COLD SPRING HARBOR SYMPOSIA ON QUANTITATIVE ......COLD SPRING HARBOR SYMPOSIA ON QUANTITATIVE BIOLOGY VOLUME XLI Origins of Lymphocyte Diversity COLD SPRINC HARBOR LAHOI{ATOHY 1977

COLD SPRING HARBOR SYMPOSIA

ON QUANTITATIVE BIOLOGY

VOLUME XLI

Origins of Lymphocyte Diversity

COLD SPRINC HARBOR LAHOI{ATOHY

1977

PFIZER EX. 1616 Page 1

COLD SPRING HAHBOH SYMPOSIA ON (~UANTITATIVE BIOLOGY VOLUME XLI

"J 1977 by The Cold Spring Harbor Laboratory

International Standard Book Number 0-879G9-040-2 (clothbound)

Library of Congress Catalog Card Number 34-8174

Printed in the United States oj'Amerim

All riuhts reserved

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! v. L/ I

,. I I 'I' >) fl NATIONAL LIBRARY fJr Mf.OICINE

~ETHESDA, MARYLAND 20014

Follll!!nl in J

Symposium Participants Foreword

Introduction

Contents

Part l

The Common Sense of Immunology N. K. Jerne

LYMPHOCYTE FUNCTION

1'-cell Marlwrs and Differentiation

'l'hymopoietin and Bursopoietin: Induction Signals Regulating Early Lympho-cyte Differentiation G. Goldstein, M. Scheid, E. A. Boyse, A. Brand and D. G. Gilmour

Normal and Neoplastic Maturation of 'I'- lineage Lymphocytes /. L. Weissman, S. Baird, R. L. Gardner, V. E. Papaioannou and W. Raschke

Regulation of Cellular and Humoral Immune Responses by '!'-cell Subclasses H. Cantor and E. A. Boyse

Surface Markers and Functional Relationships of Cells Involved in Murine B-lymphocyte Differentiation L. A. Herzenberg, L. A. Herzenberg, S. J. l3lacli, M. R. Lolwn, K. Olwmura, W. van der Loo, B. A. Osborne, D. Hewgill, -1. W. Goding, G. Gutman and N. L. Warner

An Unusual Kappa Immunoglobulin Antigen Present on the Membrane ofT and B Lymphocytes A. B. Gottlieb, M. Engelhard, H. G. Kunlwl and S. M. Fu

Rat 'I'hy-1 Antigens from Thymus and Brain: Their Tissue Distribution, Purifica-tion, and Chemical Composition A. F. Williams, A. N. Barclay, M. Letarte-Muirhead and R .. ]. Morris

Specialized DNA Polymerases in Lymphoid Cells D. Baltimore, A. E. Silverstone, P. C. Kung, 1'. A. Harrison and R. P. McCaffi·ey

Studies on the Interactions between Viruses and Lymphocytes B. R. Bloom, A. Senih, G. Stoner, G. Ju, M. Nowalwwshi, S. Kano and L. Jimenez

Helper and Suppressor 1' Cells and Their Products

The Hermaphrocyte: A Suppressor-Helper 'I' Cell R. K. Gershon, D. D. Eardley, K. F. Naidorf and W. Ptah

Suppressor 'I' Cells in Tolerance to Non-self and Self Antigens A. Basten, R. Lob-lay, E. Chia, R. Gallard and H. Pritchard-Briscoe

Tolerance: Two Pathways of Negative lmmunoregulation in Contact Sensitivity to DNFB H. N. Claman, S. D. Miller and J. W. Moorhead

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viii CONTENTS

Current Concepts of the Antibody Response: Heterogeneity of Lymphoid Cells, Interactions, and Factors M. Feldmann, P. Beverley, P. Erb, S. Howie, S. Kontiainen, A. Maoz, M. Mathies, I. McKenzie and J. Woody

Suppressive and Enhancing T-cell Factors as /-region Gene Products: Properties and the Subregion Assignment 7'. Tada, M. Taniguchi and C. S. David

B-cell Differentiation and Commitment

In Vitro Studies on the Generation of Lymphocyte Diversity .}. J. T. Owen, R. K. Jordan, J. H. Robinson, U. Singh and H. N. A. Willcox

Studies of Generation of B-cell Diversity in Mouse, Man, and Chicken M.D. Cooper, J. F. Kearney, P. M. Lydyard, C. E. Grossi and A. R. Lawton

Ontogeny of Murine B Lymphocytes: Development of Ig Synthesis and of Reac-tivities to Mitogens and to Anti-Ig Antibodies F. Melchers, .J. Andersson and R. A. Phillips

Development and Modulation of B Lymphocytes: Studies on Newly Formed B Cells and Their Putative Precursors in the Hemopoietic Tissues of Mice M. C. Ra(f

Induction of Immunoglobulin Synthesis in Abelson Murine Leukemia Virus-transformed Mouse Lymphoma Cells in Culture B. J. Weimann

The Interplay of Evolution and Environment in B-cell Diversification N. R. Klinman, N. H. Sigal, E. S. Metcalf, S. K. Pierce and P. J. Gearhart

Synthesis of Multiple Immunoglobulin Classes by Single Lymphocytes B. Per-nis, L. Forni and A. L. Luzzati

Immunoglobulin Receptors on Murine B Lymphocytes E. S. Vitetta, J. Cambier, J. Forman, J. R. Kettman, D. Yuan and J. W. Uhr

Functional and Structural Characterization of Immunoglobulin on Murine B Lymphocytes R. M. E. Parhhouse, E. R. Abney, A. Bourgois and H. N. A. Willcox

