Three-dimensional structures of the free and antigen-bound Fab from monoclonal antilysozyme antibody HyHEL-63(,)

Antigen-antibody complexes provide useful models for studying the structure and energetics of protein-protein interactions. We report the cloning, bacterial expression, and crystallization of the antigen-binding fragment (Fab) of the anti-hen egg white lysozyme (HEL) antibody HyHEL-63 in both free and antigen-bound forms. The three-dimensional structure of Fab HyHEL-63 complexed with HEL was determined to 2.0 A resolution, while the structure of the unbound antibody was determined in two crystal forms, to 1.8 and 2.1 A resolution. In the complex, 19 HyHEL-63 residues from all six complementarity-determining regions (CDRs) of the antibody contact 21 HEL residues from three discontinuous polypeptide segments of the antigen. The interface also includes 11 bound water molecules, 3 of which are completely buried in the complex. Comparison of the structures of free and bound Fab HyHEL-63 reveals that several of the ordered water molecules in the free antibody-combining site are retained and that additional waters are added upon complex formation. The interface waters serve to increase shape and chemical complementarity by filling cavities between the interacting surfaces and by contributing to the hydrogen bonding network linking the antigen and antibody. Complementarity is further enhanced by small (<3 A) movements in the polypeptide backbones of certain antibody CDR loops, by rearrangements of side chains in the interface, and by a slight shift in the relative orientation of the V(L) and V(H) domains. The combining site residues of complexed Fab HyHEL-63 exhibit reduced temperature factors compared with those of the free Fab, suggesting a loss in conformational entropy upon binding. To probe the relative contribution of individual antigen residues to complex stabilization, single alanine substitutions were introduced in the epitope of HEL recognized by HyHEL-63, and their effects on antibody affinity were measured using surface plasmon resonance. In agreement with the crystal structure, HEL residues at the center of the interface that are buried in the complex contribute most to the binding energetics (DeltaG(mutant) - DeltaG(wild type) > 3.0 kcal/mol), whereas the apparent contributions of solvent-accessible residues at the periphery are much less pronounced (<1.5 kcal/mol). In the latter case, the mutations may be partially compensated by local rearrangements in solvent structure that help preserve shape complementarity and the interface hydrogen bonding network.     

Energetic analysis of an antigen/antibody interface: alanine scanning mutagenesis and double mutant cycles on the HyHEL-10/lysozyme interaction.

Alanine scanning mutagenesis of the HyHEL-10 paratope of the HyHEL-10/HEWL complex demonstrates that the energetically important side chains (hot spots) of both partners are in contact. A plot of deltadeltaG(HyHEL-10_mutant) vs. deltadeltaG(HEWL_mutant) for the five of six interacting side-chain hydrogen bonds is linear (Slope = 1). Only 3 of the 13 residues in the HEWL epitope contribute >4 kcal/mol to the free energy of formation of the complex when replaced by alanine, but 6 of the 12 HyHEL-10 paratope amino acids do. Double mutant cycle analysis of the single crystallographically identified salt bridge, D32H/K97, shows that there is a significant energetic penalty when either partner is replaced with a neutral side-chain amino acid, but the D32(H)N/K97M complex is as stable as the WT. The role of the disproportionately high number of Tyr residues in the CDR was evaluated by comparing the deltadeltaG values of the Tyr --> Phe vs. the corresponding Tyr --> Ala mutations. The nonpolar contacts in the light chain contribute only about one-half of the total deltadeltaG observed for the Tyr --> Ala mutation, while they are significantly more important in the heavy chain. Replacement of the N31L/K96 hydrogen bond with a salt bridge, N31D(L)/K96, destabilizes the complex by 1.4 kcal/mol. The free energy of interaction, deltadeltaG(int), obtained from double mutant cycle analysis showed that deltadeltaG(int) for any complex for which the HEWL residue probed is a major immunodeterminant is very close to the loss of free energy observed for the HyHEL-10 single mutant. Error propagation analysis of double mutant cycles shows that data of atypically high precision are required to use this method meaningfully, except where large deltadeltaG values are analyzed.     

