

a

adaptation.

Mobility and Recognition in C e l l Biology © 1 9 8 3 by Walter d e Gruyter & Co., Berlin • N e w York

roles

in

chemotactic transmembrane

small

molecules face

unknown After

changes

"methyl-

key

of

outer of

the

MCP's

play

phases

multifunctional

interact

generate

response,

walk

to

chemical

probabilities

rotation, a

of

[6, 7].

accepting response

to

simple

counter-clockwise

Chemotactic

concentrations a

both

the

and

a

amenable

absence

relative

flagellar

cytoplasmic

(CW)

2]. the

events,

changes

readily

the

in a r a n d o m

response

repellent

stimulus rotational Several

[1,

modulating

detected

distinct

initiates

about

represents

is In

clockwise

rotation

by

coli

that

analysis.

of

rotation

attractant two

system

the b a c t e r i a move

flagellar

brought and

Escherichia

and b i o c h e m i c a l

alternating (CCW)

in

of

nature

initiating in

These

or the to a

methylation methylation-

J.S. Parkinson, M.K. Slocum and A.M. Callahan

564 demethylation residues

reactions

in e a c h

MCP-specific c h e R gene 14] .

and

a

products

that

the

at

[9-12]

several

and

are

methyltransferase

a methylesterase che

indicating

place

MCP m o l e c u l e

enzymes,

These

take

are

MCP

glutamic

catalyzed

by

two

by

the

s p e c i f i e d by the cheB gene

[13,

located

molecules

in

specified

acid

the

must

cell

span

cytoplasm,

the

cytoplasmic

membrane. Three

MCP

loci

are

known:

responses

to

aspartate

responses

to

serine;

ribose

and

comprise

mutations

an

bacteria. genetic

of

1.

organization

at

minute

genes,

of

42

on

of

the

cheR

the

with

gene

clusters

[20;

and

and

investigated

carries

made

In

to

to carry

addition

investigations, insight

signaling our

to such

about

the

proteins

initial

efforts

functional

MCP

loci.

col i c h r o m o s o m e ,

genes

organization

that

loci.

whose

the m a j o r

transcriptional phage

tap,

the

flagellar

including

describe

responses

has b e e n

these

products involved,

isolation of a few

considerable

we

tsr also

tactic

transmembrane

gene,

Discussion

clusters

and

to

role

in at and in

clear.

R e s u l t s and

maps

mediates

the two m a j o r MCP loci, tar and tsr,

MCP

is not yet

Genetic

mediates

product

are

from the

for b i o c h e m i c a l

article of

newly-discovered

chemotaxis

tsr

tar

in

of

furnish

evolution

In this

product

and

no a t t e m p t

studies

mutants

dissection

the

MCP,

Aside

MCP g e n e ,

genetic should

and

gene

The

cell's

indirectly,

useful

analysis

function

the

repellents.

in e a c h

detailed

generating

a

and

maltose;

[15-17] .

of

perhaps

temperature out

bulk

tar

and the trg p r o d u c t m e d i a t e s r e s p o n s e s

galactose

the

although

and

the

other

cheB of

among

[18, tar

aid of X c h e 2 2 , a s p e c i a l i z e d

the

tar

Slocum

locus &

and

several

Parkinson,

locus

several

chemotaxis-related

loci the

The tar

in

19] .

The

region

was

transducing

neighboring

che

preparation].

565

Transmembrane Signaling Mutants of E.coli

Deletion

mutants

inactivation with

a

was

of

and

used

collection

the

first

containing cheZ.

but

mutants

reduced

genes

tar

and

expression

insertion

mutagenesis

of

the

were

tar

tsr

the

known

maps

flagellar

chemotaxis

genes

that

to

Atsr70 the

about

tsr.

these

13

Kb

hosts

of

Under

these is

the

host

synthesized

on

flbB-,

in that

functions. by

Mud 1.

deletion

analyses

that

of

tar

which

gene

other

a

types

a

To

DNA

tsr, from

from

test

this

two

Xtsr

by

the

strain.

adjacent

Xtsr72

also

promoter-distal

bacterial

inserts

l a b e l e d by i n f e c t i o n of electrophoresis

polyacrylamide presence

of of

most flbB

Neither

flal-dependent

tsr

demonstrated

whereas

the

of

functional

studies

of the

cluster

12 Kb of E^ coli DNA of

no

raising

is.

carried

mapping

about end

in

contains

characterized

expression

programming any

tar

and

analyzed

the

region

located

the

host

conditions

dependent

for

vicinity,

specifically

and

a

this

be

of

sulfate-containing

genes

defects

element

several

synthesized

two p h a g e s were

dodecyl

responses.

che

in

restriction

contained

Proteins

irradiated

in

as

promoter-proximal

carried of

also

tar

were g e n e r a t e d

However,

could

both

and

are

whereas

mutations

transposable

99,

isolated

phages,

Deletion

polar

unsuitable

map

genes

we

transducing that

[21] . do

operon

phenotype.

minute

tsr

chemotaxis-related possibility, gene.

at

tar

functions

general

carry

the

an

and m a l t o s e

downstream

but were

mutations

possibility

che

compounds,

w i t h polar p r o p e r t i e s with

in

used to c o n s t r u c t a fine s t r u c t u r e

gene,

locus

to

tests

that

tar-cheR-cheB-cheY-

exhibited

proved

found

gene

these

in a s p a r t a t e

f u n c t i o n due to their C h e -

The

of all

agent

mapping

We

order:

any to

of

tar m u t a n t s

map

in

strains

ability

mutants

the

chelating

and

tar m u t a n t s .

in

only

Additional These

and

defective

some

by

promoter-proxima1)

che

defective

chemotactic

selected

complementation

che

nonchemotactic

are

However,

were

for

of

(i.e.

four

Mutants

motile,

Xche22

gels

in

end of UV-

sodium

(SDS-PAGE).

chemotaxis-related and

flal

\tsr70

proteins

activity

nor

other

Xtsr72

than

the

566

J.S. Parkinson, H.K. Slocum and A.M. Callahan

tsr

product,

related

Parkinson, E.

coli

in

loss

stimuli.

with

of

maps

dominant

at

mutants

them

were

yield

a

tar

or

was

tsr

and

ability

to

complement

we

is

tsr r e g i o n

exhibited

a

and

with

designated a

cheD

in

the tsr gene.

form

of

tsr

on

not

Only

work,

phenotype

on

a X transducing progeny centers

recombine

mutant with

obtained into

in

the the

tar

the or

of

We A

tested that

were were

relative tsr

double

cause

the grew of

mutant.

chemotactic unable The

to tar

then

crossed

for

further

position

coding

of

ability

variety

host.

chromosome

Our

either

double for

mutant

manner

a

tsr

single-

therefore

induce

a tar tsr

double

this

copy

A

were

host

In a d d i t i o n ,

within

to

phage the

tar

chemotactic

phage.

on

of tsr

but

the

In

that

type

plaque

to

[22] .

