Sep 27, 1990 - Fischmann, Graham A. Bentley, Talapady N. Bhat, Ginette Boulot, Roy A. Mariuzza,. Simon E. V. Phillips$, Diana Tello, and Roberto J. Poljak.
Vol. 266, No . 20, Issue of July 15, pp. 12915-12920,1991 Printed in U.S.A.
THEJOURNAL OF BIOLOGICAL CHEMISTRY 8 1991 by The American Society for Biochemistry and Molecular Biology Inc
Crystallographic Refinementof the Three-dimensional Structureof the FabD1.3-Lysozyme Complex at 2.5-A Resolution* (Received for publication, September 27, 1990)
Thierry 0. Fischmann, Graham A.Bentley, Talapady N. Bhat, Ginette Boulot, Roy A. Mariuzza, Simon E. V. Phillips$, Diana Tello, and Roberto J. Poljak From the Departement d’lmmunologie, Institut Pasteur, 75724 Paris-Cedew15, France and the $Department of Biochemistry and Molecular Biology, Leeds University, Leeds LS29JT, United Kingdom
The three-dimensional crystal structure of the complex between the Fab from the monoclonal anti-lysozyme antibody D1.3 and the antigen, hen egg white lysozyme, has been refined by crystallogFaphic techniques using x-ray intensity data to 2.5-A resolution. The antibody contacts the antigen with residues from all its complementarity determining regions. Antigen residues 18-27 and 117-125 form a discontinuous antigenic determinant making hydrogen bonds and van der Waals interactions with the antibody. Water molecules at or near the antigen-antibody interface mediate some contacts between antigen and antibody. The fine specificity of antibody D1.3, which does not bind ( K . < 10‘ M-’) avian lysozymes where GlnlZ1in the amino acid sequence is occupied by His, can be explained on the basis of the refined model.
The three-dimensional structure of a complex between the Fab of a mouse (BALB/c) monoclonal antibody (mAb),’ D1.3 (IgG’, K ) , and its specific antigen, hen egg white lysozyme (HEL), has been determined by x-ray diffraction at 2.8-A resolution (Amit et al., 1986), followed by a preliminary refinement. The structural model thus obtained allowed the definition of the complete antigenic determinant as well as that of the combining site of the antibody. The antigenic determinant recognized bymAbD1.3 is discontinuous in nature, consisting of two stretches of the polypeptide chain of HEL centered around amino acid residues 20 and 120. At 2.8-A resolution, no major conformational change was observed in the complexed antigen. The occurrence of conformational changes in the antibody could not be directly ascertained by a comparison with the free Fab since this had not been crystallized. Fine specificity studies using a panel of anti-HEL antibodies (Harper et al., 1987) indicate that antibody D1.3 has a very low affinity for other avian lysozymes in which Gln”’ is replaced by His. There are several possible structural explanations for the fine specificity of mAb D1.3, but selecting the correct one requires a model in which atomic contacts with the antigen can be precisely determined. Since the structure of the FabD1.3-HEL complex was re-
ported(Amit et al., 1986), FabD1.3 has been crystallized unliganded, and in a complex with the syngeneic, anti-idiotopic FabE225(Boulot et al., 1987). In addition, the Fv segment of D1.3, consisting of the variable domains of the heavy and light polypeptide chains (VH and VL, respectively), has been cloned, expressed in Escherichia coli (Ward et al., 1989), and subsequently crystallized as the free Fv, and in a complex with the antigen, HEL (Boulot et al., 1990). The three-dimensional structures of these crystclline forms have been determined (Bhat et al., 1990) to 1.9-A and 2.4-A resolution, respectively. Comparison of the free and the antigenbound Fv structures reveals a small relative displacement of the VH and VL domains. Establishing the possible occurrence of similar conformational differences between the free and the antigen-bound FabD1.3 is essential for a study of the antigen-binding reaction and for establishing apossible mechanismin the activation of effector functions by antibody molecules. This goal requires refined, high resolution structures of the free and antigen-bound FabD1.3. Thus, thecrystallographic refinement of th? atomic coordinates of the FabD1.3-HEL complex to 2.5-A resolution presented here is an essential step for structure-function correlations in this experimental antigen-antibody system. MATERIALSANDMETHODS
