AUGUST 1989
`VOLUME86
`·.· . .
`NUMBER 15
`
`:-
`
`'\
`
`Proceedings
`
`OF THE
`
`National Acadellly
`of Sciences
`
`OF THE UNITED STATES · OF AMERICA
`
`BIOEPIS EX. 1087
`Page 1
`
`

`

`Proceedings
`OF THE
`National Academy
`of Sciences
`OF THE UNITED STATES OF AMERICA
`
`Officers
`of the
`Academy
`
`Editorial Board
`of the
`Proceedings
`
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`
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`GORDON A. BA YM
`RONALD BRESLOW
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`
`IGOR B. DAWID , Chairman
`EDWARD E. DAVID, JR.
`RONALD L. GRAHAM
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`PHILIP W. MAJERUS
`DANIEL NATHANS
`
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`
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`
`BIOEPIS EX. 1087
`Page 2
`
`

`

`Proc. Nat/. Acad. Sci. USA
`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 I complementarity I discontinuous epitope)
`EDUARDO A. PADLAN *, ENID w. SILVERTON *, STEVEN SH ERIFF *t, GERSON H . COHEN *,
`SANDRA J. SMITH-GILLf, AND DAVID R. DAVIES*
`
`*Laboratory of Molecular Biology, National Institute of Diabetes, Digesti ve and Kidney Diseases, and *Laboratory of Genetics, National Cancer Institute,
`National Institutes of Health , Bethesda, MD 20892
`
`Contributed by David R . Da vies, 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(cid:173)
`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 of two tyrosines that penetrate the cleft.
`All six complementarity-determining regions of the Fab con(cid:173)
`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 of the monoclonal anti-lysozymes Dl.3 (1) and
`HyHEL-5 (2) and a Fab complex with influenza neuramini(cid:173)
`dase (3). From these data a common pattern of interaction is
`emerging (4) in which there is a high degree of complemen(cid:173)
`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 lgG1(K) antibody specific for hen egg
`white lysozyme. The affinity ofHyHEL-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), slightl y lower than that of HyHEL-5, thus
`making H yHEL-10 intermediate in affinity between HyHEL-
`5 and Dl.3 .§
`HyHEL-10 expresses a member of the VH36-60 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, H yHEL-10
`is structurally di stinct from HyHEL-5 (which expresses
`VHJ558 and VK4) and Dl.3 (which expresses VHQ52 and
`VK/2 / 13).
`
`The publication costs of this article were defrayed in part by page charge
`pay ment. Thi s article must therefore be hereby marked " advertisement"
`in accordance with 18 U.S.C. §1734 solely to indicate this fact.
`
`MATERIALS AND METHODS
`Crystals of the complex ofHyHEL-10 Fab with hen egg white
`lysozyme, grown as described (11), exhibit the symmetry of
`syace group P212121 with a= 57.47, b = 118.73, c = 137.68
`A and 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 10.0-A spacings with F
`;;;., 3a(F). These constitute about 78% of the theoretically
`observable reflections betw~en 10.0- and 3.1-A spacin&,s; 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 MERL?T
`(14). Rotation and translation searches were perfo~~ed to(cid:173)
`dependently (15) for the lysozyme , Fv (module cont~tmng V H
`and V L• the variable domains of the H and L chams), _and
`CLICH1 (constant domain ofL chain/first constant domatn of
`H chain) portions of the structure. In the search for t~e
`orientation of the lysozyme and the Fv , the highest peaks tn
`the rotation function turned out to be the correct peaks. The
`correct peak in the rotation search for the CL/ CH1 was ~nly
`the seventh highest. The translation search gave unambtgu(cid:173)
`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 l y~ozyme
`from the refinement analysis of Diamond (16) [Protem D~r
`Bank (PDB) File 6L YZ] , the Fv of McPC603 (17) (PDB Ft)e
`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) allowi~g the V L• ~H·
`CL and CHI domains and lysozyme to move mdependent Y·
`The structure was then subjected to restrained least-squares
`
`. s~
`.
`.
`Abbreviations: H , heavy ; L , hght ; V Land V ':1 • vanable ? omadnfirst
`L and H c hains; CL and CH1 , constant domam o~ L cham an VH;
`constant domain of H c hai n ; Fv , module contammg V L an~-H
`CDR, complementarity-determining region; CDRn-L or CD
`'
`nth CDR of L or H chain .
`sox
`t Present address: Squibb Institute for Medical Research , P.O.
`4000, Prince~on , NJ 08543~4~ .
`era~
`09
`§Using PEG Jmmunoprec JpJ tatiOn at pH 7 .2 , Denton and Sch ><
`1
`determined association constants of 1.5 x ~09 M- 1 an~ 2.5
`(5)
`M- 1 for HyHEL-10 and Hy HEL-5, respectively . Lav01e et 0 io•o
`1
`determined association constants of = 4 x 109 M- 1 a nd = 1.4 ~ tioO
`M -1 at pH 8.2 by the method of Friguet eta/. {6). The ass~c~ettt
`constant for Dl.3 Fab , also determined by the method of Fng entiY•
`a/. , has been reported {7) as 4.5 x 107 M- 1 at pH 7.4;_more re~ ores(cid:173)
`an association constant of 1.3 x 108 M- 1 was detenruned by u
`cence quenching {8).
`
`5938
`
`BIOEPIS EX. 1087
`Page 3
`
`