Quantitative evaluation of the chicken lysozyme epitope in the HyHEL-10 Fab complex: free energies and kinetics.

The hen (chicken) egg-white lysozyme (HEWL) epitope for the monoclonal antibody HyHEL-10 Fab (Fab-10) was investigated by alanine scan mutagenesis. The association rate constants (k(on)) for the HEWL Fab-10 complexes were obtained from the homogenous solution method described in the preceding paper (Taylor et al., 1998). A new method for determining the dissociation rate constant (k(off)) for the complex, by trapping nascent free antibody with an inactive HEWL mutant is described. The values of k(on) fall within a factor of 2 of the wild-type (WT) HEWL value (1.43+/-0.13 X 10(6)M(-1)s(-1)), while the increases in k(off)more nearly reflect the total change in free energies of the complex (deltadeltaG(D)). The dissociation constants (K(D)) were measured directly in those cases where satisfactory kinetic data could not be obtained. The Y20A, K96A, and K97A HEWL.Fab-10 complexes are destabilized by more than 4 kcal/mol compared to the WT complex. The R21A, L75A, and D101A antibody complexes are moderately destabilized (0.7 < deltadeltaG(D)< or = 1.0 kcal/mol). Additional mutations of the "hotspot" residues (Tyr20, Lys96, Lys97) were constructed to probe, more precisely, the nature of their contributions to complex formation. The results show that the entire hydrocarbon side chains of Tyr20 and Lys97, and only the epsilon-amino group of Lys96, contribute to the stability of the complex. The value of deltadeltaG(D) for the R21A mutant complex is a distinct outlier in the Arg21 replacement series demonstrating the importance of supplementing alanine scan mutagenesis with additional mutations.     

Salt links dominate affinity of antibody HyHEL-5 for lysozyme through enthalpic contributions.

The binding of murine monoclonal antibody HyHEL-5 to lysozyme has been the subject of extensive crystallographic, computational, and experimental investigations. The complex of HyHEL-5 with hen egg lysozyme (HEL) features salt bridges between Fab heavy chain residue Glu(50), and Arg(45) and Arg(68) of HEL. This interaction has been predicted to play a dominant role in the association on the basis of molecular electrostatics calculations. The association of aspartic acid and glutamine mutants at position 50(H) of the cloned HyHEL-5 Fab with HEL and bobwhite quail lysozyme (BQL), an avian variant bearing an Arg(68) --> Lys substitution in the epitope, was characterized by isothermal titration calorimetry and sedimentation equilibrium. Affinities for HEL were reduced by 400-fold (E50(H)D) and 40,000-fold (E50(H)Q) (DeltaDeltaG degrees estimated at 4.0 and 6.4 kcal mol(-1), respectively). The same mutations reduce affinity for BQL by only 7- and 55-fold, respectively, indicating a reduced specificity for HEL. The loss of affinity upon mutation is in each case primarily due to an unfavorable change in the enthalpy of the interaction; the entropic contribution is virtually unchanged. An enthalpy-entropy compensation exists for each interaction; DeltaH degrees decreases, while DeltaS degrees increases with temperature. The DeltaCp for each mutant interaction is less negative than the wild-type. Mutant-cycle analysis suggests the mutations present in the HyHEL-5 Fab mutants are linked to those present in the BQL with coupling energies between 3 and 4 kcal mol(-1).     

