`ON QUANTITATIVE BIOLOGY
`
`VOLUME XLI
`
`Origins of Lymphocyte Diversity
`
`COLD SPRING HARBOR LABORATORY
`
`1977
`
`1 of 13
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`Celltrion, Inc., Exhibit 1116
`
`
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`COLD SPRING HARBOR SYMPOSIA ON QUANTITATIVE IUOL05\r
`VOLUME XLI
`
`© 1977 by The Cold Spring Harbor Laboratory
`International Standard Book Number 0-87969-040-2 !clothbound)
`Library of Congress Catalog Card Number 34-8174
`
`Printed in the United States of America
`All rights reserved
`
`COLD SPRJN(; HARBOR SYMPOSIA ON QUANTITATIVE B/OLO<;Y
`
`Founded in 1931 by
`RE<;(NALD C. HARRIS
`JJirertor of the Biologiral Laboratory I 924 to I '116
`
`l'rn1iu/I.\ Syrtt/Josia Volumes
`
`I 11933) Surface Phenomena
`II 11934) Aspects of Growth
`III (1935) Photochemical Reactions
`IV (1936) Excitation Phenomena
`V (1937) Internal Secretions
`VI 11938) Protein Chemistry
`VII 0939) Biological Oxidations
`VIII 0940) Permeability and the Nature of Cell Membranes
`IX 0941) Genes and Chromosomes: Structure and Organization
`X ( 1942) The Relation of Hormones to Development
`XI (1946) Heredity and Variation in Microorganisms
`XII (1947) Nucleic Acids and Nucleoproteins
`XIII 0948) Biological Applications of Tracer Elements
`XIV 0949) Amino Acids and Proteins
`XV (1950) Origin and Evolution of Man
`XVI (1951) Genes and Mutations
`XVII (1952) The Neuron
`XVIII (1953) Viruses
`XIX 0954) The Mammalian Fetus: Physiological Aspects of De(cid:173)
`velopment
`XX 0955) Population Genetics: The Nature and Causes of Genetic
`Variability in Population
`
`XXI 0956) Genetic Mechanisms: Structure and Function
`XXII 0957) Population Studies: Animal Ecology and Demography
`XXIII (1958) Exchange of Genetic Material: Mechanism and Con-
`sequences
`XXIV 0959) Genetics and Twentieth Century Darwinism
`XXV 0960) Biological Clocks
`XXVI 0961) Cellular Regulatory Mechanisms
`XXVII (1962) Basic Mechanisms in Animal Virus Biology
`XXVIII 11963) Synthesis and Structure of Macromolecules
`XXIX 0964) Human Genetics
`XXX 0965) Sensory Receptors
`XXXI 0966) The Genetic Code
`XXXII 0967) Antibodies
`XXXIII 11968) Replication of DNA in Microorganisms
`XXXIV I 1969) The Mechanism of Protein Synthesis
`XXXV I 1970) Transcription of Genetic Material
`XXXVI 11971) Structure and Function of Proteins at the Three-
`dimensional Level
`XXXVII 0972) The Mechanism of Muscle Contraction
`XXXVIII (1973) Chromosome Structure and Function
`XXXIX (1974) Tumor Viruses
`XL (1975) The Synapse
`
`The Symposium Volumes are published by the Cold Spring Harbor Laboratory, Cold Spring Harbor, New York 11724, and
`may be purchased directly from the Laboratory or through booksellers. Price of Volume XLI-2-part set $60.00 line.
`postage). May be purchased only as a complete set. Price subject to change without notice.
`
`2 of 13
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`Celltrion, Inc., Exhibit 1116
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`
`Model-building Studies of Antigen-binding Sites:
`The Hapten-binding Site of MOPC-315
`
`E. A. PADLAN, D.R. DAVIES, I. PECHT,* D. GrvoL* AND C. WRIGHTt
`Laboratory of Molecular Bwlogy, Natwnal Institute nf Arthritis, Metabolism and Digestive Diseases, National Institutes of
`Health, Bethesda, Maryland 20014; *Department of Chemical Immunology, The Weizmann Institute of Science, Rehovot.