Crystals of the FabD1.3-HELcomplex were grown by macroseeding in hanging drops or capillaries, using 15 to 20% polyethylene glycol 8000, 0.1 M KPO,, p H 6.0 (Mariuzza et al., 1983). Their space group is P2,, with a = 56.0 A, b = 143.5 A, c = 49.3 A, fl = 120.4”, and one molecule of the Fomplex per asymmetric unit. For this work, a new data set to 2.5-A resolution was collected at the Biotechnology Resource, University of Virginia, Charlottesville, on the multiwire x-ray diffractometer (Sobottka et al., 1990), using two detectors placed at 87 cm from the crystal. CuKa radiation passing through a graphite monochromator was obtained from a rotating anode generator operated a t 42 kV, 150 mA, with an effective x-ray focus size of 0.5 X 0.5 mm. Three crystals, measuring about1.2 X 0.6 X 0.4 mm, were used. A total of 41,026 observations were scaled, reduced, and merged to give 20,475 independent reflections, 98%of the total expected between 5.5 and 2.5 A (see Fig. l). Intensity data between 5.5 and 2.8 A, collected with one detector, gave aoRmerge (= C h k l I Ie, - Iobs I &k.I Ie,) of 0.077; data between 3.5 and 2.5 A, collected with a second detector, were merged with the datacollected with the first detectorgivjng an overall R,,,,, of 0.115. Reflections in the range 7.0 A to 5.5 A were those previously measured(Amit et al., 1986) using afour-circle * This work was supported by the Centre Nationale de la Recherche diffractometer. In the final data set,18,460 reflections had structure Scientifique, Institut Pasteur, BAP Contract0221 from the EEC, and amplitudes IF1 2 2.5 o ( I ) and were retained for the crystallographic refinement reported here. ResearchGrant AI 25369 from theUnitedStatesPublicHealth Thestartingatomiccoordinates were thoseobtained at 2.8-A Service. The costs of publication of this article were defrayed in part by the paymentof page charges. This article must therefore hereby be resolution (Amit et al., 1986). The amino acid sequences of VH and marked “advertisement” in accordance with 18 U.S.C. Section 1734 VL have been determined (Verhoeyenet al., 1988 Wardet al., 1989); solely to indicate thisfact. those of the constant domainsof the heavy and thelight chains, CHI I The abbreviations used are: mAb, monoclonal antibody; HEL, and CL, respectively, were taken from Kabatet al. (1987). Refinement hen egg white lysozyme; TEL, turkey egg white lysozyme; CDR, proceeded by iterative use of the program PROLSQ (Hendrickson complementarity determining regions. and Konnert, 1980; Hendrickson, 1985) followed by model building
12915
Crystal Structureof FabD1.3-Lysozyme Complex
12916 0.4
0.3
I
2 t
0.2
0.1
0.0, 1
0.1
0.2
0.3
I
2SinBIL
FIG. 1. Crystallographic R-factor and percentage of theoretically possible reflections as a function of resolution. The line marked 0.35 A is taken from a Luzzati plot (Luzzati, 1952) and represents a statistical error of 0.35 A in the atomic positionsof the model. with the program FRODO (Jones, 1978; Pflugrath et al., 1984) on an Evans and Sutherland PS390 graphics system. Difference Fourier (2Fo - Fc), (Fo - Fc) and OMIT-maps (Bhat and Cohen,1984) were used to correct themodel. Individual atomic temperature factors were introduced. At the end of three cycles of rebuilding and refinement, I Fo I) was 28%, with a root the R-factor (R = I h k l 11 Fo I - I Fc II/&+i mean square deviation in bond lengths of 0.030 A. The program XPLOR (Briinger et al., 1987) was then used to perform one cycle of refinement, giving an R-factor of23.7% with a root mean square deviation in bond lengths of 0.024 A. The resulting electron density map gave better indications for manual improvements to the model. After these correctionswere applied, a second X-PLORcycle g9ve an H-factor