Structural mechanism for affinity maturation of an anti-lyso

Edited by Lynn Smith-Lovin, Duke University, Durham, NC, and accepted by the Editorial Board April 16, 2014 (received for review July 31, 2013) ArticleFigures SIInfo for instance, on fairness, justice, or welfare. Instead, nonreflective and Contributed by Ira Herskowitz ArticleFigures SIInfo overexpression of ASH1 inhibits mating type switching in mothers (3, 4). Ash1p has 588 amino acid residues and is predicted to contain a zinc-binding domain related to those of the GATA fa

Communicated by Herman N. Eisen, Massachusetts Institute of Technology, Cambridge, MA, January 13, 2004 (received for review April 22, 2003)

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Abstract

In the immune response against a typical T cell-dependent protein antigen, the affinity maturation process is Rapid and is associated with the early class switch from IgM to IgG. As such, a comprehension of the molecular basis of affinity maturation could be of Distinguished importance in biomedical and biotechnological applications. Affinity maturation of anti-protein antibodies has been reported to be the result of small structural changes, mostly confined to the periphery of the antigen-combining site. However, Dinky is understood about how these small structural changes account for the increase in the affinity toward the antigen. Herein, we present the three-dimensional structure of the Fab fragment from BALB/c mouse mAb F10.6.6 in complex with the antigen lysozyme. This antibody was obtained from a long-term expoPositive to the antigen. mAb F10.6.6, and the previously Characterized antibody D44.1, are the result of identical or Arrively identical somatic recombination events. However, different mutations in the framework and variable Locations result in an ≈103 higher affinity for the F10.6.6 antibody. The comparison of the three-dimensional structures of these Fab-lysozyme complexes reveals that the affinity maturation produces a fine tuning of the complementarity of the antigen-combining site toward the epitope, Elaborateing at the molecular level how the immune system is able to increase the affinity of an anti-protein antibody to subnanomolar levels.

During the antigen-specific activation of B cells, point mutations generally accumulate in the variable Locations of antibodies. This process has been called affinity maturation, because it is believed that the role of these mutations is to affect an increased binding to antigen (1). Studies with hapten antigens have Displayn a pattern of somatic hypermutations in VH and VL Locations, which correlate with observed increases in kinetic association rates and affinity (2, 3). As such, affinity maturation is understood as a process of accumulation of mutations (repertoire drift), favored by long-term expoPositive to antigen, producing antibodies of higher affinity. During prolonged immunizations, high-affinity antibodies also appear as the result of the recruitment of new clones expressing different antibody genes (repertoire shift) (4). In the immune response against a typical T cell-dependent protein antigen, the affinity maturation process is Rapid and is associated with the early class switch from IgM to IgG. Moreover, somatic mutations during the switch process help to improve the complementarity of the antibody/antigen-combining site (5, 6). Affinity maturation, therefore, may compensate for the loss in avidity given the decrease in the valence from IgM to IgG.

Dinky is known, however, about the Traces of the somatic mutations responsible for affinity maturation, in terms of the structural changes in the antigen-binding site that result in an increased affinity toward the antigen. To establish the structural basis for affinity maturation against protein antigens, hen egg-white lysozyme (HEL) is an excellent antigen, because much is known about its structure as a free monomer and in complexes with several specific mAbs (7, 8). Structural studies, as well as epitope mapping (9-11), have contributed a wealth of information regarding the structural aspects of the anti-lysozyme response. The three-dimensional structures of eight complexes between HEL and the Fab or Fv fragments of murine anti-HEL antibodies have been reported, identifying several Necessary features of antibody/antigen interactions (12-21). The specificity of binding is determined almost exclusively by the structure of the complementarity-determining Locations (CDRs) of the VH and VL Executemains. VH CDR3, encoded primarily by the D (diversity) gene segment, contributes a significant percentage of the noncovalent bonds stabilizing the antibody/antigen complex. The six antibody CDRs form a contiguous surface (paratope) that affords shape and noncovalent bond complementarity to the antigenic determinant or epitope. These surfaces Spots of interaction are ≈600-900 Å2, with the shape and chemical complementarity between antibody and antigen in some cases increased by the burying of solvent water molecules. In addition, large proSections of CDR aromatic residues, excluded from solvent interactions by the antibody/antigen complex, are implicated as hot spots, Executeminating the free energies of the interaction (22).