`Israel; t Laboratory of Molecular Biophysics, Department of Zoology, Oxford University, Oxford, 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 (lg) fragments (see Davies
`et al. 1975a,b for a review). These structures reveal
`that the combining site is formed by bringing to(cid:173)
`gether the three hypervariable regions (Wu and
`Kabat 1970) of V1, and of V H to form a continuous
`complementarity-providing surface. A quantitative
`comparison of the tertiary structures of a number
`of variable domains from both light and heavy
`chains has demonstrated that their nonhypervari(cid:173)
`able or framework regions are very similar, with the
`principal differences occurring in the hypervariable
`loops (Padlan and Davies 1975).
`In view of this structural invariance, the im(cid:173)
`munoglobulin variable region can be regarded as
`consisting of a rigid framework to which are at(cid:173)
`tached the hypervariable loops. These loops are
`not large, generally consisting of at most 17 resi(cid:173)
`dues; in addition, the configurations of a number of
`different hypervariable loop regions are already
`known from X-ray diffraction. Thus it might be
`possible to construct by model building new loop
`regions (and hence the lg binding sites) from a
`knowledge of the sequences alone. Since it is clearly
`impractical to determine by X-ray diffraction the
`structures of all the interesting antibodies, this
`model-building method offers an attractive alter(cid:173)
`native approach when sequence data are available.
`We have examined the potential usefulness of this
`possibility by attempting to build the V regions of
`the rabbit anti-type-III polysaccharide antibody
`(Davies and Padlan 1976) and of protein MOPC-
`315, a mouse immunoglobulin with dinitrophenyl
`(DNPl-binding specificity
`(Eisen et al. 1968),
`based on the known three-dimensional structure
`of McPC-603 Fab' (Padlan et al. 1973, 1974; Segal
`et al. 1974a,b) together with the amino acid se(cid:173)
`quences of these immunoglobulin Fab's.
`In this paper, we describe the results of two
`independent attempts to construct the model of
`MOPC-315, one at the National Institutes of
`Health and the other at the Weizmann Institute.
`The two models were ultimately merged to give a
`final model that is presented and is discussed in
`terms of the known binding specificity, the kinetic
`mapping of the binding site (Pecht et al. 1972a,b;
`
`Haselkorn et al. 1974), and the results of affinity
`labeling with various reagents (Goetz! and Metzger
`1970a,b; Givol et al. 1971; Haimovich et al. 1972).
`We discuss some of the limitations and the potential
`of this method of model building, the assumptions
`that have to be made, and some of the additional
`information that will be needed to put this tentative
`approach on a firmer footing.
`
`MATERIALS AND METHODS
`
`Sequence Alignment
`
`The amino acid sequences of the V 11 domains of
`proteins McPC-603 (Rudikoff and Potter 1974)
`and MOPC-315 (Francis et al. 1974; L. Hood,
`M. Margolies, D. Givol and R. Zakut, unpubl.)
`were aligned for maximum sequence homology
`and structural analogy, as presented in Table la.
`The sequence of New V H (Poljak et al. 1974) is
`included for comparison. A similar alignment of
`the 603 (Segal et al. 1974a) and 315 V1, (Dugan et al.
`1973) sequences is given in Table lb. However,
`only the sequence of the first 49 residues is known
`for 603, and the residues given for the rest of the
`sequence are those which most frequently occur in
`mouse kappa chains (McKean et al. 1973). Included
`in Table lb are the sequences of the V L domains of
`REI (Epp et al. 1974), Meg (Fett and Deutsch
`1974), and New (Chen and Poljak 1974) proteins.
`The sequence numbering schemes used in these
`tables are those obtained from the original refer(cid:173)
`ences. Whenever a residue position is referred to in
`the text, however, the quoted number corresponds
`to the 315 sequence. The hypervariable regions of
`the light chain are: Ll, residues 23-36; 12, residues
`52-58; and L3, residues 91-99; and those of the
`heavy chain are: Hl, residues 31-36; H2, residues
`49-66; and H3, residues 99-104.
`
`Model Building
`
`Model 1 was constructed at the Weizmann Insti(cid:173)
`tute from Nicholson molecular models (Labquip,
`Reading, England), which have a scale of 1 cm c= 1
`A, and CPK space-filling models (1.25 cm = 1 AJ.
`Model 2 and the final model were constructed at
`the National Institutes of Health from Kendrew
`
`627
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`628
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`PADLAN ET AL.