Origin and Differentiation of Lymphocytes Involved in the Secretory IgA Re-sponse J. J. Cebra, P .• ]. Gearhart, R. Kamal, S. M. Robertson and J. Tseng

Mechanism of B-cell Activation and Self-Non-self Discrimination G. Moller Growth and Maturation of Single Clones of Normal Murine T and B Lymphocytes

In Vitro J. Andersson, A. Coutinho, F. Melchers and T. Watanabe Hapten-specific B Lymphocytes: Enrichment, Cloning, Receptor Analysis, and

Tolerance Induction G. J. V. Nossal, B. L. Pihe, J. W. Stoclwr, .]. E. Layton and J. W. Goding

Regulation of Clonal B-lymphocyte Proliferation by Anti-immunoglobulin or Anti-Ia Antibodies P. W. Kincade and P. Ralph

Cellular and Molecular Interactions in Control ofB-cell Immunity and Tolerance E. Diener, C. Shiozawa, B. Singh and K.-C. Lee

Receptors

Lymphocyte Surface Immunoglobulins: Evolutionary Origins and Involvement in Activation J. J. Marchalonis, .}. M. Decher, D. DeLuca, .}. M. Moseley, P. Smith and G. W. Warr

Antigen-binding, Idiotypic Receptors from T Lymphocytes: An Analysis of Their Biochemistry, Genetics, and Use as Immunogens To Produce Specific Im-mune Tolerance H. Binz and H. Wigzell

On the Structure of the T-cell Receptor for Antigen U. Krawinhel, M. Cramer, C. Berch, G. Hiimmerling, S. J. Blach, K. Rajewslly and K. Eichmann

The Immune Response to Staphylococcal Nuclease: A Probe of Cellular and Humoral Antigen-specific Receptors D. H. Sachs, J. A. Berzofshy, C. G. Fathman, D. S. Pisetshy, A. N. Schechter and R. H. Schwartz

Functional Characterization of Rabbit Lymphocytes Carrying Fe Receptor P.-A. Cazenave, D. Juy and C. Bona

Structural and Functional Heterogeneity of Fe Receptors H. M. Grey, C. L. Anderson, C. H. Heusser, B. K. Borthistle, K. B. Von Eschen and J. M. Chiller

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201 217

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CONTENTS

THE MAJOR HISTOCOMPATIBILITY COMPLEX

Structure of the Molecular Products of the MHC

Structure of HL-A A and B Antigens Isolated from Cultured Human Lympho-cytes J. L. Strominger, D. L. Mann, P. Parham, R. Robb, T. Springer and C. Terhorst

Structural Studies of (32-Microglobulin-associated and Other MHC Antigens P. A. Peterson, H. Anundi, B. Curman, L. Klareslwg, S. Kvist, L. Ostberg, /,. Rash, L. Sandberg and K Sege

Comparative Chemical Analyses and Partial Amino Acid Sequences of the Heavy Chains of HL-A Antigens E. Appella, N. Tanigalli, 0. Henrillsen, D. Press-man, D. F. Smith and T. Fairwell

Structul'al Differences between Parent and Variant H-2K Glycoproteins from Mouse Strains Carrying H-2 Gene Mutations S. G. Nathenson, .]. L. Brown, B. M. Ewenstein, T. Nisizawa, D. W. Sears and J. H. Freed

Structure of Murine Histocompatibility Antigens B. A. Cunningham, R. Hen-ning, R. J. Milner, K Resile, J. A. Ziffer and G. M. Edelman

Structural Studies of H-2 and TL Alloantigens J. W. Uhr, E. S. Vitetta, J. Klein, M. D. Poulih, D. G. Klapper and ,J. D. Capra

Chemical Characterization of Products of the H-2 Complex J. Silver, J. M. Ceclw, M. McMillan and L. Hood

Human Ia Antigens-Purification and Molecular Structure D. Snary, C. Bam-stable, W. F. Boclrner, P. Goodfellow and M. ,], Crumpton

Chemiral and Immunological Characterization of l-IL-A-linked B-lymphocyte Alloantigens 7'. A. Springer, J. F. Kaufman, L. A. Siddoway, M. Giphart, D. L. Mann, C. Terhorst and J. L. Strominger

The Guinea Pig MHC: Functional Significance and Structural Characterization B. D. Schwartz, A.M. Kash and E. M. Shevach

Partial Amino Acid Sequences of MHC Products J. Silver Analysis of Lymphocyte Surface Antigen Expression by the Use of Variant Cell

Lines R. Hyman and I. Trowbridge

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331

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343

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387

397 405

407

lX


Model-building Studies of Antigen-binding Sites: 'The 1-lapten-binding Site of MOPC-315

E. A. PADLAN, D. R DAVIES, I. PECHT,* D. GIVOL* AND c. WRIGHT"!" Laboratmy of" Molecular Biology, National Institute of" Arthritis, Metabolism and Digestive Diseases, National Institutes of" Ilmlth, Bcthesd~r, J'vfm:yland 20014; ·''Department of" Chcmiml Immunology, The Weizmann Institute of" Science, Relwvot

Ismel; T Laborat01:y oj"J'v!olccular Biophysics, Department of" Zoology, Oxford University, Oxf'ord, England '

The molecular basis for the structural diversity of antibody combining sites has become apparent through the recent X-ray diffraction studies on several immunoglobulin

628 PADLAN ET AL.

(2 em = 1 AJ molecular models mepetition Engi-neers, Cambridge, England).