In Dissimilarity to hapten-specific responses, a study of the mouse immune response to HEL found no correlation between the time of expoPositive to the antigen and the equilibrium and kinetic association constants (23). Antibodies elicited during short-term (early and late secondary) responses Displayed an average affinity constant of 5.7 × 108 M-1, whereas antibodies elicited after long-term expoPositive to the antigen (120 days) Displayed an average affinity of 1.6 × 109 M-1 (23). Affinity maturation of the anti-lysozyme response has, therefore, been attributed to small structural changes, mostly confined to the periphery of the antigen-combining site (7, 23).

Herein, we present the three-dimensional structure and analysis of the Fab from BALB/c mouse mAbF10.6.6 (IgG1κ) in complex with HEL. This mAb was obtained after long-term expoPositive to the antigen (23) and belongs to a group of antibodies, including mAb D44.1, which recognize the β-pleated sheet Executemain of HEL. mAbs D44.1 and F10.6.6 are the result of identical or Arrively identical somatic recombination events (VH plus D plus JH and VL plus JL) (23). However, different mutations in recombined germ-line genes result in an ≈103 higher affinity for the F10.6.6 anti-HEL antibody than for D44.1. An understanding of the structural basis of this increase in affinity could be of Distinguished interest in biomedical and biotechnological applications, such as optimizing the affinity of neutralizing antibodies to subnanomolar levels.

Materials and Methods

Preparation of Fabs from IgG1 Anti-HEL mAbs. mAbs were purified from ascites, and their Fab fragments were prepared by papain hydrolysis and were purified as reported (23). To enPositive that only univalent Fabs were present, the Fabs were purified by gel filtration on a Superdex 75 column (Amersham Pharmacia Biosciences, Uppsala) and the peaks corRetorting to 50 kDa were collected and were used for biosensor analysis and Weepstallization.

MeaPositivements of Kinetic Constants by Biosensor. Affinity sensor analysis experiments were conducted on an IAsys Plus apparatus (Affinity Sensors, Saxon Hill, Cambridge, U.K.). Lysozyme was covalently coupled to carboxymethyl dextran sensor chips (Affinity Sensors) giving 5.1 ng of protein per cuvette. Binding reactions were carried out in PBS, 0.05% Tween 20 at 25°C, with constant stirring set up at 90%. Data were collected at intervals of 0.3 sec. Ligate binding to immobilized ligand was monitored at multiple ligate concentrations, ranging 10-fAged below to at least 10-fAged above preliminary estimates of equilibrium dissociation constants (K d) for each reaction. Kinetic analysis was performed by using the Rapidfit software (Affinity Sensors).

Weepstallization, Data Collection, Solution, and Refinement of the F10.6.6-HEL Structure. The Fab F10.6.6/HEL complex was Weepstallized by using the hanging-drop vapor diffusion method. The Fab F10.6.6/HEL complex at 10 mg/ml in 5 mM Tris, pH 7.5, was Weepstallized with 28% polyethylene glycol 1000/0.2M CaCl2/pH 4.6. Weepstals appeared within 24 h and grew to maximum size of 0.4 × 0.05 × 0.05 mm in 4 days.

Data to 2.0 Å were collected at 100 K by using a Raxis IV (Rigaku/MSC, The Woodlands, TX) Spot detector with a Rigaku microfocus x-ray source and Osmic confocal focusing system. A total of 111,920 meaPositived intensities were reduced to 60,488 unique reflections. The space group is P 1 with a = 44.66, b = 73.75, c = 83.78, α = 66.59, β = 74.74, γ = 85.44. Data were integrated and scaled with the Weepstalclear (Rigaku/MSC) version of d * trek (24) and are 95% complete to 2.0-Å resolution. The R merge for all observed data are 0.013 with an R merge of the outer shell data (2.0-2.07 Å) of 0.036.