`
`(2 cm = 1 Al molecular models (Repetition Engi(cid:173)
`neers, Cambridge, England).
`The general principle used for model building was
`to construct first the framework part of the variable
`region, based on the structure of 603 Fab'. The
`hypervariable loops were then constructed, leaving
`the structure as little changed as possible except
`when forced by amino acid insertions and deletions.
`An attempt was made to maximize the structural
`stability within each loop by forming hydrogen
`bonds whenever possible and maintaining the phi
`and psi peptide angles within reasonable limits
`(Ramakrishnan and Ramachandran 1965 ). The
`interactions between loops were then maximized,
`leaving no large holes in the domain interior, while
`minimizing steric hindrance between groups.
`The Ll regions in the kappa chains 603 and REI
`are simple loops, whereas the corresponding re(cid:173)
`gions are helical in New (Poljak et al. 1974) and
`Meg (Schiffer et al. 1973), which are both lambda
`chains. Since the light chain of protein 315 is of the
`lambda type and since this region has the same
`number of residues as New and Meg with no gross
`differences in 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 Ll loop in 315 was built
`to conform as closely as possible to the correspond(cid:173)
`ing region in the lambda chains with the aid of the
`atomic coordinates of the Meg backbone kindly
`provided by M. Schiffer and coworkers (pers.
`comm.).
`L2 was built by assigning to it the same back(cid:173)
`bone conformation as in protein 603. In this region,
`603 and REI are not significantly different (Padlan
`and Davies 1975). Moreover, kappa and lambda
`light chains have the same number of residues in
`this part of the molecule (Dayhoff 1972); an excep(cid:173)
`tion is New (Table lb), 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
`(Edmundson et al. 1974). Thus it is reasonable to
`assume that 315 L2 will have the same configura(cid:173)
`tion as that of 603.
`L3 was built essentially as in 603, the main dif(cid:173)
`ference being the possibility of forming more hy(cid:173)
`drogen bonds in 315. The L3 loop in 315 was built
`as two, fully antiparallel, segments, the loop in 603
`being less regular. Although the sequence of 603
`is not known in this region, the six mouse kappa
`chains sequenced so far (McKean et al. 1973) all
`have a proline at position 94. The possible occur(cid:173)
`rence of this residue in 603 could explain the fewer
`hydrogen bonds formed in 603 L3.
`Protein 315 has one more residue than 603 in Hl
`(Table la). Originally, the sequence of 315 Hl con(cid:173)
`tained a lysine in position 35 (Francis et al. 1974).
`Structurally, this residue is analogous to Met 34
`of 603 since both occur two positions from the struc(cid:173)
`turally invariant Trp 37. In 603, Met 34 is buried in
`
`the domain interior. Its replacement by a lysine
`side chain would cause a significant rearrangement
`of the Hl backbone in order to expose the charged
`amino group to solvent. A reexamination of the
`sequence of this part of 315 led to the assignment of
`a Trp instead of the Lys in position 35 ( L. Hood,
`M. Margolies, D. Givol and R. Zakut, unpubl.).
`A Trp side chain can easily be accommodated in the
`domain interior without significantly changing
`the configuration from that observed in 603.
`The alignment shown in Table la leads us to con(cid:173)
`clude that the additional residue in Hl of 315 can
`be best accommodated in the exposed loop at the
`beginning of Hl. The glycine at position 32 permits
`the construction of a sharp turn in this part of 315.
`H2 has three more residues in 603 than in 315,
`whereas 315 and New are of the same length in this
`region. An initial model of 315 H2 was built by
`simply excising three residues from the end of the
`603 H2 loop. Minor adjustments were then made to
`make the 315 H2 loop resemble as closely as possi(cid:173)
`ble the corresponding region in New (Poljak et al.
`1974).
`Basically the same procedure was employed in
`building the 315 H3 region, which has the same
`length as New and which is two residues shorter
`than 603. Here, however, the C-terminal segment
`of 315 H3 was made to approximate the correspond(cid:173)
`ing region in 603, rather than in New. The differ(cid:173)
`ence between 603 and New, aside from the two(cid:173)
`residue insertion in 603, is the configuration of the
`segment immediately preceding the phenylalanine
`at position 105, which almost always contains a
`large hydrophobic residue (Dayhoff 1972). In 603,
`the side group of Trp 104a, which is structurally
`analogous to Leu 103 of 315, projects into the
`hapten-binding cavity. On the other hand, the back(cid:173)
`bone of this segment in New appears to be different
`(Poljak et al. 1974). In view of the greater structural
`similarity of residues 103-105 of 315 (Leu-Tyr(cid:173)
`Phe) to those of 603 (Trp-Tyr-Phe) rather than to
`those of New (Gly-Cys-Ile), the configuration of
`603 was followed in building this segment of 315.