Refined structures of Bobwhite quail lysozyme uncomplexed and complexed with the HyHEL-5 Fab fragment

The HyHEL-5 antibody has more than a thousandfold lower affinity for bobwhite quail lysozyme (BWQL) than for hen egg-white lysozyme (HEL). Four sequence differences exist between BWQL and HEL, of which only one is involved in the interface with the Fab. The structure of bobwhite quail lysozyme has been determined in the uncomplexed state in two different crystal forms and in the complexed state with HyHEL-5, an anti-hen egg-white lysozyme Fab. Similar backbone conformations are observed in the three molecules of the two crystal forms of uncomplexed BWQL, although they show considerable variability in side-chain conformation. A relatively mobile segment in uncomplexed BWQL is observed to be part of the HyHEL-5 epitope. No major backbone conformational differences are observed in the lysozyme upon complex formation, but side-chain conformational differences are seen in surface residues that are involved in the interface with the antibody. The hydrogen bonding in the interface between BWQL and HyHEL-5 is similar to that in previously determined lysozyme-HyHEL-5 complexes. © 1996 Wiley-Liss, Inc.     

Structural consequences of mutations in interfacial Tyr residues of a protein antigen-antibody complex. The case of HyHEL-10-HEL

Tyrosine is an important amino acid in protein-protein interaction hot spots. In particular, many Tyr residues are located in the antigen-binding sites of antibodies and endow high affinity and high specificity to these antibodies. To investigate the role of interfacial Tyr residues in protein-protein interactions, we performed crystallographic studies and thermodynamic analyses of the interaction between hen egg lysozyme (HEL) and the anti-HEL antibody HyHEL-10 Fv fragment. HyHEL-10 has six Tyr residues in its antigen-binding site, which were systematically mutated to Phe and Ala using site-directed mutagenesis. The crystal structures revealed several critical roles for these Tyr residues in the interaction between HEL and HyHEL-10 as follows: 1) the aromatic ring of Tyr-50 in the light chain (LTyr-50) was important for the correct ternary structure of variable regions of the immunoglobulin light chain and heavy chain and of HEL; 2) deletion of the hydroxyl group of Tyr-50 in the heavy chain (HTyr-50) resulted in structural changes in the antigen-antibody interface; and 3) the side chains of HTyr-33 and HTyr-53 may help induce fitting of the antibody to the antigen. Hot spot Tyr residues may contribute to the high affinity and high specificity of the antigen-antibody interaction through a diverse set of structural and thermodynamic interactions.     

Association and dissociation kinetics of anti-hen egg lysozyme monoclonal antibodies HyHEL-5 and HyHEL-10.

The immunoglobulin G1 (IgG1) kappa antibodies HyHEL-5 and HyHEL-10 interact with nonoverlapping epitopes on hen egg lysozyme (HEL); the HyHEL-5/HEL interface has two energetically and structurally important salt links, whereas the HyHEL-10/HEL interface involves predominantly hydrogen bonds and van der Waals interactions. The kinetics of association and dissociation of antibodies HyHEL-5 and HyHEL-10 with HEL under a variety of conditions were investigated in this study. The association of each antibody with HEL follows second-order kinetics. The association process is significantly diffusion-limited, as indicated by the viscosity dependence of the interaction of both antibodies with HEL, although detailed energetics suggest that the association process may be more complex. The association rate constant for the HyHEL-5/HEL system is within a factor of 2 of the modified Smoluchowski estimate for proteins of this size, whereas HyHEL-10 interacts with HEL with an association rate an order of magnitude lower. The association reactions are insensitive to ionic strength, showing only a twofold decrease in the association rate constant when the ionic strength was increased from 27 mM to 500 mM. Interestingly, the association rate constant for the interaction of HyHEL-5 with HEL varies with pH in the range 6.0-10.0, whereas HyHEL-10/HEL association is not affected by pH in the same range. The dissociation of the HyHEL-5/HEL and HyHEL-10/HEL complexes follow first-order kinetics with half-lives at 25 degrees C of approximately 3,150 s and approximately 21,660 s, respectively.     