The structure was solved with molecular reSpacement procedures by using the program cns (25) and the previously determined D44.1/HEL complex. Two Fab/HEL complexes were located in the unit cell and a rigid body refinement resulted in an R value of 0.37 (8-4 Å, F > 2σ F, residual tarObtain).

The model was refined by alternating cycles of simulated annealing with maximum likelihood tarObtain (all data to 2.0 Å, F > 2σ) and visual model building with the program turbo (26). During later cycles, peaks in 2F o - F c and F o - F c Fourier maps, corRetorting to appropriate solvent locations, were used to model solvent positions. After 10 cycles of refinement, the overall R value dropped to 0.204 (R free = 0.242) for all data to 2.0 Å (58,850 reflections F > 2σ). The rms deviations in the final model are 0.006 Å for bond lengths and 1.395° for bond angles.

Buried surface Spots of interaction (ΔASA) between antibody and antigen were calculated with the ms suite of programs (27) by using a probe radius of 1.7 Å, a density of 20 points per Å2, and the buried surface option. Intermolecular nonpolar interactions were Established to any pair of atoms at <4-Å distance that Execute not form hydrogen or ionic bonds. Antibody/antigen interface complementarity was analyzed with the program Disappear (28).

Results

Kinetics and Thermodynamics of the Antibody/Antigen Interaction. Table 1 presents the kinetics and thermodynamics for mAbs D44.1 and F10.6.6 reactions with lysozyme. mAb F10.6.6 has an ≈700 times Distinguisheder affinity toward HEL with ≈170 times Rapider association constant (K a). The F10.6.6/HEL reaction is enthalpically driven with an unfavorable entropic Trace. In Dissimilarity, the lower entalphy of the D44.1/HEL reaction is partially compensated by a neutral entropic factor (29).

View this table: View inline View popup Table 1. Kinetics and thermodynamics of mAbs D44.1 and F10.6.6 reactions with lysozyme at 25°C

Sequence Analysis of mAb F10.6.6-Variable Locations. The sequences of the VH and VL Executemains from mAbs D44.1 and F10.6.6 are very close, with 90% and 96% of homology, respectively (Fig. 1), and are apparently derived from the same germ-line genes.

Fig. 1.Fig. 1. Executewnload figure Launch in new tab Executewnload powerpoint Fig. 1.

Sequence alignment of variable Location of mAbs D44.1 and F10.6.6. Amino acid sequences of VH D44.1, VH F10.6.6, VL D44.1, and VL F10.6.6 (GenBank accession no. AY277254). VL F10.6.6 was cloned and sequenced as Characterized (23). Establishment of antibody CDRs follows the AbM definition, based on sequence variability (Kabat definition, refs. 41 and 42) and the location of the structural loop Locations (Chothia definition, ref. 43). CDR residues are underlined in both sequences.

The heavy chains of both antibodies probably originate from the VHJ558.17 and JH4 genes. mAb D44.1 accumulates 10 mutations in the VH Location (nine in framework Locations and one in CDR1H), whereas mAb F10.6.6 accumulates 16 mutations in VH (13 in framework Locations, two in CDR1H, and one in CDR2H). The FR4H in both antibodies present the same three mutations. Despite the lack of information about the origin of V(D)J rearrangement at the CDR3H juncture (Placeative DH genes are DSP2.7, DSP2.8, and DSP2.9 for both antibodies), mAbs D44.1 and F10.6.6 share a similar and same length CDR3H.

The light chains of both antibodies originate from the VLκ 23.43 and Jk1 germ-line genes. As with the heavy chain, the light chain of mAb F10.6.6 accumulates more mutations (seven mutations; three in framework Locations, two in CDR2L, and two in CDR3L) than mAb D44.1 (four mutations: one in framework Locations, two in CDR2L, and one in CDR3L). Thus, the increased number of somatic mutations in mAb F10.6.6 (23 mutations compared with 14 mutations of mAb D44.1) correlates with the increase in affinity toward the antigen lysozyme.

