`Vol . 258, No. 23, IS$Ue of December lO, pp. 14433- 14437, 1983
`Printed in l}.S.A.
`
`Molecular Anatomy of the Antibody Binding Site*
`
`Jii'i Novotny, Robert Bruccoleri, John Newell, David Murphy, Edgar Haber, and Martin Karplus
`From the Cellular and Molecular Research Laboratory and Cardiac Computer Center, Massachusetts General Hospital and
`Haruard Medical School, Boston, Massachusetts 02114 and the Department of Chemistry, Haruard University,
`Cambridge, Massachusetts 02138
`
`(Received for publication, June 10, 1983)
`
`The bindin.g region of immunoglobulins, which in·
`cludes the portion of the molecule having the most
`variability in its amino a.cid sequence, is shown to have
`a surprisingly constant structure that can be charac(cid:173)
`terized in terms of a simple, well-defined model. The
`binding region is composed of the antigen combining
`site plus its immediate vicinity and arises by noncova(cid:173)
`lent association of the light and heavy chain variable
`domains (VL and VH, respectively) . The antigen com(cid:173)
`bining site itself consists of six polypeptide chain seg(cid:173)
`ments ("hypervariable loops") which comprise some 80
`amino acid residues and are attached to a framework
`of VL and VH P-sheet bilayers. Having analyzed re(cid:173)
`fined x-ray crystallographic coordinates for three an(cid:173)
`tigen-binding fragments (Fab KOL (Marquart, M.,
`Deisenhofer, J., and Huber, R. (1980) J. Mol. Biol.
`141, 369-391), MCPC 603 (Segal, D., Padlan, E. A.,
`Cohen, G. H., Rudikoff, S., Potter, M., and Davies, D.
`R. (1974) Proc. Natl. Acad. Sci. U. S. A. 71, 4298-
`4302), and NEW (Saul, F. A., Amzel, L. M., and Poljak,
`R. J. (1978) J. Biol. Chem. 253, 585-597)) we use the
`results to introduce a general model for the VL-VH
`interface forming the binding region. The region con(cid:173)
`sists of two closely packed P-sheets, and its geometry
`corresponds to a 9-stranded, cylindrical barrel of av(cid:173)
`erage radius 0.84 nm with an average angle of -53°
`between its two constituent P-sheets. The barrel forms
`the bottom and sides of the antigen combining site. The
`model demonstrates that the structural variability of
`the binding region is considerably less than was
`thought previously. Amino acid residues which are
`part of the domain-domain interface and appear not to
`be accessible to solvent or antigen contribute to anti(cid:173)
`body specificity.
`
`Immunoglobulins have a modular structure in that both the
`light chains (M, = 25,000) and the heavy chains (M. = 50,000)
`(4, 5) are composed of several domains (6-8), each of which
`consists of approximately 110 amino acid residues. The NHr
`terminal variable domains (9, 10) of both chains (VV and
`VH) are formed from two layers of ~-sheet (1- 3, 11- 13) and
`have been shown to fold independently of the rest of the
`polypeptide chain (14). Six hypervariable loops of the VL and
`VH domains (Ll, L2, L3, Hl, H2, and H3) were predicted by
`
`•The work carried out in the Department of Chemistry was sup(cid:173)
`ported in part by a grant from the National Institutes of Health. The
`costs of publication of this article were defrayed in part by the
`payment of page charges. This article must therefore be hereby
`marked "aduertisement" in accordance with 18 U.S.C. Section 1734
`solely to indicate t his fact.
`1 The abbreviations used are: VL, light chain variable domain; VH,
`heavy chain variable domain.
`
`Wu and Kabat (15, 16) to have a role in antigen binding. The
`loops are relatively short segments of primary structure that
`occur in homologous positions in all light and heavy chains,
`but have amino acid sequences and lengths which differ from
`one antibody molecule to another. The x-ray structure shows
`that each of the hypervariable loops forms a connection
`between two antiparallel strands of P-sheet. By noncovalent
`association of the VL and VH domains, all six loops come
`into close contact and form a contiguous area on the surface
`of the VL-VH dimer from which the binding site is con(cid:173)
`structed (Fig. 1) (2, 11, 17-20).
`To obtain a consistent set of refined atomic coordinates
`from the crystallographic data (1-3, 29), we discarded atoms
`which form the constant domains of the Fab fragment and
`energy-minimized the resulting Fv fragments (i.e. VL-VH
`domain dimers, the domains being defined as in Ref. 16) with
`CHARMM, an empirical energy function program designed
`for the study of macromolecules (30). The root-mean-square
`differences between the original and energy-minimized coor(cid:173)
`dinates were 0.031 nm (0.31 A) for MCPC 603 and 0.021 nm
`for NEW; such small shifts indicate that the original crystal(cid:173)
`lographic structures were satisfactory and t hat acceptable
`values for the potential energy can be achieved by small
`adjustments of the coordinates.
