`Vol. 86, pp. 5938-5942, August 1989
`Immunology
`
`Structure of an antibody-antigen complex: Crystal structure of the
`HyHEL-10 Fab-lysozyme complex
`(x-ray crystallography/complementarity/discontinuous epitope)
`EDUARDO A. PADLAN*, ENID W. SILVERTON*, STEVEN SHERIFF*t, GERSON H. COHEN*,
`SANDRA J. SMITH-GILLt, AND DAVID R. DAVIES*
`*Laboratory of Molecular Biology, National Institute of Diabetes, Digestive and Kidney Diseases, and *Laboratory of Genetics, National Cancer Institute,
`National Institutes of Health, Bethesda, MD 20892
`Contributed by David R. Davies, April 24, 1989
`
`The crystal structure of the complex of the
`ABSTRACT
`anti-lysozyme HyHEL-10 Fab and hen egg white lysozyme has
`been determined to a nominal resolution of3.0 A. The antigenic
`determinant (epitope) on the lysozyme is discontinuous, con-
`sisting of residues from four different regions of the linear
`sequence. It consists of the exposed residues of an a-helix
`together with surrounding amino acids. The epitope crosses the
`active-site cleft and includes a tryptophan located within this
`cleft. The combining site of the antibody is mostly flat with a
`protuberance made up oftwo tyrosines that penetrate the cleft.
`All six complementarity-determining regions of the Fab con-
`tribute at least one residue to the binding; one residue from the
`framework is also in contact with the lysozyme. The contacting
`residues on the antibody contain a disproportionate number of
`aromatic side chains. The antibody-antigen contact mainly
`involves hydrogen bonds and van der Waals interactions; there
`is one ion-pair interaction but it is weak.
`
`The interaction of antibodies with protein antigens has been
`the subject of several recent crystallographic investigations.
`These include complexes of hen egg white lysozyme with the
`Fab fragments ofthe monoclonal anti-lysozymes D1.3 (1) and
`HyHEL-5 (2) and a Fab complex with influenza neuramini-
`dase (3). From these data a common pattern of interaction is
`emerging (4) in which there is a high degree of complemen-
`tarity between the interacting surfaces of the antibody and
`antigen; the epitope is made up of several small, discrete
`segments of the polypeptide chain; and relatively small
`conformational changes occur in the antigen as a result of
`binding. Here we report the x-ray analysis of HyHEL-10
`Fab-lysozyme, in which the antigenic site differs from the
`two previous examples. The results complement the previous
`studies but differ from them in several ways.
`HyHEL-10 is an IgGl(K) antibody specific for hen egg
`white lysozyme. The affinity of HyHEL-10 for hen egg white
`lysozyme, as estimated by PEG immunoprecipitation, is 1.5
`x 109 M-1 (M. E. Denton and H. A. Scheraga, personal
`communication), slightly lower than that of HyHEL-5, thus
`making HyHEL-10 intermediate in affinity between HyHEL-
`5 and D1.3.§
`HyHEL-10 expresses a member of the VH36-6O variable
`gene segment family, the DQ52 diversity gene segment, and
`the JH3 joining gene segment in the heavy (H) chain and a
`VK23 gene and JK2 in the light (L) chain (9). Thus, HyHEL-10
`is structurally distinct from HyHEL-5 (which expresses
`VHJ558 and VK4) and D1.3 (which expresses VHQ52 and
`VK12/13).
`
`The publication costs of this article were defrayed in part by page charge
`payment. This article must therefore be hereby marked "advertisement"
`in accordance with 18 U.S.C. §1734 solely to indicate this fact.
`
`MATERIALS AND METHODS
`Crystals ofthe complex ofHyHEL-10 Fab with hen egg white
`lysozyme, grown as described (11), exhibit the symmetry of
`space group P212121 with a = 57.47, b = 118.73, c = 137.68
`Aand one Fab-lysozyme complex per asymmetric unit.
`Intensity data were collected with the Mark II multiwire
`detector system at the University of California, San Diego
`(12). The R factor relating the intensities of symmetry-related
`reflections (12) was 0.066. The data set used in the structure
`analysis had 12,501 reflections beyond io.0-A spacings with F
`, 30(F). These constitute about 78% of the theoretically
`observable reflections between 10.0- and 3.1-A spacings; an
`additional 5% of the reflections between 3.1 and 3.0 A are
`present in this data set.