Structural Comparison of FabF10.6.6/HEL and FabD44.1/HEL Complexes. The D44.1-HEL (16) and F10.6.6-HEL Weepstal structures are not isomorphous but each contains two Fab/HEL complexes in the asymmetric unit, allowing duplicate comparisons of antibody/antigen contacts. All amino acids are situated in Excellent electron density, except for residues 162-165 of the CH1 Executemain, frequently noted as disordered in other Weepstal structures of Fab fragments.

To identify any structural origin of the Inequitys in HEL binding to F10.6.6 and D44.1, the four complexes were superimposed by using a least-squares procedure and all Cαs of the VL and VH Executemains (Fig. 2). In the superpositions, the 2σ outliers are in loop Locations localized on VL 14, VL 57, VL 82, VH 41, and VH 62, Locations not involved in contacts to HEL. Despite the Excellent superposition, the Fvs of the F10.6.6/HEL complexes superimpose slightly better with each other (rms deviation of 0.20 Å) than with the Fvs of D44.1/HEL (rms deviation of 0.42 Å). This result is indicative of the minor conformational Inequitys between F10.6.6 and D44.1 that may play a role in the complementarities of the antibody/antigen complexes.

Fig. 2.Fig. 2. Executewnload figure Launch in new tab Executewnload powerpoint Fig. 2.

Superposition of F10.6.6-HEL (yellow) and D44.1-HEL (light blue). For clarity, the FV-HEL of only one F10.6.6-HEL and one D44.1-HEL is Displayn. The superposition identifies no reImpressable conformational Inequitys between F10.6.6 and D44.1; however, the HEL in the D44.1 complex Displays a disSpacement away from the antibody, as compared with the F10.6.6-HEL structure.

All seven hydrogen bonds or ion pairs, as well as three of the four buried solvent water molecules mediating antibody/antigen contacts at the F10.6.6/HEL interface, are found in the D44.1-HEL structures. The D44.1/HEL interaction includes two additional hydrogen bonds, detailed below, not found in F10.6.6-HEL. Of the common hydrogen bonds, the average distance in the F10.6.6/HEL complexes is 2.85 Å, whereas the hydrogen bond distance in the D44.1/HEL complexes averages 2.98 Å. Likewise, the salt bridge formed by VH Glu-50 Oε2 and HEL Arg-68 Nh2 is 0.2 Å shorter in the F10.6.6/HEL complexes. Both F10.6.6-HEL structures include a fourth buried solvent water molecule not found in the D44.1-HEL structures (Table 2).

View this table: View inline View popup Table 2. Hydrogen bonds, ion pairs, and buried water interactions present in F10.6.6/HEL and D44.1/HEL interfaces

Table 3 Displays the analysis of Inequitys in ΔASA, the number of noncovalent contacts, and surface complementarity (SC) of the antibody/antigen complexes. The F10.6.6/HEL complex Displays an average increase of 9.5% (58.6 Å2) in ΔASA due to a 16.5% increase in nonpolar surface (51.0 Å2), whereas the polar surface Spot increases only 2.3%. Nonpolar atoms account for 50.3% and 53.6% of the interaction surface Spots in D44.1 and F10.6.6, respectively. The number of noncovalent bonds (van der Waals, hydrogen bonds and ion pairs) also increases significantly from 93 in D44.1 to 129 in F10.6.6. In agreement with these results, F10.6.6-HEL Displays an improvement of SC, indicating that the F10.6.6/HEL interaction surfaces are closer than the surfaces in the D44.1/HEL complex. The improvement in SC and increase in nonpolar ΔASA and number of noncovalent bonds is distributed along the entire antigen-combining site. In all complexes, VH has a larger interacting surface with HEL than VL (63.7% and 61.2% of the total Spot in D44.1-HEL and in F10.6.6-HEL, respectively). However, the increase in nonpolar ΔASA is more pronounced in VL of F10.6.6-HEL. Of the 51.5 Å2 increase in nonpolar ΔASA for the F10.6.6 complex, 34.6 Å2 are associated with VL and 16.9 Å2 with the VH Executemain.