`However, it should be kept in mind that an alterna(cid:173)
`tive configuration might be that observed in New.
`As soon as a tentative model of the .binding site
`was completed, a likely site for DNP-binding was
`located using the criteria that the nitro groups of
`the DNP moiety must be hydrogen-bonded to the
`protein and that the stacking van der Waals inter(cid:173)
`action between the DNP ring and the side group of
`Trp 93 (LJ must be maximal. The model was then
`adjusted to accommodate the DNP-hapten while
`maintaining the general stability of the various loop
`structures. Further adjustments were then made to
`ensure the feasibility of labeling Tyr 33 (L) and
`Lys 52 (H) by specific affinity reagents (Givol et al.
`1971; Haimovich et al. 1972). The adjustments
`made to the tentative model to accommodate the
`ligand were minor and involved only slight displace-
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`Table I. Amino Acid Sequences of V H Domains of Proteins McPC-603
`and MOPC-315
`
`(a) Alignment of V11 Sequences
`
`McPC-603
`MOPC-315
`New
`
`0
`2
`0
`4
`0
`2
`0
`4
`0
`0
`1 Glu-Val -Lys - Leu-Val-Glu-Ser-Gly-Gly -Gly
`1 Asp-Val -Gln-Leu-Gln-Glu -Ser -Gly-Pro -Gly
`1 Pea -Val -Gln-Leu-Pro - Glu -Ser -Gly-Pro -Glu
`
`0
`4
`0
`0
`0
`0
`4
`0
`4
`0
`11 Leu-Val -Gin-Pro -Gly - Gly - Ser -Leu-Arg -Leu
`11 Leu-Val -Lys-Pro -Ser-Gin - Ser -Leu-Ser -Leu
`11 Leu-Val -Ser-Pro -Gly- Glx - Thr-Leu-Ser -Leu
`0
`4
`0
`4
`1
`0
`4
`0
`4
`0
`21 Ser -Cys -Ala-Thr-Ser - Gly-Phe-Thr-Phe -Ser
`21 Thr -Cys -Ser - Val -Thr -Gly -Tyr -Ser -Ile -Thr
`21 Thr-Cys -Thr-Gly-Ser - Thr- Val-Ser -Thr -Phe
`c
`c
`0
`4
`2
`4
`2
`4
`3
`31 Asp-
`- Phe-Tyr -Met - Glu -Trp-Val -Arg -Gin
`31 Ser -Gly -Tyr-Phe-Trp-Asn-Trp-Ile -Arg -Gin
`31 Ala-
`-Val-Tyr-Ile -Val-Trp-Val-Arg-Gln
`c
`c
`0
`0
`0
`0
`2
`4
`4
`2
`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
`2
`1
`4
`0
`0
`0
`0
`0
`2
`0
`50 Ala -Ser -Arg-Asn-Lys-Gly -Asn-Lys-Tyr -Thr
`50 Phe-Ile -Lys-Tyr -Asp-Gly -
`-Ser
`50 Tyr -Val -Phe-Tyr -His - Gly -
`-Thr
`
`0
`3
`2
`0
`0
`1
`4
`0
`0
`1
`58bThr-Glu -Tyr-Ser -Alii-Ser -Val-Lys -Gly -Arg
`57 Asx -(Tyr , Gly) Asx - Pro - Ser -Leu- Lys -Asn -Arg
`57 Ser -Asp -Thr- Asp-Thr-Pro -Leu-Arg-Ser -Arg
`4
`0
`4
`0
`2
`0
`0
`0
`3
`0
`68 Phe -Ile - Val -Ser-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
`1
`4
`0
`4
`0
`0
`4
`0
`78 Ile -Leu -Tyr-Leu-Gln-Met-Asn-Ala - Leu -Arg
`78 Gln-Phe -Phe-Leu-Lys-Leu -Asp-Ser -Val -Thr
`78 Gin -Phe -Ser-Leu-Arg-Leu-Ser -Ser -Val -Thr
`c
`0
`0
`2
`3
`4
`c
`4
`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
`c
`0 cc c c
`3
`3
`2
`0
`98 Arg-Asn-Tyr-Tyr-Gly-Ser -Thr-Trp-Tyr -Phe
