Crystal Structure of an Fv–Fv Idiotope–Anti-idiotope Complex at 1.9 Å Resolution

J. Mol. Biol. (1996) 264, 137–151

Crystal Structure of an Fv–Fv Idiotope–Anti-idiotope Complex at 1.9 Å Resolution Bradford C. Braden1, Barry A. Fields1, Xavier Ysern2 William Dall’Acqua1, Fernando A. Goldbaum1, Roberto J. Poljak1* and Roy A. Mariuzza1 1

Center for Advanced Research in Biotechnology University of Maryland Biotechnology Institute 9600 Gudelsky Dr., Rockville MD 20850, USA 2

Center for Drug Evaluation and Research, F.D.A. 5600 Fishers Lane, Rockville MD 20857, USA

Anti-idiotopic antibodies react with unique antigenic features, usually associated with the combining sites, of other antibodies. They may thus mimic specific antigens that react with the same antibodies. The structural basis of this mimicry is analyzed here in detail for an anti-idiotopic antibody that mimics the antigen, hen egg-white lysozyme. The crystal structure of an anti-hen-egg-white lysozyme antibody (D1.3) complexed with an anti-idiotopic antibody (E5.2) has been determined at a nominal resolution of 1.9 Å. E5.2 contacts substantially the same residues of D1.3 as lysozyme, thus mimicking its binding to D1.3. The mimicry embodies conservation of hydrogen bonding: six of the 14 protein–protein hydrogen bonds bridging D1.3-E5.2 are structurally equivalent to hydrogen bonds bridging D1.3-lysozyme. The mimicry includes a similar number of van der Waals interactions. The mimicry of E5.2 for lysozyme, however, does not extend to the topology of the non-polar surfaces of E5.2 and lysozyme, which are in contact with D1.3 as revealed by a quantitative analysis of the contacting surface similarities between E5.2 and lysozyme. The structure discussed herein shows that an anti-idiotopic antibody can provide an approximate topological and binding-group mimicry of an external antigen, especially in the case of the hydrophilic surfaces, even though there is no sequence homology between the anti-idiotope and the antigen. 7 1996 Academic Press Limited

*Corresponding author

Keywords: antibody; anti-idiotypic antibody; protein structure; protein crystallography; molecular mimicry

Introduction Special cases of antibody-antigen reactions are those in which the antigen (Ag) is an antibody (Ab). In these reactions, an idiotope (Id) is an antigenic determinant (or epitope) specific for an antibody. It Present address: F. A. Goldbaum, IDEHU-CONICET, Ca´tedra de Immunologı´a, FFYB-UBA, Junı´n 956, 1113 Buenos Aires, Argentina. Abbreviations used: mAb, monoclonal antibody, HEL, hen egg-white lysozyme, CDR, complementarity determining region; VH , VL , variable domains of the antibody heavy (H) and light (L) polypeptide chains; L1, L2, L3, H1, H2, H3; the CDRs of the light and heavy variable domains; Id, idiotope; Ag, antigen; anti-Id, Ab2, the anti-idiotopic antibody; anti-anti-Id, Ab3, the anti–anti-idiotopic antibody; r.m.s., root-mean-square. 0022–2836/96/460137–15 $25.00/0

is defined by the reaction of an anti-idiotopic antibody (anti-Id; Ab2) with the antibody (Id; Ab1) bearing the idiotope. The sum of idiotopes of an antibody constitute its idiotype. Extensive studies on the amino acid sequences of antibodies, and correlation with serology and function have shown that idiotypes are predominantly associated with the complementarity determining regions (CDRs) of antibody molecules. Thus, antiidiotypic antibodies bind to the Ag recognition site of specific antibodies (Ab1) in competition with specific antigens. Given the variability of CDRs, idiotypes have a large potential for diversity and constitute an important system of unique selfantigens. Since many anti-Id antibodies (Ab2) bind the CDRs of antibodies (Ab1), as do antigens specific to the Ab1, it has been postulated that some anti-Id 7 1996 Academic Press Limited

138 antibodies may resemble the antigen and thus carry its ‘‘internal image’’ at the molecular level (Jerne, 1974). Mimicry of ligands of biological receptors by anti-Id antibodies has been described in several systems (reviewed by Gaulton & Green, 1986; Greenspan, 1992; Pan et al., 1995). Furthermore, the concept of anti-Id mimicry has led to proposals to use anti-Id antibodies as surrogate antigens (Nisonoff & Lamoyi, 1981; Roitt et al., 1981; Sacks et al., 1983). Based on these proposals, anti-idiotopic antibodies have been used in attempts to evoke immune responses to pathological antigens. An understanding of how an antigen may be mimicked, at a stereochemical level, by an anti-Id antibody requires the elucidation of the three-dimensional structures of at least two complexes. The first complex, between the Ab1 and its specific antigen, would define the epitope that may be mimicked, and its interactions with the combining site of the Ab1. The second complex, that between the Ab1 and the internal image anti-Id Ab2, would define the surface of the anti-Id Ab2 that binds the Ab1 and its binding interactions. If the anti-Id antibody bears an internal image of the antigen its CDR structure should, in some way, mimic the epitope and its interactions with the combining site of the Ab1. With the aim of testing the internal image postulate, the three-dimensional structures of several anti-Id antibodies, either alone or in complex with the idiotope, have been determined (Bentley et al., 1990; Garcia et al., 1992; Ban et al., 1994; Evans et al., 1994). However, these structures have not led to firm conclusions concerning the mimicry of external antigens. This was due to the fact that in some studies the structure of only one of the complexes (the Ab1-Ab2) was determined and the structure of the antigen specific for the Ab1 is not known (Ban et al., 1994), or the complex was that of an anti-anti-Id with only a part of the antigen (Garcia et al., 1992). In other cases in which the structures of the Ab1-Ag and the Ab1-Ab2 complexes were determined, the anti-Id Ab2 did not conform to the internal image postulate, either because the anti-Id was apparently not appropriate for that purpose (Tello et al., 1994) or the anti-Id antibody could not bind in the deep cleft between the Ab1 antibody VH and VL chains as did the original antigen (Evans et al., 1994). A recent study, based on a comparison of the three-dimensional structures of the complexes between an Ab1-Ag and Ab1-anti-Id Ab2, provides an example of how anti-Id antibodies can functionally mimic external antigens (Fields et al., 1995). In this system, the Ab1 is the murine anti-hen-eggwhite lysozyme (HEL) antibody mAb D1.3 (BALB/ c, IgG1, k). Here, we describe in detail the solution and refinement of the crystal structure of the Fv D1.3-Fv E5.2 complex. We discuss and quantify the molecular mimicry of E5.2 for HEL as demonstrated by similar intermolecular interactions and the altered affinities for HEL and E5.2 of single-site Fv D1.3 mutants.