`The structure was determined by molecular replacement
`(13) using a predecessor of the program package MERLOT
`(14). Rotation and translation searches were performed in-
`dependently (15) for the lysozyme, Fv (module containing VH
`and VL, the variable domains of the H and L chains), and
`CL/CH1 (constant domain of L chain/flrst constant domain of
`H chain) portions of the structure. In the search for the
`orientation of the lysozyme and the Fv, the highest peaks in
`the rotation function turned out to be the correct peaks. The
`correct peak in the rotation search for the CL/CH1 was only
`the seventh highest. The translation search gave unambigu-
`ous results in all three cases. Details of the molecular
`replacement analysis will be published elsewhere (S.S.,
`E.A.P., G.H.C., and D.R.D.). The molecular probes that
`proved useful in the analysis were hen egg white lysozyme
`from the refinement analysis of Diamond (16) [Protein Data
`Bank (PDB) File 6LYZ], the Fv of McPC603 (17) (PDB File
`1MCP), and the CL/CH1 of HyHEL-5 (2) (PDB File 2HFL).
`The orientations and positions of the various parts of the
`complex were refined with CORELS (18) allowing the VL, VH,
`CL and CH1 domains and lysozyme to move independently.
`The structure was then subjected to restrained least-squares
`
`Abbreviations: H, heavy; L, light; VL and VH, variable domains of
`L and H chains; CL and CH1, constant domain of L chain and first
`constant domain of H chain; Fv, module containing VL and VH;
`CDR, complementarity-determining region; CDRn-L or CDRn-H,
`nth CDR of L or H chain.
`tPresent address: Squibb Institute for Medical Research, P.O. Box
`4000, Princeton, NJ 08543-4000.
`§Using PEG immunoprecipitation at pH 7.2, Denton and Scheraga
`determined association constants of 1.5 x 109 M-1 and 2.5 x 109
`M-1 for HyHEL-10 and HyHEL-5, respectively. Lavoie et al. (5)
`determined association constants of -4 x 109 M-1 and -1.4 x 1010
`M-1 at pH 8.2 by the method of Friguet et al. (6). The association
`constant for D1.3 Fab, also determined by the method of Friguet et
`al., has been reported (7) as 4.5 x 107 M-1 at pH 7.4; more recently,
`an association constant of 1.3 x 108 M-1 was determined by fluores-
`cence quenching (8).
`
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`PETITIONER'S EXHIBITS
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`refinement using the program PROLSQ (19, 20) and model
`rebuilding on the basis of OMIT maps (21) using the graphics
`
`Proc. Natl. Acad. Sci. USA 86 (1989)
`
`5939
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`program FRODO (22). The final R value was 0.24 with devi-
`ations from ideality of 0.011 A for bond lengths and of 0.034
`
`Stereo diagrams. (a)
`FIG. 1.
`ca-Carbon trace of the HyHEL-10
`Lyso-
`Fab-lysozyme complex.
`zyme is shown in white, VL in
`yellow, VH in light blue, CL in red,
`and CH1 in dark blue. (b) Same as
`a and showing the interacting sur-
`faces: the surface covering the
`epitope in green and the surface
`covering the contacting residues
`from the Fab in magenta. At left,
`the complex is as it is in the crystal
`structure; in the middle and at
`right, the lysozyme has been sep-
`arated from the Fab by 7 A and by
`14 A, respectively. (c) Backbone
`of HyHEL-10 Fv and lysozyme
`with the contacting side chains
`from HyHEL-10 shown in red and
`those from the lysozyme shown in
`yellow. The rest of the helical re-
`gion (lysozyme residues 88-99)
`and VL are shown in light blue,
`and VH is shown in dark blue. (d)
`HyHEL-10 Fv showing the CDRs
`in yellow and the contacting resi-
`dues in red. VL is on the left (light
`blue) and VH is on the right (dark
`blue). (e) The HyHEL-10 epitope
`on lysozyme showing the contact-
`ing residues in red. The helical
`region 88-99 is shown in yellow
`and the rest of the lysozyme in
`light blue.
