TH£ JOURNAL OF BIOLOGICAL CHEMISTRY
`Vol. 258. No. 23, Issue of De<>ember 10, pp. 14433-14437, 1983
`Printed in U.S.A.
`
`Molecular Anatomy of the Antibody Binding Site*
`
`JiH 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
`Harvard Medical School, Boston, Massachusetts 02114 and the Department of Chemistry, Harvard University,
`Cambridge, Massachusetts 02138
`
`
`
`(Received for publication, June 10, 1983)
`
`Wu and Kabat (15, 16) to have a role in antigen binding. The
`The binding region of immunoglobulins, which in­
`
`cludes the portion of the molecule having the most
`loops are relatively short segments of primary structure that
`in its amino acid sequence, is shown to have
`variability
`occur in homologous positions in all light and heavy chains,
`
`a surprisingly constant structure that can be charac­
`but have amino acid sequences and lengths which differ from
`
`terized in terms of a simple, well-defined model. The
`one antibody molecule to another. The x-ray structure shows
`binding region is composed of the antigen combining
`that each of the hypervariable loops forms a connection
`site plus its immediate vicinity and arises by noncova­
`between two antiparallel strands of ,8-sheet. By noncovalent
`
`lent association of the light and heavy chain variable
`association of the VL and VH domains, all six loops come
`
`domains (VL and VH, respectively). The antigen com­
`into close contact and form a contiguous area on the surface
`
`chain seg­bining site itself consists of six polypeptide
`
`of the VL-VH dimer from which the binding site is con­
`
`ments ("hypervariable loops") which comprise some 80
`structed (Fig. 1) (2, 11, 17-20).
`amino acid residues and are attached to a framework
`To obtain a consistent set of refined atomic coordinates
`of VL and VH P-sheet bilayers. Having analyzed re­
`from the crystallographic data (1-3, 29), we discarded atoms
`
`fined x-ray crystallographic coordinates for three an­
`which form the constant domains of the Fab fragment and
`
`tigen-binding fragments (Fab KOL (Marquart, M.,
`energy-minimized the resulting Fv fragments (i.e. VL-VH
`J., and Huber, R. (1980) J. Mol. Biol.
`Deisenhofer,
`domain dimers, the domains being defined as in Ref. 16) with
`141, 369-391), MCPC 603 (Segal, D., Padlan, E. A.,
`CHARMM, an empirical energy function program designed
`Cohen, G. H., Rudikoff, S., Potter, M., and Davies, D.
`R. (1974) Proc. Natl. Acad. Sci. U. S. A. 71, 4298-
`for the study of macromolecules (30). The root-mean-square
`differences between the original and energy-minimized coor­
`4302), and NEW (Saul, F. A., Amzel, L. M., and Poljak,
`dinates were 0.031 nm (0.31 A) for MCPC 603 and 0.021 nm
`R. J. (1978) J. Biol. Chem. 253, 585-597)) we use the
`for NEW; such small shifts indicate that the original crystal­
`results to introduce a general model for the VL-VH
`interface forming the binding region. The region con­
`lographic structures were satisfactory and that acceptable
`sists of two closely packed P-sheets, and its geometry
`values for the potential energy can be achieved by small
`
`
`corresponds to a 9-stranded, cylindrical barrel of av­
`adjustments of the coordinates.
`erage radius 0.84 nm with an average angle of -53°
`As a first step in deriving the geometric characteristics of
`
`
`between its two constituent P-sheets. The barrel forms
`the binding region, the boundaries between the parts of the
`the bottom and sides of the antigen combining site. The
`VL-VH interface forming ,8-sheet strands and hypervariable
`
`
`model demonstrates that the structural variability of
`loops were determined. The /j-sheets were defined by their
`the binding region is considerably less than was
`
`interstrand backbone (C==O· · -H-N) hydrogen-bonding
`
`thought previously. Amino acid residues which are
`pattern. A hydrogen bond list was generated with CHARMM
`
`part of the domain-domain interface and appear not to
`(30) for the four polypeptide chain segments which contribute
`
`be accessible to solvent or antigen contribute to anti­
`to the VL-VH interface (Fig. 2), and amino acids with hydro­
`body specificity.
`gen bond energy of -4.2 kJ /mol or less were taken to be parts
`of the .B-sheets. By this criterion, portions of the hypervariable
`loops (as defined in Ref. 16) form part of /j-sheets (Fig. 2).
`Examination of the four .B-sheet segments with adjacent by­
`pervariable loops by computer graphics showed that they
`consist of nine antiparallel fl-strands; the light chain P-sheet
`is 4-stranded, and the heavy chain .B-sheet 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
`/j-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
`• The work carried out in the Department of Chemistry was sup­
`
`ported in part by a grant from the National Institutes of Health. The
`
`"back-to-back ,8-barrels that share one wall occur in the
`
`
`costs of publication of this article were defrayed in part by the
`variable half of immunoglobulin Fab structures."