`As a first step in deriving the geometric characteristics of
`the binding region, the boundaries between the parts of the
`VL-VH interface forming P-sheet strands and hypervariable
`loops were determined. The ~-sheets were defined by their
`interstrand backbone (C=O .. -H-N) hydrogen-bonding
`pattern. A hydrogen bond list was generated with CHARMM
`(30) for the four polypeptide chain segments which contribute
`to the VL-VH interface (Fig. 2), and amino acids with hydro(cid:173)
`gen bond energy of-4.2 kJ/mol or less were taken to be parts
`of the P-sheets. By this criterion, portions of the hypervariable
`loops (as defined in Ref. 16) form part of ti-sheets (Fig. 2).
`Examination of the four P-sheet segments with adjacent hy(cid:173)
`pervariable loops by computer graphics showed that they
`consist of nine antiparallel ti-strands; the light chain P-sheet
`is 4-stranded, and the heavy chain .B-shcet is 5-stranded. The
`atom-packing density (32) of the sheet-sheet interface is on
`the order of that found in interiors of other proteins (33, 34).
`The sheets are strongly curved and wrap counterclockwise
`around a nearly perfect cylindrical surface (Fig. 3). Although
`.fl-barrels are frequently found among protein structures (35),
`it is unusual to find one formed of sheets contributed by
`different polypeptide chains. Richardson (35) has noted that
`"back-to-back .B-barrels that share one wall occur in the
`variable half of immunoglobulin Fab structures."
`To provide a quantitative assessment of the geometry of
`the binding region, a cylindrical surface was least-squares(cid:173)
`fitted to the three VL-VH interface barrels (see Fig. 4 for
`details). Radii of the least-squares-fitted cylinders are very
`
`14433
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`1 of 5
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`14434
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`Antibody Binding Site
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`Fie. 1. Antibody binding region. A stereo a-carbon drawing of VL-VH domain dimer of mouse Fab fragment
`MCPC 603 (2). VL domain is at right; VH domain is at 1.eft. The interface fJ·sheets, represented by heauy lines
`(segments Sl, 82, S3, and S4 of Fig. 2), are highly curved and their noncovalent contact forms a nearly perfect
`cylindrical barrel the upper surface of which constitutes the antigen combining site. Ca atoms of hypervariable
`residues are represented as heauy dots. The nine ,S·strands which form the VL· VH interface are numbered ,Sl
`through fJ9, in correspondence with Fig. 2.
`
`KOL S1
`NEW S 1
`HCPC603
`
`S1
`
`40
`28 30
`25 27a b c
`60
`50
`45
`35
`55
`s c s GT S S N I
`0 v p D R
`- G S D S V N W Y Q Q L p 0 H A p K L L I y ROAM R p s
`-
`- GAG N B v J[ w y Q Q L p G T A P K L L I
`s c T GS SS N I
`H N N A R
`f' -
`-
`-
`-
`-
`-
`-
`s c Its s 0 s LL H S G N 0 J[ N p L A W Y Q Q K p G Q p p K L L I y GASTRE s
`G V P D R
`I
`Ll
`81
`82
`l - -L2 - [
`
`KOL S2
`NEW S2
`HCPC603
`
`S2
`
`95 97a b 98 100
`90
`85
`s D y y c A SW N NS DNA y v
`f' G T G T K v
`A D y y c 0 S Y D R S -
`- L R v f' 0 G G T K L
`A v y Y C 0 N D B S Y P L -
`- T f' 0 A G T K L
`84
`83
`l - - L3 - - I
`
`KOL S3
`NEW S3
`HCPC603
`
`S3
`
`30
`40
`45
`60
`52a b c 53
`50
`55
`65
`35
`f' s s y AMY .,, v R Q A P G K G L E W V A I
`I W DD G s - - D 0 B YA D s v It G R
`F D D y Y S T W V R Q p p G R G L E w I G YVPYBGT s OTOT PL RS
`- R
`F S 0 F Y M B W V R Q p p G K R L E W I A A S R N It G N It y TT B Y SAS V It G R
`86 1 - - 82
`BS
`87
`~Bl-!
`
`KOL S4
`NEW S4
`HCPC 603
`
`S4
`
`100a b c d e r g h
`105
`101
`90
`95
`T G v y F C A R DGGBGFC s s A S c F G P D 'l W 0 Q G T p v
`'{ c A R D L
`- - - c I 0 V W G Q G s L V
`T A v y
`I AG
`'{ 'f C A R N y Y GS T w - -
`T A I
`y F 0 V W G A G T T V
`-
`-
`68
`69
`83
`
`FIG. 2. Polypeptide-chain segments which constitute the binding region and the VL-VH interface.