`98 Gly -Asp -Asn-Asp-His -
`-Leu-Tyr -Phe
`98 Arg-Asx -Leu-Ile -Ala-
`-Gly-Cys -Ile
`c
`1
`4
`4
`0
`2
`4
`0
`4
`0
`106 Asp-Val -Trp-Gly -Ala-Gly -Thr-Thr-Val -Thr
`106 Asp-Tyr -Trp-Gly -Gln-Gly -Thr-Thr-Leu -Thr
`106 Asx-Val -Trp-Gly-Gln-Gly-Ser-Leu-Val -Thr
`4
`0
`0
`116 Val -Ser -Ser
`116 Val -Ser -Ser
`116 Val -Ser -Ser
`
`(a ) Above each residue in the McPC-603 sequence is its structural location designated
`by: 0, complete ly exposed to solvent; 1, mainly exposed; 2, partly exposed, partly buried
`in the domain interior; 3, 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 origina l publica(cid:173)
`tions.
`
`629
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`Table I. (continued)
`
`(b) Alignment of V L sequences
`
`McPC-603
`REI
`MOPC-315
`Meg
`New
`
`0
`0
`0
`1
`4
`0
`4
`0
`3
`0
`1 Asp-Ile -Val-Met-Thr-Gln -Ser -Pro -Ser -Ser
`1 Asp-Ile -Gln-Met-Thr-Gln -Ser -Pro -Ser -Ser
`1 Pea-Ala -Val-Val-Thr-Glu -Glu-
`-Ser -Ala
`1 Pea -Ser -Ala-Leu-Thr-Gln -Pro -Pro-
`-Ser
`1 Pea-Ser -Val-Leu-Thr-Gln-Pro-Pro-
`-Ser
`
`0
`4
`0
`0
`0
`0
`0
`4
`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-(Val, Ile)
`10 Ala -Ser -Gly-Ser -Leu-Gly -Gin-Ser -Val -Thr
`10 Val -Ser -Gly-Ala-Pro -Gly-Gln-Arg-Val -Thr
`
`0
`3
`0
`0
`0
`4
`0
`4
`0
`4
`21 Met-Ser -Cys-Lys -Ser -Ser -Glx-Ser -Leu -Leu
`21 Ile -Thr -Cys-Gln-Ala -Ser -Gln-
`20 Leu-Thr -Cys-Arg-Ser -Ser -Thr-Gly-Ala -Val
`20 Ile -Ser - Cys-Thr - Gly -Thr - Ser -Ser -Asp - Val
`20 Ile -Ser -Cys-Thr-Gly-Ser -Ser -Ser -Asn -Ile
`0 c c c
`c
`4
`3
`1
`0
`e
`27 Asx-Ser -Gly-Asx-Glx -Lys-Asx-Phe-Leu -Ala
`28
`-Asp-Ile -Ile -Lys -Tyr -Leu -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
`c
`c
`c c
`0
`0
`0
`3
`4
`35 Trp -Tyr -Glx-(G]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-His -Leu -Phe
`37 Trp -Tyr -Gin-Gin-His -Ala -Gly-Lys-Ala -Pro
`34 Trp -Tyr -Gin-Gin -Leu-Pro -Gly-Thr-Ala -Pro
`c
`c
`4
`4
`0
`0
`1
`0
`1
`1
`45 Lys -Leu -Leu-Ile
`-Tyr - X - X - X - X - X
`45 Lys -Leu -Leu-Ile
`-Tyr -Glu -Ala -Ser -Asn -Leu
`47 Thr-Gly -Leu-Ile
`-Gly -Gly -Thr-Ser -Asp -Arg
`47 Lys - Val -Ile -Ile
`-Tyr -Glu -Val -Asn-Lys -Arg
`44 Lys -Leu -Leu-Ile
`-Phe-His -Asn-Asn-Ala -Arg
`
`4
`0
`4
`1
`0
`1
`4
`0
`0
`0
`55 X - X -Gly-Val -Pro -Ala -Arg-Phe-Ser -Gly
`55 Gin -Ala -Gly-Val -Pro -Ser -Arg-Phe-Ser -Gly
`57 Ala -Pro -Gly-Val -Pro -Val -Arg-Phe-Ser -Gly
`57 Pro -Ser -Gly- Val -Pro -Asp-Arg-Phe-Ser -Gly
`61
`-Phe-Ser -Val
`
`0
`4
`0
`4
`0
`2
`0
`0
`4
`0
`65 Ser - Gly - Ser -Arg-Thr -Asp- Phe-Thr - Leu -Thr
`65 Ser -Gly --Ser-Gly -Thr -Asp-Tyr-Thr-Phe -Thr
`67 Ser -Leu -Ile -Gly-Asp-Lys-Ala-Ala-Leu -Thr
`67 Ser - Lys -Ser - Gly -Asn -Thr -Ala - Ser - Leu -Thr