`

`Immunology: Padlan et a/.
`
`Proc. Nat/. A cad. Sci. USA 86 ( 1989)
`
`5939
`
`using the program PROLSQ (19, 20) and model
`on the basis of OMIT maps (21) using the graphics
`
`program FRODO (22). The final R value was 0.24 with devi(cid:173)
`ations from ideality of 0.011 A for bond lengths and of 0.034
`
`FIG . 1. Stereo diagrams . (a)
`a-Carbon trace of the HyHEL-10
`Fab-lysozyme complex. Lyso(cid:173)
`zyme is shown in white, VL in
`yellow, VH in light blue , CL in red,
`and CHI in dark blue . (b) Same as
`a and showing the interacting sur(cid:173)
`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(cid:173)
`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(cid:173)
`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(cid:173)
`dues in red . V Lis on the left (light
`blue) and VH is on the right (dark
`blue). (e) The HyHEL-10 epitope
`on lysozyme showing the contact(cid:173)
`ing residues in red . The helical
`region 88-99 is shown in yellow
`and the rest of the lysozyme in
`light blue.
`
`BIOEPIS EX. 1087
`Page 4
`
`

`

`5940
`
`Immunology: Padlan et a/.
`
`Proc. N at/. A cad. Sci. USA 86 ( / 989)
`
`constitute the external surface of the helix; Ser-100, Asp-101
`~n? Gl y-102.' wh~ch extend beyond the heli x; Trp-63, which
`IS m the act1ve-s1te cleft; and Arg-73 and Leu-75, which are
`on the other side of the cleft (Fig. 1e). 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 Gl y-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(cid:173)
`sion is formed by the side chains ofTyr-33 from CDR1-H and
`Tyr-53 from CDR2-H (Fig . 1b). The interacting surface of the
`antibody contains a disproportionate number of aromatic side
`chains that point outward and that interact with the antigen
`(Fig. 1c; Table 1). Large numbers of aromatic residues have
`also been observed in the combining sites of McPC603 (17)
`and Dl.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(cid:173)
`zyme . The CDRs of the L chain contribute 8 residues to the
`contract and those of the H chain contribute 10. One addi(cid:173)
`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(cid:173)
`mational changes occur in the structure of the lysozyme when
`it binds to HyHEL-10. Comparison of the complexed lyso(cid:173)
`zyme with the uncomplexed structure (coordinates of tetrag·
`onallysozyme courtesy of D. 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
`Ci3-C"Y bond presumabl y 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(cid:173)
`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. 1c) 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 Waal s radii (26) . Atomic contacts were defined according
`to the criteria of Sheriff et a/. (27). The various domains of
`HyHEL-10 Fab were compared with the following immuno(cid:173)
`globulin structures: McPC603 and J539 (28) (PDB File 1FBJ),
`HyHEL-5 and Dl.3 (courtesy of R. Poljak, 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 structurall y equivalent pairs of atoms. The number(cid:173)
`ing scheme used here for the HyHEL-10 residues follows the
`convention of Kabat et a/. (32).
`
`RESULTS
`Overall Structure. Fig. 1a shows the a-carbon trace of the
`HyHEL-10 Fab-lysozyme complex . The contact between
`lysozyme and HyHEL-10 involves the complementarity(cid:173)
`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. 1b) are
`strikingly complementary so that solvent is completely ex(cid:173)
`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
`di scontinuous, consisting of residues coming from distant
`part s of the linear sequence but made contiguous by the
`folding of the protein. The area of lysozyme that is in contact
`with the antibod y 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
`
`Table 1. HyH EL-10 residues in contact with lysozyme
`HyHE L-10 residue*
`Lysozyme residue(s)
`
`Gl y-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
`
`V L
`Gly-30
`Asn-31 (h)
`Asn-32 (h)
`Tyr-50
`Gln-53 (h)
`Ser-91 (m)
`Asn-92 (m,h)
`Tyr-96 (h)
`VH
`Arg-73
`Thr-30t
`Arg-73 , Leu-75
`Ser-31 (h)
`Lys-97
`Asp-32 (s)
`Trp-63, Lys-97, lle-98, Ser-100, Asp-101
`Tyr-33 (h)
`Tyr-50 (h)
`Arg-21, Ser-100
`Asp-101
`Ser-52 (h)
`Trp-63, Leu-75 , Asp-101
`Tyr-53 (h)
`As p-101
`Ser-54
`As p-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.
`t Framework residue.
`
`Asn-31 ODl
`Asn-32 ND2
`Gln-53 OE1
`Gln-53 NE2
`
`Lys-96 NZ
`Gly-16 0
`Asn-93 ND2
`Asn-93 OD1
`
`Table 2. Hydrogen bonds between HyHE L-10 and lysozym_:__
`Lysozyme
`Lysozyme
`V L
`VH
`Thr-30 0
`Arg-73 NHl
`Arg-73 NHl
`Ser-31 OG
`Lys-97 0
`Tyr-33 OH
`Arg-21 NHL
`Tyr-50 OH
`Ser-100 0
`Asp-101 ODl
`Gl y-102 N
`
`Tyr-53 0
`Ser-91 0
`Tyr-20 OH
`Asn-92 0
`Arg-21 N
`Tyr-58 OH
`Tyr-96 OH
`Arg-21 NH1
`~----------~------------------------------
`
`BIOEPIS EX. 1087
`Page 5
`
`