`Homologous segments from different structures are aligned to maximize amino acid homology. Boldface letters
`indicate hypervariable loops (as defined in Ref. 16). Boxes denote fJ·sheet strands as defined in this paper (see
`text). Hypervariable loops as redefined in this paper are denoted by horizontal bars. Segments Sl and S2 are parts
`of light chain variable domain; segments S3 and S4 are parts of heavy chain variable domain. The numbering
`system is that of Ref. 16. Amino acids are given in the one· letter code (31).
`
`2 of 5
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`Antibody Binding Site
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`14435
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`H1
`
`H3
`
`J Se<jment
`
`Fie. 3. Topology of the binding site barrel. Four hairpin
`segments, SI to $4, primary structures of which are given in Fig. 2,
`organize themselves along a cylindrical.surface. Segments.Si and S2
`form the light chain fJ-sheet (on the r1Hht, counterclockw1Se, tJ2. {H,
`/33 and fl4) as well as the three light chain hypervariable loops LI,
`Li and L3. Segments $4 and $3 form the heavy chain tJ-sheet, consist
`of five /3-strands (on the left, clockwise, pg, {JS, {J5, fJ6, and ff7), and
`the three heavy chain hypervariable loops HI, H2, and HJ. Geneti(cid:173)
`cally, the strand fJ4 corresponds to the light chain J gene (21, 22); ~
`corresponds to the heavy chain J gene (23- 26); and the H3 loop 1s
`coded for by the D gene segment (27, 28). The shapes of the hyper(cid:173)
`variable loops are indicated schematically, and only their most general
`features are given. In all three structures studied, the domain-domain
`interface 1s closely packed, leaving no free space between the two fJ·
`sheets
`
`similar (0.82 nm for KOL, 0.86 nm for MCP C 603, and 0.84
`nm fo r NEW), and the root-mean-square deviation of back(cid:173)
`bone atoms from the fitted surface (0.25 nm for KOL, 0.22
`nm fo r MCP C 603, and 0.21 nm for NEW) is small considering
`that t he fit includes parts of the hypervariable segments. For
`all three binding-site cylinders, we also computed angles
`between the backbone atoms of adjacent /J·strands; the back·
`bone atoms were represented by straight, least-squares-fitted
`lines (axes of inertia). The average twist angle between adja·
`cent strands in the same fl-sheet is -20•, a value correspond·
`ing to that found for s heets in other proteins (36-38). If the
`binding site barrels were perfect cylinders, strands which form
`sheet-sheet junctions would be expected to subtend the same
`angle (i.e. - 20"); the angles are, in fact, significantly larger
`(the fJ4-fJ7 angle is - 57• and the /J2- {39 angle is 33°).
`Interacting fJ·sheets of a single domain normally pack with
`the relative orientation of the sheet axes equal to either -30•
`or -oo· (36-38); the sheet -sheet angle of the VL-VH interface
`by contrast is -52· in KOL, -54• in MCPC 603, and -52• in
`NEW.
`The KOL, MCPC 603, and NEW interface cylinders closely
`resemble each other (Fig. 4), and their interface /J·sheet
`segments which are adjacent to hypervariable loops have
`virtually identical primary structures. The same, or very sim·
`ilar, amino acid sequences occur in corresponding segments
`of all immunoglobulins (16). This suggests t hat the geometry
`of the VL-VH interface is an impon.ant feature of the anti·
`body binding region and that structurally similar 9-stranded
`cylinders form the essential element of all the binding regions;
`the antigen combining site is the concave part of the "upper"
`surface of the cylinder (Fig. 3).
`To ascertain the role which hyperva.riable residues play in
`domain-domain association (17, 39, 40), we computed the
`total contact surface between VL and VH domains. The
`
`interdomain contact surfaces were obtained as differences
`between total solvent-accessible surface (41, 42) of isolated
`domains and of the domains involved in the dimer. The VL(cid:173)
`VH contact surfaces, obtained as sums of the individual VL
`and VH interfaces, are 17.56 nm2 in KOL, 19.39 nm 2 in
`MCPC, and 603 and 17.09 nm2 in NEW. Thus, the average
`interface area of a single variable domain is 9 nm2 (900 A2
`),
`36% of which consists of polar residues. The values are on
`the order of those reported for other protein-protein interfaces
`(e.g., afJ oxyhemoglobin dimer) (34). The hypervariable loo~s
`contribute about half of the total contact surface (47% m
`KOL, 53% in MCPC 603, and 423 in NEW).