Antigen Mimicry by an Anti-idiotope

Results and Discussion Crystal structure of the Fv D1.3-Fv E5.2 complex The crystal structure of the Fv D1.3-Fv E5.2 complex was determined by molecular replacement using the known Fv D1.3 crystal structure (Bhat et al., 1990, 1994) as the initial model for both Fvs. Refinement of the model, including 157 bound solvent molecules and three zinc ions, as described in Materials and Methods, resulted in an R value of ˚ (F > 2sF ) range. 0.194 for data in the 7 to 1.9 A Details of the data collection and refinement are given in Table 1. The Id-anti-Id interaction involves all six CDRs of each molecule (Figure 1), although the interaction between E5.2 VH CDR1 and D1.3 is achieved only through bridging water molecules. The complex thus formed is a dimer of VH-VL dimers, exhibiting roughly 222 symmetry defined by the VH-VL pseudo-dyad and a pseudo-dyad relating the VH (or VL ) domains. From sequence comparisons and three-dimensional structure, the E5.2 hypervariable loops fit the canonical structures L1-1, L2-1, L3-1, H1-1 and H2-2. The canonical structures for the CDR loops of D1.3 have been described (Chothia et al., 1989). The H3 loop of E5.2 is 13 residues long and forms a long turn stabilized by hydrogen-bond interactions between atoms VH Tyr99 OH-VH Asp101 Od1, VH Met100e O-VH Trp103 Ne1, VH Tyr102 O-VH Thr94 N, VH Gly100c O-VL Gln89 Ne2 and VH Gln100 Oe1-VL Arg53 Nh1 (sequence numbers as in Kabat et al., 1991). The model for the Fv D1.3 in the Id-anti-Id complex is quite similar to that in the Fv D1.3-HEL complex. Root-mean-square (r.m.s.) deviations between all main-chain atoms and Cb atoms of the ˚. Fv D1.3 domains in the two complexes is 0.56 A The largest deviations in Fv D1.3 between the complex with Fv E5.2 and the complex with HEL occur at the amino and carboxy termini (VL Asp1, VL Lys107, VH Gln 1 and VH Ser115), VL residues Ser7, Gln70, Leu78, Pro80, Trp92 and Ser93, and VH residues Ala13, Ser15, Gln16, Thr30, Thr31, Gly42, Table 1. Data collection and refinement statistics Total reflections Unique reflections ˚) Resolution (A Redundancya Completeness (%) Rmerge (%) Refinement data set ˚ , 2s) Reflections (7-1.9 A Protein atoms Water molecules Zinc ions Final R Free R ˚) r.m.s.d. bond lengths (A r.m.s.d. bond angles (deg.) a

Siemens data

R-axis data

66,725 31,484 2.0 2.1 77 9.0

105,969 41,404 1.9 2.6 82.5 8.5

Average number of observations per reflection.

32,539 3495 157 3 0.194 0.235 0.016 2.0

139

Antigen Mimicry by an Anti-idiotope

Figure 1. Stereo view of the ribbon representation of the D1.3-E5.2 structure. The complex exhibits roughly 222 symmetry through a pseudo-dyad normal to the pseudo-dyad that relates antibody domains VH and VL . The complex is formed via contacts from all six CDR loops of each Fv, although the protruding VH CDR3 (labeled as H3) of E5.2 is predominant.

Ser84 and Thr87 all having positional differences ˚ after superpositioning. Removal greater than 1.0 A of the outlier residues, listed above, reduces the ˚ . D1.3 VL residues Trp92 r.m.s. difference to 0.38 A and Ser93 deviate from the positions in the D1.3-HEL structure due to a 145° difference in the orientation of the carbonyl group of D1.3 VL Trp92, as noted in Materials and Methods. This conformational change in VL CDR3 is very important in the Id-anti-Id interaction (see below). In a comparison of the free and HEL-complexed Fv D1.3 (Bhat et al., 1990), a small change in the relative positions of the VL and VH domains of D1.3 was found to occur on binding of HEL. By contrast, least-squares superpositions show a difference of ˚ and 0.06 A ˚ for the centers of mass of the only 0.05 A D1.3 VL and VH domains, respectively, in the complexes with HEL and E5.2. Moreover, rotations of only 1.2° and 1.5° are necessary to bring the VL domains and the VH domains, respectively, into coincidence. Therefore, the relative arrangement of the D1.3 VL and VH domains in the Id-anti-Id complex is essentially identical with that in the Ab1-Ag complex, suggesting that the intermolecular interactions responsible for this domain rearrangement of Fv D1.3 upon the binding of HEL are equivalent upon the binding of Fv E5.2. Only six of the 409 non-glycyl residues in the Fv D1.3-Fv E5.2 complex have main-chain torsion angles usually considered as disallowed due to steric effects. Among these, residues D1.3 VL Asn31 (f = 73°, c = 10°) and E5.2 VL Asn31 (f = 76°, c = 0°) are components of I'-type turns, residues D1.3 VL Thr51 (f = 68°, c = −44°) and E5.2 VL Thr51 (f = 76°, c = −62°) are in position i + 1 of g-turns, and residues D1.3 VH Ser15

Glu42 (f = 89°, c = −26°) and E5.2 VH (f = 71°, c = 22°) are in type II turns. Although glycine is quite often a preferred residue in tight turns (Chou & Fasman, 1977), the tight turns linking antibody b-strands often do not include glycine residues (Milner-White et al., 1988).

Structure of the Id-anti-Id interface The Fv D1.3-Fv E5.2 complex is stabilized by contacts from all six CDRs of each Fv, although the VH of D1.3 and of E5.2 are predominant in such contacts. The surface area buried by the Fv D1.3-Fv E5.2 interaction, excluding bound solvent mol˚ 2 (711 A ˚ 2 from D1.3, 706 A ˚ 2 from ecules, is 1417 A E5.2; see Materials and Methods). It should be noted that the D1.3-E5.2 buried surface area is ˚ 2 smaller than previously published (Fields 470 A et al., 1995) due to the different algorithms used to calculated buried surface area. With the inclusion of ˚ of the D1.3-E5.2 water molecules within 1.4 A buried surfaces, the interaction area expands to ˚ 2 (1031 A ˚ 2 from D1.3, 1019 A ˚ 2 from E5.2), 2050 A demonstrating the solvation of otherwise empty cavities in the interface. The total buried surface area of the D1.3-E5.2 interaction is approximately 15% larger than for the D1.3-HEL interaction ˚ 2, unsolvated; 1740 A ˚ 2, solvated). Excluding (1234 A contributions to the buried surfaces by bound water molecules, hydrophilic atoms (nitrogen and oxy˚ 2 ) of the D1.3 buried gen) account for 48% (341 A 2 ˚ surface and 46% (325 A ) of the E5.2 buried surface. ˚ 2 ) of the For the D1.3-HEL complex, 46% (278 A ˚ 2 ) of the buried buried D1.3 surface and 52% (331 A HEL surface are hydrophilic. The buried surfaces of