The general principle used for model building was to c.onstruct first the framework part of the variable regiOn, b.ased on the structure of 603 Fab'. The hypervanable loop~ were then constructed, leaving the structure as l~ttle changed as possible except when forced by ammo acid insertions and deletions

An attempt was made to maximize the structurai stability within eacl~ loop by forming hydrogen bonds ";hene~er possible and maintaining the phi and psi peptide angles within reasonable limits (Ramaknshnan and Ramachandran 1965 ). The interactions between loops were then maximized leaving no large holes in the domain interior whil~ minimizing sterie hindrance between groups:

The L1 regions in the kappa chains 60:3 and REI a:e simple lo_ops, . whereas the corresponding re-gwns are hehcal m New (Poljak ct a!. 1974) and Meg (Schiffer et a!. 1973), which are both lambda chains. Since the light chain of protein a 15 is of the lambda type and since this region has the same n.umber of r.esidues as New and Meg with no gross differences m the nature of the amino acid side chains, it is most likely that this region in 315 will have a similar configuration to that found in New and Meg. Accordingly, the L1 loop in :315 was built to conform as closely as possible to the correspond-ing region in the lambda chains with the aid of the ato~ic coordinates of the Meg backbone kindly provided by M. Schiffer and coworkers ( pers. comm.).

L2 was built by assigning to it the same back-bone conformation as in protein 603. In this region, 603 and REI are not significantly different rPadlan and Davies 1975). Moreover, kappa and lambda light chains have the same number of residues in this part of the molecule may hoff 1972); an excep-tion is New ('fable 1b), which has a seven-residue deletion in this part of the molecule. The L2 region in Meg appears to have the same structure as 603

Table I. Amino Acid Sequences of V 11 Domains of Proteins McPC-603 and MOPC-:315

McPC-GO:l MOI'C-:l15 New

(a) Ali;;nment of V11 Sequences

0 2 0 4 0 2 0 4 0 0 1 Glu-Val -Lys-Leu-Val-Glu-Ser-Gly-Gly -Gly 1 Asp- Val -Gin-Leu -Gln-Glu -Ser -Gly -Pro -Gly I Pea- Val -Gin-Leu -Pro-Glu -Ser -Gly -Pro -Glu

0 4 0 0 0 0 0 4 0 4 ll Leu- Val -Gin-Pro -Gly-Gly -Ser -Leu-Arg -Leu II Leu-Val-Lys-Pro-Ser-Gln-Ser-Leu-Ser -Leu 11 Leu- Val -Ser -Pro -Gly-Glx -Thr-Leu-Ser -Leu

0 4 0 4 014 0 4 0 21 Ser -Cys -Ala-'I'hr -Ser -Gly -Phe-Thr-Phe -Scr 21 Thr-Cys -Ser-Val-'I'hr-Gly-Tyr-Ser -Ile -Thr 21 Thr -Cys -Thr-Gly -Ser -Thr- Val-Ser -Thr -Phe

0 4 2 4 2 4 c 3 c 31 Asp- -Phe-'I'yr -Met-Glu -Trp- Val-Arg -Gin 31 Ser -Gly -Tyr-Phe-Trp-Asn -'l'rp-Ile -Arg -Gin 31 Ala- -Val-Tyr-Ile -Val-Trp-Val-Arg-Gln

2 0 0 0 0 c 2 c 4 4 40 Pro -Pro -Gly-Lys -Arg-Leu-Glu-Trp-Ile -Ala 40 Phe -Pro -Gly-Asn-Lys-Leu -Glu -Trp -Leu -Gly 40 Pro -Pro -Gly-Arg-Gly-Leu -Glu-Trp-Ile -Ala

4 2 0 0 0 0 0 2 0 50 Ala -Ser -Arg-Asn-Lys-Gly -Asn-Lys -Tyr -Thr 50 Phe- lie -Lys-Tyr -Asp-Gly- -Ser 50 'l'yr- Val -Phe-'l'yr -His -Gly- -Thr

2 0 3 0 0 4 0 01 filibThr-Glu -Tyr-Ser -Ala-Ser - Val-Lys -Gly -Arg 57 Asx- !Tyr , Gly) Asx -Pro - Ser -Leu- Lys- Asn - Arg 57 Ser -Asp -Thr-Asp-Thr-Pro -Lcu-Arg-Ser -Arg

4 0 4 0 2 0 0 0 0 3 68 Phc-Ile - Val-Scr-Arg -Asp-Thr-Ser -Gin -Ser 68 Val-Ser -Ile -Thr -Arg-Asp -Thr-Ser -Glu -Asn 68 Val -Thr -Met-Leu- Val-Asn -Thr-Ser -Lys -Asn

0 4 4 0 4 0 0 4 0 78 Ile -Leu -'l'yr-Leu-Gln-Met -Asn-Ala -Leu -Arg 78 GIn -!'he - Phe- Leu- Lys- Leu -Asp- Ser -Val - Thr 78 Gin -Phe -Scr-Leu-Arg-Leu -Ser -Ser-Val -Thr

0 0 2 3 4 c 4 c 4 c 88 Ala -Glu -Asp-Thr -Ala-Ile -Tyr -Tyr -Cys -Ala 88 Thr-Glx -Asx-Thr-Ala-Thr -Tyr-Tyr-Cys -Ala 88 Ala -Ala -Asp- Thr- Ala- Val - Tyr-Tyr- Cys -Ala

