THERMODYNAMIC ANALYSIS OF AN ANTIBODY FUNCTIONAL EPITOPE

We have probed the relative contribution of polar and nonpolar interactions to antibody-antigen interaction by measuring the effect of single amino acid substitutions in an humanized anti-p185HER2 antibody (hu4D5-5) on the thermodynamics of antigen binding. First we mapped the functional epitope by complete alanine-scan mutagenesis of the antibody complementarity-determining region (CDR). Four residues, H91 in V(L) and R50, W95, and Y100 and V(H), make large contributions to the free energy of binding (DELTADELTAG > 3 kcal mol-1) and have DELTADELTAG > DELTADELTAH. These residues are clustered in a shallow pocket on the antibody surface in the X-ray structure determined for hu4D5 Fv. The majority of other CDR residues make less energetically important contributions (DELTADELTAG < 1 kcal mol-1) to binding but have DELTADELTAH > DELTADELTAG, suggesting that the wild-type side chain does contact antigen but the loss in entropy, perhaps through restriction of side-chain conformational freedom, offsets the favorable enthalpic term. Effects of Phe and Ala substitutions on the DELTAG and DELTAC(p) of antigen binding indicate that the favorable contribution of antibody tyrosine residues to binding results primarily from burial of the aromatic ring in the interface with antigen. Burial of the phenyl ring has a favorable DELTAH at 25-degrees-C but at least for one site (V(L)-Y92) is opposed by DELTAS. This latter feature is inconsistent with the thermodynamics predicted for the hydrophobic effect based on hydrocarbon-transfer experiments. In addition, the magnitude of DELTAC(p) for burial of the phenyl ring is greater than would be predicted from the model compound data. These results suggest that the aromatic ring of antibody Tyr residues may contribute other interactions to antigen binding, such as aromatic hydrogen bonding, in addition to the contribution from the hydrophobic effect.