140 the D1.3-HEL and D1.3-E5.2 complexes are shown in Figure 2. The residues of E5.2 in contact with D1.3 are listed in Table 2. VH CDR3 of E5.2 accounts for 76% of the total contacts to D1.3. As noted above, this loop is stabilized by internal hydrogen bonds. It penetrates into the central cavity of D1.3 and makes contacts with all the CDRs of D1.3, except VL CDR2 (Table 2). With the exception of VL Tyr49, no ‘‘framework’’ residues (Kabat et al., 1991) are involved in interface contacts. This is true also for the Ab1 D1.3, in which only one framework residue, also VL Tyr49, is in contact with the anti-Id. However, Tyr49 makes contacts with the Ag, and is thus part of the combining site of D1.3. Of the 18 D1.3 residues that contact the anti-Id and the 17 that contact the Ag, 14 are in contact with both the anti-Id and the Ag. These 14 residues of the Ab1 ˚ 2 ) of the total contacting area make up 75% (533 A ˚ 2 ) of that with the with the anti-Id and 87% (526 A Ag. The Fv D1.3-Fv E5.2 interaction features 13 intermolecular hydrogen bonds, plus two polar interactions mediated by a zinc ion. By comparison, the D1.3-HEL interaction includes 15 intermolecular hydrogen bonds (Table 2). Zinc ions are not required for the formation of the Id-anti-Id complex, since the rate constants and affinity of the D1.3-E5.2 reaction are unaffected by the absence or the presence of zinc (unpublished results). The presence of this cation in the Id-anti-Id interface is probably due to the high concentration of zinc acetate in the crystallization buffer. Since the D1.3 and E5.2 atoms in the polar interactions with the zinc ion are within hydrogen-bonding distance, or

Antigen Mimicry by an Anti-idiotope

in close contact, the conformation of these interacting residues may not be changed by deletion of the zinc ion. Thus, D1.3 VH atom Asp100 Od1 would likely form a hydrogen bond to E5.2 VH atom His33 Ne2. Figures 3 and 4 demonstrate the hydrogen bond network between the interacting residues of D1.3 and E5.2. A significant difference between the HEL-bound and the E5.2-bound Fv D1.3 occurs in the backbone conformation of VL residues Trp92 and Ser93 (Figure 5). In the Fv D1.3-HEL complex, the carbonyl group of VL Trp92 points away from the antigen making a hydrogen bond to VL His90. The amide nitrogen atom of VL Ser93 is thus exposed at the surface of D1.3 and forms a hydrogen bond to HEL atom Gln121 Oe1. In the D1.3-E5.2 complex, the peptide orientation of Trp92-Ser93 has changed such that the amide group of Ser93 points away from the anti-Id and the carbonyl oxygen atom of Trp92 is accessible at the surface, forming a hydrogen bond to E5.2 atom VH Arg100b Nh2. An identical ‘‘peptide flip’’ at D1.3 Trp92-Ser93 was observed in the crystal structure of D1.3 complexed with turkey egg-white lysozyme (TEL: Braden et al., 1996). Thus, as in the D1.3 complexes with the lysozymes, the conformation of D1.3 VL CDR3 in the complex with E5.2 is dependent on the electrostatic nature of the residue in contact with the L3 backbone. If the conformation of the VL Trp92-Ser93 peptide link was the same as in the D1.3-HEL complex, the amide nitrogen atom of Ser93 would be exposed at the interface and unable to form a hydrogen bond to the E5.2 arginine.

Figure 2. Buried surface representations of the D1.3-HEL and D1.3E5.2 complexes. The hydrophilic surfaces (defined by nitrogen and oxygen atoms) are in lighter color and the hydrophobic surfaces (defined by carbon atoms) are shaded dark. Black represents the exterior of the molecules or the solvated cavities in the molecular interface. The surfaces for each complex are separated by a translation such that the D1.3 molecule would be behind the page and the HEL and E5.2 molecules would be in front. Mapped onto each surface are the CDR loop designations or the HEL residue numbers of the atoms that form the buried surfaces.

E5.2

92 93 93 93

Ob N Ca Cb

H3100bNh2

H3 100b Cz H3 100bNh2 H3 100b Cz L3 92 O H3 100bNh2 L3 93 Cg2 L3 92 O

121 121 125 125

125 125 125 125 Oe1 Oe1 Cd Ne

Ca Cg Cg Cd

22 Ca 18 Cg 19 Cb 18 Cg 18 Od1 18 Od2 18 Od2 18 Od1 18 Cg 119 Cb 119 Cg 119 Nd2 119 Nd2 121 Ne2 121 Cd 121 Cd 121 Ne2 121 Cd

121 Ne2

HEL

D1.3

32 33 52 52

Cz N Cb Cg

H2 52 Cd1 H2 52 Ne1

H2 52 Ce3

H2 52 Ce2

H2 52 Cd2

H1 H1 H2 H2

H1 32 C

H1 32 N H1 32 Ca

H1 31 C H1 31 O

H1 30 O

98 98 98 98 98

Cb Cd1 Cb Cb Cd1

N Cb Cg2 C Ca Cb Cb

H3 H3 H3 H3 H3 H3 H3 H3 H3 H3 H3 H3 H3

100 Ca 100 Cb 100 Ca 100 Cb 100 Ca 100 Cg 98 Cd1 98 Ce1 99 C 99 O 100 Ca 100 Cg 100 Cg

H3 98 Cd1

H3 H3 H3 H3 H3

98 97 97 97 97 98 98

E5.2 H3 H3 H3 H3 H3 H3 H3

118 C 119 Ca 119 N

119 Cb

117 O

116 Ce

117 Ca 116 Cg 117 Ca

HEL

D1.3

56 58 58 98 98

Nd2 Cg Od1 Cg Cd

H3 99 Nh1

H3 98 Oe1

H2 H2 H2 H3 H3

H2 56 Cg

H2 56 Cb

H2 54Od2

H2 53 C H2 54 Cb H2 54 Cg

H2 53 N H2 53 Ca

H2 52 Ch2

H2 52 Cz2

H2 52 Cz3

E5.2

H3 98 O H3 98 Cb H3 98 O H3 97 Cg2 H3 99 Cd2 L2 49 OH H3 100 N H3 100 N L2 49 OH L2 49 Cz L2 49 Ce1 H3 100 Cd H3 100 Oe1 H3 100 Ne3 H3 100 Cd H3 100 Oe1 H3 100 Oe1 H3 100 Ne2 H3 100 Ne2 H3 98 OH H3 98 Ce1 H3 98 OH H3 98 OH H3 98 Cz H3 98 Ce1 H1 30 O H2 53 Cb