The u n d e r l y i n g

clear,

and

designed

method.

observation

host

tar

screening

a wild

the

of

tar and

collection

entirely

plated

phage

large

general

mutD

that

and m o s t of

procedures

furnishing a

Evidently

product

a handful

mutant

nonchemotactic.

on

that

rotation.

selection

the

are

flagellar

demonstrated

in p r e v i o u s

a

generally

defects

CCW bias

by

mutations

mutation

tsr-dependent

produces

studies

altered

a more

identify or

other

mutations,

a l l e l e s of

an

isolated

phage

characterization. new

&

a severe

generated

using

gene

from

the

chemotaxis[Callahan

but

mapping

through

based

\tsr+

and

tsr

other locus

a less b i a s e d view of the range of

restored

Lysogens

from

serine

preconceived

are g e n e r a l l y

mutations

and

no tsr

chemotaxis

have

special

obtained

behavior

Atar+

the

locus,

and

been

mutants

strategy

be

of

new M C P m u t a n t s .

of

have

possible,

MCP

this

were

to o b t a i n

can

the

[21] . M u t a n t s

structure

specific,

defects

mutants

tsr

type

to

of

synthesize

Isolation

step

are

of

i n t e r f e r e s w i t h CW f l a g e l l a r

tsr m u t a n t s

order

class

the

Fine

cheD mutations

2.

there

deletions

phenotype

to wild

rotation.

actively

that

vicinity

chemotaxis

Another

nonchemotactic

cheD

the

in p r e p a r a t i o n ] .

mutants

specific cheD,

suggesting

genes

of

each

region

was

567

Transmembrane Signaling Mutants of E .coli

established

by

comparing

restriction

the

location

phenotype

it

functional

domains within

Three

produced,

classes

independent

of

chemotaxis, Unlike

to

type

some

were

found

null

mutations

throughout

the

of

the

clustered if

to

the

of

were

the

Tar

phenotype

were

generally

were

defective

normal

tsr, the C h e appeared

in

responses -

have

mutations

or

in tar were

polar

region.

We

have

not

Mal+

mutations

to

know

whether

tar

coding

region,

specifically

defects; Both

mutations

coding

the

maltose.

deletions.

(Che-)

polar

aspartate to

mutations

to

14/24

(null)

tar

were

discrete

obtained:

-

Asp-

within

they

evidence

7/24

the

By

with

MCP's.

nonsense

and

mapping.

allele

strains;

and

be

see

mutations

a l l e l e s of

deletion

mutant

to

essentially

wild

the

enough

these

3/24

had

cheD

the

recessive

hoped

exhibited

and

but

and

each

tara

of

nonchemotactic;

we

tar

isolates

characteristic

analysis of

yet

as m i g h t

defective

mapped examined

they

be

are

expected

in

aspartate

30/50

independent

chemoreception.

Two

classes

isolates tsr

A

of

strains;

mutants.

null

The

cheD

a pronounced

mutants

mutants

pattern

of

these

detail; mutants and

were CCW

mapped

were

two

were

classes

to

respond

to

in

those the

only

has

not

studies

flagellar

stimuli. sorts

the

tests

and

whereas

the

the

yet

In of

been

wild

responses

stimuli

type

behavior

studied

that

contrast,

coding

All of

response

indicated

cheD

in a r e g i o n

rotation,

exhibited

of

exception,

sequence.

The s t i m u l u s

of

tsr

one

complementation

and

initiate

these

with

flagellar

rotation.

tsr-processed

to

half of the tsr gene

recessive

initial

unable

in

characteristic

throughout

of the coding

dominant

mutant

similar

mutations,

bias

flagellar

however,

other

could

alleles

about one-fourth

new

obtained:

phenotypes

cheD-like

had

of

had

were

(null) p h e n o t y p e

in the p r o m o t e r - d i s t a l

comprising

null

Tsr-

the

20/50

The

region. mapped

tsr m u t a t i o n s

exhibited

the to

cheD under

in null

serine mutants certain

568

J.S. Parkinson, M.K. Slocum and A.M. Callahan

conditions,

suggesting

that

they

cheraoreception f u n c t i o n s of the 3.

Post-translational

MCP's. by

The

the

We

relative

mutants

to

these

cheR cheB

A

sensory

strains

of

either

Xtar

these or

The

methylation

up

or

the

unable

is

is

[23] .

slow

down

typically

presence

methylated

to

cheR6

cheB

MCP2*

contain

varying

of

extents

the

with

results

cheB

of

synthesized is

has

in are

responses. and

cheB

properties methylation

subjected

CW

present,

with by

to

UV

SOS-

different this

work

in

form

a

signaling MCP2*

is

is also u n m e t h y l a t e d ,

but

stimuli

conversion,

very little of e i t h e r

c h e R and

out

MCP b a c k b o n e s

and

to

to carry

cheBA hosts

and

Chemotactic

to MCPl*

able

to

signaling

then

function

1*, w h i c h

in

extreme

response

cheR

molecules

initially

properties.

of b o t h

were The

bias

an

that c h a n g e s

assess the

unmethylated

If

the

labeled

resolves

to a form c a l l e d signaling

infected

molecules

MCP

the To

and

type

for e x c i t a t i o n , b u t

patterns

reflect

CW were

in

of

swimming the

had

mutants

rotation

contain.