of 20.5% with a root mean squaredeviation of 0.017 A from ideal bond lengths. Thirty watermolecules for which there were clear electron densities andacceptable hydrogen bonds were subsequently included in the model, and their coordinates and temperature factors were refined withX-PLOR.Three cycles of X-PLORconjugate gradient minimization positional (without simulated annealing) and individual B-factor refinementwere performed giving a final R-factor of 18.4% (19.5% for all reflections) with a root mean square deviation of 0.013 A and 3.3" from ideal bond lengths and angles. The root mean square deviation of the angle x1 from its nearest ideal value is 16.3" (Bhat et al., 1979; Hendrickson and Konnert,1980). An estimate of theintrinsic accuracy of thpatomicpositions(Luzzati, 1952) indicated a mean error of0.35 A (see Fig. 1). A list of the atomic coordinates has been sent for deposit at theBrookhaven Data Bank. The calculation of interatomiccontacts between atoms of the antigen and the antibody was performed with the same maximum contacting distances used by Sheriff et al. (1987) and Tulip et al. (1989) (see Table I). Enzyme-linked immunosorbentassaystotestdirectbinding of monoclonal antibodies to hen and turkey egg white lysozymes were performed as described (Harper et al., 1987).
disulfide, which is difficult to trace in many Fab structures, is stabilized by contacts between neighboring molecules and could be easily interpreted in the maps. In the variable domains, a major correction was made in the orien>ation of VH Leuz9, which was exposed to solvent in the 2.8-A model and is now buried. This change inVH affects the adjacent residue Thr3' in that it is not in contact with HEL as in the initial model (see below). Also, the VL Prog5previously built astrans has now been corrected to the cis form; this modification does not lead to significant changes in the antigen-antibody contacts previously reported. These two changes in the complementarity determining regions (CDRs) bring them into conformitywiththecanonicalstructures derivedfrom other immunoglobulins andwiththeirpredictedconformation (Chothia et al., 1986). Within the limits of the current resolution, the V regions and, in particular, the antigen-antibody interface are well defined throughout without obvious ambiside chains. guities in the positioningof the main chain or the The Ramachandran plots (Ramachandran and Sasiskharan, 1968) (Fig. 2) indicate that the angular distributionconforms well with those seen in highly refined high resolutioq structyres. Thus, the extensionof the resolution from 2.8 A to 2.5 A and the refinement reported here result in an improvement in the R-factor (18.4% with a foot mean square deviation ideal bond lengthsof 0.013 A,compared to 28% and 0.03 4) and in the estimFted errorof the atomic coordinates(0.35 A compared to0.6 A). As described before (Amit et al., 1986), the angle made by the pseudo-2-fold axes relating VH to VL and CHI to cLis of about 176",giving an elongated Fab structurewith few intra-
tram
Psi
(')
Phi (") a
Psi ( O )
RESULTS AND DISCUSSION
Most of the carbonyl groups of the main chains of the FabD1.3-HEL complex can be oriented with confidence in the electron density maps. The major corrections applied to the preceding 2.8-A resolution model were in the CHI and CL domains. The polypeptide chains can be traced throughout, except for cH1 residues 130to 136, formingpart of an external loop exposed t o solvent, for which there is no clear electron density. In addition, several other loops near the terminal cystine of the cH1 and CL chainsarebasedonirregular Phi (") density, namely residues CL 154 to 158, CL 199 t o 203, and b C H I 187 to 194. For the latter,a ~ i s - P r o seems ' ~ ~ to fit better FIG. 2. Ramachadran plotsof the model at 2.5-A resolution. in the electron density than a trans, but this interpretation is 0,Gly; x, other residues. a, FAB D1.3 (432 residues); b, lysozyme in not definitive. By contrast, the region around the interchain the Fab-HEL complex (129 residues).