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`PETITIONER'S EXHIBITS
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`
`constitute the external surface of the helix; Ser-100, Asp-101,
`and Gly-102, which extend beyond the helix; Trp-63, which
`is in the active-site cleft; and Arg-73 and Leu-75, which are
`on the other side of the cleft (Fig. le). In addition, Asn-19,
`Asn-103, and Ala-107 are partly buried by the interaction with
`the antibody, although not in actual contact by the criteria we
`have used. Four of these residues participate in the contact
`with the antibody only through their main-chain atoms (His-
`15, Gly-16, Ile-98, and Gly-102). Most of the contacting
`residues are polar and five of them are charged.
`Structure of the Combining Site. The surface of HyHEL-10
`that interacts with lysozyme is unusual in that it is not
`noticeably concave and contains no pronounced grooves or
`cavities. On the contrary, the surface has a large protrusion,
`which fits into the active-site cleft of lysozyme. This protru-
`sion is formed by the side chains of Tyr-33 from CDR1-H and
`Tyr-53 from CDR2-H (Fig. lb). The interacting surface of the
`antibody contains a disproportionate number ofaromatic side
`chains that point outward and that interact with the antigen
`(Fig. ic; Table 1). Large numbers of aromatic residues have
`also been observed in the combining sites of McPC603 (17)
`and D1.3 (1) and in the presumed binding site of the human
`class I major histocompatibility antigen A2 (33).
`All six CDRs participate in the interaction with the lyso-
`zyme. The CDRs of the L chain contribute 8 residues to the
`contract and those of the H chain contribute 10. One addi-
`tional residue from the H chain, Thr-30, comes from the
`framework. CDR2-H has the largest number of contacting
`residues with 6, while CDR3-H has only 1 (Table 1). For 3 of
`the residues (Gly-30, Ser-91, and Asn-92, all from the L
`chain), only their main-chain atoms are involved in the
`contact. Seven of the contacting residues have aromatic side
`chains: Tyr-50 and -96 from the L chain; Tyr-33, -50, -53 and
`-58 and Trp-95 from the H chain. Only one side chain, that of
`Asp-32 of the H chain, is charged. In addition to the 19
`contacting residues, Ser-93 and Trp-94 of the L chain are
`partly buried by the interaction with the antigen. The surface
`area on the antibody that is buried by the interaction with the
`lysozyme is 720 A2.
`Conformational Changes in the Antigen. No major confor-
`mational changes occur in the structure of the lysozyme when
`it binds to HyHEL-10. Comparison of the complexed lyso-
`zyme with the uncomplexed structure (coordinates of tetrag-
`onal lysozyme courtesy ofD. C. Phillips) gives a rms deviation
`of 0.47 A for corresponding a carbons, with significant differ-
`ences occurring at positions 47, 101, and 102 having deviations
`of 1.44, 1.80, and 2.13 A, respectively. Larger differences are
`found for the side chains, most notably with the aromatic ring
`of Trp-62, which has been rotated by 150 degrees about the
`CO-Cy bond presumably in order to avoid close steric inter-
`actions with a tyrosine side chain from the antibody.
`Forces Between the Antibody and the Antigen. The com-
`plementarity of the contacting surfaces of HyHEL-10 and
`lysozyme is so great that there are no cavities in the interface
`large enough to accommodate a water molecule. The inter-
`action between the two proteins (Fig. ic) consists of polar
`and apolar interactions; of the 126 pairwise atomic contacts
`
`A for angle distances and with a deviation from planarity of
`0.004 A. The refined coordinates have been deposited in the
`Protein Data Bank (23) (File 3HFM). The error in atomic
`positions was estimated (24) to be 0.4 A.
`Molecular surface representations were computed with the
`program MS (25) using a probe radius of 1.5 A and standard van
`der Waals radii (26). Atomic contacts were defined according
`to the criteria of Sheriff et al. (27). The various domains of
`HyHEL-10 Fab were compared with the following immuno-
`globulin structures: McPC603 and J539 (28) (PDB File 1FBJ),
`HyHEL-5 and D1.3 (courtesy of R. Poijak, Pasteur Institute),
`KOL (29) (PDB File 1FB4), NEW (30) (PDB File 3FAB), and
`REI (31) (PDB File 1REI). Least-squares superposition of
`structures was accomplished with the program ALIGN (written
`by G.H.C.); only a carbons were used in the superpositions.