[9-14] .

which

properties

cells

MCP

that

2*,

we

which

states

demonstrated

processed

they

MCP's,

analysis,

CCW

strains

but were

or b o t h

of

pronounced of

insertion

one

results demonstrate

presumably

Atsr. phage

programming

has

a

mutant

products.

their

Mutants

types

rotation

the

controlled

Mudl

lacking

are not r e q u i r e d

flagellar

MCP m o l e c u l e s

called

of

examined

cheR&cheB+

stimuli,

in

and

is

for the a d a p t a t i o n p h a s e of c h e m o t a c t i c

states

PAGE

and

three

These

state

and

strains

flagellar

chemotactic

aberrant the

type

MCP m o l e c u l e s

exhibited

All in

adaptation.

deletion of

A

whereas

bias.

changes

essential

wild

responses.

cheR cheB

MCP methylation

The

deletion

A

rotational

appropriate

of

Ache22

functions,

rotation,

initiate

of

stimulus

and

flagellar CCW

construct and

+

of

defective

a c t i v i t i e s of the c h e R and c h e B gene

derivatives

enzymatic

patterns

state

not

product.

processing

methylation

utilized

were

tsr

activity,

depending

on

can

but w i l d

speed type

form, b e c a u s e MCPl*

the

is

in

rapidly

concentrations

569

Transmembrane Signaling Mutants of E.col i

of

attractants

24].

The

increase The

CW

analysis

deletions

this

type

else

nonsense

Missense

was

type.

By

be

an

cases

intrinsic

of

mutations. MCP's

we

and

cheR cheB as

be

in

the

such +

hosts,

¿

with

grossly

band

wild

hosts,

we

related

seemed In

those being was

patterns of

in

intrinsic

follows. the

null

phenotype

a gene p r o d u c t of wild about

contained half

Since to the

of

the they

missense

these

mutant

2* f o r m of could

the

not

1* f o r m , these

altered

in type

be

mutant

conformations

that

the c h e R or c h e B gene p r o d u c t s .

null-type with

missense a

fairly

to

of

complex

mention

that

to

not

capable

banding

resembling

conversion

have

of

mutants

MCP.

patterns

entire

One these

substitutions

cheR cheB

still

exhibited

type

probably of

not

comparison ¿

in

eliminated

bands

interaction

banding

the

single

undergo

type

of

that

In

wild

remainder

or

patterns.

early acid

in

were

synthesized

+

corresponding

preclude

amino

description

have

tsr

often

methylated proteins

proteins

assumed

nor

could

or c h e B - p r o c e s s i n g , but r a t h e r

we

migrated

with changes

in the d i s c u s s i o n

tests

and

of

product

that

apparent

synthesis

position

tar

rather

tsr m u t a n t s

gene

m i g r a t i o n d i f f e r e n c e s were

facilitate

mutants,

chemotaxis size,

MCP

mutant

mobility changes Mutants

proteins

became

mobility

these

the To

and

to

mutations

p r o p e r t y of the m u t a n t p o l y p e p t i d e s .

the

methylated,

tar

detectable

[23,

seem

but

more diverse banding

that

slight that

where

different

All

no

fusion

displayed

methylation

shifted.

or

with

interpretable,

patterns.

examining

to e i t h e r

mutants

that MCP m o l e c u l e s

demonstrated

MCP's

nonsense

MCP

easily

factor

showed

environment

methylated.

mutants

analyses

cells'

methylated

of

fragments

complicating

of

state.

synthesized

c h e B - p r o c e s s e d or

the

to their m e t h y l a t i o n

banding

either

in

properties

gave

uninformative,

often

repellents

in p r o p o r t i o n

SDS-PAGE

or

and

signaling

mutants normal

exhibited

The wild

representation

of

J.S. Parkinson, M.K. Slocum and A.M.

570 the

various methylation

more

subtle

signaling

defects

states.

that

functions,

These

probably

but

not

the

mutants

affect

Callahan

evidently

have

chemoreception

or

methyl-accepting

ability

CCW

bias

of

MCP. Dominant

tsr

mutants

characterized Since

in

these

mode.

mutants

cheB

activities

the

flagella.

methylation balance

of

the

lead

the the

from

of

the

In in

bypass

mutants

in

mutants

an

that

function

tsr

the

scrS

tsr p r o d u c t

further

properties genetic

explanation

was

and

for

the

for

to

of

is this

cancel

lines

of

system

identify

we

second-

defects

in

Parkinson,

in

the

revertants

from that

mutations

expected of

that

cheR

these

in

that i m p r o v e d

cheR enzyme.

studies

We

chemotactic

&

in a way

First,

to

system.

compensate

suppressor

biochemical

incorrect.

effort

defective

the

mutants

feedback

suppressor

altered

For

signal

hundred

initially

(designated the

CCW

a

[25].

[Sherris

We

MCP.

signal,

methylation

one-third

contained

locus.

of

that

a

and from

modulate

Several

such

could

cheR

to

attempt

MCP. of

several

mutations substrate

futile

in a

to m a i n t a i n

input

until

mutant

the

states

CCW

we

observed

control

seems

dominant a

might

have

a

of

in order

signaling

increases

Approximately

missense at

CW

states.

"locked"

feedback

system

existence

mutations

methyl transferase cheR

imbalance

were

behavior,

overmethylation

in a r e c e n t review

cheR

preparation].

an

receive

the

the

sort m u s t be

activity

characterized

suppressor

mapped

and

methylation

CW signaling

through

overmethylation

and

revertants

to

feedback

m u t a t i o n s -that

isolated site

due

counter-balanced. coming

MCP

this

about

CCW

supporting

MCP

be

flagella

have been summarized

4.

of

rotational

paradoxical

methylation

to

signal

evidence

high

generates

demethylation

MCP

effectively would

may

This

and

when

The

brought

between

example, level

normally

that m u t a n t . M C P ' s

signaling

a

abnormally

methylation

conclude CCW

by

with

tsr) its

However,

showed

that

this

found

that

scrS

Transmembrane S i g n a l i n g

mutations

did

mutation result

not

could

exhibit

be

implied

Second,

could

be

we

restoration

of

of

a

detected

no

increase

scrS

alternative the In

chemotactic in

which

the

cheR cheR

and

was of

on

scrS

gene

dependent

that

on in

possibility

that

been

the

Third,

MCP m e t h y l a t i o n

somehow

a

mutants

suggesting

not

the

had

point This

deletion

methyltransferase.

out

methyltransferase

dependent

mutations,

level

ruled

mutation.

the

ability

active

cheR

not

that

scrS

any

scrS

was

between by

partially

strains,

any

discovered

suppressed

presence

by

suppression

interaction

products. also

allele-specificity;

suppressed

that

stereospecific

571

M u t a n t s o f E .col i

we

cheR an

activated

in

revertants. cheR+

a

flagellar

background rotation

scrS

even

lower methylation

levels

mutations

some

are

but

with

tsr

protein;

an

properties scrS

opposite the

and

hunts.