H3 98 Ce1 H3 100 Ca H3 99 O

102 O 102 C

Wat

118 Cb 118 Cg2

Wat

C N Ca Cb Cb Cg Ca Cb Cg Od1 O O

HEL 118 119 119 119 119 119 119 119 119 119 117 117

D1.3

101 101 101 101 101

N Cg Cd2 Ce2 Ce1

H3 102Nh2

H3 101 Cz

H3 101 OH

H3 H3 H3 H3 H3

H3 100 Od2

H3 100Od1

H3 100 Cg

E5.2

98 98 98 98

OH OH OH OH H3 100a O Wat H3 100bCa H3 100bCb

H3 H3 H3 H3

H1 33 Ce1

H1 33 Ne2 H2 52 Od1

H1 33 Ce1 H1 33 Ne2

HEL

121 Cb 121 Cb 120 Cg2 119 Ca 119 Od1 120 N 119 C 121 N 119 C 121 Cb 119 Ca 121 Cb 119 Od1 22 O

24Cb 24 N 24 Og 27 Nd2 24 N 22 O 23 C 23 Ca 24 Og 27 Nd2 24 Cb WAT

˚ ) less than or equal to: C-C, 4.1; C-N, 3.8; C-O, 3.7; N-N, 3.4; N-O, 3.4; and O-O, 3.3. Atom pairs in the D1.3-E5.2 Intermolecular contacts were defined by atom pair distances (A complex that mimic hydrogen-bond interactions in the D1.3-HEL complex are shown in bold type. a Hydrogen bond interaction to the phenyl ring of Tyr32. b Mimicry of E5.2 residue H3 100b for HEL 121 is maintained through a peptide flip at D1.3 VL Trp92 (see Figure 5).

L3 L3 L3 L3

L3 92 Cz2

L3 92 Ch2

L3 92 Cz3

L3 92 Ce3

L2 53 Og1 L3 91 O

L2 50 Cd2

H2 58 Ne2

100b Cd 54 Nd2 54 Nd2 58 Ne2

L2 50 OH

H3 H2 H2 H2

L3 93 Cg2 H3 100b Cd

H2 58 Ne2

30 Ce1 32 Cz Tyr 32a 32 OH 49 Cz 49 OH 50 Ce2

L2 50 Cz

L1 L1 L1 L1 L2 L2 L2

D1.3

Table 2. Intermolecular contacts in the FvD1.3-FvE5.2 and FvD1.3-HEL crystal structures

142 Solvation in the Id-anti-Id complex In total, 157 water molecules were located in the Fv D1.3-Fv E5.2 crystal structure. Of these, 60 are bound to the Fv D1.3, 91 to Fv E5.2 and six to both D1.3 and E5.2. Of 66 water molecules bound to D1.3 (60 exclusively bound plus six bound to both Ab1 and anti-Id Ab2), 45 are located in equivalent positions in the Fv D1.3-HEL crystal structure. The intermolecular interactions and hydrogen bonds that stabilize the D1.3-E5.2 complex are further enhanced by a network of D1.3-water-E5.2 hydrogen bonds. This network is composed of 27 water molecules that directly, or through other

Antigen Mimicry by an Anti-idiotope

water molecules, bridge the Id and anti-Id (Figure 6). Peripheral to the 27 bridging water molecules are nine others that, although not involved in hydrogen bonds between the Ab1 and anti-Id, are integral to the interface, since they are in contact with the D1.3-E5.2 buried surface. From the comparison of six Fv D1.3-HEL crystal structures (the wild-type Fv D1.3-HEL complex and five single-site Fv D1.3 mutants complexed with HEL), a consensus of 25 solvent sites that bridge the Ab1 and Ag were found (Braden et al., 1995). Additionally, 23 water molecules are peripheral to the bridging water structure. Moreover, a physicochemical study of the association rates and equilibrium binding constants of the D1.3-HEL

Figure 3. Stereo diagrams of the hydrogen-bond network linking E5.2 to the D1.3 light chain CDR1 and CDR2 (a) and D1.3 CDR3 (b). D1.3 atoms are shown with thick bonds, E5.2 atoms with thin bonds. Water molecules are represented by two concentric circles, zinc by a filled circle. CDR residues are number-coded by chain, D1.3 30 to 32 (VL CDR1), 49 to 53 (VL CDR2), 91 to 94 (VL CDR3); E5.2 residues 530 to 532 (VL CDR1), 550 (VL CDR2), 592-593 (VL CDR3), 880 to 888 (VH CDR2), 900b (VH CDR3).

143

Antigen Mimicry by an Anti-idiotope

Figure 4. Stereo diagrams of the hydrogen-bond network linking E5.2 to the D1.3 heavy chain CDR1 and 2 (a) and CDR3 (b). D1.3 atoms are shown with thick bonds, E5.2 atoms with thin bonds. Water molecules are represented by two concentric circles, zinc by a filled circle. CDR residues are number-coded by chain: D1.3 330 to 332 (VH CDR1), 352 to 354 (VH CDR2), 399 to 402 (VH CDR3); E5.2 549 (VL CDR2), 831 to 833 (VH CDR1), 850 to 852 (VH CDR2), 895 to 900 (VH CDR3).

reaction, under conditions of reduced water activity, has demonstrated that about 13 to 14 additional water molecules are bound when the antibody-antigen complex is formed (Goldbaum et al., 1996). The solvation of the Id-anti-Id and Ab1-Ag complexes appears to be substantially similar. Ten of the 27 bridging water molecules positions in the D1.3-E5.2 complex are found in the crystal structure of the D1.3-HEL complex. Moreover, five of these water molecules common to the two Ab1 complexes are found in the crystal structure of the free Ab1 and, as such, these water molecules are not lost upon the formation of the D1.3-HEL or D1.3-E5.2 complexes. In all, the 27 bridging water molecules in the Id-anti-Id complex are involved in 64 potential hydrogen bonds, an average of slightly more than two hydrogen bonds per bound water molecule.