Structural evidence for entropic contribution of salt bridge formation to a protein antigen-antibody interaction: the case of hen lysozyme-HyHEL-10 Fv complex

A structural and thermodynamic study of the entropic contribution of salt bridge formation to the interaction between hen egg white lysozyme (HEL) and the variable domain fragment (Fv) of anti-HEL antibody, HyHEL-10, was carried out. Three Fv mutants (HD32A, HD96A, and HD32AD96A) were prepared, and the interactions between the mutant Fvs and HEL were investigated. Crystallography revealed that the overall structures of these mutant complexes were almost identical to that of wild-type Fv. Little structural changes were observed in the HD32AD96A mutant-HEL complex, and two water molecules were introduced into the mutation site, indicating that the two water molecules structurally compensated for the complete removal of the salt bridges. This result suggests that the entropic contribution of the salt bridge originates from dehydration. In the singly mutated complexes, one water molecule was also introduced into the mutated site, bridging the antigen-antibody interface. However, a local structural difference was observed in the HD32A Fv-HEL complex, and conformational changes occurred due to changes in the relative orientation of the heavy chain to the light chain upon complexation in HD96A Fv-HEL complexes. The reduced affinity of these single mutants for the antigen originates from the increase in entropy loss, indicating that these structural changes also introduced an increase in entropy loss. These results suggest that salt bridge formation makes an entropic contribution to the protein antigen-antibody interaction through reduction of entropy loss due to dehydration and structural changes.     

Involvement of water molecules in the association of monoclonal antibody HyHEL-5 with bobwhite quail lysozyme.

Fluorescence polarization spectroscopy and isothermal titration calorimetry were used to study the influence of osmolytes on the association of the anti-hen egg lysozyme (HEL) monoclonal antibody HyHEL-5 with bobwhite quail lysozyme (BWQL). BWQL is an avian species variant with an Arg-->Lys mutation in the HyHEL-5 epitope, as well as three other mutations outside the HyHEL-5 structural epitope. This mutation decreases the equilibrium association constant of HyHEL-5 for BWQL by over 1000-fold as compared to HEL. The three-dimensional structure of this complex has been obtained recently. Fluorescein-labeled BWQL, obtained by labeling at pH 7.5 and purified by hydrophobic interaction chromatograpy, bound HyHEL-5 with an equilibrium association constant close to that determined for unlabeled BWQL by isothermal titration calorimetry. Fluorescence titration, stopped-flow kinetics, and isothermal titration calorimetry experiments using various concentrations of the osmolytes glycerol, ethylene glycol, and betaine to perturb binding gave a lower limit of the uptake of approximately 6-12 water molecules upon formation of the HyHEL-5/BWQL complex.     

Unusual joining sites in the H and L chains of an anti-lysozyme antibody

Nucleotide sequences of HyHEL-5, an antibody specific for chicken lysozyme (HEL), indicated unusual joins in the third complementarity-determining region of both the H and L chains. The VK-JK recombination site is unusual in that codon 96, normally derived from the JK gene segment, is deleted entirely, making the L3 one amino acid shorter than normal. Examination of the HyHEL-5 Fab-HEL x-ray structure suggests that the conformation of L3 is clearly important for Ag specificity. A comparison of the HyHEL-5 L3 with that of the structurally related antibody J539 indicates that the deleted residue significantly alters the conformation of the L3 turn. The H chain VH-DH join is also unusual; the VH junction site has probably occurred between the second and third nucleotides of codon 92, with the addition of five random nucleotides that encode for unusual amino acids Leu93 and His94. Although the conformation of H3 is different from what would be predicted from other H3 conformations and is clearly important to the complementarity of HyHEL-5 to HEL, the specific residues at the VH-DH join do not appear to directly contribute to Ag binding. It is not possible to attribute the main chain conformation of H3 to the particular sequence produced by the join; the structural features of H3 may be due to interactions with HEL and/or with other antibody residues.     