It

is

suppression mutations

which

enable

modulating

have o p p o s i n g

unusually cheR

clear

opposing

a

studies

on

large

number

strains

a

to

CW

of

CW

of

to

mutant

and

on

to

to cheR

flagellar

pattern.

stimuli

MCP2*

cheD,

lead

scrS

effects

the

chemotactic

properties

swimming

scrS

signaling

unlike

on

that

to a d a p t

conversion

Thus

in c o n v e n t i o n a l

these

in

mutations,

behavior

have

However,

appears type

bias

exhibited

cheD

effects

additive

wild

strains

of

signaling

but

in

scrS

rate

of

how It

to

signaling seem

subtle

defects.

result

border.

deletion

the

undermethylated.

yet

cheR

Properties

genetic

on

a

products

type tsr p r o d u c t .

have b e e n o v e r l o o k e d

cheR

the

scrS

analogous

products

fairly

not

of have

rotation

5.

are

have

and w o u l d

than wild

effect

produced

the

respects

scrS

yet

mutants

ability,

might

in

mutations

though

This

simply

MCP1*,

by

which

properties.

cryptic the

tar

MCP

of d e l e t i o n s

Subsequent revealed

the

locus.

operon,

In

we

seemed

restriction existence

r e g i o n " b e t w e e n the tar and c h e R g e n e s .

the

to end mapping

of UV

course

of

that

an

noticed

a

1.8

at

the

tar-

of

Ache22

Kb

"spacer

programming

J.S. Parkinson, M.K. Slocum and A.M. Callahan

572 experiments protein, also

showed

which

tar

in

genes.

by

amount

The

cannot

of

be

several

A

mutants

in wild

type

strains;

to

tar a

aspartate coding

clear

by

homology

[26 , 27] .

are A

that

_tar _tap the

as

well not

strains

had

+

tap

The

probably

taxis.

Evidently

product.

appeared

between

to

the

We

have

tar

been

and

tap

t a r - t a p fusion p r o t e i n s

and

exhibited

and

maltose,

sequence

deleted.

divergence

underwent

related

they

that

synthesized

and

DNA sequence

and m a l t o s e

mediated

patterns,

protein" product

strains;

that

deletions

Kd

gene

duplication

found

65

strains.

recombination

methylation

demonstrate

We

a

tap

antigenically

in a s p a r t a t e

homologous

chemotaxis

are

indicate

function.

These

aberrant

tandem

products

defects

obtained

formed

cheR cheB

through

encoded

"taxis-associated

[26]. A

in

studies

responses

also

of

gene

our

severe

these

the

al,

it w a s m e t h y l a t e d

form

arisen

tap

but

homologous very

to

et

region

tar g e n e s share c o n s i d e r a b l e

have

and

[27],

2*

spacer

to a 1* form in c h e R A c h e B +

tap and

and m a y

Boyd,

an M C P :

as

conversion The

by

to be

migrated

the

corresponds

observed

appears

that

between

varying

depending

on

These

the

tar

with

degrees the

findings

and

tap

gene

products. To

investigate

chemotaxis,

what

we

mutants.

role,

isolated

To

do

if

any,

and

this,

looked

revertants

Several

revertants

that

had

obtained;

those

that

deletion

were

indistinguishable lack

of

failure S.

any to

from

detectable find

typhimurium

from

wild

tap m u t a t i o n s does

not

seem

for

tap::Mudl

type

mutant

tap

gene

characterized

we

chemotactic) such

the

lost

tap

retained

Mud_l tar

respects

phenotype

would

(i.e., mutants.

prophage

have

c l e a r that tap serves any useful p u r p o s e

a

tap in

by

function

were

tested.

The

account

in p r e v i o u s m u t a n t h u n t s . to

in

deletion

nonpolar

insertion

the

in all

plays

gene, coli.

it

for

the

Since is

not

573

Transmembrane Signaling Mutants of E.coli Conclusions These

preliminary

complexity

of

Mutations

their

notably

seem

to

we

form?

to

the

methylated,

function

in

separate yet

characterized correspond

of MCP

molecules

adds

possibility assemble

is

or

to

further

that

MCP2* was

The by

opposing

system

regulate

is

of

tsr,

not

its

different molecules;

mutations

to

segments

of

before of

stimuli, flagellar

its cheB

swimming of

MCP

no

in the

longer

the by

2* and This

used

revertants

by of

MCP

adaptation

2*

reversible subject

and

1*

Thus

to

forms

the

cell

controlling

the

1* forms col^;

2*

cytoplasmic

of is

rudimentary mutants

this

cheB-dependent

rotation.

behavior

processing.

second-site

the

advent

and

to

in the

nascent

the

reaction

effects

on

complexity

a primitive

the

this

post-translational

into

that

chemotactic

proportion

in

used rate

is p r o b a b l y

used

MCP

enables

inserted

mechanism

be

mutations,

synthesized

this

that be

to M C P 1 * r e p r e s e n t s

and

lost

and

discrete

initially

possibility

synthesis

the

methylation-independent

of

relative

have

sequences.

Another

could

some

these

enough

to

conversion

have

MCP

mutant in

alleles

that

of

membrane.

modulation

of

likely

but

Other

cheD

domains

arc MCP m o l e c u l e s

methylation.

different

in

molecules

activity.

It seems

in E^ coli.

defects MCP

signaling

not

of

to

be

proteins

the

of

dominant

domains

One

subunits

The

to e m p h a s i z e

to a v a r i e t y

the

such

discovery Why

reflect

chemoreception

have

the MCP coding

lead

MCP.

and

served

signaling

loci

ability

scrS

reside

processing

of

activity.

whether

story.

to

or

affect

activities

The

seem

the

chemoreceptor

know

tsr

their

signaling

most

however,

tar or

activities

retain

s t u d i e s have

transmembrane

the which

functional mutants

the

of

phenotypes

genetic

through

new

adaptation

however, lacking

it m a y the

MCP

methyltransferase.