`

`Immunology: Padlan et a/.
`
`Proc. Nat/. A cad. S ci. USA 86 ( 1989)
`
`594I
`
`1), 111 are van der Waals contacts and 14 are hydro(cid:173)
`ing contacts (Table 2).
`6 of the contacting residues-Asp-32 from the H
`and Arg-21 and -73, Lys-96 and -97, and Asp-101 from
`~ 0 ~-<> ·rp probably charged under the conditions of
`we find only one salt bridge, between Asp-32
`and Lys-97 from lysozyme, with a separa(cid:173)
`.6 A between the side-chain nitrogen of the lysine and
`oxygen in the carboxyl group of the aspartate side
`This salt bridge is exposed to the solvent and could be
`by interaction with water molecules.
`of the hydrogen bonds are between side-chain and
`· atoms, including several involving the hydroxyl
`tyrosine (Tyr-33 , -50, and -58 from the H chain of
`10 and Tyr-20 of the lysozyme). There is one prob-
`main-chain/main-chain hydrogen bond, involving the
`oxygen of Thr-30 of the H chain and the amide
`of Gly-102 of the lysozyme (Table 2).
`side chain of Tyr-53 from CDR2-H of HyHEL-10
`into the catalytic cleft of the antigen and interacts
`, which has been implicated in the enzymatic
`of lysozyme (34).
`-·Ph•r"" of the Individual Domains and Conformational
`in the Antibody. The structure of the uncomplexed
`not yet available. Nevertheless, we believe that no
`conformational changes in the structure of the com(cid:173)
`site could have occurred because of the overall sim(cid:173)
`of the HyHEL-10 domain structures to those of other
`and the similar ways in which they associate.
`Fv (V L/V H) and CLI CHl modules of the Fab have the
`structures observed in other Fabs. The V H of
`is related to the V L by a pseudodyad axis (a
`of170.7 degrees and a translation of -0.3 A along this
`These values fall within the range of values for other Fvs
`structure: 165.9 to 172.6 degrees of rotation and -0.9
`of translation. The pseudodyad axis relating the CHI
`CL of HyHEL-10 yields values of 166.6 degrees of
`and -1.6 A of translation along this axis. Again, these
`are comparable to those for other CLI CHl modules:
`to 173.8 degrees of rotation and -3.1 to 3.0 A of
`N~••mvu. The angle between these two pseudodyad axes-
`the el
`bend of HyHEL-10 Fab-is 147 degrees.
`son of the framework structure of HyHEL-10 V L
`those of other immunoglobulins reveals that HyHEL-10
`similar to McPC603 (rms deviation of 0.49 A), with
`it has 53 sequence identities in the 80 framework
`, and to the human myeloma protein REI (rms
`of 0.55 A) , with which it has 46 identical residues
`uoJmoJJog,ol s positions. The V L domains of HyHEL-5 and
`53 sequence identities with HyHEL-10 in the
`mp·wn•+, but the structural differences are slightly greater
`domains (rms deviations of 0. 71 and 1.02 A,
`than for those of McPC603 and REI. The
`CDRs of REI and Dl.3 have the same number of
`as those of HyHEL-10, and the superposition of
`CDRs gives rms deviations of 0.54 and 1.04 A, respec(cid:173)
`. The sequence similarities of REI and Dl.3 to HyHEL(cid:173)
`their L-chain CDRs are 13 and 11 residues , respectively,
`common out of a total of 27.
`Comparison of the framework structure of HyHEL-10 V H
`those of other Fabs reveals that HyHEL-10 is most
`to HyHEL-5, McPC603, and NEW, with rms devia-
`of 0.83, 0.84, and 0.86 A and sequence identities of 39,