`Single amino acid exchanges in hypervariable positions at
`the bottom of the binding site and inaccessible to solvent or
`antigen have a dramatic effect on antibody specificity. Partic(cid:173)
`ularly, side chains of residues Leu L96, Glu H35, and Asp
`H lOl, known to be indispensable for the specificity of phos·
`phorylcholine-binding myelomas (43-45), are either totally or
`partially buried in the VL-VH interface. Other hypervariable
`residues (COOH -terminal parts of Ll and Hl, NH1-terminal
`parts of J gene-coded segments, i.e. L3 and H3) also form part
`of the VL· VH contact surface. Thus the domain-domain
`contact surface, even though it is not accessible in the free
`antibody structure , is directly involved in antibody specificity.
`This suggests that, due to the close packing at the interface,
`changes in volumes of buried side chains may induce changes
`in the bottom surface of the binding site. Such a result would
`be consistent with the structural variability found in hemo·
`globin (46) and the core of immunoglobulin domains (47).
`Alternatively, structural changes induced by antigen binding
`(48-58) could expose some of these buried residues.
`Visual inspection of the Fv fragments (Figs. 1 and 3) shows
`that some of the 80 amino acid residues which constitute the
`hypervariable loops are distant from the binding site surface
`and may not be directly involved in antigen binding. Others,
`as indicated in Fig. 2, participate in the well-defined /J·sheet
`secondary structures, so that their conformational freedom is
`rather limited. Those which are distant from the site include
`the whole L2 loop (it has been observed in the past that L2
`is not directly involved in antigen binding (2, 59, 60)), two
`NHr terminal residues of Ll, and five COOH-terminal resi(cid:173)
`dues of H2. The NH1 and COOH termini of Ll, L3, H3, and
`the middle of H2 are involved in backbone-backbone hydrogen
`bonds and have an extended con formation with backbone
`torsion a ngles (cf> and 'It) in t he /J-sheet range. Of the 80 amino
`acids in the hypervariable loops (16), only 47 are intimately
`involved in the binding site, having at the same time confor·
`mations which vary in such a way that they cannot be inferred
`by analogy to known structures. This greatly reduces the
`problem of determining the binding site geometry of immu·
`noglobulins with unknown structure, although, due to varia·
`tions in the lengths of hypervariable loops, the exact number
`of structurally variable positions is expected to vary somewhat
`in different molecules.
`The model proposed here represents the first part of a
`project aimed at developing a method for the analysis of the
`antigen binding region including prediction of its structure
`and evaluation of the interactions between the antibodies and
`the antigen.
`
`Acknowledgments- We lhank Ors. F. Saul, R. Poljak (Pasteur
`Institute, Paris), E. Padlan (Johns Hopkins University, Baltimore).
`D. Davies (National Institutes of Health, Bethesda), M. Marquart,
`R. Huber (Max-Planck lnstitut, Munich), and E. Abola (Brookhaven
`Data Bank) for crystallographic coordinates. Ors. F. M. Richards and
`
`3 of 5
`
`Celltrion, Inc., Exhibit 1061
`
`
`
`a)
`
`d)
`
`-.i:..
`
`.i:..
`~
`O')
`
`b) (~ ~
`
`e)
`
`~ ~
`
`~\i._ ,.
`
`;;:s ....
`~
`~
`tl::l
`[
`~·
`ff)
`~:
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`c) ~. ~~
`
`f)
`
`...
`
`I
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`
`F10. 4. Binding site barrels w ith cylindrical surfaces least squares fitted into them. Only the backbone atoms N, Ca, and C of the interface .8-sheets (i.e. of all the nine
`ft-strands including the hypervariable loops L3 and H3 of KOL, MCPC 603, and NEW) were subjected to the surface-fitting algorithm. The cylindrical surface is defined by an axis
`vector pMsing through point a with unit direction vector, X, and a radial distance, r, from the axis (7 parameters). The error being minimized is }:,(r - r.)2, where r, is the distance
`(p ,·X)X), where p ; = x, - a, and x, is the position of an atom in the polypeptide chain backbone. Stereo views of a-carbon drawings parallel
`of the i-th particle from the axis (r, = p , -
`to (a-cl and perpendiculer to (d-f) the cylinder axis a.re given. Thin lines represent fitted results (cylinder axis, cylinder radius, and the cylindrical surface); medium lines correspond
`to the light chain P·sheet; and heavy lines correspond to the heavy chain ft-sheet. a) KOL (cylinder radius, R "" 0.82 nm, root-mean-square distance of backbone atoms from the fitted
`cylindrical surface is d = 0.25 nm); b) MCPC 603 (R = 0.86 nm, d = 0.22 nm); c) NEW (R • 0.84 nm, d = 0.21 nm); d) KOL; e) MCPC 603; /)NEW.
`
`4 of 5
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`Celltrion, Inc., Exhibit 1061
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`Antibody Binding Site
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`14437
`
`J. Matthews (Yale University) for computer programs, and R. Ladner
`and J. Ladner (Harvard University) for computer graphics.
`
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`Celltrion, Inc., Exhibit 1061
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