Structure Crystal
of
FabD1.3-Lysozyme Complex
chain, interdomain contacts. In the crystal lattice, the longer axis of the Fab-HELcomplex is roughly parallel to theb axis of the unit cell. Contacts between neighboring molecules of the complex along that axis occur between the C-terminal part of the Fab residues near the N and C terminus of HEL (Arg', Cys', Gly'", CyslZ7,and ArglZ8). Antigen-Antibody Contacts-Contacts between atoms of antigen and antibody are listed in Table I. The list is based on maximum van der Waals radii and hydrogen bond distances for the atoms involved in the contacts asexplained in Table I. These distances are thesame as used by Sheriff et al. (1987) and Tulip et al. (1989) so that the epitopes defined in different crystallographic studies can be consistently compared. Direct contacts with the antigenare made by VL residues T y P , Tyr", Thrs3, Phe",Trp'', and Serg3,and by VH residues Gly", Trps2, Gly53,Asp54,Asplo",Tyr'", and Arg"'. Some of these and, inaddition, VH Am", contact antigen residues through water molecules 805, 806, 809, 810, 815, 816, and 822 (water molecules are numbered 801 to 830; Fig. 3b). Since at 2.5-A resolution not all watermolecules may be unequivocally located, there could be additional interactions mediated by them. A comparison with antigen contacts
12917
made by FvD1.3 (Bhat et al., 1990) shows substantial agreement although with some differences which can mostly be explained by the estimated error (about 0.35 A) in the atomic coordinates. Other differences with Amit et al. (1986) are due to the crystallographic refinement of atomic coordinates reported here and to the distances used in defining contacting atoms. Twelve lysozyme residues make contact with the antibody combining site. They are Asp",Am", Gly2*,SerZ4, Gin'*', IlelZ4,and ArglZs(Table Gly'I7, Thr1I8, Asp"', I). This list of contacting residues is in agreement with that of Bhat et al. (1990), except for T y P and Lys"'. In the Fv structure, TyrZ3makes a single van der Waals coatact with VH AsplW,and Lys"' makes a hydrogen bond (3.2 A) with 0 7 of FvD1.3 Tyr3'. In the FabD1.3-HEL complex, the corre:pending hydrogen-bonded atoms occur at a distance of 4.0 A. The HELresidues in contact with the antibody contribute to adiscontinuous epitope. Additional contactsare made through water molecules, as discussed below. A least squares fit of the Ca atoms of FabD1.3-bound HEL with those of free HEL in its tetragonal form at 1.6-A resolution (Blake etd . , 1965) gives a root mean square difference
TABLEI List of antigen-antibody contacting atoms Maximum contact distancesof C-C, 4.1 A; C-N, 3.8 A, C-0, 3.7 0 - 0 , 3.3 A; 0 - N , 3.4 N-N, 3.4 occurring in the FvD1.3-HELor the FabD1.3-HELcomplexes are listed. Hydrogen-bond interactions, defined as the contacts between hydrogen bond acceptors and donors within maximum contact distances, are shownbold in numbers.
A;
Fah VL
HEL
Distance
A;
Fah VH
HEL
A Ne2
Tyr:" Cy T y P C61 Tyr"' Ce1 Gln'" Tyr"' C62 T y P Ce2 T y P C[ Tyr:" C5 Tyr'" C62 Tyrs" C62 Tyr" C5 Tyr" OTJ Tyr'" OTJ Thr"] Oy 1 Phe'l 0 Trpy2C a Trpg' Ct3 Trpg' Ce3 Trps' C52 Trpg' C(2 Trp" C53 Trp" C53 Trpg' C53 Trpg' C53 Trpy' C T J ~ Trpy' C T J ~ Ser':' N