Mimicry of the antigen by the anti-idiotope As an immunogen, E5.2 elicits an anti-E5.2 response in which anti-HEL activity can be detected in BALB/c mice. The induced antibodies are not strictly D1.3-like, thus ruling out the possibility that the anti-Id simply stimulated clones expressing the Ab1 (Fields et al., 1995). Furthermore, the antibodies with anti-HEL activity constituted only a small fraction of the total anti-anti-Id. By these criteria, the Ab2 (E5.2) behaved like a typical internal image anti-idiotope. Thus, the structural comparison of the Ag and anti-Id, and their binding to the Ab1, should establish the molecular basis of anti-idiotopic mimicry of the antigen. However, procedures to quantify molecular mimicry have not been established. A general perspective of the mimicry of E5.2 for HEL can be seen in the buried surface represen-

144

Antigen Mimicry by an Anti-idiotope

Figure 5. Stereo view of the peptide flip in the D1.3 VL CDR3 backbone conformation in the D1.3-HEL (light) and D1.3-E5.2 (dark) crystal structures. The main-chain Trp92 c-torsion angle difference in the two complexes is 145°. The VL peptide conformation in each complex ensures electrostatic complementarity of the interacting atoms and mimicry of HEL residue Gln121 by E5.2 residue VH Asp100b (see the text). In this and the following Figures, the D1.3-HEL and D1.3-E5.2 models were superimposed on all main-chain and Cb atoms of the D1.3.

tations (Figure 2). As would be expected from the similarity of the D1.3 structures in its complexes with HEL and E5.2, the D1.3 interface surfaces appear to exhibit very similar topologies. Solvated cavities of roughly equal size and location are found at the conjunction of the L1, L2 and H3 CDRs, at the conjunction of the L3, H2 and H3 CDRs and in the L1 CDR. The topologies differ, however, at the conjunction of the H1, H2 and H3 CDRs, where in the D1.3-HEL complex a large solvated cavity is present, while in the D1.3-E5.2

complex this cavity is filled by the side-chain of E5.2 VH Tyr98. The topology of the HEL buried surface complements that of the D1.3 quite well. The large solvated cavities between D1.3 H1, H2 and H3 and in D1.3 L1 continue into the surface of the HEL epitope (see Figure 2). The solvated cavity at the conjunction of D1.3 L3, H2 and H3 is opposed by the hydrophilic surface formed by the side-chains of HEL residues Asp119 and Arg125. Similar observations can be made in the D1.3-E5.2 interface. Solvated cavities in D1.3 continue into the E5.2

Figure 6. Stereo view of the 27 water molecules (spheres) bridging the D1.3 (light)-E5.2 (dark) complex. These water molecules fill otherwise empty cavities in the Id-anti-Id interface (see Figure 2) as well as contributing hydrogen-bond donors/acceptors to the stability of the complex (see the text). Five water molecules (open spheres) are in positions equivalent to water molecules in all crystal structures of Fv D1.3 (see the text).

Antigen Mimicry by an Anti-idiotope

surface, and the general size and location of the hydrophobic and hydrophilic surfaces are complementary (see Figure 2). With this correspondence of hydrophobicity, hydrophilicity and solvated cavities in the Ab1-Ag and Id-anti-Id complexes, it might be expected that the surfaces of the Ag and anti-Id Ab2 bound to the Ab1 would exhibit similar structural features. It is difficult, however, to conclude much about molecular mimicry by simple comparison of the graphic representations of these interfaces. The buried surfaces of HEL and E5.2 are not as similar to each other as are the D1.3 interfaces in the D1.3-HEL and D1.3-E5.2 complexes. For example, there is a large hydrophobic area at residue Ile124 on the HEL surface that matches a hydrophobic patch, spanning CDRs L1 and L2, on the surface of D1.3; however, on the E5.2 surface this area, spanning CDRs L3 and H3 is hydrophilic in nature. Quantitative analysis of the mimicry In order to assess hydrophobic/hydrophilic surface complementarity of the Ab1-Ag and Id-anti-Id complexes, the similarity of the D1.3, HEL and E5.2 interfaces was assessed. To do this, the fraction of buried surface points of one surface whose nearest neighbors on the other surface are of the same hydrophobic/hydrophilic character was calculated (see Materials and Methods). From this analysis, the hydrophobic/hydrophilic similarity of the two D1.3 buried surfaces in the two complexes is 0.78. That is, more than three-quarters of the D1.3 surface points in the complexes with Ag and with anti-Id are of the same hydrophobic/hydrophilic character. Since few conformational differences exist between the D1.3 CDRs in the complexes with HEL and E5.2, aside from the peptide flip in VL CDR3, this value for surface similarity is the benchmark for the comparison of antibody-antigen and idiotope-anti-idiotope surface complementarity. We should consider this value as the maximum of similarity that can be attained in this comparison of complexing surfaces. The same procedure can be used to assess the similarity of the surfaces of the Ag and the anti-Id that contact the Ab1; that is, to assess the binding mimicry of the Ag by the anti-Id. The calculation shows that the complementarity of the D1.3-HEL and D1.3-E5.2 buried surfaces are 0.58 and 0.55, respectively. Thus, slightly more than half of the surface points of D1.3 are opposed by points on the HEL and E5.2 surfaces that are of the same hydrophobic/hydrophilic character. By the same procedure, the similarity of the HEL and E5.2 surfaces is 0.53, a value that is reasonably close to the complementarity of the buried surfaces in the two complexes. The similarity of the hydrophilic surfaces of HEL and E5.2 is quite high (0.75). In addition, the Ag and the anti-Id Ab2 have close to the same number of van der Waals contacts to the Ab1 (58 and 52, respectively, Table 2). Whereas hydrogen bonds have strict geometric requirements, the only geometric requisite in the van der