Critical contribution of VH-VL interaction to reshaping of an antibody: the case of humanization of anti-lysozyme antibody, HyHEL-10

To clarify the effects of humanizing a murine antibody on its specificity and affinity for its target, we examined the interaction between hen egg white lysozyme (HEL) and its antibody, HyHEL-10 variable domain fragment (Fv). We selected a human antibody framework sequence with high homology, grafted sequences of six complementarity-determining regions of murine HyHEL-10 onto the framework, and investigated the interactions between the mutant Fvs and HEL. Isothermal titration calorimetry indicated that the humanization led to 10-fold reduced affinity of the antibody for its target, due to an unfavorable entropy change. Two mutations together into the interface of the variable domains, however, led to complete recovery of antibody affinity and specificity for the target, due to reduction of the unfavorable entropy change. X-ray crystallography of the complex of humanized antibodies, including two mutants, with HEL demonstrated that the complexes had almost identical structures and also paratope and epitope residues were almost conserved, except for complementary association of variable domains. We conclude that adjustment of the interfacial structures of variable domains can contribute to the reversal of losses of affinity or specificity caused by humanization of murine antibodies, suggesting that appropriate association of variable domains is critical for humanization of murine antibodies without loss of function.     

Mutations of an epitope hot-spot residue alter rate limiting steps of antigen-antibody protein-protein associations

The antibodies, HyHEL-10 and HyHEL-26 (H10 and H26, respectively), share over 90% sequence homology and recognize with high affinity the same epitope on hen egg white lysozyme (HEL) but differ in degree of cross-reactivity with mutant lysozymes. The binding kinetics, as measured by BIAcore surface plasmon resonance, of monovalent Fab from both Abs (Fab10 and Fab26) to HEL and mutant lysozymes are best described by a two-step association model consistent with an encounter followed by docking that may include conformational changes. In their complexes with HEL, both Abs make the transition to the docked phase rapidly. For H10, the encounter step is rate limiting, whereas docking is also partially rate limiting for H26. The forward rate constants of H10 are higher than those of H26. The docking equilibrium as well as the overall equilibrium constant are also higher for H10 than for H26. Most of the free energy change of association (Delta G degrees) occurs during the encounter phase (Delta G1) of both Abs. H10 derives a greater amount and proportion of free energy change from the docking phase (Delta G2) than does H26. In the H10--HEL(R21Q) complex, a significant slowing of docking results in lowered affinity, a loss of most of Delta G2, and apparently faster dissociation. Slower encounter and docking cause lowered affinity and a loss of free energy change primarily in the encounter step (Delta G1) of H26 with mutant HEL(R21Q). Overall, in the process of complex formation with lysozyme, the mutations HEL(R21X) affect primarily the docking phase of H10 association and both phases of H26. Our results are consistent with the interpretation that the free energy barriers to conformational rearrangement are highest in H26, especially with mutant antigen.     

Crystal structure of anti-Hen egg white lysozyme antibody (HyHEL-10) Fv-antigen complex. Local structural changes in the protein antigen and water-mediated interactions of Fv-antigen and light chain-heavy chain interfaces.

In order to address the recognition mechanism of the fragments of antibody variable regions, termed Fv, toward their target antigen, an x-ray crystal structure of an anti-hen egg white lysozyme antibody (HyHEL-10) Fv fragment complexed with its cognate antigen, hen egg white lysozyme (HEL), was solved at 2.3 A. The overall structure of the complex is similar to that reported in a previous article dealing with the Fab fragment-HEL complex (PDB ID code,). However, the areas of Fv covered by HEL upon complex formation increased by about 100 A(2) in comparison with the Fab-HEL complex, and two local structural differences were observed in the heavy chain of the variable region (VH). In addition, small but significant local structural changes were observed in the antigen, HEL. The x-ray data permitted the identification of two water molecules between the VH and HEL and six water molecules retained in the interface between the antigen and the light chain complementarity determining regions (CDRs) 2 and 3 (CDR-L2 and CDR-L3). These water molecules bridge the antigen-antibody interface through hydrogen bond formation in the VL-HEL interface. Eleven water molecules were found to complete the imperfect VH-VL interface, suggesting that solvent molecules mediate the stabilization of interaction between variable regions. These results suggest that the unfavorable effect of deletion of constant regions on the antigen-antibody interaction is compensated by an increase in favorable interactions, including structural changes in the antigen-antibody interface and solvent-mediated hydrogen bond formation upon complex formation, which may lead to a minimum decreased affinity of the antibody Fv fragment toward its antigen.     