The m o s t

important u n s o l v e d p r o b l e m

in the

field of

bacterial

574

J.S. Parkinson, M.K. Slocum and A.M. Callahan

chemotaxis signals

difficult structural with

concerns

modulated to deduce studies

genetic the way

nature

oE

the

MCP's.

It

the nature of

these

of

the

MCP m o l e c u l e s .

analyses,

structure-function pave

the

by

it

should

relationships

to e v e n t u a l

for

understanding

flagella-controlling

may

prove

exceedingly

s i g n a l s solely However,

be MCP

in

through

conjunction

possible

to

molecules

derive

that

of their role

in

may

signal

transduction.

Acknowledgements The a u t h o r s w i s h to thank Ruth F r e d r i c k for a s s i s t a n c e in p r e p a r i n g this m a n u s c r i p t . T h i s work was s u p p o r t e d by U.S. P u b l i c H e a l t h Service G r a n t G M - 1 9 5 5 9 from the N a t i o n a l I n s t i t u t e of G e n e r a l M e d i c a l S c i e n c e s . M.K.S. was s u p p o r t e d b y a U.S. Public H e a l t h S e r v i c e p r e d o c t o r a l t r a i n e e s h i p from the N a t i o n a l Institute of G e n e r a l M e d i c a l S c i e n c e s .

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3.

L a r s e n , S. H., R e a d e r , R. W., Kort, E. N., Tso, A d l e r , J.: N a t u r e L o n d o n 249, 74-77 (1974)

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M a c n a b , R. • W. , K o s h l a n d , D. E., Jr.: Proc. N a t l . Sci. USA ^ 9 , 2 5 0 9 - 2 5 1 2 (1972).

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B r o w n , D. A., B e r g , H. C.: Proc. Natl. Acad. Sci. 71, 1 3 8 8 - 1 3 9 2 (1974).

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B e r g , H. C., T e d e s c o , P. M.: Proc. N a t l . Acad. 72, 3235-3239 (1975).

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S p u d i c h , J. L., K o s h l a n d , D. E., Jr.: Proc. Natl. Sci. USA 12, 710-713 (1975).

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S p r i n g e r , M. S., Goy, M. F., A d l e r , J.: N a t u r e 279-284 (1979).

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B o y d , A., S i m o n , M. I.: J. B a c t e r i o l . ( 1980) .

L o n d o n 249,

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Transmembrane S i g n a l i n g

M u t a n t s o f E .col i

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Chelsky, D., Dahlquist, 77, 2434-2438 (1980).

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11.

D e F r a n c o , A. L., K o s h l a n d , D. E. , J r . : Sci. USA 77, 2429-2433 (1980).

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S p r i n g e r , W. R . , K o s h l a n d , D. Sci. USA 74, 533-537 (1977).

14.

S t o c k , J. B . , K o s h l a n d , D. E., J r . : S c i . U S A 75, 3 6 5 9 - 3 6 6 3 ( 1 9 7 8 ) .

15.

S p r i n g e r , M. S., G o y , M. F . , A d l e r , J r . : A c a d . Sci. U S A 7 4 , 3 3 1 2 - 3 3 1 6 ( 1 9 7 7 ) .

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S i l v e r m a n , M., Simon, M.: 3317-3321 (1977).

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K o n d o h , H . , B a l l , C. B . , A d l e r , J . : Sci. USA 76, 260-264 (1979).

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Silverman, ( 1977) .

M . , S i m o n , M . : J. B a c t e r i o l .

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Parkinson,

J. S.: J. B a c t e r i o l .

20.

P a r k i n s o n , J. S . , H o u t s , p r e s s (1982).

21.

Parkinson,

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R e a d e r , R. W . , T s o , W . - W . , S p r i n g e r , M. S . , G o y , M. A d l e r , J . : J. G e n . M i c r o b i o l . Ill, 3 6 3 - 3 7 4 ( 1 9 7 9 ) .

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S h e r r i s , D., P a r k i n s o n , 78, 6051-6055 (1981).

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Rollins,

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P a r k i n s o n , J. S.: G e n e t i c s of B a c t e r i a l C h e m o t a x i s . In: S o c i e t y for G e n e r a l M i c r o b i o l o g y S y m p o s i u m 31: G e n e t i c s a s a T o o l in M i c r o b i o l o g y (S. W. G l o v e r , D. A. H o p w o o d , Eds.), Cambridge University Press (1981).

26.

Boyd, A., Krikos,

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W a n g , E. A . , M o w r y , J r . : J. B i o l . C h e m .

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(1981). D.

E.,

576

J.S.

P a r k i n s o n , M . K . Slocum and A . M .

Callahan

DISCUSSION Boos: Does the attractant or repellent have to be present to maintain the response in a ahe R mutant? Parkinson: Yes. ahe R mutants stop responding as soon as the stimulating chemical is removed. As long as it is present, however, they respond without adapting. Macnab: What do you think provides the tCP-specificity of the postulated feedback mechanism during adaptation? Parkinson: I think there must be specific conformational changes in the MCP molecules engaged in signalling, that alter their substrate properties for the methyltransferase and methylesterase enzymes. Macnab: You are postulating protein-protein interactions between MCP I and II (for example). Given the distance between the 2 genes on the chromosome would you not then expect a gene dosage effect on the probability of complex formation? Parkinson: Not necessarily. It is not inconceivable that the fCP mRNA's are actually translated in close proximity so their products could form complexes as they are synthesized. Hcwever, even if dosage effects did occur, they might have no easily observable behavioral consequences. Dahlquist: A carment: We observe ooirplete lack of specificity of methylation in a ahe B cell. This suggests that methylation is new specific and that the specificity may reside in the rrethylesterase. Schweizer: You described the cryptic mutant in tor which is completely ohe~ in the tsr background. According to your idea this altered tar product needs the interaction of the tsv and tar products with their -COOH ends to exert function. Is this interaction dependent on the presence of serine or aspartate or both? Parkinson; I do not think so. The mutants behave the same way in either the presence or absence of these cortpounds. Voordouw: Is there a physiological logic to the fact that maltose and serine are attractants and leucine is a repellent? Parkinson: Yes. E.ooli is attracted to compounds such as sugars and amino acids, or their structural analogs, that are potential carbon and energy sources. These substances have detection thresholds in the micrcmolar range. Leucine and other hydropholic amino acids repel cells at high concentration, in the millimolar range. At these levels they can impair the cells' growth, perhaps due to seme deleterious effect on the cytoplasmic membrane. This must be why the cells are repelled by such potentially noxious cctrpounds.