145 Waals interactions is the distance of the interacting atoms, a requirement that can be satisfied even though the topological similarity of non-polar surfaces of the Ag and the anti-Id is only 0.30. Thus, topological similarity of the hydrophobic surfaces of the Ag and anti-Id is not a necessary requirement for binding mimicry. The picture that emerges from this analysis is that the anti-Id mimics the Ag by a strong topological similarity in hydrophilic interactions and by making a comparable number of van der Waals contacts to the combining site of the Ab1. This mimicry is well exemplified by the patterns of hydrogen bonding: six of the 14 protein-protein interface hydrogen bonds in the Ab1-anti-Id Ab2 complex are superimposable with hydrogen bonds in the Ab1-Ag interface (Table 2, bold type). As the atoms that form these hydrogen bonds are superimposable in the Ab1-Ag and Id-anti-Id complexes, the geometry of the hydrogen bonds is equivalent, suggesting that the relative contribution of these bonds to the stability of the complexes is similar. Moreover, five of the six equivalent hydrogen bonds are formed by identical atom types. For example, the amide nitrogen atom of D1.3 VH Gly53 and Nh1 atom of D1.3 VH Arg99 bind to carbonyl oxygen atoms in both the Ag and anti-Id. The only mimicked interacting pairs that do not conserve atom type are the interactions to D1.3 VL Tyr50. In the Ab1-Ag complex the hydroxyl group of D1.3 VL Tyr50 forms a hydrogen bond to HEL atom Asp18 Od2, whereas in the Ab1-anti-Id Ab2 complex the hydroxyl group is hydrogen bonded to E5.2 atom Gln58 Ne2. Nonetheless, as an hydroxyl group can be either a proton donor or acceptor, the functional mimicry of these interactions is maintained. As can be inferred from Figure 2 and the interactions listed in Table 2, much of the mimicry of E5.2 for HEL resides in the similar interactions made by the VH CDR3 (H3) loop of the anti-Id and residues 116 to 121 of the Ag. Figure 7 demonstrates the mimicry of HEL residues 117 to 121. Shown therein are two of the six mimicked hydrogen-bond interactions (D1.3 VH Tyr101-HEL Asp119; D1.3 VH Tyr101-E5.2 VH Gly100a and D1.3 VH Gly53 N-HEL Gly117 O; D13 VH Gly53 N-E5.2 VH Tyr98 O). In addition, the anti-Id functionally substitutes for several water molecules important in the Ab1-Ag interaction. Specifically, E5.2 VH residue Tyr98 substitutes for three Ab1-Ag water molecules that bridge Ab1 atoms VH Glu98 Oe1 and VH Tyr101 N to Ag atoms Thr118 O and Gly117 N. Mimicry of the solvent structure is exemplified by the position equivalence of the tyrosine hydroxyl group, which interacts with D1.3 in a manner identical with that of the water molecules in the D1.3-HEL structure. Furthermore, the amide nitrogen atom of E5.2 VH residue Gln100 is positionally equivalent to a water molecule that bridges D1.3 VH Asp54 and HEL Asp119. The solvent structure of the Id-anti-Id complex contributes to the mimicry of the Ag. As shown in Figure 7, a water molecule in the

146

Antigen Mimicry by an Anti-idiotope

Figure 7. Stereo view of molecular mimicry of HEL residues 117-121 by VH CDR3 of the anti-Id, E5.2. The D1.3-E5.2 complex is shown in blue, D1.3-HEL complex in yellow. D1.3 VH Glu98 (omitted for clarity) also interacts with E5.2 VH Tyr98 and the water molecules (spheres) in the D1.3-HEL complex.

Ab1-anti-Id Ab2 complex structurally and functionally substitutes for the amide nitrogen atom of HEL Val120, making the same bond to the hydroxyl group of D1.3 VH Tyr101. The mimicry of E5.2 for HEL residues Asp18, Gly22, Tyr23 and Ser24 is shown in Figure 8. Here, E5.2 residue VH Gln58 substitutes for HEL residue Asp18 in the formation of a hydrogen bond to the hydroxyl group of D1.3 VH Tyr50. Additionally, E5.2 atom VH His33 Ne2 substitutes for the amide nitrogen atom of HEL Ser24 and E5.2 atom VH Asp52 Od1 substitutes for the carbonyl oxygen of HEL Gly22 in interactions with D1.3 VH Asp100. These last two interactions are mediated by a zinc ion as discussed above.

Effect of mutations The contribution of each Fv D1.3 CDR side-chain to the affinity of the D1.3-HEL and D1.3-E5.2 reactions has been probed by single-site alanine mutations (Dall’Acqua et al., 1996). The results of these experiments further exemplify the structural/ functional mimicry of E5.2 for HEL. It is quite revealing that only three of the Fv D1.3 alanine mutants, VH Trp52Ala, VH Asp54Ala and VH Glu98Ala, greatly reduce (i.e. by more than three orders of magnitude) the affinity of the D1.3-E5.2 reaction while minimally affecting the D1.3-HEL reaction. D1.3 VH Trp52 makes an equal number of contacts to the Ag and to the anti-Id (Table 2).

Figure 8. Stereo view of molecular mimicry of HEL residues Arg18, Gly22, Tyr23 and Ser24 by E5.2 VH His33 (CDR1) and VH Asp52 and Gln58 (CDR2). Color code as for Figure 7. The zinc ion in the D1.3-E5.2 interface is shown in red. This ion is not necessary for complex formation and its deletion should not change the conformation of the D1.3-E5.2 interaction (see the text).

147

Antigen Mimicry by an Anti-idiotope

Figure 9. Stereo view of the areas of interaction in the D1.3-E5.2 complex (blue) and D1.3-HEL complex (yellow) involving D1.3 VH Trp52. The surface of VH Trp52 in contact with E5.2 is depicted as Xs, and the surface in contact with HEL as Os.

However, the interaction surface area of the VH ˚ 2) Trp52 interaction with E5.2 is much greater (108 A than the interaction surface area in the D1.3-HEL ˚ 2, see Figure 9). The affinity of the VH complex (26 A Trp52Ala mutant-E5.2 reaction with D1.3 is approximately 103 less than the wild-type-E5.2 reaction, which translates to an approximately 4 kcal mol−1 difference in free energy. Estimates of the contribution by hydrophobic forces to the free energy of complex formation range from 25 to ˚ −2 (Chothia, 1974; Eisenberg & 50 cal mol−1 A McLachlan, 1986; Nicholls et al., 1991). Therefore, the 4 kcal mol−1 difference in the free energy between the Trp52Ala and wild-type D1.3 reactions ˚ 2 of with E5.2 can be ascribed to the loss of 108 A ˚2 interaction surface area. With the loss of only 26 A of interaction surface area, the affinities of the D1.3 Trp52Ala -HEL and wild-type D1.3-HEL reactions are substantially the same. Thus, the anti-Id mimics the Ag in the number of van der Waals contacts to D1.3 Trp52 and surpasses the Ag in the contribution of these contacts to complex stability. This feature is in agreement with the observed preponderance of aromatic residues in antibody combining sites (Amit et al., 1986; Janin & Chothia, 1990; Padlan, 1990). A D1.3 VH Asp54Ala substitution decreases the affinity of the Id-anti-Id reaction, while having little effect on the Ab1-Ag reaction. Asp54 makes two hydrogen bonds to E5.2 (D1.3 VH Asp54 Od2-E5.2 VH Gln100 N; D1.3 VH Asp54 Od2-E5.2 VL Tyr49 OH). In the D1.3-HEL complex, VH Asp54 interacts with the amide nitrogen atom of HEL Asp119 through a bound water molecule (see Figure 6). Likewise, the replacement of D1.3 VH Glu98 with alanine decreases the affinity of the D1.3-E5.2 reaction by