`64 Ser - Lys -Ser - Gly - Ser - Ser - Ala -Thr - Leu -Ala
`
`4
`2
`1
`0
`0
`2
`2
`0
`0
`4
`75 Ile -Asx -Pro-Val -Glx -Ala -Asx-Asp-Val -Ala
`75 Ile -Ser -Ser-Leu-Gin -Pro -Glu-Asp-Ile -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-Glu -Ala
`c
`c
`c
`0
`0
`2
`4
`4
`4
`e
`85 Thr-Tyr -Phe-Cys- X - X - X - X - X - X
`85 Thr-Tyr -Tyr-Cys-Gln-Gln-Tyr-Gln-Ser -Leu
`87 Met-Tyr - Phe- Cys -Ala - Leu -Trp - Phe -Arg -Asx
`87 Asp-Tyr -Tyr-Cys-Ser -Ser -Tyr-Glu-Gly -Ser
`84 Asp-Tyr -Tyr-Cys-Gln -Ser -Tyr-Asp-Arg -Ser
`c
`c
`0
`4
`0
`e
`4
`3
`0
`- X - X - X -Phe-Gly-Gly-Gly-Thr -Lys
`
`95
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`MODELS OF ANTIGEN-BINDING SITES
`
`631
`
`Table I. r continued)
`
`(b) Alignment of V L sequences
`
`95
`-Pro -Tyr-Thr-Phe-Gly-Gln-Gly-Thr -Lys
`97
`-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
`104 Leu-Glu -Ile -Lys-Arg
`104 Leu-Gin - Ile -Thr-Gly
`106 Val -Thr - Val-Leu-Gly
`107 Val-Thr - Val-Leu - Gly
`105 Leu-Thr -- Val-Leu-Arg
`
`Cb) See footnote to a. The McPC-603 sequence is known only to residue 49; for the rest
`of the sequence, the most frequently occurring residue in other mouse kappa chains (Mc·
`Kean et al. 1973) is given for each position except for the hypervariable residues, which
`are simply designated by X. Ala 34, Thr 85, and Gly 100 in 603 project into the V11 :V1. inter·
`face but are not in actual contact with V,,; 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 315 hypervariable
`residues were measured from the model and ad(cid:173)
`justed using Diamond's model-building program
`(Diamond 1966). In building the DNP-hapten into
`the model, the nitro groups were assumed to be
`coplanar with the phenyl ring. Although the crystal
`structureof2,4-dinitroaniline has not been reported,
`theoretical arguments (Pauling 1948) would pre(cid:173)
`dict a planar molecule. This is consistent with the
`observat~ons of Trotter (Trotter 1961; Trotter and
`Williston 1966) on the crystals of various nitro(cid:173)
`phenyl compounds; Trotter found small departures
`from coplanarity but attributed these to intermo(cid:173)
`lecular forces in the crystal. In DNP-lysine and its
`derivatives, a hydrogen bond was assumed between
`one oxygen of the 2-nitro group and the NH of the
`1:-amino group of the lysine, thus fixing the position
`of the C1:.
`
`RESULTS
`
`Sequence Comparisons and Correlation with
`Structure
`Included in Table la,b is the structural classifi(cid:173)
`cation of each residue, as observed in 603, according
`to whether it is buried in the domain interior, ex(cid:173)
`posed to solvent, or involved in the contact with
`the homologous domain.