Gln"' Ne2 Gln"' Ne2 Gln'" Ne2 Gln"' Ne2 Gln"' Ne2 Gln"' Cy Asn" Cy AsnIg N62 Asp" 062 Asp" Cy Asp" 062 Asnlg N62 Gln"' Ne2 G1n''l Nt2 Gln"' C6 Gln"' Ne2 ArglZsCy Arglzs C6 Gin'" Cy Gin'" C6 Gln'" Ne2 1 ~ c641 1 1 e 4c61 ArglZSCy Gln"' O t l
3.3" 3.5" 3.6" 3.3" 3.4" 3.5" 4.0 4.1 3.7 3.6 3.6
2.9 3.1 3.1 3.8 3.5 3.0 Asp''' 3.6 4.0 Asp""' 3.9 Asp"" 3.6 3.4 3.9 Asp"'" 3.9 Asp'"' 3.5
3.2
A
Distance
A Cly"' c Gly:" 0 Trp"' Ce3 Trp"' Ct3 Trp"' C(2 Asp"' Trp" C[2 Trp"' C(3 Trp"' C53 CTJZ Trp"' Trp"' C T J ~ Trp"' C T J ~ Trp"' C T J ~
N
~ 1 ~ , 5 : ~
Gly"' Cn GlY":' c Asps4 N Asps4 0 6 1 Cy Asp"' Cy Cy Cy Asp'" 0 6 1 Asp'"o 0 6 1 061 0 61 Asp""' 0 6 2 Asp""' 0 6 2 Tyr"" Ce 1 Tyr'"' Cel Tyr"" Ct2 Tyr'"' '25 Tyr'"' OTJ Tyr"' OTJ Tyr'" OTJ Tyr'"' OTJ Tyr"' OTJ Gln"' Tyr"' OTJ Arg"" N T J ~
Gly"' Ca ~ 1 ~ca 1 1 ~ Thr"' C Asp"' Ca Asp"' Cp Cy Asp"' Ca Asp"' Cp Asp"' Ca Asp"' C@ Asp"' Cy Asp1190 6 1 Gly"' 0 Gly"' 0 Gly"' 0 Gly"' 0 Thr"" Cy2 SerL4N Ser" Cp Ser'4 Oy Am2' N62 Ser14N S e P Ca Ser" C@ Ser" Oy SerA4Oy Amz7N62 Val"" Cy2 Gln'" Cp Gln"' Cp Gin'" Cp Asp"' Ca Asp"' 061 Asp"' C Val"' N Gln"' N Cg Gly" 0
3.9 3.7 3.9 4.0 4.0 4.1 3.7 4.0 4.0 3.8 3.9 3.7
2.9 3.2 3.7
3.2 3.6 3.8 3.9 3.2 3.5
2.8 3.6 3.2
3.0 2.6 3.0 3.9 3.9 4.0
3.5 3.4
3.0 3.3
3.3 3.0 3.4
3.2
a Contacts between the HEL Gln"' Ne2 and the aromatic ringof the side chainof VI, Tyr"' can be taken (Levitt and Perutz, 1988) as a hydrogen bond interaction.
Crystal Structure of FabDl.3-Lysozyme Complex
12918
A.
A,
of 0.48 The largest difference, 1.1 is observed for the Ca of Leu'". This C-terminal residue is very mobile as reflected by a large temperature factor. Thus, as noted before (Amit et al., 1986), no major change of conformation can be observed in the bound HEL. Changes in the orientation of its amino acid side chains relative to free lysozyme (Blake et al., 1965) can be observed in areas that arein contact with the antibody as well as in other areas which are not. However, a change in the conformation of Asnlg (a rotation of about 100" around a Ca-CP axis) can be explained by the formation of the complex with the antibody. In the crystal structureof native HEL, the N62 of Asnlg makes a hydrogen bond with the 0 6 1 of Asp". This conformation of Asnlg could not be kept in the complex with D1.3 since it would lead to disallowed van der Waals contacts with VL Tyr". Using the program MS (Connolly, 1983), the sum of the areas of the solvent-buried surfaces of HEL and D1.3 after complex formation was found to be 1290 A'. For this calculation, the radius of the probe was taken to be 1.4 A. HEL
Fab D1.3 V, Tyr32
Tyr50 TM3
Fab DL3
%e91
Trp92 sea93 V"Gly31 Trp52 GIy53 Asp54
'Arg125
Asp100 TyrlOl AIKlM
a
b
FIG. 3. Antigen-antibody contacts. Residuesorwater
molecules which form at least one hydrogen bond are joined by a solid line; dotted lines indicate residues which make only van der Waals contacts. a, directcontacts between Fabandantigen residues; b, contacts mediatedby water molecules.