`ALIGN reports the individual deviations and the rms deviation
`between structurally equivalent pairs of atoms. The number-
`ing scheme used here for the HyHEL-10 residues follows the
`convention of Kabat et al. (32).
`
`RESULTS
`Overall Structure. Fig. la shows the a-carbon trace of the
`HyHEL-10 Fab-lysozyme complex. The contact between
`lysozyme and HyHEL-10 involves the complementarity-
`determining regions (CDRs) of the antibody with the exterior
`of the lysozyme helix (residues 88-99) and some surrounding
`amino acid residues. The two interacting surfaces (Fig. lb) are
`strikingly complementary so that solvent is completely ex-
`cluded from the interface. The helix in the epitope is oriented
`diagonally across the combining site so that its N terminus
`interacts with the second CDR of the L chain (CDR2-L)
`whereas its C terminus and the segment beyond it interact
`mainly with CDR1-H and CDR2-H (Fig. 1 c and d; Table 1).
`The Epitope. The lysozyme epitope for HyHEL-10 is quite
`discontinuous, consisting of residues coming from distant
`parts of the linear sequence but made contiguous by the
`folding of the protein. The area of lysozyme that is in contact
`with the antibody is 774 A2.
`The lysozyme residues that contact the antibody are His-
`15, Gly-16, Tyr-20, and Arg-21, which are on one side of the
`helix; Thr-89, Asn-93, Lys-96, Lys-97, and Ile-98, which
`
`Gly-16
`His-15, Gly-16, Lys-96
`Gly-16, Tyr-20
`Asn-93, Lys-96
`Thr-89, Asn-93
`Tyr-20
`Tyr-20, Arg-21
`Arg-21
`
`Table 1. HyHEL-10 residues in contact with lysozyme
`Lysozyme residue(s)
`HyHEL-10 residue*
`VL
`Gly-30
`Asn-31 (h)
`Asn-32 (h)
`Tyr-50
`Gln-53 (h)
`Ser-91 (m)
`Asn-92 (m,h)
`Tyr-96 (h)
`VH
`Thr-30t
`Arg-73
`Ser-31 (h)
`Arg-73, Leu-75
`Asp-32 (s)
`Lys-97
`Trp-63, Lys-97, Ile-98, Ser-100, Asp-101
`Tyr-33 (h)
`Arg-21, Ser-100
`Tyr-50 (h)
`Ser-52 (h)
`Asp-101
`Tyr-53 (h)
`Trp-63, Leu-75, Asp-101
`Ser-54
`Asp-101
`Asp-101, Gly-102
`Ser-56
`Arg-21, Ser-100, Gly-102
`Tyr-58 (h)
`Arg-21, Lys-97, Ser-100
`Trp-95
`*Nature of interaction is indicated in parentheses: m, main-chain
`atoms only; h, hydrogen bonding; s, salt bridge.
`tFramework residue.
`
`Table 2.
`VL
`Asn-31 OD1
`Asn-32 ND2
`Gln-53 OE1
`Gln-53 NE2
`
`Ser-91 0
`Asn-92 0
`Tyr-96 OH
`
`Hydrogen bonds between HyHEL-10 and lysozyme
`VH
`Lysozyme
`Lysozyme
`Thr-30 0
`Lys-96 NZ
`Arg-73 NH1
`Gly-16 0
`Arg-73 NH1
`Ser-31 OG
`Lys-97 0
`Tyr-33 OH
`Asn-93 ND2
`Arg-21 NH1,
`Tyr-50 OH
`Asn-93 OD1
`Ser-100 0
`Asp-101 OD1
`Gly-102 N
`
`Tyr-20 OH
`Arg-21 N
`Arg-21 NH1
`
`Tyr-53 0
`Tyr-58 OH
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`(Table 1), 111 are van der Waals contacts and 14 are hydro-
`gen-bonding contacts (Table 2).
`Although 6 of the contacting residues-Asp-32 from the H
`chain and Arg-21 and -73, Lys-96 and -97, and Asp-101 from
`the lysozyme-are probably charged under the conditions of
`our experiment, we find only one salt bridge, between Asp-32
`from HyHEL-10 and Lys-97 from lysozyme, with a separa-
`tion of 3.6 A between the side-chain nitrogen of the lysine and
`the nearer oxygen in the carboxyl group of the aspartate side
`chain. This salt bridge is exposed to the solvent and could be
`weakened by interaction with water molecules.
