Hydrogen Bonding and Solvent Structure in an Antigen-Antibody Interface. Crystal Structures and Thermodynamic Characterization of Three Fv Mutants Complexed with Lysozyme

Using site-directed mutagenesis, X-ray crystallography, and titration calorimetry, we have examined the structural and thermodynamic consequences of removing specific hydrogen bonds in an antigen−antibody interface. Crystal structures of three antibody FvD1.3 mutants, VLTyr50Ser (VLY50S), VHTyr32Ala (VHY32A), and VHTyr101Phe (VHY101F), bound to hen egg white lysozyme (HEL) have been determined at resolutions ranging from 1.85 to 2.10 A. In the wild-type (WT) FvD1.3−HEL complex, the hydroxyl groups of VLTyr50, VHTyr32, and VHTyr101 each form at least one hydrogen bond with the lysozyme antigen. Thermodynamic parameters for antibody−antigen association have been measured using isothermal titration calorimetry, giving equilibrium binding constants Kb (M-1) of 2.6 × 107 (VLY50S), 7.0 × 107 (VHY32A), and 4.0 × 106 (VHY101F). For the WT complex, Kb is 2.7 × 108 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, respectively. In all thr...