103, as a result of the loss of the Glu98 Oe1-E5.2 VH Tyr98 OH hydrogen bond, but has little effect on the D1.3-HEL reaction, where the Ab1-Ag interaction is mediated by water molecules. In these two cases, the substitution of Ab1-Ag interface water molecules by anti-Id protein atoms enhances the Ab1-anti-Id complex stability while maintaining electrostatic complementarity. Comparing the effect of the D1.3 CDR alanine mutants on the affinity of the D1.3-HEL and D1.3-E5.2 reactions with the crystal structures rationalizes the lack of molecular mimicry of E5.2 for several of the D1.3-HEL hydrogen bonds. Of the eight D1.3-HEL hydrogen bonds that are not mimicked in the D1.3-E5.2 crystal structure, four are at the periphery of the D1.3-HEL interface and are exposed to the solvent continuum, decreasing the importance of these interactions to the overall stability of the antibody-antigen complex. However, the remaining four nonmimicked hydrogen bonds (D.13 VL Phe91 O-HEL Gln121 Ne2, D1.3 VH Asp100 Od2-HEL Ser24 O, D1.3 VH Asp100 Od2-HEL Asn27 Nd2 and D1.3 VH Tyr101 OH-HEL Gln121 N) are buried in the D1.3-HEL interface and consequently afford considerable stability to the antibody-antigen complex.

Conclusions The Id-anti-Id complex described here gives an indication of how the CDRs of an anti-Id can ‘‘mimic’’ a protein antigen such as HEL. In general, the closeness of molecular mimicry will depend on the structure of the epitope, the choice of Id and of anti-Id, and on many other parameters that affect

148 immune responses. The D1.3-E5.2 idiotope-anti-idiotope complex strongly indicates that antigen mimicry by anti-Id Ab2 is functional. This means that the anti-Id Ab2 provides similar binding interactions rather than exact ‘‘topological’’ replicas of the antigen at a molecular level. This will be particularly so when anti-Id antibodies ‘‘mimic’’ molecules that are not antibodies nor even proteins. Furthermore, it is concluded that mimicry does not depend on amino acid sequence homologies between the antigen and anti-Id, supporting a conclusion proposed on the basis of immunochemical studies of anti-Id antibodies (Erlanger, 1989) and molecular modeling of major histocompatibilty class II-peptide complexes (Quaratino et al., 1995) and peptide mimicry of myelin basic protein binding to T cell receptor (Wucherpfennig & Strominger, 1995). Other biological systems, such as that of the growth hormone and its receptor, provide interesting examples of molecular mimicry that are not associated with the immune system. A monomer of the human growth hormone binds to the same area of two receptor molecules by non-covalent bonding interactions made by two different areas of the hormone structure (De Vos et al., 1992). In this case, it could be said that one of these hormone areas mimics the other, not structurally but in a functional way, by binding to the same site of the receptor. In addition, crystal structures of uracilDNA glycosylase (UDG), in complex with inhibitor Ugi, which mimics DNA, have shown that the functional mimicry of the Ugi for DNA is managed by shape and electrostatic complementarity to the UDG binding site (Savva & Pearl, 1995; Mol et al., 1995). This is the way in which an anti-Id may mimic an Ag, by binding to the same area of the Ab1 and by making similar bonding interactions to those made by the Ag.

Materials and Methods Production, crystallization and X-ray data collection The Fv fragments from antibodies D1.3 and E5.2 were expressed in Escherichia coli and crystallized as a complex from PEG8000 as described (Goldbaum et al., 1994). Crystals are monoclinic, space group C2, with unit cell ˚ , b = 79.4 A ˚ , c = 51.5 A ˚ , b = 100.2°. dimensions a = 152.8 A The asymmetric units consists of one Fv-Fv complex with approximately 60% (v/v) solvent content. Data were collected from two crystals at ambient temperature. Data from the first crystal were collected on a Siemens multi-wire area detector as described (Goldbaum et al., ˚ resolution 1994). Merging 66,725 observations to 2.0 A using the program XENGEN (Howard et al., 1987) resulted in 31,489 unique reflections with an Rmerge of 9.0% (77% completeness). This data set was used for the molecular replacement calculations and initial refinement of the structure. Subsequently, data were collected to higher resolution from a second crystal on a Rigaku R-AXIS image plate instrument. A total of 105,969 ˚ resolution were merged to give observations to 1.79 A 41,404 unique reflections with F > 0.0 (Rmerge = 8.5%). This ˚ data set is 82.5% complete in the resolution range 7.0 A

Antigen Mimicry by an Anti-idiotope ˚ (38,579 reflections) and 42.1% complete in the to 1.9 A ˚ to 1.9 A ˚ (2845 reflections). range 2.0 A Structure determination The structure was solved using the molecular replacement method with the program AMoRe (Navaza, 1994). The search model consisted of the backbone and Cb atoms of Fv D1.3 taken from the structure of the Fv D1.3-HEL complex (Bhat et al., 1994; PDB accession code 1VFB, Bernstein et al., 1977). Refined individual temperature factors were retained in the search model. To aid interpretation of the translation function, the coordinates of the search model were translated such that the center of mass was at the origin. Structure factors were calculated in an artificial triclinic cell (space group ˚, P1) with unit cell parameters a = b = c = 120 A a = b = g = 90°. The cross-rotation function was calcu˚ to 5 A ˚ and a lated using reflections in the range 10 A ˚ . The highest two peaks Patterson sphere radius of 30 A had correlation coefficients that were 29% (peak 1) and 25% (peak 2) greater than the first noise peak that had a correlation coefficient only 4% greater than the next noise peak. The top 25 peaks obtained from the cross-rotation function were used as input to the translation function, ˚ to 5 A ˚. also calculated using reflections in the range 10 A Cross-rotation peaks 1 and 2 gave correlations in the translation function that were 60% and 28% greater, respectively, than the next highest translation peak. The position obtained for peak 1 was then fixed and the translation function repeated in order to establish the position of the second Fv fragment of the complex. The correct solution had a correlation coefficient 55% greater than the highest incorrect solution and the R value was 0.45 compared with 0.49 for the highest incorrect solution. Rigid-body refinement of the orientation and position of each Fv fragment reduced the R value from ˚ to 4 A ˚. 0.44 to 0.42 for data in the range 8 A Structure refinement Refinement of the structure was initiated with the Siemens data using XPLOR with parameter and topology sets param19x and toph19x, respectively (Bru¨nger et al., 1987). The first 100 cycles of conventional positional ˚ to 3.0 A ˚ and refinement employed data in the range 7.0 A resulted in a reduction of R from 0.459 to 0.334. At this stage the Fv-Fv model consisted only of the main-chain and Cb atoms from the molecular replacement solution. Side-chains corresponding to the conformations of those in Fv D1.3 were added to both Fv fragments of the complex in order to establish the correct identity of each Fv subunit. Positional refinement (100 cycles) followed by inspection of a 2Fo − FC electron density map showed unambiguously which fragment was Fv D1.3 due to well defined electron density for side-chains present in the D1.3 sequence and not in the E5.2 sequence. Side-chains for Fv D1.3 were retained while those for E5.2 were trimmed back to Cb (Ca for Gly) unless the amino acid was conserved with D1.3 and good electron density was present for the side-chain. Refinement of the structure consisted of positional refinement, refinement of temperature factors (B, overall and individual atom) and, less frequently, simulated annealing, interspersed with map inspection and model building. Map inspection and model building were carried out with the program TURBO-FRODO (Roussel & Cambillau, 1989) running on a Silicon Graphics Indigo2 workstation. The structure

