HYDROGEN-BONDING AND SOLVENT STRUCTURE IN AN ANTIGEN-ANTIBODY INTERFACE - CRYSTAL-STRUCTURES AND THERMODYNAMIC CHARACTERIZATION OF 3 FV MUTANTS COMPLEXED WITH LYSOZYME

Using site-directed mutagenesis, X-ray crystallography, and titration calorimetry, we have examined the structural and thermodynamic consequ ences of removing specific hydrogen bonds in an antigen-antibody inter face. Crystal structures of three antibody FvD1.3 mutants, V(L)Tyr50Se r (V(L)Y50S), V(H)Tyr32Ala (V(H)Y32A), and V(H)Tyr101Phe (V(H)Y101F) b ound to hen egg white lysozyme (HEL) have been determined at resolutio ns ranging from 1.85 to 2.10 Angstrom. In the wild-type (WT) FvD1.3-HE L complex, the hydroxyl groups of V(L)Tyr50, V(H)Tyr32, and V(H)Tyr101 each form at least one hydrogen bond with the lysozyme antigen. Therm odynamic parameters for antibody-antigen association have been measure d using isothermal titration calorimetry, giving equilibrium binding c onstants K-b (M(-1)) of 2.6 x 10(7) (V(L)Y50S), 7.0 x 10(7) (V(H)Y32A) , and 4.0 x 10(6) (V(H)Y101F). For the WT complex, K-b is 2.7 x 10(8) M(-1); thus, the affinities of the mutant Fv fragments for HEL are 10- , 4-, and 70-fold lower than that of the original antibody, respective ly. In all three cases entropy compensation results in an affinity los s that would otherwise be larger. Comparison of the three mutant cryst al structures with the WT structure demonstrates that the removal of d irect antigen-antibody hydrogen bonds results in minimal shifts in the positions of the remaining protein atoms. These observations show tha t this complex is considerably tolerant, both structurally and thermod ynamically, to the truncation of antibody side chains that form hydrog en bonds with the antigen. Alterations in interface solvent structure for two of the mutant complexes (V(L)Y50S and V(H)Y32A) appear to comp ensate for the unfavorable enthalpy changes when protein-protein inter actions are removed. These changes in solvent structure, along with th e increased mobility of side chains near the mutation site, probably c ontribute to the observed entropy compensation. For the V(H)Y101F comp lex, the nature of the large entropy compensation is not evident from a structural comparison of the WT and mutant complexes. Differences in the local structure and dynamics of the uncomplexed Fv molecules may account for the entropic discrepancy in this case.