Transmembrane S i g n a l i n g

Mutants of

E.coli

577

Manson: Can de CCW bias of star strains be explained by residual polarity on the che genes of the tar-che Z operon? Parkinson: I think not. We have examined many different tar deletion strains, at least sane of which should be nonpolar, and all had the same behaviour. Their behaviour does not change if we supply a A transducing phage that furnishes fully active copies of the dcwnstream genes in the tar operon. Manson: If MCP's interact within the membrane, shouldn't there be an effect of tsr or tar gene dosage or level of gene expression on seme aspect of signalling or adaptation? Parkinson: This is possible; however, even if such interactions do occur, it is not obvious that they are essential for proper signalling, so changing the stoichicmetry of the system might have little or no effect on behaviour.

INDEX OF CONTRIBUTORS Ackers, G.K.

63,210,237*,

332 Adam, G.

195*

Haiml, L. 49 6

Akeroyd, R. Ausio, J.

317*

von Hippel, P.H. 281*

1 1 6, 1 34, 1 71 , 21 1 ,21 3*, 278, Houts, S.E.

448 291*

Bennett, Jr., W.S.

21*

563*

Huber, R.

21*,79,101

Hucho, F.

80,134,263,393*

Berg, H.C.

469,485*,515,516

Hutticher, A.

Bond, M.W.

533*

Jaenicke, R.

Boos, W.

Bosing-Schneider, R. Boyd, A.

Jovin, T.M.

372

332,372,407,426,446,480,49 7

551*

Bradbury, E.M.

173*,210,279,

332,390 Brass, J.M.

563* 389

Corin, A.F.

Kennedy, C.

335*,372

485* 357*

Knippers, R.

23 6

Koltmann, A.

333

Konings, W.N.

119?,263,278

Dahlquist, F.W.

533*

Kijne, J.W.

65,103*,152

Callahan, A.M.

Kehry, M.R. Khan, S.

447,530

Cheney, C.M.

20,65,78,119*,171,

209,234,251,263,281*,316,

170,211,372

Bryant, R.G.

471* 18,19,47,67*,100

116,154,211,278,279,356,427

236,530,562,576

Bosma, H.J.

19,46,64,65,

291*,332,390,426,469,548

19 5*

Bear, D.G.

471*

Hawley, D.K.

2 53*

Arndt-Jovin, D.J. Bade, E.

Haik, Y.

117,529,533*,

Kreil, G.

170,445,446,471*

Krikos, A.

576

449*

551*

170,235,375* , 515 Kroneck, P. 101 Lageveen, R.G. 449* 253*

Dedman, J.R.

van Deenen, L.L.M. Eisenberg, H.

Engelhardt-Altendorf, D. Fairfield, F.R. Ghisla, S.

213*

18,19

Gregory, R.B.

Lauffer, L.

195*

49*

562

Lengeier, J.W. Macnab, R.M.

517* 100,134,426,470,

480,496,499*,530,562,576 Manson, M. 562,577

*Symposium paper

393*

373,497,515,548,

Index o f

580 Massey, V.

3*,101,153

Matayoshi, E.D. Mazurek, N.

119* 46,47,116,210,

235,317*,356,480,530,548 Mollay, C.

83*

Muhn, P.

83*

Simon, M.

137*

Munjaal, R.P. Mutoh, N. Neupert, W.

375*

Pecher, A. Pecht, I.

Sund, H.

56 3*

517* 18,47,79,133,134,

Pohl, F.M. Poolman, B. Renner, I.

137*

65,235,267* 449*

551* 563*

47,80,153,252,264, 80,101,194,236,263,

372,406,426,446,447,480,497 471*

Visser, A.J.W.G. Voordouw, G.

21 1 ,445,481 ,516, 576 Westerman, J.

293*

Wirtz, K.W.A.

80,171,253*,389,

281* 48,133,154,

Xavier, A.V. Zarling, D.A.

Zidovetzki, R. 264, 279, 31 6,407,449*, 497 , 529 Robson, R.L.

20,137*

4 6,64,81,154,193,

407,446

517*

Robert-Nicoud, M. Robillard, G.T.

563*

Veeger, C. Vilas, U.

155*,279,389,409* Penners, N.H.G.

237*

529

48,279,427,429*

Parkinson, J.S.

447,480,576

Slocum, M.K.

551*

357*

19 5*

Sherris, D.

393*

Müller, F.

119*

Schweizer, H. Shea, M.A.

Moura, J.J.G.

409*

van der Schaal, I.A.M. Seger, D.

471*

Moura, I.

19,49*,81,496

Sagi-Eisenberg, R. Sawyer, W.H.

409*

McClure, W.R.

Rosenberg, A.

Contributors

335*

20,83*,171 281* 119*

SUBJECT

INDEX

Acetylation of histones 190 Acetylcholine receptor 407 ADP/ATP carrier

39 3 -

434-442

A g g r e g a t i o n of p r o t e i n s 73 Allergy

176-

72,

B-Z transition Calcium

267-279

Anaesthetics

555-557

405

Antagonist/agonist n a t i o n 404

discrimi-

CheB dependent modification (chemotaxis) 5 3 3 - 5 4 9 517-531

485-5 77

Chromatin structure 189,197-200,204

175,187-

Chromosomal proteins

against Z-DNA 269-272, 282-288 h a p t e n interaction 159-168 Arginine modification

258,264

A s s o c i a t i o n of p r o t e i n s

72-75

Bacterial chemotaxis 533-549

517-531,

Bacteriophage

237-252

lambda

282

Binding cooperativity

Citrate synthetase 48 Conformational 49-51

Cromoglycate 419-423

284,286

38-43,46-

fluctuations

Correlation times

Cytochrome c 216-222

174-194

Chromosomes, polytene

112

(DSCG)