Understanding antibody–antigen associations by molecular dynamics simulations: Detection of important intra- and inter-molecular salt bridges

1 NSec molecular dynamics (MD) simulation of anti-hen egg white antibody, HyHEL63 (HH63), complexed with HEL reveals important molecular interactions, not revealed in its X-ray crystal structure. These molecular interactions were predicted to be critical for the complex formation, based on structure–function studies of this complex and 3-other anti-HEL antibodies, HH8, HH10 and HH26, HEL complexes. All four antibodies belong to the same structural family, referred to here as HH10 family. Ala scanning results show that they recognize ‘coincident epitopes’. 1 NSec explicit, with periodic boundary condition, MD simulation of HH63-HEL reveals the presence of functionally important salt-bridges. Around 200 ps in vacuo and an additional 20 ps explicit simulation agree with the observations from 1 Nsec simulation. Intra-molecular salt-bridges predicted to play significant roles in the complex formation, were revealed during MD simulation. A very stabilizing salt-bridge network, and another intra-molecular salt-bridge, at the binding site of HEL, revealed during the MD simulation, is proposed to predipose binding site geometry for specific binding. All the revealed salt-bridges are present in one or more of the other three complexes and/or involve “hot-spot” epitope and paratope residues. Most of these charged epitope residues make large contribution to the binding free energy. The “hot spot” epitope residue Lys97_Y, which significantly contributes to the free energy of binding in all the complexes, forms an intermolecular salt-bridge in several MD conformers. Our earlier computations have shown that this inter-molecular salt-bridge plays a significant role in determining specificity and flexibility of binding in the HH8-HEL and HH26-HEL complexes. Using a robust criterion of salt-bridge detection, this inter-molecular salt-bridge was detected in the native structures of the HH8-HEL and HH26-HEL complexes, but was not revealed in the crystal structure of HH63-HEL complex. The electrostatic strength of this revealed salt-bridge was very strong. During 1 Nsec MD simulation this salt-bridge networks with another inter-molecular salt-bridge to form an inter-molecular salt-bridge triad. Participation of Lys97_Y in the formation of inter-molecular triad further validates the functional importance of Lys97_Y in HH63-HEL associations. These results demonstrate that many important structural details of biomolecular interactions can be better understood when studied in a dynamic environment, and that MD simulations can complement and expand information obtained from static X-ray structure. This study also highlights “hot-spot” molecular interactions in HyHEL63-HEL complex. The publisher or recipient acknowledges right of the U.S. Government to retain a non exclusive, royalty-free license in and to any copyright covering the article.     

Association and dissociation kinetics of bobwhite quail lysozyme with monoclonal antibody HyHEL-5

The anti-hen egg lysozyme monoclonal antibody HyHEL-5 and its complexes with various species-variant and mutant lysozymes have been the subject of considerable experimental and theoretical investigation. The affinity of HyHEL-5 for bobwhite quail lysozyme (BWQL) is over 1000-fold lower than its affinity for the original antigen, hen egg lysozyme (HEL). This difference is believed to arise almost entirely from the replacement in BWQL of the structural and energetic epitope residue Arg68 by lysine. In this study, the association and dissociation kinetics of BWQL with HyHEL-5 were investigated under a variety of conditions and compared with previous results for HEL. HyHEL-5-BWQL association follows a bimolecular mechanism and the dissociation of the antibody-antigen complex is a first-order process. Changes in ionic strength (from 27 to 500 mM) and pH (from 6.0 to 10.0) produced about a 2-fold change in the association and dissociation rates. The effect of viscosity modifiers on the association reaction was also studied. The large difference in the HEL and BWQL affinities for HyHEL-5 is essentially due to differences in the dissociation rate constant.     