F I G . 4. Stereoscopic view of water molecules superimposed on the Ca skeleton of the model of Fab D1.3 and the lysozyme. The 30 water molecules (only those close to Fab D1.3 or lysozyme are shown here) are represented by black points.
The pattern of hydrogen bonds (Fig. 3a) reveals the complexity and complementarity of the antigen-antibody interaction. Through these bonds, antibody main and side chains contact the epitope main and side chains. For example, main chain atomsof VL CDR3 (0of Phegl,N of Serg3)are hydrogenbonded to atoms of a side chain (Ne2 and 0 6 1 of Gln'*l) of the antigen. In addition, the close approach of antigen and antibody allows main chain atomsof VH CDR2 to make close hydrogen bonds with main chainatoms of the antigenic determinant. Water molecules buried in theantigen-antibody interface contributeto thehydrogen bonding pattern between antigen and antibody (see below). Water Molecules-Peaks in difference electron density maps consistently appearing at the later stages of crystallographic refinement have been interpreted as indicating the presence of solvent molecules. Since the current study does not have the resolution required for an unequivocal assignment, only those showing good electron density, low temperature factors, and close proximity to hydrogen bond donors or acceptors were retained. As a consequence, only 30 well ordered, buried, or partially buried water molecules, most of which have also been observed in the FvD1.3-HEL complex (Bhat et al., 1990), were added to the model (Fig. 4). Some, such as 805, 809, and 822 (Fig. 3b), are in contact with both the antigen andthe antibody and areburied in theirinterface. Water molecules 815 and 816,which also make a bridge between antigen and antibody (Fig. 3b), are largely exposed to the bulk solvent. Other possible water molecules such as 806,809, and 810 fill a cavity in the VH-VL interface opening onto the antigen-antibody contacting area. However, an alternative explanation for some of the electron density peaks assigned to these water molecules is that they correspond to one of two partially occupied positions of VL Tyr50. No buried water molecules were found inside any of the domqins of FabD1.3 e5cept for 802, H-bonqed to VL Ncl Trp35 (3.0 A), 0 ThrS1(2.8 A), and 0 Ser65(3.1 A), and for 803, Hbonded to VH 0 Tyr3' (3.2 A), 0 Ile51 (2.9 A), Nt Lys71 (3.1
16,
Crystal Structure of FabD1.3-Lysozyme Complex
vLPhe91
12919
VLTyr32
HEL Gln121
VLTrp92
FIG.5. Stereoscopic view of the residues forming the environmentof Gln121of lysozyme. The amide group of the side chain of Gln'" makes hydrogen bonds to VLPhegl and VLSerg3(thin lines).It makes, in addition, a hydrogen bond with the aromatic side chain of VL Tyr3'(see Table I). The side chain of HEL Gln''l is surrounded on one side bv the aromatic residues V,. Tyr", .~VI. T d ' , and VH Tyr"", and on the other side by HEL Asp"' and HEL ArgiZ5which together form a salt bridge.