Crystallography of 21-48

124-128

Base modification

55

Che protein 507,5 34-546,565, 568-573,576

Chemotaxis

155-171

Band 3 protein

375-390

Chemosensors

Anisotropy decay a n t i b o d i e s 157-159 p r o t e i n s 137-154

503,504

375-390

Catalyst penetration

Amino acid sequence (chemotaxis) 545,555-558 transducer proteins

268,269,288

b i n d i n g to f l a g e l l a fluxe 409-427 t r a n s p o r t 46 2

mRNA

Allosteric DNA

56

375,376,388,409-427

Calmodulin

414

Antibodies

Bond percolation

414-416, proteins

438-442,445,446

Subject

582 Disorder in proteins 28-31 Dipeptidyl aminopeptidase 475-477 Dithiol-disulfide interchange 449-470 DNA, allosteric 26 7-279 protein interaction 237-252, 291-316

Index

Phosphotransferase Pyruvate dehydrogenase Ribonuclease RNA polymerase Signal peptidase Transhydrogenase Trypsin Trypsinogen Tryptophan synthase Enzymes II 517-531

Domains in proteins 6 7,71

Epidermal growth factor 128131

Domain motion in proteins 31-43

Ethidium bromide 270-27 2,275

Dynamics of cell surface 119134 of gene regulation 237-252 Electrochemical proton gradient 449-470 Electron paramagnetic resonance of iron sulfur proteins 83,91,93,95 Electron transfer proteins, related to nitrogen fixation 336 Electron transferase 5 Enzymes see Citrate synthetase Cytochrome c Dipeptidyl aminotransferase Electron transferase Enzymes II Ferredoxin Flavodoxin p-hydroxybenzoate hydroxylase Lactate dehydrogenase Lysozym Malate dehydrogenase Multienzyme complexes Nitrogenase Oxidase Oxygenase

FAD 14 7,149 Fe protein 343,356 [3Fe-3S] centres 83-101 [4Fe-4S] centres 83-101 Ferredoxin 83-101 Flagella, rotation of bacterial 485-497 Flagellar-rotary motor 485497,499-516,563-577 switching 499-516 Flavin hydroperoxides 12-15, 19,20 oxygen reactions 13-15 sulfite adducts 10,18 Flavodoxin 6 Flavoproteins 3-20,139,144151,153,154 classification 5,18,19 Flexibility of antibodies 155-159 of chromosomal proteins 174-194 of proteins 21-48,80,137151,155-159

Subject

583

Index

Fluorescence decay of proteins 148 Folding of protein 67-71,81 Gene 32 protein 236

215-232,235,

Gene regulation 237-252 Glucose transport

450,452-460

Glutathione maleimide 461 Heteroprotein assembly 232,234 Histamin release 413 Histones

174-194,196-211

acetylation 176-190 DNA interactions 182-185, 196-211 -histone interactions 196211 HMG proteins 1 and 2: 185, 186,193 14 and 17: 186-189 Hydrogen bond 6,305-309 rearrangements 56

Ion channel 393-407 fluxes 409-427 Iron-sulfur proteins 83-101 EPR 83,91,93,95 Mössbauer spectra 84,86,88, 96 Kinetics of hydrogen isotope exchange 49-65 lac operator 29 2-303 repressor 297-304 Lactate dehydrogenase 79,80 Lactose transport 450 Lectins

bond percolation 56 temperature dependence 58 viscosity dependence 60-6 2

359-362

recognition

359-362,367

Legumes, symbiosis with Khizobium 358-373 Ligand binding

393-427

Lipid binding site 257-261 Local unfolding of proteins 55 Lysozym

Hydrogen exchange

107-112,116

Magnetic relaxation

MCP mutants 577

Hydrophobicity

Melittin 471-481

p-hydroxygenzoate hydroxylase 139,144-151,153,154 Immunofluorescence

283-288

Immunoglobulin 31-37,47 Interconversion between ironsulfur centres 83-101

105-112

Malate dehydrogenase 7 5

Hydrogen isotope exchange, kinetics of 49-65 254

74,75,

(Chemotaxis) 56 3-

Membrane potentials 410-414 Methylation (Chemotaxis) 533549 ,552,557,570-574 Mitochondria precursor proteins 4 31-442, proteins 429-448 445

Subject

584 Mobile defect hypothesis Mobility of biopolymers Modulator

56 3-134

322-325,328

MoFe protein

Photoactivable analogues of p h o s p h a t i d y l c h o l i n e 257-261 poly(dG-dC)

268-279

B-Z transition

336

Index

268,269

Polytene chromosomes

284,286

M ô s s b a u e r s p e c t r a of i r o n sulfur proteins 84,86,88,96

Post-translational processing in c h e m o t a x i s 568

Multienzyme complexes

Precursor proteins 445

nif gene r e g u l a t i o n

76

345

Prepromelittin

s t r u c t u r e 3 37 Nitrogen fixation Nitrogenase

335-356

179—

175

Nucleic acid-protein a c t i o n s 213-236 Oligomeric enzymes

inter-

67-81

Operators 222-225,241-248 see a l s o lac o p e r a t o r Oxidase 5 Oxygenase 5 Phosphatidylcholine p r o t e i n 253-264

transfer

binding site 256,257 c o n f o r m a t i o n 255,256 ionic interaction 256,257 p r i m a r y s t r u c t u r e 253-264 Phosphorescence of 119-134

proteins

Phosphorylation/dephosphoryl a t i o n i n c h e m o t a x i s 517-531 Phosphotransferase 517-531

471-481 387,388

Prolin isomerization

335-348

N M R s p e c t r a of h i s t o n e s 187,193 Nucleosomes

Proliferation

431-442

system

transport Promoter 333

70

460-463

29 3 , 3 0 4 - 3 0 9 , 3 1 7 -

g e o m e t r y 325 m u t a t i o n s 309-312 o v e r l a p p i n g 328 recognition by RNA r a s e 317-333

polyme-

Proteins see a l s o Acetylcholine receptor Antibodies Band 3 protein Calmodulin Che protein Chromosomal proteins Fe p r o t e i n G e n e 32 p r o t e i n Histones HMG proteins Melittin Phosphatidylcholine transfer p r o t e i n Prepromelittin Ribosomal proteins Signaling proteins Tobacco mosaic virus a n i s o t r o p y d e c a y 137-154 a r g i n i n e m o d i f i c a t i o n 258, 264

Subject

Index

Proteins

585

...