:3 3 2 c 0 0 98 Arg-Asn -Tyr-Tyr -Gly-Ser 98 Gly -Asp -Asn-Asp-His-98 Arg-Asx -Leu-lie -Ala-

c c c c -Thr-Trp-Tyr -Phe

-Leu-Tyr -Phe -Gly-Cys -lie

14 c 4 0 4 2 0 4 0 lOG Asp- Val -Trp-Gly -Ala-Gly -Thr-Thr-Val -Thr lOG Asp -Tyr -Trp-Gly -Gln-Giy -Thr-Thr-Leu -Thr lOG Asx- Val -Trp-Gly -Gln-Gly -Ser -Leu- Val -Thr

4 0 0 IIG Val -Ser -Ser llG Val -Ser -Ser 116 Val-Ser -Ser

(a) Above each residue in the Mci'C-603 sequence is its structural location designated by: 0, completely exposed to solvent; I, mainly exposed; 2, partly exposed, partly buried in the domain interior; :l, mainly buried; 4, completely buried; or C, in contact with the homologous domain. The numbers at the left alongside the sequences correspond to the sequence number of the first residue in each row as obtained from the original publica-tions.

629


MePC-603 REI MOPC-315 Meg New

Table I. ( eontinuedJ

lbi Alignment of' v~. sequence'S

0 3 0 4 0 4 () () 1 Asp-Ile - Val-Met-Thr-Gln -Ser -Pro -Ser 1 Asp-lle -Gln-Met-Thr-Gln -Ser -Pro -Ser 1 Pea -Ala- Val- Val-Thr-Glu -Glu- -Ser 1 Pea -Ser -Ala-Leu-Thr-Gln -Pro -Pro-

0 -Ser -Ser -Ala -Ser

1 Pea -Ser -Val- Leu -Thr- Gin -Pro- Pro- -Ser

4 () 4 0 () () 0 0 4 () 11 Leu-Ser - Val-Ser -Ala-Gly -Glu -Arg- Val -Thr 11 Leu-Ser -Ala-Ser- Val-Gly -Asp-Arg- Val -Thr 10 Leu-Thr -Thr-Ser -Pro -Gly -Gly -Thr-IVal, Ilel 10 Ala -Ser -Gly-Ser -Leu-Gly -Gin -Ser- Val -Thr 10 Val -Ser -Gly-Ala -Pro -Gly -Gin -Arg- Val -Thr

4 () 4 0 4 () () 0 :l () 21 Met-Scr -Cys-Lys -Ser -Ser -Glx -Ser -Leu -Leu 21 Ile -Thr -Cys-Gln-Ala -Scr -Gln-20 Leu-Thr -Cys-Arg-Ser -Ser -Thr-Gly -Ala- Val 20 Ile -Ser -Cys-Thr-Gly-Thr-Ser -Ser -Asp -Val 20 Ilc -Scr -Cys-Thr-Gly -Ser -Ser -Ser -Asn -lie

() 0 27 Asx -Ser 28

c c c c 3 4 e -Gly-Asx-Glx -Lys -Asx-Phe-Leu -Ala

-Asp-Ile -Ile -Lys-Tyr-Lcu -Asn 30 -Thr -Thr -Ser -Asn-Tyr -Ala -Asn 30 -Gly -Gly -Tyr -Asn-Tyr- Val -Ser 27e -Gly -Ala -Gly -Asn-His- Val - Lys

4 c 3 c 0 0 0 c c 35 Trp -Tyr -Glx-IG]x)-Lys-Pro -Gly -Glx- Pro -Pro 35 Trp-Tyr -Gln-Gln-Thr-Pro -Gly-Lys-Ala -Pro :37 Trp -Ile -Glx-Glx -Lys -Pro -Asx-IIis -Leu -Phc 37 Trp -Tyr -Gin-Gin -His -Ala -Gly -Lys -Ala -Pro 34 Trp -Tyr -Gin-Gin -Leu-Pro -Gly -Thr -Ala -Pro

0 c 4 4 45 Lys -Leu -Leu-Ile 45 Lys -Leu -Leu-lie 47 Thr-Gly -Leu-Ile 47 Lys- Val - Ilc -Ilc 44 Lys -Leu -Leu- Ilc

c 1 1 () 1 () - Tyr - X - X - X - X - X -Tyr -Glu -Ala -Scr -Asn -Leu -Gly -Gly -Thr-Scr -Asp -Arg -Tyr -Glu- Val -Asn-Lys -Arg -Phc-IIis -Asn-Asn-Ala -Arg

() 0 () 41 () 14 () 4 -Gly 55 X - X -Gly- Val -Pro -Ala -Arg-Phc-Scr

55 Gin -Ala -Gly- Val-Pro -Scr -Arg-Phc-Sc1· 57 Ala -Pro -Gly- Val-Pro- Val -Arg-I'hc-Scr 57 Pro -Ser -Gly- Val-Pro -Asp-Arg-I'hc-Scr 61 -I'hc-Scr

-Gly -Gly -Gly -Val

() 4 () () 2 () 4 () 4 () 65 Scr -Giy -Ser -Arg-Thr -Asp- I'hc-Thr-Lcu -Thr 65 Ser -Gly -Scr-Gly-Thr-Asp-Tyr-Thr-Phc -Thr 67 Scr -Leu -Ile -Gly -Asp-Lys -Ala -Ala -Leu -Thr 67 Ser -Lys -Ser -Gly -Asn-Thr -Ala -Scr -Leu -Thr 64 Ser -Lys -Ser-Gly-Scr -Sc1· -Ala-Thr-Lcu -Ala