`54 among the 87 residues in the framework , respec(cid:173)
`. HyHEL-IO does not have aU three H-chain CDRs with
`lengths as any of the other V H domains of known
`However, CDRl-H and CDR2-H of HyHEL-IO
`same number of residues as the corresponding CDRs
`W and Dl.3. Superposition of these CDRs gives rms
`of 1.51 and 1.36 A, respectively; the sequence
`
`similarities are 6 and 7 identical residues out of 21 correspond(cid:173)
`ing CDR positions for NEW and Dl.3, respectively. HyHEL(cid:173)
`IO V H has a tyrosine at position 47 instead of the more usual
`tryptophan (32). The structure of the region around position 47
`in HyHEL-IO is essentially unaltered compared to that found
`in the other V H domains of known structure, which all have
`tryptophan at this position.
`The CL of HyHEL-IO has the same sequence as those of
`HyHEL-5, McPC603 , and 1539. Superposition of these do(cid:173)
`mains gives rms deviations of 0.60, 0.66 and 0.79 A, respec(cid:173)
`tively. The CL ofD1.3 has 4 amino acid differences relative to
`HyHEL-IO; supel_l)osition of these domains gives a rms de(cid:173)
`viation of 1.35 A. The CHI domains of HyHEL-IO and
`HyHEL-5 have identical sequences and superposition of these
`domains gives a rms deviation of 0.78 A. The CHI of Dl.3
`differs from the sequence ofHyHEL-IO at 2 positions and the
`corresponding a carbons differ with arms deviation ofi.25 A.
`The CDRs of HyHEL-IO are short. CDRI-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 CDRI-H with 5 have the usual number of
`amino acids in these regions . CDR2-H with 16 residues
`(longest known has I9 ; ref. 32) and CDR3-H with 5 (longest
`known has I9) represent the shortest of these regions in the
`structures of Fabs. The CDR residues of HyHEL-IO provide
`a total hypervariable surface area of 2220 A 2 .
`DISCUSSION
`There is a close similarity in structure between the V L
`domains of HyHEL-IO and REI , even in their CDRs. Also,
`there are similar structures for the framework parts of the V H
`domain of HyHEL-IO and of the other Fabs. Further, the
`H-chain CDRs of HyHEL-IO, Dl.3, and NEW, which have
`the same number of residues , have similar backbone struc(cid:173)
`tures. This leads us to conclude that no major conformational
`changes have occurred in the structure of HyHEL-10 anti(cid:173)
`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(cid:173)
`ever, the determination of these changes would require a
`structural analysis of the uncomplexed Fab .
`The differences observed at position 47 and around posi(cid:173)
`tion IOI between the lysozyme structure in the complex with
`HyHEL-IO 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 oflysozyme 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(cid:173)
`lysozyme complex .
`Several factors contribute to the energy of interaction
`between HyHEL-10 and lysozyme. The complete exclusion of
`solvent molecules from the HyHEL-IO-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 ofthese proteins (35 , 36). Further, the involvement of
`many aromatic residues in this antibody-antigen interaction
`minimizes the loss of conformational 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-IO and lysozyme is at the edge of the
`interface and is exposed to solvent, so that it is probably weak.
`
`BIOEPIS EX. 1087
`Page 6
`
`