Contribution of Asparagine Residues to the Stabilization of a Proteinaceous Antigen-Antibody Complex, HyHEL-10-Hen Egg White Lysozyme

Many germ line antibodies have asparagine residues at specific sites to achieve specific antigen recognition. To study the role of asparagine residues in the stabilization of antigen-antibody complexes, we examined the interaction between hen egg white lysozyme (HEL) and the corresponding HyHEL-10 variable domain fragment (Fv). We introduced Ala and Asp substitutions into the Fv side chains of l-Asn-31, l-Asn-32, and l-Asn-92, which interact directly with residues in HEL via hydrogen bonding in the wild-type Fv-HEL complex, and we investigated the interactions between these mutant antibodies and HEL. Isothermal titration calorimetric analysis showed that all the mutations decreased the negative enthalpy change and decreased the association constants of the interaction. Structural analyses showed that the effects of the mutations on the structure of the complex could be compensated for by conformational changes and/or by gains in other interactions. Consequently, the contribution of two hydrogen bonds was minor, and their abolition by mutation resulted in only a slight decrease in the affinity of the antibody for its antigen. By comparison, the other two hydrogen bonds buried at the interfacial area had large enthalpic advantage, despite entropic loss that was perhaps due to stiffening of the interface by the bonds, and were crucial to the strength of the interaction. Deletion of these strong hydrogen bonds could not be compensated for by other structural changes. Our results suggest that asparagine can provide the two functional groups for strong hydrogen bond formation, and their contribution to the antigen-antibody interaction can be attributed to their limited flexibility and accessibility at the complex interface.     

Calculation of HyHel10-lysozyme binding free energy changes: Effect of ten point mutations

The change in free energy of binding of hen egg white lysozyme (HEL) to the antibody HyHel-10 arising from ten point mutations in HEL (D101K, D101G, K96M, K97D, K97G, K97G, R21E, R21K, W62Y, and W63Y) was calculated using a combination of the finite difference Poisson-Boltzmann method for the electrostatic contribution, a solvent accessible surface area term for the non-polar contribution, and rotamer counting for the sidechain entropy contribution. Comparison of experimental and calculated results indicate that because of pKa shifts in some of the mutated residues, primarily those involving Aspartate or Glutamate, proton uptake or release occurs in binding. When this effect was incorporated into the binding free energy calculations, the agreement with experiment improved significantly, and resulted in a mean error of about 1.9 kcal/mole. Thus these calculations predict that there should be a significant pH dependence to the change in binding caused by these mutations. The other major contributions to binding energy changes comes from solvation and charge charge interactions, which tend to oppose each other. Smaller contributions come from nonpolar interactions and sidechain entropy changes. The structures of the HyHel-10-HEL complexes with mutant HEL were obtained by modeling, and the effect of the modeled structure on the calculations was also examined. “Knowledge based” modeling and automatic generation of models using molecular mechanics produced comparable results. Proteins 33:39–48, 1998. © 1998 Wiley-Liss, Inc.     

The role of hydrogen bonding via interfacial water molecules in antigen-antibody complexation. The HyHEL-10-HEL interaction

To study the role of hydrogen bonding via interfacial water molecules in protein-protein interactions, we examined the interaction between hen egg white lysozyme (HEL) and its HyHEL-10 variable domain fragment (Fv) antibody. We constructed three antibody mutants (l-Y50F, l-S91A, and l-S93A) and investigated the interactions between the mutant Fvs and HEL. Isothermal titration calorimetry indicated that the mutations significantly decreased the negative enthalpy change (8-25 kJ mol(-1)), despite some offset by a favorable entropy change. X-ray crystallography demonstrated that the complexes had nearly identical structures, including the positions of the interfacial water molecules. Taken together, the isothermal titration calorimetric and x-ray crystallographic results indicate that hydrogen bonding via interfacial water enthalpically contributes to the Fv-HEL interaction despite the partial offset because of entropy loss, suggesting that hydrogen bonding stiffens the antigen-antibody complex.     