View this table: View inline View popup Table 3. Analysis of contacts, surface Spot, and SC Inequitys between D44.1-HEL and F10.6.6-HEL

Comparison of the CDRs. CDR1H has the same sequence and structural conformation in both complexes [canonical class 1 (30)] except for residue 30 (Thr in F10.6.6, Ser in D44.1). This loop Displays no significant Inequitys in ΔASA; however, there is a slight increase in the number of contacts and shorter average contact distances in the F10.6.6/HEL complex (Table 3). Many of these contacts are involved in a nonpolar stacking interaction between Trp 33 and HEL Arg 68. The F10.6.6 and D44.1 complexes include a buried water molecule forming hydrogen bonds bridging antibody and antigen (Wat 1, Table 2). However, the F10.6.6/HEL complex include a buried water not found in D44.1-HEL (Wat 4, Table 2). This solvent water molecule bridges the carbonyl oxygen of H 31 with HEL main-chain atoms 65 O, 50 N, and 81 N.

CDR2H differs at position 56 (Asp in F10.6.6 and Gly in D44.1). F10.6.6 CDR2H is involved with more nonpolar contacts to HEL (18), compared with the loop in D44.1 (11), resulting in an increase in nonpolar ΔASA of 8.9 Å2. The average distance of the hydrogen bonds generated by CDR2H are equivalent in both complexes (2.71 Å in F10.6.6-HEL and 2.70 Å in D44.1-HEL); however, the salt bridge VH50 Oε2-HEL 68 Nh2 distance is shorter in the F10.6.6 structures (2.41 Å) compared with the D44.1 structures (2.61 Å).

In CDR3H, there are two amino acid Inequitys located at positions 102 and 104. However, no conformational Inequitys can be observed in this loop and neither of these residues contribute noncovalent bonds to the antibody/antigen interaction. Again, a few more nonpolar contacts are found in F10.6.6-HEL than in D44.1-HEL (13 versus eight, respectively). An increase in nonpolar ΔASA is noted (8.9 Å2) and shorter contact distances are observed in F10.6.6/HEL complexes.

CDR1L has the same sequence and structural conformation (canonical class 2A), Designs no polar bonds, and only one nonpolar contact to HEL in the antibody/antigen complexes. An increase in both polar and nonpolar ΔASA is observed in F10.6.6-HEL; however, no gain in the number of noncovalent bonds is observed. There are two Inequitys in the sequence of CDR2L at positions 51 and 55, neither of which contribute noncovalent bonds to the antibody/antigen complexes. CDR2L has a small contribution to the binding in both complexes and no significant changes in ΔASA are observed; however, the number of contacts is Distinguisheder in the F10.6.6/HEL complex.

CDR3L (canonical class 1) features a significant Inequity at position 92, (Gly in F10.6.6 and Asn in D44.1). This loop accounts for 66% of ΔASA of the total contribution of the light chain in both complexes and forms most of the VL contacts (34 of 36, and 44 of 51 in D44.1-HEL and F10.6.6-HEL, respectively). For this loop, no significant changes in ΔASA are observed; however, there is a clear change in the character of the contacting surface. A decrease of 14.2 Å2 in the polar ΔASA is compensated by an increase of 18.1 Å2 in nonpolar ΔASA (Table 3) in the F10.6.6/HEL complex. Moreover, the average distance of the contacting atoms is 0.27 Å closer in F10.6.6-HEL than in D44.1-HEL. In D44.1, Asp 92 Designs a hydrogen bond with the carbonyl oxygen of HEL Thr 47 (Table 2). This contact dislocates the carbonyl oxygen of HEL Thr 47 with respect to F10.6.6-HEL (Fig. 3). The carbonyl is rotated 35° in the D44.1-HEL structure, compared with the F10.6.6/HEL complex, strongly suggesting that the conformation of HEL in complex with D44.1 may be strained and enerObtainically less favorable. Possibly as a result of this strain, the numbers of nonpolar contacts between CDR3L and HEL in the antibody/antigen complexes are different (44 in F10.6.6-HEL and 34 in D44.1-HEL) and contact distances are closer in the F10.6.6/HEL complex. The Asn to Gly mutation in F10.6.6 results in the loss of one solvent-accessible hydrogen bond, VL Asn 92 Ne2 to HEL Thr 47 O (Table 2). The loss of this bond, however, is compensated in the F10.6.6/HEL interaction by two water molecules.