4 () () 2 2 () () 12 4 75 Ilc -Asx -Pro- Val -Glx -Ala -Asx--Asp- Val -Ala 75 Ile -Scr -Scr-Lcu-Gln -Pro -Glu--Asp-Ilc -Ala 77 Ile -Thr -Gly-Ala -Glx -Thr -Glx -Asp-Asp -Ala 77 Val -Ser -Gly-Leu-Gln -Ala -Glu-Asp-Glu -Ala 74 Ile -Thr -Gly-Leu-Gln -Ala -Glu-Asp-Giu -Ala

e 4 c 4 c 4 c 2 () () 85 Thr-Tyr -Phc-Cys- X - X - X - X - X - X 85 Thr -Tyr -Tyr-Cys -Gin -Gin -Tyr -Gin -Scr -Leu 87 Mct-Tyr -Phc-Cys-Ala-Leu-Trp-Phc-Arg -Asx 87 Asp-Tyr -Tyr-Cys-Ser -Scr -Tyr-Glu-Gly -Scr 84 Asp-Tyr -Tyr-Cys -Gin -Scr -Tyr -Asp-Arg -Scr

() c 3 c 4 e () 4 () 95 - X -X- X -Phc-Gly-Gly-Gly-Thr -Lys

630


MODELS OF ANTIGEN-BINDING SITES 631

Table I. (continued)

I b) Alignment of V, sequences

D5 -Pro -Tyr-Thr-I'he-Gly-Gln-Gly-Thr -Lys H7 -His -Phe- Val-Phe-Gly -Gly -Gly -Thr -Lys 97 Asp -Asn -Phe- Val -Phe-Gly -Thr-Gly -Thr -Lys 94 -Leu -Arg- Val-Phe-Gly -Gly -Gly -Thr -Lys

4 0 3 0 0 10,1 Leu -Glu -lie -Lys -Arg 104 Leu-Gin -lie -Thr-Gly lOG Val-Thr -Val-Leu-Gly 107 Val-Thr -Val-Leu-Gly 105 Leu-Thr -Val-Leu-Arg

I b) Sec footnote to"· The Mci'C-603 sequence is known only to residue 49; for the rest of the Hequencc, the most frequently occurring residue in other mouse kappa chains I Me-l< can ct al. IH7:li is given fi>r each position except for the hypervariable residues, which are simply designated by X. Ala :!4, Thr 85, and Gly 100 in 603 project into the V11 :V, inter-face but are not in actual contact with V11 ; this structural classification is designated by the lower case c.

ments of main-chain atoms and rotation of side groups about single bonds.

Atomic coordinates of the :Jl5 hypervariable residues were measured from the model and ad-justed using Diamond's model-building program miamond 1966). In building the DNP-hapten into the model, the nitro groups were assumed to be coplanar with the phenyl ring. Although the crystal structure of2,4-dinitroaniline has not been reported, theoretical arguments (Pauling 1948) would pre-dict a planar molecule. This is consistent with the observations of Trotter

632 PADLAN ET AL.

by the Phe 1315) for Leu (603) change at position 79. The cluster involving Met 83, Leu 86, and Val 114 in 603 is replaced by the grouping of Leu 83, Val86, and Leu 114 in 315. Furthermore, the replacement of Val 107 in 603 by a Tyr in 315 is counterbal-anced by the substitution of a Gly in 315 for the Arg 98 in 603. In V~,, Leu 68 and Ala 73 of 315 re-place Gly 66 and Phe 71, respectively, of 603.

Position 6 in 603 V~. is occupied by a Gin which is completely buried, whereas a Glu exists at this posi-tion in 315. The substitution is structurally feasible since the Ca-Cf3 bond of this residue is oriented such that the side group could swing in or out of the domain interior. Exposing the side group of 315 Glu 6 to solvent creates a void that could be filled by a structural rearrangement of the flexible seg-ment Gly-Gly-Gly at positions 101-103. In 315, Gly 101 is probably closer to the main body of VL, as in V11 where a Glu also occurs at position 6.

Comparisou of residues iuvolved iu the V1.:V11 con-tact. It is remarkable that the V11 residues in-volved in the contact with V~. are virtually invari-ant. Of the eight contacting residues, seven are identical and the other involves an lie for Val inter-change at position 38. Of the eight v~. residues in contact with V11 , three are the same in 315 and 603. Substitutions at the other contact positions gen-erally complement each other so that the relative disposition of the two variable domains is prob-ably the same in 603 and 315. For example, in the light chain, the replacements of 603 Tyr 36, Leu 46, and Tyr 49 by the smaller 315 lie 38, Gly 48, and Gly 51, respectively, could cause VL to move closer to V11 in 315. This tendency is counteracted, on the other hand, by the replacement of 603 Pro 43 and Pro 44 by 315 Leu 45 and Phe 46, respectively, as well as the replacement of the hypervariable residue Ala 34 in 603 by Asn 36 in 315. Furthermore, the substitution of 603 Thr 85 by 315 Met 87 in V~. com-plements the change from 603 Lys 43 to 315 Asn 43 in V11 •

Changes iuvolving structural hypervariable resi-dues. Certain changes in framework residues ap-pear to be necessary to accommodate substitutions at hypervariable positions. For example, the re-placement in VL of 603 lie 2 by 315 Ala helps to ac-commodate the bulky side group of Phe 94 from L3. Also in VL, the substitution of 603 Met 4 by 315 Val helps accommodate the buried Leu 92. In addition, this Val-for-Met substitution at position 4, as well as the Ala-for-Phe replacement at position 73 and the Ala-for-Leu interchange at the hypervariable position 35, all facilitate the formation of a helical L1 region in 315. In V11 , the replacement of the con-tact residue Val 37 by lie in 315 complements the Asn for Glu substitution at the hypervariable posi-tion 35 in Hl.

