Hydrogen bonding and solvent structure in an antigen-antibody interface. Crystal

FIELDS BA, GOLDBAUM FA, DALL'ACQUA W, MALCHIODI EL, CAUERHFF A, SCHWARZ FP, YSERN

BIOCHEMISTRY

Año: 1996 vol. 35 p. 15494 - 15503

0006-2960

Using site-directed mutagenesis, X-ray crystallography, and titrationcalorimetry, we have examined the structural and thermodynamic consequences ofremoving specific hydrogen bonds in an antigen-antibody interface. Crystalstructures of three antibody FvD1.3 mutants, VLTyr50Ser (VLY50S), VHTyr32Ala(VHY32A), and VHTyr101Phe (VHY101F), bound to hen egg white lysozyme (HEL) havebeen 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 eachform at least one hydrogen bond with the lysozyme antigen. Thermodynamicparameters for antibody-antigen association have been measured using isothermaltitration calorimetry, giving equilibrium binding constants Kb (M-1) of 2.6 x10(7) (VLY50S), 7.0 x 10(7) (VHY32A), and 4.0 x 10(6) (VHY101F). For the WTcomplex, Kb is 2.7 x 10(8) M-1; thus, the affinities of the mutant Fv fragmentsfor HEL are 10-, 4-, and 70-fold lower than that of the original antibody,respectively. In all three cases entropy compensation results in an affinity lossthat would otherwise be larger. Comparison of the three mutant crystal structureswith the WT structure demonstrates that the removal of direct antigen-antibodyhydrogen bonds results in minimal shifts in the positions of the remainingprotein atoms. These observations show that this complex is considerablytolerant, both structurally and thermodynamically, to the truncation of antibody side chains that form hydrogen bonds with the antigen. Alterations in interfacesolvent structure for two of the mutant complexes (VLY50S and VHY32A) appear tocompensate for the unfavorable enthalpy changes when protein-protein interactionsare removed. These changes in solvent structure, along with the increasedmobility of side chains near the mutation site, probably contribute to theobserved entropy compensation. For the VHY101F complex, the nature of the largeentropy compensation is not evident from a structural comparison of the WT andmutant complexes. Differences in the local structure and dynamics of theuncomplexed Fv molecules may account for the entropic discrepancy in this case.