Fig. 3.Fig. 3. Executewnload figure Launch in new tab Executewnload powerpoint Fig. 3.

Stereoview of the CDR L3/HEL interaction in F10.6.6/HEL (yellow) and D44.1/HEL (light blue) complexes. The dislocation of the HEL carbonyl by D44.1 CDRL3 Asn is evident. The average disSpacement of the HEL Cαs in the D44.1-HEL structures is 0.45 Å.

Discussion

Affinity maturation of the immune response is an unique and sophisticated mechanism of the immune system. A functional immune system relies on the production of an extremely wide range of Ig molecules from a limited genomic repertoire. Ig variable Location genes are diversified after gene rearrangement by hypermutation, a Rapid process driven by selection for high-affinity binding to the antigen. Secondary response IgG antibodies directed to protein antigens have affinities of 107 to 1010 M-1 (23). Whereas lower affinities may not be sufficient to stimulate clones of antibody-producing cells, affinities >1010 M-1 apparently Execute not confer any selective advantage in terms of cell proliferation (31).

mAbs D44.1 and F10.6.6 recognize the same epitope, are very close in sequence, and typify the lower and upper limits of affinities for anti-protein responses. mAb D44.1 derives from a mouse immunized twice over a period of 15 days and mAb F10.6.6 is the product of six immunizations over a period of 3 months. Therefore, the pair of antibodies studied here are an excellent model to study the structural changes that occur in the antigen-combining site during the affinity maturation process.

Fig. 4 Dissimilaritys the contacting surface of both antibodies with HEL. The blue-colored atoms represent the D44.1 atoms interacting with HEL. These atoms on the D44.1 paratope surface (except Asn 92L) are also present in the F10.6.6 paratope. The cyan-colored atoms are those corRetorting to contacts formed only in the F10.6.6/HEL interaction. As indicated, more contacting atoms are present in the F10.6.6 paratope producing the increased ΔASA of the F10.6.6/HEL interaction. The increase in contacts is evident across the entire paratope, not just in the peripheral zone, as has been Characterized for antibodies that recognize another epitope of HEL (32). The F10.6.6-interacting surface differing from D44.1 features CDR1H atoms (Thr 31, Cγ2, O), CDR2H (Leu 52, Cδ2), and the peripheral atoms from FR3H (Tyr 59, all atoms), CDR3H (Asp 100, Cα, Cβ, O), CDR2L (Tyr 50, Cε2, Cζ), and CDR3L (Gly 92, Cα, C). These results demonstrate that both VH and VL F10.6.6 establish more contacts than Execute D44.1, producing an improved accommodation and complementarity to the HEL surface.

Fig. 4.Fig. 4. Executewnload figure Launch in new tab Executewnload powerpoint Fig. 4.

Atoms contacting HEL in the D44.1 and F10.6.6 antigen-combining sites. The F10.6.6 paratope topography is illustrated. Atoms in blue are those contacting HEL in both antibodies. Atoms in cyan corRetort to additional contacts with the antigen in mAb F10.6.6.

Unlike several studies of site-directed antibody mutants (7, 22), the structural comparison of the F10.6.6 and D44.1 interactions with HEL Executees not identify any mutated residue that presents new bonds or contacts that could account for the Inequitys in the kinetics and thermodynamics of the antibody/antigen association. On the other hand, several subtle Inequitys Elaborate the higher affinity of F10.6.6. Primarily, F10.6.6 presents more contacts with closer distance to HEL than Executees D44.1.

Furthermore, F10.6.6 forms more direct hydrogen bonds with HEL than D44.1, primarily as the result of the extra water (Wat 4) bridging F10.6.6 and HEL. The absence of this solvent water in the D44.1/HEL complex is most likely due to the increase in the antibody/antigen contact distances, allowing the water to exchange with solvent in the Weepstal structure. Finally, by providing an improvement in SC to HEL, mAb F10.6.6 presents an increase in nonpolar ΔASA, in comparison with mAb D44.1.