`

`5942
`
`Immunology: Padlan et a/.
`
`Proc. Nat/. A cad. S ci. USA 86 ( 1989)
`
`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 ofHyHEL-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 1539 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(cid:173)
`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(cid:173)
`ences in the nature of the contacts exist. In HyHEL-5, for
`example, the importance of electrostatic interactions is em(cid:173)
`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 ofHyHEL-10 with lysozyme, the one ion-pair inter(cid:173)
`action observed is weak. In the complex of D1.3 with lyso(cid:173)
`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(cid:173)
`tribute to the binding of the antigen. In Dl.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 VL-VH interactions (39, 40).
`The three lysozyme epitopes constitute > 40% of the total
`surface of the lysozyme (4). This observation, together with
`the known existence of antibodies 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(cid:173)
`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(cid:173)
`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(cid:173)
`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(cid:173)
`ticus cells and hexasaccharide (ref. 43; J. R. Rupley, personal
`communication). However, we have been unable to demon(cid:173)
`strate competitive inhibition ofHyHEL-10 binding to hen egg
`
`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 roorn
`temperature with smaller saccharides .
`
`I
`
`1. Ami! , A. G., Mariuzza R. A., Phillips, S. E. V. & Poljak , R. J. (1986)
`Science 233, 747-753.
`2. Sheriff,S ., Silverton , E. W., Padlan, E. A., Cohen. G. H., Smith-Gill,
`S. J. , Fmzel, B. C. & Dav1es, D. R. (1987) Proc. Nat/. A cad. Sci. US.4
`84, 8075-8079.
`3. Colman, P.M. , Laver, W. G., Varghese , J. N., Baker, A. T., Tulloch
`P. A., Air, G. M. & Webster, R. G. (1987) Nature (London) 326,358-363:
`4. Davies, D. R. , Sheriff, S. & Padlan , E. A. (1988) 1. Bioi. Chern. 263
`10541-10544.
`5. Lavoie, T. B., Kam-Morgan , L. N. W., Mallett, C. P., Schilling, J. w.,
`Prager, E. M., Wilson, A. C. & Smith-Gill , S. J. (1989) in The Use of
`X-ray Crystallography in the Design of Antiviral Agents, eds. Laver,
`G. W. & Air, G. M. (Academic, New York), in press.
`6. Friguet, B., Chaffotte, A. F., Djavadi-Ohaniance, L. & Goldberg, M. E.
`(1985) 1. lmmunol. Methods 77, 305-3