`
`Comparison of framework residues. As expected,
`the residues that are completely or mainly buried
`in the domain interiors are more conserved than
`those that are completely or mainly exposed to
`solvent. In V H, 16 of the 29 residues that are com(cid:173)
`pletely or mainly buried are identical in 315 and
`603. Of the buried residues that are different, a
`Val in 315 replaces a Thr (position 24), a Tyr re(cid:173)
`places a Phe (position 27l, and 2 Leu replace an Ile
`(position 48) and a Met (position 83) in 60::!. Of the
`
`residues that are completely or mainly exposed to
`solvent, 18 of 44 are identical, 5 involve Ser-Thr
`interchanges (positions 21, 25, 28, 30, and 71), a
`Lys replaces an Arg (position 44), and a Glu re(cid:173)
`places a Gin (position 76). The residues involved in
`beta bends (Venkatachalam 1968) are quite con(cid:173)
`served. The bend around position 14-15 is facili(cid:173)
`tated by the sequence Pro-Gly in 603; Pro 14 is also
`present in 315. The other hairpin bend around posi(cid:173)
`tion 41-42 is due to the sequence Pro-Gly which is
`present in both 315 and 603.
`The lack of complete sequence information for 603
`precludes a detailed correlation of the V1, sequences
`with structure. On the basis of the tentative se(cid:173)
`quence shown in Table lb, 17 of the 26 completely
`or mainly buried residues are identical in 315 and
`603 V1,. Of the buried residues that are different in
`315 and 603, a Thr in 315 replaces a Val in 603 at
`position 12 and a Leu replaces a Met at position 20.
`Of the completely or mainly exposed residues, 16 of
`41 are identical, and 2 Thr for Ser interchanges oc(cid:173)
`cur at positions 11 and 21. The Gly at position 16,
`which is involved in a beta bend, is present in both
`315 and 603, the sharp bend in 315 being further
`facilitated by Pro 15. Another bend around positions
`42-43 involves the sequence Pro-Gly in 603; Pro is
`also present at position 42 in 315. The additional
`residue at position 8 or 9 (Table lb) produces a kink
`in this region in kappa chains (Epp et al. 197 4; Segal
`et al. 1974a,b). However, the a-carbons ofresidues 8
`and 10 are only about 5 A apart, so that the excision
`of this additional residue requires only minor struc(cid:173)
`tural alterations. The substitutions at positions 45
`and 46 result in one more potential hydrogen bond
`in 315 compared to 603.
`The replacement of an interior residue that in(cid:173)
`volves a change in side-group size is generally
`accompanied by a compensatory substitution else(cid:173)
`where so that no large voids are created in the do(cid:173)
`main interiors. For example, in V H• the Ile (315)
`for Phe (603) substitution at position 29 is balanced
`
`7 of 13
`
`Celltrion, Inc., Exhibit 1116
`
`
`
`632
`
`PADLAN ET AL.
`
`by the Phe (315) 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, Val 86,
`and Leu 114 in 315. Furthermore, the replacement
`of Val 107 in 603 by a Tyr in 315 is counterbal(cid:173)
`anced by the substitution of a Gly in 315 for the
`Arg 98 in 603. In V1.. Leu 68 and Ala 73 of 315 re(cid:173)
`place Gly 66 and Phe 71, respectively, of603.
`Position 6 in 603 V1. is occupied by a Gin which is
`completely buried, whereas a Glu exists at this posi(cid:173)
`tion in 315. The substitution is structurally feasible
`since the Co:-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(cid:173)
`ment Gly-Gly-Gly at positions 101-103. In 315,
`Gly 101 is probably closer to the main body of Vi.,
`as in VH where a Glu also occurs at position 6.
`
`Comparison of residues involved in the V L:V11 con(cid:173)
`It is remarkable that the V H residues in(cid:173)
`tact.
`volved in the contact with V1. are virtually invari(cid:173)
`ant. Of the eight contacting residues, seven are
`identical and the other involves an Ile for Val inter(cid:173)
`change at position 38. Of the eight V1. residues in
`contact with V 11 , three are the same in 315 and 603.