`Some of the hydrogen bonds are between side-chain and
`main-chain atoms, including several involving the hydroxyl
`group of tyrosine (Tyr-33, -50, and -58 from the H chain of
`HyHEL-10 and Tyr-20 of the lysozyme). There is one prob-
`able main-chain/main-chain hydrogen bond, involving the
`carbonyl oxygen of Thr-30 of the H chain and the amide
`nitrogen of Gly-102 of the lysozyme (Table 2).
`The side chain of Tyr-53 from CDR2-H of HyHEL-10
`penetrates into the catalytic cleft of the antigen and interacts
`with Trp-63, which has been implicated in the enzymatic
`activity of lysozyme (34).
`Structures of the Individual Domains and Conformational
`Changes in the Antibody. The structure of the uncomplexed
`Fab is not yet available. Nevertheless, we believe that no
`gross conformational changes in the structure of the com-
`bining site could have occurred because of the overall sim-
`ilarity of the HyHEL-10 domain structures to those of other
`Fabs and the similar ways in which they associate.
`The Fv (VL/VH) and CL/CH1 modules of the Fab have the
`canonical structures observed in other Fabs. The VH of
`HyHEL-10 is related to the VL by a pseudodyad axis (a
`rotation of 170.7 degrees and a translation of -0.3 A along this
`axis). These values fall within the range of values for other Fvs
`ofknown structure: 165.9 to 172.6 degrees ofrotation and -0.9
`to 0.8 A of translation. The pseudodyad axis relating the CH1
`to the CL of HyHEL-10 yields values of 166.6 degrees of
`rotation and -1.6 A oftranslation along this axis. Again, these
`values are comparable to those for other CL/CH1 modules:
`167.4 to 173.8 degrees of rotation and -3.1 to 3.0 A of
`translation. The angle between these two pseudodyad axes-
`i.e., the elbow bend of HyHEL-10 Fab-is 147 degrees.
`Comparison of the framework structure of HyHEL-10 VL
`with those of other immunoglobulins reveals that HyHEL-10
`is most similar to McPC603 (rms deviation of 0.49 A), with
`which it has 53 sequence identities in the 80 framework
`residues, and to the human myeloma protein REI (rms
`deviation of 0.55 A), with which it has 46 identical residues
`in homologous positions. The VL domains of HyHEL-5 and
`D1.3 also have 53 sequence identities with HyHEL-10 in the
`framework, but the structural differences are slightly greater
`for these two domains (rms deviations of 0.71 and 1.02 A,
`respectively) than for those of McPC603 and REI. The
`L-chain CDRs of REI and D1.3 have the same number of
`residues as those of HyHEL-10, and the superposition of
`these CDRs gives rms deviations of 0.54 and 1.04 A, respec-
`tively. The sequence similarities of REI and D1.3 to HyHEL-
`10 in their L-chain CDRs are 13 and 11 residues, respectively,
`in common out of a total of 27.
`Comparison of the framework structure of HyHEL-10 VH
`with those of other Fabs reveals that HyHEL-10 is most
`similar to HyHEL-5, McPC603, and NEW, with rms devia-
`tions of 0.83, 0.84, and 0.86 A and sequence identities of 39,
`43, and 54 among the 87 residues in the framework, respec-
`tively. HyHEL-10 does not have all three H-chain CDRs with
`the same lengths as any of the other VH domains of known
`structure. However, CDR1-H and CDR2-H of HyHEL-10
`have the same number of residues as the corresponding CDRs
`of NEW and D1.3. Superposition of these CDRs gives rms
`deviations of 1.51 and 1.36 A, respectively; the sequence
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`Proc. Natl. Acad. Sci. USA 86 (1989)
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`5941
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`similarities are 6 and 7 identical residues out of 21 correspond-
`ing CDR positions for NEW and D1.3, respectively. HyHEL-
`10 VH has a tyrosine at position 47 instead of the more usual
`tryptophan (32). The structure ofthe region around position 47
`in HyHEL-10 is essentially unaltered compared to that found
`in the other VH domains of known structure, which all have
`tryptophan at this position.