The Antigen-binding Site of MOPC-315

As soon as a tentative model was completed, even before any adjustments were made to fit hapten, several features of the binding site became ap-parent. The middle of the hypervariable surface was dominated by a rather pronounced cavity. Com-pared to 603, the 315 cavity contained substantially more aromatic residues. It seemed most likely that the DNP moiety would be bound within this cavity.

A schematic drawing of the hypervariablc loops of 315 is shown in Figure lA. For comparison, the hypervariable loops of 603 arc shown in Figure lB. The disposition of the hypervariable residues of 315, including the side-chain positions obtained from the model, is shown in Figure 2.

The hapten-binding site is bounded by Ll on one side and by HI and H2 on the other. The floor is formed by L3, and the roof is formed primarily by I-I:J. The hypervariablc residues that project into the hapten-binding cavity include Phe 34, Asn 36, Asp 99, and Leu 103 of the heavy chain and Tyr 34, Asn 36, Trp 93, and Phe 98 of the light chain. Ser 32 and Asn 96 of the light chain and Phe 50, Lys 52, and Asp 101 of the heavy chain ring the opening of the pocket. As in 603, L2 is screened from the hapten-binding site by L1 and H3, and no residues from L2 project into the binding cavity.

The distorted 11-helix in Ll iPoljak et a!. 1974) of 315 involves residues 26-31, with a hydrogen bond between the carbonyl oxygen of Thr 26 and the amide nitrogen of Thr 31. The helix is initiated by a sharp bend at position 26-27, with a hydrogen bond between the carbonyl oxygen of Ser 25 and the amide nitrogen of Ala 28. The helical configuration places Ser 24, Ala 28, Val 29, and Ala 35 in the domain interior, with Ala 28 juxtaposed against Phe 94 of L3. Aside from Tyr 34 and Asn 36, which project into the cavity, the other residues in L1 are either completely or mainly exposed to solvent. A pair of hydrogen bonds can be formed between main-chain atoms of Asn ;36 and Ala 91 ofL3. All the resi-dues in L2 are exposed to solvent, and a beta bend is formed with residues in positions 51-54. In L3, Leu 92, Phc 94, and Val 99 arc buried, Ala 91 pro-jects into the v~.:VII interface, and Arg 95, Asn 96, and His 97 are exposed. The L3 loop has a beta bend involving residues 94-97. Phe 98 of L3 and the non-hypervariable Phc 105 of the heavy chain delimit the maximum depth for hapten-binding. Trp 93 lines the floor of the binding cavity and probably serves to orient the planar hapten.

The restructuring of H1 leaves Tyr 33 of 315 in the domain interior, in an analogous position to 603 Phe 32. Phe 34, Asn 36, and, to a lesser extent, Phe 50 and the nonhypervariable Trp 4 7 form one side of the binding pocket. A beta bend is formed with residues 30-33, and hydrogen bonding is possible between the carbonyl oxygen of Gly 32 and the



A

~ 66H

B

Figure l. Stereo drawings of the hypervariable loops of ( AJ 315 and (B) 603 in approximately the same orientation. The numbers indicate the first and last hypervariable positions in each loop. These and other figures were drawn using the OH TEP program of Carroll K. ,Johnson rOHNL-3794).

amide nitrogen of Asp 101, as well as between the amide nitrogen of Phc 34 and the carbonyl oxygen of Asp 99. Phc 34 from 1-11 introduces a rather bulky hydrophobic side group into the binding cavity. Beta bends arc fi:>rmcd with residues 52-55 and 61-64 in H2. Leu lO:l from H3 contributes to the hydrophobic nature of the hapten-binding site of 315 and with L1 residues Tyr 34 and Asn 36 forms

the other side of the cavity. The roof of the cavity is formed mainly by the backbone of H3, the side group of Asp 99, and partly by Phe 34 of J-11 and Leu 103 of H3. A beta bend involves positions 100-103 in H3. As in 603, J-13 is in contact with Ll.

The residues that would appear to interact most with the hapten are Phc 34, Asn 36, and Leu 103 from the heavy chain and Asn 36 and Trp 93 from

Figure 2. Stereo drawing of the 315 antigen-binding site showing allnonhydrogen atoms. The orientation and scale are the same as in Fig. lA. This figure is rotated through 90'' in relation to the frontispiece.


634 PADLAN ET AL.

~us 52

~E 50

Q

~LIS 52

_N'riE so

!j

Figure 3. Stereo drawing of the hypervariable residues projecting into the ;315 hapter~-bim!in~ cavity. II. drawingof BADE (without the bromine atom) is included in the figure to show the hapten-protem mteractwns and the possilnhty of affinity-labeling 'l'yr :34 (L). The figure is rotated 15"' relative to Fig. lA.

the light chain. The nitro groups of the DNP are then at suitable distances for hydrogen bonding to the amide of the L-chain Asn 36 side group and to the amide of the H-chain Asn 36. The aromatic side group of Trp 93 is essentially parallel to the planar DNP moiety and is 3.5-4 A from it. There is considerable overlap of the two conjugated ring systems. The hapten-protein interactions are illus-trated in Figure 3. In our model of DNP binding to 315, the specific affinity reagents used by I-Iaimo-vich et al. !1972) can react with Lys 52 of the heavy chain and Tyr 34 of the light chain, but with no other.