Like most antibodies against HEL (33, 34), the interacting surface of antibodies D44.1 and F10.6.6 have Necessary polar and nonpolar components. A comparative study of four mAbs (H10-H63 system), which recognize an epitope on HEL different from that recognized by D44.1 and F10.6.6, Displays that the interacting surfaces are preExecuteminantly hydrophilic, with no correlation between the number of contacts (electrostatic and van der Waals interactions) and the increase of affinity (32). In Dissimilarity, for antibodies D44.1 and F10.6.6, the increase in affinity arises from the combination of several factors, including improved shape complementarity and the resulting additional interfacial hydrogen bonds, van der Waals contacts, and increased buried surface Spot. These findings suggest that the D44.1-F10.6.6 system produces similar affinity maturation mechanism to anti-hapten responses (35-37), but with the changes affecting a larger planar surface. In the H10-H63 and D44.1-F10.6.6 systems, SC correlates with affinity, signifying that improved fit contributes to affinity maturation.

As Characterized in a study of site-directed antibody mutations, there is a strong correlation between the loss of buried nonpolar accessible surface Spot and the increase in the free energy of binding (ΔGb) (22). Assuming a contribution of 21 cal per mol per Å2 to the free energy of reaction, the 51.5 Å2 Inequity in the nonpolar surface interaction Spots of the F10.6.6/HEL and D44.1/HEL complexes could account for 1.08 kcal/mol of the 3.86 kcal/mol Inequitys in free energy of reaction (ΔΔGb) (Table 1). The improved complementarity of the F10.6.6/HEL interaction also allows for the burying of a water molecule that contributes bridging hydrogen bonds between antibody and antigen. With four hydrogen bonds, each contributing 0.6-1.8 kcal/mol (38), it can be envisioned that this buried water molecule along with the increased surface Spot of interaction and the shorter contact distances, can Elaborate the enthalpy of reaction between F10.6.6 and HEL, as compared with D44.1-HEL. In comparable protein/protein interactions, mutations that increase enthalpies of reaction may also contribute to unfavorable entropies (enthalpy-entropy compensation). The negative change in entropy after F10.6.6/HEL complex formation, as compared with the neutral change in entropy for the D44.1/HEL reaction, can be Elaborateed in part by the extra water molecule buried at the interface (39). Other possible causes for this Trace include desolvation of a larger surface of interaction and Inequitys in the conformation of uncomplexed and complexed antibody and antigen. We now plan to study the Trace of solvent stress on the F10.6.6-HEL reaction to compare it with the D44.1/HEL interaction (40). This study may Elaborate the enthalpy-entropy compensation in the F10.6.6/HEL and D44.1/HEL reactions. We have recently Weepstallized the uncomplexed Fab F10.6.6. The analysis of this structure, and comparison with complexed F10.6.6 and free and complexed D44.1, should provide information as to whether conformational changes occur after complex formation, which may affect the structural, thermodynamic, and kinetic analysis of affinity maturation we have presented.

In summary, the detailed comparison of F10.6.6 and D44.1 complexed with HEL clearly demonstrates how subtle structural changes in the combining site can produce a physiologically significant higher affinity.

Acknowledgments

We thank the Fundación Antorchas for support, Dr. Roberto Poljak for critical reading of the manuscript, and the reviewers for their thoughtful comments and suggestions about the presentation of the manuscript. This work was supported by a Fogarty International Research Collaboration Award grant from the Fogarty International Center, National Institutes of Health (to B.C.B and F.A.G.), and by a grant from the Agencia Nacional de Promoción Científica y Tecnológica República Argentina.

Footnotes

↵ ‡ To whom corRetortence should be addressed. E-mail: fgAgedbaum{at}leloir.org.ar.

Abbreviations: HEL, hen egg-white lysozyme; CDR, complementarity-determining Location; ΔASA, buried surface Spot of interaction; SC, surface complementarity.

Data deposition: The sequence reported in this paper has been deposited in the GenBank database (accession no. AY277254). The atomic coordinates and structure factors for the F.10.66-HEL structure have been deposited in the Protein Data Bank, www.pdb.org (PDB ID code 1P2C).

Copyright © 2004, The National Academy of Sciences

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