`The CL of HyHEL-10 has the same sequence as those of
`HyHEL-5, McPC603, and J539. Superposition of these do-
`mains gives rms deviations of 0.60, 0.66 and 0.79 A, respec-
`tively. The CL of D1.3 has 4 amino acid differences relative to
`HyHEL-10; superposition of these domains gives a rms de-
`viation of 1.35 A. The CH1 domains of HyHEL-10 and
`HyHEL-5 have identical sequences and superposition ofthese
`domains gives a rms deviation of 0.78 A. The CH1 of D1.3
`differs from the sequence of HyHEL-10 at 2 positions and the
`corresponding a carbons differ with a rms deviation of 1.25 A.
`The CDRs of HyHEL-10 are short. CDR1-L with 11
`residues and CDR3-L with 9 are both only one residue longer
`than the shortest of these regions known so far (32). CDR2-L
`with 7 residues and CDR1-H with 5 have the usual number of
`amino acids in these regions. CDR2-H with 16 residues
`(longest known has 19; ref. 32) and CDR3-H with 5 (longest
`known has 19) represent the shortest of these regions in the
`structures of Fabs. The CDR residues of HyHEL-10 provide
`a total hypervariable surface area of 2220 A2.
`DISCUSSION
`There is a close similarity in structure between the VL
`domains of HyHEL-10 and REI, even in their CDRs. Also,
`there are similar structures for the framework parts of the VH
`domain of HyHEL-10 and of the other Fabs. Further, the
`H-chain CDRs of HyHEL-10, D1.3, and NEW, which have
`the same number of residues, have similar backbone struc-
`tures. This leads us to conclude that no major conformational
`changes have occurred in the structure of HyHEL-10 anti-
`body upon binding to lysozyme. Minor changes may have
`occurred in the backbone structures of the CDR loops but
`these would be obscured by the relatively low resolution of
`the present work. Movements of side chains, most notably
`the ones exposed to solvent, may also have occurred. How-
`ever, the determination of these changes would require a
`structural analysis of the uncomplexed Fab.
`The differences observed at position 47 and around posi-
`tion 101 between the lysozyme structure in the complex with
`HyHEL-10 and that of lysozyme by itself may represent an
`adjustment of the structure of the antigen upon binding to the
`antibody. Alternatively, these differences may simply reflect
`the flexibility of lysozyme in these regions. Indeed, the
`crystallographic B factors, which are frequently used as a
`measure of structural mobility, for the a carbons of Thr-47
`and Asp-101 of lysozyme are 2.7 and 2.4 standard deviations,
`respectively, above the mean for all the a carbons in the
`uncomplexed structure. Nevertheless, no major changes in
`the structure of the antigen are observed in this antibody-
`lysozyme complex.
`Several factors contribute to the energy of interaction
`between HyHEL-10 and lysozyme. The complete exclusion of
`solvent molecules from the HyHEL-10-lysozyme interface
`contributes a large hydrophobic component to the binding
`energy in the form of an increase in entropy due to the release
`of water molecules that would normally be bound to the
`surface of these proteins (35, 36). Further, the involvement of
`many aromatic residues in this antibody-antigen interaction
`minimizes the loss ofconformational entropy when side chains
`are fixed upon complex formation. Additional energy comes
`from the polar interactions. In this instance, charge-charge
`interactions contribute very little, since the only ion-pair
`between HyHEL-10 and lysozyme is at the edge of the
`interface and is exposed to solvent, so that it is probably weak.
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`The hydrogen-bond interactions contribute significantly to the
`binding energy; some of these hydrogen bonds involve
`charged groups and should be strong (37), and the hydrogen
`bonds that involve main-chain atoms should serve to anchor
`the two proteins more firmly to each other.
`In general terms, the structure of HyHEL-10 Fab is similar
`to what has been found in other Fabs (1, 2, 17, 28-30). The
`elbow bend of 147 degrees for this liganded Fab is essentially
`the same as that found for the unliganded J539 Fab (28); this
`is further evidence that the variation in the elbow bend is not
`correlated with the ligand state of the antibody molecule (38)
`but, instead, is simply an indication of the flexibility of this
`part of the structure.