Many hypervariable residues in one chain are in contact with hypervariable residues in the other chain. These include Asn 36, Phe 50, and Leu 103 of the heavy chain and Tyr 34, Asn 36, Asx 96, and Phe 98 of the light chain.

DISCUSSION

Correlation of the Model with Known Chemical Data

A number of chemical studies of the affinity label-ing !Metzger and Potter 1968; Goetz! and Metz-ger 1970a,b; Haimovich et al. 1970, 1972; and Givol et a!. 1971) and kinetic mapping of the combining site !Haselkorn eta!. 1974) of protein 315 have been reported. The results of these studies are for the most part consistent with our model. The afiinity labeling with a m-nitrobenzene diazonium com-pound !Goetz! and Metzger 1970a,b) and with BADE !Haimovich et a!. 1970, 1972; Givol et a!. 1971) both indicated that Tyr 34 !L) was close to the binding site. In the model of 315, examination of the positions of the reactive groups when the re-agents are bound through the nitrophenyl groups indicates that they are in a suitable position for them to react with this tyrosine. Similarly, the re-action of BADE (Haimovich eta!. 1970, 1972; Givol et a!. 1971) with Lys 52 !H) may be accounted for by its position in the model. The reaction of a bifune-tional reagent !Givol eta!. 1971) with protein 315 to cross-link the light and heavy chains is in accord with the positions of the Tyr 34 !L) and Lys 52 !H).

It should be noted that although these results are

quite consistent, to a large extent they derive from coarse ratlwr than fine detail in the model. The CrY-to-Ca separation of 20.9 A f(>r Tyr ~!4 IL) and Lys 52 (f-l) is principally due to the f~lct that they occur in the first and second hypervariable regions of the light and heavy chains, respectively. Since these loops occur on opposite sides of the binding cavity, they will produce a significant separation of the residues regardless of the fine detail in the structure. However, if the orientation of Tyr 34 ( L) had been similar to that of the corresponding resi-due in 60:3 IPhe :3:3), then no reaction with these reagents would have been possible, since here the group points away from the binding pocket. The difference in orientation of Tyr 34 in :!15 and Phe 33 in 603 lies in the diflerence in the configuration of the L1 loops of these two proteins. In this model, the phenolic oxygen of Tyr :34 !L) and the epsilon nitrogen of Lys 52


binding of the p-nitro derivatives lacks the ad-ditional stabilizing force of one hydrogen bond (presumably to Asn 36 [L]l. The complex with 2,6-dinitrophenyl compounds would permit the f(>rma-tion of only one hydrogen bond t presumably to Asn 36 I L I), since an attempt to form hydrogen bonds to both Asn 36 ( L) and Asn 3() LE-I) would pr'J-duce an impossible contact between the substituent at the !-position and the side chain of Phc 34 (I-I).

St1bstitutions at the !-position of the 2,4-dinitro-benzene moiety add to the interaction with the ring of Trp 93 (L), and longer substituents permit addi-tional interactions with Tyr 34 IL), Leu 103 (H), main-chain atoms in the H3 loop, and protein side groups farther away. Thus alkyl derivatives of 2,4-dinitroaniline as well as derivatives with branched cyclic or aromatic side chains are gen-erally found to bind better than the parent corn-pound IHaselkorn et al. 1974). Branched side chains that arc too bulky, however, apparently interfere with binding. Thus, though the I-N-dimethyl derivative of 2,4-dinitroaniline is a stronger ligand, the diethyl derivative is a weaker binder than the parent riiaselkorn et al. 1974). In our mod0l, it is clear that whereas the methyl groups in the former compound would add favorably to the hapten-protein interaction by increased van dcr Waals con tact with Trp 9:3 ( L ), Leu 103 (!-!), and the main chain of H3, the ethyl groups in the latter derivative would cause too close contacts with Trp 93 (L) and H:3, leading to the instability of the complex. Simi-lar close contacts with Trp 93 IL) would occur in the binding of dialaninc derivatives of DNP in which the first alanine residue is in the L-configuration, which explains the lower affinity of 315 for these compounds IHaselkorn ct al. 1974). This interfer-ence would occur only when another amino acid residue is linked to the first alanine, if the peptide dihedral angles are kept within reasonable limits.

For DNP derivatives with side chains that have a hydrogen-bonding capacity, there are a number of polar amino acids nearby with which they may interact. These include Lys 52 (fl), Asp 101 !H), His 102 I.Hl, Ser 32 IL), Tyr 34 (L), and Asn 96 IL). This additional potential hydrogen bonding between hapten and protein could explain the observed high afiinity of ;315 for DNP derivatives with hydroxyl and carboxyl groups !l-Iaselkorn et a!. 1974). In addition, Lys 52 (l-l) is located where it could ac-count for the positive subsite observed by I-lasel-korn ct a!. !Hl74).

Although Otlr model of the binding site of 315 can account, at least qualitatively, for most of the avail-able chemical data on the binding of hapten to 315, we cannot rule out the possibility that other hapten-binding configurations may exist. The other con-figurations that seem plausible to us are not grossly difl"erent from our present model. They retain the present location of the DNP-hapten while reorien-tating it, as well as the Trp 93 ILl side group, rela-

tive to the other groups in the cavity. Here again, only minor adjustments in the binding-site struc-ture would be required.