A),and 0 Trp5*(3.2 A). No buried water molecules are found in the interface between CHI and CLand between VH and VL, except for 802,806, and 810, noted above. A t places where the complementarity of the antigen and antibody surfaces is not very close, water molecules fill the gaps as discussed above for 805, 807, 811, 818, 822, and 826. Water molecules 811, 818, 822, and 826 are in a pocket between the antigen and the antibody, close to VH Gly'". Antigen Variability and Antibody Specificity-The reactivity patterns of monoclonal anti-lysozyme antibodies were studied using a panel of eight different avian lysozymes (Harper et al., 1987). Antibody D1.3 reacts with high affinity only with HEL and bobwhite quail lysozyme which have a Gln residue at position 121. In the avian egg white lysozymes from partridge, turkey, California quail, pheasant, and guinea hen that wereused to study the fine specificity of mAb D1.3, position 121 is replaced by His. As can be seen in Table I and Fig. 5 , Gln"' makes a large number of close contacts with the antibody. It penetratesa hydrophobic pocket where it is surrounded by the aromatic side chains of VLTyr3*and Trpg2 and that of VH Tyr'". The atoms of its side chain make a large number of van der Waals contacts with atoms of VL Tyr:'?,Trp'', and VH Tyr"'. In addition, its side chain amide nitrogen and hydroxyl make three buried H bonds with the antibody. The hydrophobic cavity penetrated by Gln'*l, lined by the aromatic side chains of the antibody, is closed toward the solvent side by Arglz5and Asp"' of HEL, which are linked to each other by a salt bridge. Among the possible explanations for the very low affinity of D1.3 for the heterologous lysozymes are: 1) His'*l may betoo bulky for the tight pocket occupied by Gln in the FabD1.3-HEL complex; 2) His'" could be charged and consequently unstablein the hydrophobic pocket described above; and 3) at least one hydrogen bond would be lost when His'" is substituted for Gln'*l. The second explanation has been ruled out by performing direct binding enzyme-linked immunosorbent assays. These indicate no binding of D1.3 to plates coated with turkey egg white lysozyme (TEL) at pHvalues from 4.5 to 9.0, a range that goes from a predominantly charged to anuncharged form of the imidazole side chain of His. As a control, the recognition of HEL by mAb D1.3 and that of HEL and TEL by mAb D122.13 (Harper et al., 1987) are not affected in that pH range. In addition, the loss of H bonds resulting from the replacement Gln by His at position 121 could contribute to the much lower affinity of D1.3 for the variant lysozymes. It
is interesting to note in this context that VH D1.3 can bind HEL by itself with an affinity constant only one order of magnitude smaller than thatof the whole mAbD1.3 or its Fv (Ward et al., 1989). In the VH-HEL interaction, some of the hydrogen bonds made by the side chain of GlnIz1 with VL residues (see Table I and Fig. 5 ) may not be formed. The explanation invoking steric hindrance would be fully compatible with the refined structure reported here. A histidine residue could not fit into the hydrophobic cavity formed by VL Tyr3*, Trp9*, and VH Tyr'" and further enclosed by lysozyme residues Asp"' and ArgIz5.The 2 latter residues are linked to each other by a salt bridgewhichwould in all likelihood prevent adisplacement of the side chain 121 toward the solvent. In this tightly packed cavity, the histidine side chain would make a number of disallowed contacts with antibody atoms. Thus, a simple interpretation of the lack of recognition by antibody D1.3 of heterologous antigens in which position 121 is occupied by His could be steric hindrance. We cannot, however, preclude the possibility of small structural adjustments aroundthe pocket for Gln121to accommodate the larger amino acid side chain. Should this be the case, the loss of one hydrogen bond and the hydrogen-aromatic interaction would lead to a less favorable free energy gain in comparison to thecomplex formed with HEL. Whatever the true mechanism may be, this structure provides a basis for explaining the frequently observed escape of viral and microbial antigens from antibody neutalization through the occurrence of point mutations affecting their antigenic determinants. Acknowledgment-We thank the Convex France Co. for access to their C210 supercomputer. REFERENCES Amit, A. G., Mariuzza, R., Phillips, S. E. V., and Poljak, R. J. (1986) Science 228,1280-1284 Bhat, T. N., and Cohen, G. H.(1984) J. Appl. Crystallogr. 1 1 , 268272 Bhat, T. N., Sasisekharan, T., and Vijayan, M. (1979) Znt. J. Peptide Protein Res. 1 3 , 170-184 Bhat, T. N., Bentley, G. A., Fischmann, T. O., Boulot, G., and Poljak, R.J. (1990) Nature 347,483-485 Blake, C. C. F., Koenig, D. F., Mair, G. A., North, A. C. T., Phillips, D. C., and Sarma, V. R. (1965) Nature 206, 757-763 Boulot, G., Rojas, C., Bentley, G. A., Poljak, R. J., Barbier, E., Le Guern, C., and Cazenave, P. A. (1987) J. Mol. Biol. 194,577-579 Boulot, G., Eisek, J.-L., Bentley, G. A., Bhat, T. N., Ward, E. S., Winter, G., and Poljak, R. J. (1990) J. Mol. Biol. 213, 617-619
12920
Crystal Structure of FabDl.3-Lysozyme Complex
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