Analysis of the interactions of human ribonuclease inhibitor with angiogenin and ribonuclease A by mutagenesis: importance of inhibitor residues inside versus outside the C-terminal "hot spot"

Ribonuclease inhibitor (RI) binds diverse mammalian RNases with extraordinary avidity. Here, we have investigated the structural basis for this tight binding and broad specificity by mutational analysis of the complexes of RI with angiogenin (Ang) and RNase A (K(D)=0.5 fM and 43 fM, respectively). Both crystal structures are known; the interfaces are large, and the ligands dock similarly, although few of the specific interactions formed are analogous. Our previous mutagenesis studies focused primarily on one contact region, containing RI 434-438 and the enzymatic active site. Many single-residue replacements produced extensive losses of binding energy (2.3-5.9 kcal/mol), suggesting that this region constitutes a "hot spot" in both cases. We have now explored the roles of most of the remaining RI residues that interact with Ang and/or RNase A. One major cluster in each complex lies in a Trp-rich area of RI, containing Trp261, Trp263, Trp318, and Trp375. Although the energy losses from individual replacements in this portion of the Ang complex were small-to-moderate (0-1.5 kcal/mol), the changes from multiple substitutions were much greater than additive, and the binding energy provided by this region is estimated to be approximately 6 kcal/mol (30 % of total). Effects of replacing combinations of hot spot components had also been found to be superadditive, and this negative cooperativity is now shown to extend to the neighboring contact residue RI Ser460. The overall contribution of the hot spot, taking superadditivity into account, is then approximately 14-15 kcal/mol. The hot spot and Trp-rich regions, although spatially well separated, are themselves functionally linked. No other parts of the RI-Ang interface appear to be energetically important. Binding of RNase A is more sensitive to substitutions throughout the interface, with free energy losses>/=1 kcal/mol produced by nearly all replacements examined, so that the sum of losses greatly exceeds the binding energy of the complex. This discrepancy can be explained, in part, by positive cooperativity, as evident from the subadditive effects observed when combinations of residues in either the hot spot or Trp-rich region are replaced. These findings suggest that the binding energy may be more widely distributed in the RNase A complex than in the Ang complex.     

Modeling implicit reorganization in continuum descriptions of protein-protein interactions

The determination of free energies that govern protein-protein recognition is essential for a detailed molecular understanding of biological specificity. Continuum models of macromolecular interactions, in which the solvent is treated by an implicit representation and the proteins are treated semi-microscopically, are computationally tractable for estimating free energies, yet many questions remain concerning their accuracy. This article reports a continuum analysis of the free-energy changes underlying the binding of 31 interfacial alanine substitutions of two complexes of the antihen egg white lysozyme (HEL) antibody D1.3 bound with HEL or the antibody E5.2. Two implicit schemes for modeling the effects of protein and solvent relaxation were examined, in which the protein environment was treated as either homogeneous with a "protein dielectric constant" of epsilon(p) = 4 or inhomogeneous, with epsilon(p) = 4 for neutral residues and epsilon(p) = 25 for ionized residues. The results showed that the nonuniform dielectric model reproduced the experimental differences better, with an average absolute error of +/-1.1 kcal/mol, compared with +/-1.4 kcal/mol for the uniform model. More importantly, the error for charged residues in the nonuniform model is +/-0.8 kcal/mol and is nearly half of that corresponding to the uniform model. Several substitutions were clearly problematic in determining qualitative trends and probably required explicit structural reorganization at the protein-protein interface.