The rationale for the presence of Asp 99 (l-1) in the binding cavity is not apparent. This residue is deep in the pocket and is strategically positioned so that it may play an important role in determin-ing the hapten-binding specificity of the protein. Its presence here raises the possibility that another, more tightly bound hapten might be found or de-signed for 315. Such a hapten would probably be positively charged to interact with Asp 99 (I-ll, with an aromatic ring to interact maximally with Trp 93 (L) and the other hydrophobic residues in the cavity, and with side groups that could utilize the hydrogen-bonding potential of either or both Asn 36 !L) and Asn 36 !I-I). A similar case is found in protein New IAmzel et al. 1974), where a Lys and an Arg are found deep in the binding site but are apparently not utilized in the binding of menadione haptens.

I-Iypervariable residues have been found to par-ticipate in the contact between V1. and Vu in 603 where almost the entire H3 is in intimate contact with the large loop of Ll. In our model, the homolo-gous regions in 315 are also in contact. In the ab-sence of an actual structure determination, the effect of the interaction of hypervariable residues on the size and shape of the antigen-binding site of 315 can only be guessed. We have built the model or the combining site of 315 assuming that these hypervariable interactions will have only a modu-lating effect on the structure of the binding cavity and will not affect significantly the disposition of the pertinent side groups.

Comparison with Other Model-building Studies

Poljak et al. (1974) have briefly discussed the binding site or 315 relative to the structure of Fab New, a human myeloma protein. Their analysis is based on the unrevised sequence of H1, with Lys at position 35, but they do not discuss the effect of this group on the conformation of I-ll. Nevertheless, many of their conclusions regarding the probable nature of the amino acids lining the predicted hap-ten-binding cavity are similar to ours.

A previous attempt involved construction or a model of a homologous protein from a known three-dimensional structure for a-lactalbumin and chicken lysozyme !Browne et al. 1969). In that case, there was a striking degree of sequence homology between the two proteins, including four S-S bridges; as a result, much of the molecule could readily be constructed to the lysozyme framework.

Here, by contrast, although the degree of sequence homology between 603 and 315 is high for the framework part of the two molecules, the hyper-variable loops that form the binding surface are quite different in sequence. The invariance of the


636 PADLAN ET AL.

framework is an essential assumption in order for the construction of the model of the 315 binding site to be feasible. The other assumption that was roughly adhered to in both studies was that loops of the same lenf,rth would have essentially the same conformation. We know that this need not be pre-cisely true, and even when the same amino acid sequences occur in the loops, as in the two variable domains of the immunoglobulin light-chain dimer Meg, the loops have in places somewhat difl'erent conformations (Edmundson et a!. 1974). When the amino acids are different, then this assumption is even less likely to be true. Nevertheless, this pro-vided a reasonable starting model for loops of the same size, and the biggest difficulties were encoun-tered with insertions and deletions. It is interest-ing to note that in two human VK domains, REI and Au, the entire backbone conformations, including the hypervariable regions, appear identical despite their having 16 amino acid differences CFehlhammer et a!. 1975).

Discussion of the Present Approach

This study was limited in several ways. First, it was based on a very small number of known struc-tures. Although these structures provide a reason-able basis for assuming framework in variance, they do not as yet provide enough data to enable us to make specific structural statements about the na-ture of the hypervariable surface. In particular, they leave us with no good feeling for the changes produced by insertions and deletions. To this extent, more structure determinations of immunoglobulins from diflerent insertion and deletion subgroups are needed.

Second, the study has been restricted to the use of mechanical molecular models. At the initial stages, it might be beneficial to examine particularly large loops by methods such as those of Chou and Fasman (1974) or Wu and Kabat (1971) in order to predict the possible changes in secondary structure. In the final stages, it is clear that the use of a general energy minimization program which takes into ac-count the bad contacts and attempts to alleviate them would be of benefit. Nevertheless, until more sophisticated procedures are carried out, model-building techniques of the kind used in this study will be the only way in which an understanding of the nature of the binding sites of most immuno-globulins can be obtained.

Acknowledgment

Part of this work was supported by National Institutes of Health Grant R01 Al-11453 to D.G.

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EPP, 0., P. CoLMAN, II. FEHLHAMMEit, W. BonE, M. Scm~·nm, R. HuBER and W. PALM. 1974. Crystal and molecu-lar structure of a dimer comrxJsed of the variable por-tions of the Hence-Jones protein HE I. Eur. J. Biochem. 45:513.

FEHLHAMMER, II., M. ScmFFER, 0. EPP, P. M. CoLMAN, E. E. LATTMAN, P. ScHWAGER, W. STEIGEMANN and H .• J. ScmtAMM. 1975. The structure determination of the variable portion of the Bence-.Jones protein Au. Biophys. Struct. Mechanism 1: 139.

FETT, J. W. and H. F. DEUTSCH. 1974. Primary structure of the Meg lambda chain./1iochemist1y 13: 4102.

FnANCIS, S. H., H. G. q. LEsLm, L. Hoon and H. N. EISEN. 1974. Amino-acid sequence of the variable region of the heavy !Alpha) chain of a mouse myeloma protein with anti-hapten activity. Proc. Nat. Acad. Sci. 71: 1123.

G!VOL, D., P. H. STitAUSBAUCH, E. HURWITZ, M. WILCHEK, J. HAIMOVICJI and H. N. EISEN. 1971. Affinity labeling and cross-linking of the heavy and light chains of a myeloma protein with anti-2,4-dinitrophenyl activity. Biochemistry 10: 3461.

GLASER, M. and S . • ]. SINGER. 1971. Extrinsic Cotton effects



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