149

Antigen Mimicry by an Anti-idiotope was modeled iteratively into Fo − Fc and 2Fo − Fc maps calculated with gradually increasing high-resolution limits. While E5.2 VH CDR2 (insertion of one residue) and CDR3 (insertion of five residues) required model building into electron density, none of the CDR loops in D1.3 required manual rebuilding at this stage. The remaining CDRs of E5.2 did not differ in length from the corresponding CDRs of D1.3 and modeling was restricted to amino acid side-chain substitutions. As the refinement progressed persistent positive and negative Fo − Fc density on opposite sides of the peptide group of D1.3 VL Trp92-Ser93 became more pronounced. This density was modeled successfully by flipping the backbone peptide group linking residues 92 and 93. The first cycles of restrained individual B refinement were run ˚ to 2.5 A ˚ data and resulted in when R was 0.282 for 7.0 A the reduction of R to 0.259. Water molecules were incorporated into the atomic model only when the conformations of the four polypeptide chains in the complex were modeled with confidence. A peak in an Fo − Fc map was modeled as water only if it: (1) was greater than 3.5s; (2) made at least one potential hydrogen-bonding interaction; (3) made no contact with ˚ and no contact with N or O atoms C or S atoms < 3.0 A ˚ ; and (4) persisted in subsequent 2Fo − Fc electron < 2.4 A density at the ls level. Two large (09s) peaks in Fo − Fc ˚ ) contacts maps that made unacceptably close (02.0 A with protein atoms persisted during the refinement. These peaks were ultimately modeled as zinc ions. One of these zinc ions was present in the Fv-Fv interface and bridged a D1.3 side-chain (VH Asp100) with two E5.2 side-chains (VH His33 and Asp52). The other Zn site lies on the crystallographic 2-fold axis and is bound to the N terminus of E5.2 VL . To test whether the Zn sites could be modeled equally well as water molecules, the atomic scattering factors of Zn were temporarily substituted with the scattering factors of oxygen. Subsequent temperature factor refinement reduced B for both ˚ 2 (the lower limit) and residual electron positions to 2.0 A density was visible indicating that a more electron-rich atom should be placed at each site. Thus, with the absence of other heavy-atom reagents in the crystallization medium, we are confident that these two sites are occupied by zinc ions. An additional Zn site was modeled at a later stage that bridged the side-chains of His86 and Asp88 on the surface of D1.3 VL . Since the peak (6s) corresponding to this site was smaller than the other Zn sites, it was modeled with occupancy 0.5. The observation of zinc ions in the structure was not surprising, since the presence of 0.2 M zinc acetate in the crystallization medium facilitated the growth of larger crystals. Refinement progressed with the electronic area ˚ detector data until the R value was 0.196 for 7.0 to 2.2 A data (19,808 reflections) and 89 water molecules present in the model. At this stage the R-axis data became available and, because of its higher resolution and superior merging statistics, refinement was continued with this data set alone. The initial R calculated with the image plate data using the final coordinates from the refinement with the Siemens data was 0.249 for the 30,748 ˚ . The reflections within the resolution range 7.0 to 2.0 A parameter and topology sets param19x and toph19x were replaced by those of Engh & Huber (1991) when the R ˚ data. Refinement was value was 0.201 for 7.0 to 2.0 A continued until convergence, giving a final R of 0.194 for ˚ to the 32,539 reflections with F > 2sF in the range 7.0 A ˚ (data completeness 69.6%). The R value for all 1.9 A ˚ and 1.9 A ˚ is 0.213. The 38,579 reflections between 7.0 A

free-R value (Bru¨nger, 1992), computed after a cycle of simulated annealing against a subset of 1000 reflections, is 0.235, close to the intial R value computed for the R-axis data set. The 157 water molecules and three zinc ions are included in the final model. Atomic coordinates of the D1.3-E5.2 crystal structure have been deposited in the Brookhaven Protein Data Bank (code 1DVF). Molecular surface areas buried by the Fv D1.3-Fv E5.2 interaction were calculated using the MS suite of ˚. programs (Connolly, 1983) using a probe radius of 1.7 A Water molecules associated with the interface were ˚ (minimum defined by those water molecules within 1.4 A radius of a water molecule) of the buried molecular surfaces. The complementarity of the hydrophobic and hydrophilic surfaces of the D1.3-HEL and D1.3-E5.2 complexes was quantified by the percentage of surface points on the D1.3 buried surface whose nearest neighbors on the HEL and E5.2 buried surfaces are of the same hydrophobicity/hydrophilicity. In this procedure, hydrophilic surfaces were simply defined as those generated by nitrogen or oxygen atoms. Surface points ˚ distant where the nearest neighbor was more than 1 A were not included in the analysis. Similarity of the D1.3 surfaces and of the HEL and E5.2 surfaces were calculated in the same manner. All calculations of buried surfaces and surface complementarity were performed on D1.3-HEL and D1.3-E5.2 models, which were superimposed on all main-chain and Cb atoms of the D1.3 moiety.

Acknowledgements This research was supported by grants from the NIH (to R.A.M.) and from the Human Frontier Science Program and the W. E. Elkins Professorship at the University of Maryland (to R.J.P.).

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Edited by A. R. Fersht (Received 24 June 1996; received in revised form 9 September 1996; accepted 12 September 1996)