`payment of page charges. This article must therefore be hereby
`To provide a quantitative assessment of the geometry of
`with 18 U.S.C. Section 1734
`marked "advertisement" in accordance
`the binding region, a cylindrical surface was least-squares­
`
`solely to indicate this fact.
`fitted to the three VL-VH interface barrels (see Fig. 4 for
`
`
`details). Radii of the least-squares-fitted cylinders are very
`
`heavy chain variable domain.
`
`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 (VL1 and
`VH) are formed from two layers of /j-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
`
`1 The abbreviations used are: VL, light chain variable domain; VH,
`
`14433
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`

`14434
`
`Antibody Binding Site
`
`Fie. 1. Antibody binding region. A stereo a· carbon drawing of VL· VH domain dimer of mouse Fab fragment
`by heavy lines
`MCPC 603 (2). VL domain is at right; VH domain is at left. The interface
`
`P·sheets, represented
`(segments 81, 82, 83, and S4 of Fig. 2), are highly curved and their noncovalent contact forms a nearly perfect
`
`barrel the upper surface of which constitutes the antigen combining site. Ca atoms of hypervariable
`
`cylindrical
`
`
`residues are represented as heavy dots. The nine P·strands which form the VL-VH interface are numbered Pl
`
`
`
`through {J9, in correspondence with Fig. 2.
`
`60
`
`KO!. 51
`
`NEW 51
`
`25 27a b c
`28 30
`35
`40
`50
`55
`45
`- G S D S V N w y
`c s GT s s N I
`R p s
`RD AM
`Q Q I. p G H A p
`5
`K I. L I y
`G V p D R
`- - - -
`Q Q I. p G T A p
`- G AGN B V It w y
`c T GS s s N I
`H N N A R
`K I. L I F
`5
`c It s s 0 s LL NS G N Q It N p LA w y
`K I. I. I y
`GASTRBS
`51 5
`HCPC603
`Q Q K p G Q p p
`G V P D R
`Ll
`I
`Bl
`l-- L2 -[
`82
`
`KO!. 52
`
`NEW 52
`
`
`
`HCPC603 52
`
`98 100
`95 97a b
`85
`90
`5 D y y c A SW N N S D N A y v
`F G T G T I< v
`A D y y c 0 SYD RS - - L R v
`F G G G T K I.
`A v y y c Q N D B S Y P L - - T F G A G T K I.
`B3
`B4
`J-- L3--J
`
`KO!. 53
`
`50 52a b c 53
`30
`40
`65
`45
`35
`60
`55
`- -D 0 B
`5 s YAM y w v R Q A P G K G I. E W V
`I I W DP GS
`YA D S V It G R
`A
`F
`p L R S -- - R
`D D Y Y S T W V R Q p p G R G I. E W I
`Y V I!' Y B G T S
`D TD T
`F
`G
`53 F 5 0 p YM E W V R Q P p G K R I. E W I A A S R N It G N It y T TB y S A S v It G R
`HCPC603
`1-- 82
`BS
`B6
`tBl-j
`B7
`
`NEW 53
`
`KOL 54
`
`NEW 54
`
`HCPC603
`54
`
`1ooa b c d e r g h 101
`90
`105
`95
`T G v y F C A R D G GB G PC s s A s c p G p D Y W c Q G T p v
`- - - c I D V W G Q C 5 I. v
`T A v y y c A R D L I A G
`- - y F 0 V W c A G T T V
`T A I y y c A R N y Y G S T W --
`83
`BS
`B9
`
`F10. 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 P-sheet strands as defined in this paper (see
`
`text). Hypervariable loops as redefined in this paper are denoted by horizontal bars. Segments Sl and 82 are parts
`
`
`of light chain variable domain; segments 83 and 84 are parts of heavy chain variable domain. The numbering
`code (31).
`system is that of Ref. 16. Amino acids are given in the one-letter
`
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`Antibody Binding Site
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`14435
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`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
`VH contact surfaces, obtained as sums of the individual VL
`and VH interfaces, are 17.56 nm2 in KOL, 19.39 nm2 in
`MCPC and 603 and 17.09 nm1 in NEW. Thus, the average
`interfa�e area of a single variable domain is 9 nm2 (900 A 2),
`36% of which consists of polar residues. The values are on
`the order of those reported for other protein-protein interfaces
`(e.g., a/3 oxyhemoglobin dimer) (34). The hypervariable loops
`contribute about half of the total contact surface (4 7% in
`KOL, 53% in MCPC 603, and 42% 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
`ularly, side chains of residues Leu L96, Glu H35, and Asp
`HlOl, known to be indispensable for the specificity of phos­
`Fie. 3. Topology or the binding site barrel. Four hairpin
`segments, Sl to S4, primary structures of which are given in Fig. 2,
`
`phorylcholine-binding myelomas (43-45), are either totally or
`Segments.SI and S2
`
`organize themselves along a cylindrical.surface.