`There are now three epitopes on lysozyme that have been
`located by crystallographic analyses. Of these, the HyHEL-
`10 epitope is the most discontinuous. Whereas the HyHEL-5
`and D1.3 epitopes both consist essentially of two stretches of
`polypeptide chain, the HyHEL-10 epitope is most easily
`described as the exposed surface of a helix plus some of the
`surrounding structure. The central location of the helix in the
`HyHEL-10 epitope and the involvement of all the exposed
`residues in the contact suggest that the helix by itself might
`suffice to block the binding of HyHEL-10 to lysozyme. The
`epitope for NC41 Fab on the influenza virus neuraminidase
`is also rather discontinuous, consisting of four segments of
`polypeptide chain (3).
`The anti-lysozyme-lysozyme complexes studied have com-
`parable areas of interaction between antibody and antigen
`(about 700 A2 per molecule). Further, the binding constants
`are comparable (between 107 and 1010 M-1). However, differ-
`ences in the nature of the contacts exist. In HyHEL-5, for
`example, the importance of electrostatic interactions is em-
`phasized by the presence of two salt bridges in the center of
`the antibody-antigen interface, involving two arginines from
`the antigen and two glutamic acids from the antibody (2). In the
`complex of HyHEL-10 with lysozyme, the one ion-pair inter-
`action observed is weak. In the complex of D1.3 with lyso-
`zyme, no ion pairs were found (1). In all three complexes,
`many hydrogen bonds exist between antibody and antigen and
`several aromatic residues are involved in the contact. Also, in
`all three complexes, framework residues were found to con-
`tribute to the binding of the antigen. In D1.3 and HyHEL-10,
`the framework residue was immediately adjacent to a CDR; in
`HyHEL-5, the contacting framework residue involved was a
`very highly conserved tryptophan (32) that probably plays an
`important role in VLVH interactions (39, 40).
`The three lysozyme epitopes constitute >40%o of the total
`surface of the lysozyme (4). This observation, together with
`the known existence ofantibodies to other regions oflysozyme
`(41), strongly supports the conclusion that all accessible parts
`of the molecule may be antigenic (42). There is a slight overlap
`of the HyHEL-10 and D1.3 epitopes (around the main chain of
`Asn-19) that probably would preclude the simultaneous bind-
`ing of these two antibodies to lysozyme. There is no overlap
`of the HyHEL-10 and HyHEL-5 epitopes or of the HyHEL-5
`and D1.3 epitopes. Although these three epitopes are generally
`accessible to large probes, only parts of them encompass
`residues of high mobility as determined from tetragonal lyso-
`zyme (D. C. Phillips, personal communication) (4).
`The epitope for HyHEL-10 includes part of the catalytic
`cleft of lysozyme, suggesting that binding of antibody could
`interfere with the enzyme's ability to bind and cleave sub-
`strate. Modeling of a hexasaccharide in the catalytic cleft of
`lysozyme suggests that the first two subsites are unavailable
`for binding in the presence of antibody. This prediction
`agrees well with the observation that HyHEL-10 is an
`efficient inhibitor of catalysis of both Micrococcus lysodeik-
`ticus cells and hexasaccharide (ref. 43; J. R. Rupley, personal
`communication). However, we have been unable to demon-
`strate competitive inhibition of HyHEL-10 binding to hen egg
`
`Proc. Natl. Acad. Sci. USA 86 (1989)
`
`2.
`
`3.
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`4.
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`5.
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`6.
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`7.
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`8.
`9.
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`10.
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`11.
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`12.
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`13.
`
`14.
`15.
`16.
`17.
`
`18.
`19.
`
`20.
`21.
`22.
`23.
`
`white lysozyme utilizing oligosaccharide substrates, either at
`low temperature with hexa- or pentasaccharide under con-
`ditions in which both these oligosaccharides competitively
`inhibited binding of HyHEL-5 to lysozyme (10) or at room
`temperature with smaller saccharides.
`Amit, A. G., Mariuzza R. A., Phillips, S. E. V. & Poljak, R. J. (1986)
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`
`24.
`25.
`26.
`27.
`
`28.
`
`29.
`
`30.
`31.
`
`32.
`
`33.
`
`34.
`35.
`36.
`37.
`
`38.
`39.
`
`40.
`
`41.
`
`42.
`
`43.
`
`PETITIONER'S EXHIBITS
`
`Exhibit 1087 Page 5 of 5