`Substitutions at the other contact positions gen(cid:173)
`erally complement each other so that the relative
`disposition of the two variable domains is prob(cid:173)
`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 Ile 38, Gly 48, and
`Gly 51, respectively, could cause VL to move closer
`to V H 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 L com(cid:173)
`plements the change from 603 Lys 43 to 315 Asn
`43 in VH.
`
`Changes involving structural hypervariable resi(cid:173)
`dues. Certain changes in framework residues ap(cid:173)
`pear to be necessary to accommodate substitutions
`at hypervariable positions. For example, the re(cid:173)
`placement in V L of 603 Ile 2 by 315 Ala helps to ac(cid:173)
`commodate the bulky side group of Phe 94 from L3.
`Also in Vi., 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
`Ll region in 315. In V H, the replacement of the con(cid:173)
`tact residue Val 37 by Ile in 315 complements the
`Asn for Glu substitution at the hypervariable posi(cid:173)
`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(cid:173)
`parent. The middle of the hypervariable surface was
`dominated by a rather pronounced cavity. Com(cid:173)
`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 hypervariable loops
`of 315 is shown in Figure lA. For comparison, the
`hypervariable loops of 603 are 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 Hl and H2 on the other. The floor is
`formed by L3 , and the roof is formed primarily by
`H3. The hypervariable 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 Ll and H3, and no residues
`from L2 project into the binding cavity.
`The distorted fl-helix in Ll <Poljak et al. 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 Ll are
`either completely or mainly exposed to solvent. A
`pair of hydrogen bonds can be formed between main(cid:173)
`chain atoms of Asn 36 and Ala 91 ofL3. All the resi(cid:173)
`dues in L2 are exposed to solvent, and a beta bend
`is formed with residues in positions 51-54. In L3,
`Leu 92, Phe 94, and Val 99 are buried, Ala 91 pro(cid:173)
`jects into the V1.:V 11 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(cid:173)
`hypervariable Phe 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 Hl 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 47 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
`
`8 of 13
`
`Celltrion, Inc., Exhibit 1116
`
`
`
`MODELS OF ANTIGEN-BINDING SITES
`
`633
`
`99H
`
`36
`
`d~ '""~i31H Hl
`/36L
`~'~
`2JLSJ
`'"
`
`L3
`
`d'
`
`2
`SQ
`
`A
`
`B
`
`L 1
`
`Figure I. Stereo drawings of the hypervariable loops of ( AJ 315 and (BJ 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
`OR TEP program of Carroll K. Johnson (ORNL-3794).
`
`amide nitrogen of Asp IOI, as well as between the
`amide nitrogen of Phe 34 and the carbonyl oxygen
`of Asp 99. Phe 34 from HI introduces a rather bulky
`hydrophobic side group into the binding cavity.
`Beta bends are formed with residues 52-55 and
`6I-64 in H2. Leu 103 from H3 contributes to the
`hydrophobic nature of the hapten-binding site of
`3I5 and with LI 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 HI and
`Leu I03 of H3. A beta bend involves positions 100-
`I03 in H3. As in 603, H3 is in contact with LI.
`The residues that would appear to interact most
`with the hapten are Phe 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 all nonhydrogen atoms. The orientation and scale are
`the same as in Fig. lA. This figure is rotated through 90° in relation to the frontispiece.
`
`9 of 13
`
`Celltrion, Inc., Exhibit 1116
`
`
`
`634
`
`PADLAN ET AL.
`
`LEU\03
`
`PHE 34
`
`ASP 99
`I
`ASH 36 :< Y /r~
`--\~-~ '
`1TA-;". ~)
`~
`
`TAP 93PttE 98
`
`~ L TS 52
`
`~E 50
`Q
`
`ASP 99
`1 PHE ~4
`
`LEU\03
`
`RSH3K y Ar
`-\.~ .t
`1TA~~·~P~
`
`~ LTS 52
`
`I/HE 50
`
`1RP SJ PHE 98
`
`Figure 3. Stereo drawing of the hypervariable residues projecting into the 315 hapten-binding cavity. A drawing of
`BADE (without the bromine atom) is included in the figure to show the hapten-protein interactions and the possibility
`of affinity-labeling Tyr 34 (L). The figure is rotated 15° relative to Fig. IA.
`
`the light chain. The nitro groups of the DNP are
`