a s s o c i a t i o n 72-75 b i o s y n t h e s i s 213-236 c i s - t r a n s i s o m e r i z a t i o n 70, 75, 79 conformational fluctuation 49-51 c r y s t a l l o g r a p h y 21-48 d i s o r d e r 28-31 d i s s o c i a t i o n 73,74 d o m a i n s 6 7,71 d o m a i n m o t i o n 31-43' dynamics 49-65,103-117 flexibility 21-48,80,137-151 f o l d i n g 67-71,81 f l u o r e s c e n c e 138-151 f l u o r e s c e n c e d e c a y 144-151 high pressure dissociation 73,74 l i g a n d b i n d i n g 393-427 local u n f o l d i n g 55 modulators 322-325,328 n u c l e i c a c i d i n t e r a c t i o n 213— 252 p r o l i n i s o m e r i z a t i o n 70 p r o t e i n a s s o c i a t i o n 137-154, 196-211 r e c o n s t i t u t i o n 72-74 refolding 68,80 r o t a t i o n 104 self o r g a n i z a t i o n 67-81 s t r u c t u r e 21-48 s u r f a c e m o t i o n 113,114 transfer 429-448

Repressors see a l s o lac

237-245

repressor

Response regulator model (Flagella) 5 0 4 - 5 0 6 Rhizobium-legume 358-373 r e c o g n i t i o n 36 8 Ribonuclease

69-71

Ribosomal proteins 235 Ribosome assembly

Rotation, bacterial 485-497

359-362

Reconstitution of proteins 74

72-

R e f o l d i n g of p r o t e i n s 6 8 , 6 9 , 8 0 R e g u l a t i o n of g e n e s of n i f gene

345

237-252

times

S e l f - o r g a n i z a t i o n of 67-81

protein

Sensory transduction

551-562

Solute-solvent 104

R e c o g n i t i o n of l e c t i n s

358

flagella

Rotational correlation 143,148

Pyruvate dehydrogenase 128-131

225-232

R o o t h a i r s of R h i z o b i u m

Signal peptidase

Receptors

225-232,

R N A p o l y m e r a s e 2 4 1 - 2 4 5 , 2 9 5, 303-305,311,317-333

P r o t o n m o t i v e force of f l a g e l l a r r o t a t i o n 490-49 4,49 6 , 5 0 1 - 5 0 7 , 511 76

symbiosis

472,473

Signaling proteins

563-577

interaction

Stochastic process 504,508,509

(Flagella)

S u p e r o x i d e a n i o n 15 Surface, dynamics of cell 119-134 motion

113,114

T4 b a c t e r i o p h a g e 236

215-232,235,

586

Subject

Taxis 509

Trypsin 24,48

Thorin 454

Trypsinogen 24,46,48

Tobacco mosaic virus protein 76

Tryptophan synthase 76

Index

Transhydrogenase 5

Tumbling (Flagellar motor) 508

Translational operator 222225

Viscosity effects on hydrogen exchange 60-6 2

Transport of calcium 462 glucose 450,452-460 lactose 450 proline 460-46 3 proteins 449-470

Water surface dynamics 103117

Triphenylmethylphosphonium ion (TPMP+) 395-405,407

Z-DNA 267-279,282-288 antibodies 269,279

w DE

G H. Sund G. Blauer (Editors)

Walter de Gruyter Berlin-New York Protein-Ligand Interactions Proceedings of a Symposium on Protein-Ligand Interactions. Konstanz, West Germany, September 1974. 1975.17 cm x 24 cm. XVI, 486 pages. Numerous illustrations. Hardcover. DM 145,-; approx. US $72.50 ISBN 311 004881 7

H. Sund (Editor)

Pyridine Nucleotide-Dependent Dehydrogenases Proceedings of the 2nd International Symposium on Pyridine Nucleotide-Dependent Dehydrogenases. Konstanz, West Germany, March 28-April 1,1977. FEBS Symposium No. 49 1977.17 cm x 24 cm. IV, 529 pages. Numerous illustrations and tables. Hardcover. DM 155,-; approx. US $77.50 ISBN 311 007091X

G. Blauer H. Sund (Editors)

Transport by Proteins

Proceedings of a Symposium on Transport by Proteins. Konstanz, West Germany, July 9-15,1978. FEBS Symposium No. 58

1978.17 cm x 24 cm. XV, 420 pages. Numerous figures and tables. Hardcover. DM 145,-; approx. US $72.50 ISBN 311007694 2

D. Pette (Editor)

Plasticity of Muscle

Proceedings of a Symposium on Plasticity of Muscle. Konstanz, West Germany, September 23-28,1979. 1980.17 cm x 24 cm. XXVI, 625 pages. Numerous figures. Hardcover. DM 160,-; approx. US $80.00 ISBN 311007961 5

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w DE

G W. Pfleiderer (Editor)

Walter de Gruyter Berlin-New York Chemistry and Biology of Pteridines Proceedings of the 5th International Symposium on Chemistry and Biology of Pteridines. Konstanz, West Germany, April 14-18,1975. 1975.17 cm x 24 cm. VII, 949 pages. Numerous illustrations. Hardcover. DM 190,-; approx. US $95.00 ISBN 311 005928 2

K. Keck P. Erb (Editors)

Basic and Clinical Aspects of Immunity of Insulin Proceedings of an International Workshop on Basic and Clinical Aspects of Immunity of Insulin. Konstanz, Germany, October 1980. 1981.17 cm x 24 cm. XIV, 442 pages. Numerous illustrations. Hardcover. DM 140,-; approx. US $70.00 ISBN 311 0084406

H. Wächter H. Ch. Curtius W. Pfleiderer (Editors)

Biochemical and Clinical Aspects of Pteridines Volume 1 Cancer • Immunology • Metabolic Diseases 1982.17 cm x 24 cm. XVI, 373 pages. Numerous illustrations. Hardcover. DM 150,-; approx. US $75.00 ISBN 311 008984 X

J. A. Blair (Editor)

Chemistiy and Biology of Pteridines Proceedings of the 7th International Symposium on Pteridines and Folic Acid Derivatives. St. Andrews, Scotland, September 21-24,1982. 1983.17 cm x 24 cm. About 900 pages. Numerous illustrations. Hardcover. Approx. DM 200,-; approx. US $100.00 ISBN 311008560 7 (To appear in spring 1983)

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