`partially buried in the VL-VH interface. Other hypervariable
`form the light chain P-sheet (on the right, counterclockwise, {32, PI,
`residues (COOH-terminal parts of L l and Hl, NHrterminal
`
`tJ3 and P4) as well as the three light chain hypervariable loops LI,
`parts of J gene-coded segments, i.e. L3 and H3) also form part
`
`Li and L3. Segments S4 and S3 form the heavy chain ti-sheet, consist
`of the VL-VH contact surface. Thus the domain-domain
`of five tJ-strands (on the lR/t, clockwise, pg, PB, fJ5, /16, and n'T), and
`contact surface, even though it is not accessible in the free
`
`the three heavy chain hypervariable loops Hl, H2, and H3. Geneti­
`
`cally, the strand ,:34 corresponds to the light chain J gene (2I, 22); �
`antibody structure, is directly involved in antibody specificity.
`
`corresponds to the heavy chain J gene (23-26); and the H3 loop 1s
`This suggests that, due to the close packing at the interface,
`
`coded for by the D gene segment (27, 28). The shapes of the hyper­
`changes in volumes of buried side chains may induce changes
`
`
`
`variable loops are indicated schematically, and only their most general
`in the bottom surface of the binding site. Such a result would
`
`
`
`features are given. Jn all three structures studied, the domain-domain
`be consistent with the structural variability found in hemo­
`
`interface is closely packed, leaving no free space between the two fJ·
`globin (46) and the core of immunoglobulin domains (47).
`sheets.
`Alternatively, structural changes induced by antigen binding
`(48-58) could expose some of these buried residues.
`Visual inspection of the Fv fragments (Figs. l 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 P-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
`NHrterminal residues of Ll, and five COOH-terminal resi­
`dues of H2. The NHi and COOH termini of Ll, L3, H3, and
`the middle of H2 are involved in backbone-backbone hydrogen
`bonds and have an extended conformation with backbone
`torsion angles ( 4> and 'I') in the fj-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.
`
`similar (0.82 nm for KOL, 0.86 nm for MCPC 603, and 0.84
`nm for NEW), and the root-mean-square deviation of back­
`bone atoms from the fitted surface (0.25 run for KOL, 0.22
`nm for MCPC 603, and 0.21 nm for NEW) is small considering
`that the fit includes parts of the hypervariable segments. For
`all three binding-site cylinders, we also computed angles
`between the backbone atoms of adjacent fJ-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 P-sheet is -20°, a value correspond­
`ing to that found for sheets 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 fJ2-f39 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 -90· (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 that the geometry
`of the VL-VH interface is an important 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 hypervariable residues play in
`domain-domain association (17, 39, 40), we computed the
`total contact surface between VL and VH domains. The
`
`Acknowledgments-We thank Ors. F. Saul, R. Poljak (Pasteur
`
`Baltimore). Institute, Paris). E. PadJan (Johns Hopkins University,
`
`
`
`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
`
`BI Exhibit 1061
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`

`

`a)
`
`d)
`
`b) (�
`
`�
`
`e)
`
`1CJ\� ��
`
`c) �A
`
`�A
`
`f)
`
`....
`
`I I
`
`...
`
`. I
`
`..... ,,,..
`,,,..
`�
`0)
`
`)..
`�
`g:
`�
`to
`;:;· R..
`�-
`VJ .....
`�
`
`F10. 4. Binding site barrels with cylindrical surfaces least squares fitted into them. Only the backbone atoms N, Ca, and C of the interface P-sheets (i.e. of all the nine
`Jt-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 passing through point a with unit direction vector, A, and a radial distance, r, from the axis (7 parameters). The error being minimized is r,(r - r,)2, where r1 is the distance
`of the i-th particle from the axis (r. = p, - {p,·A)A), where p, = x; - a, and x, is the position of an atom in the polypeptide chain backbone. Stereo views of a-carbon drawings parallel
`to (a-c) and perpendicular to (d-f) the cylinder axis are given. Thin lines represent fitt.ed 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 ti-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
`
`BI Exhibit 1061
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`

`Antibody Binding Site
`
`14437
`
`J. Matthews (Yale University) for computer programs, and R. Ladner
`and J. Ladner (Harvard University) for computer graphics.
`
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