A MUTATIONAL ANALYSIS OF BINDING INTERACTIONS IN AN ANTIGEN-ANTIBODY PROTEIN-PROTEIN COMPLEX

Alanine scanning mutagenesis, double mutant cycles, and X-ray crystall ography were used to characterize the interface between the anti-hen e gg white lysozyme (HEL) antibody D1.3 and HEL. Twelve out of the 13 no nglycine contact residues on HEL, as determined by the high-resolution crystal structure of the D1.3-HEL complex, were individually truncate d to alanine. Only four positions showed a Delta Delta G (Delta G(muta nt) - Delta G(wild-type)) of greater than 1.0 kcal/mol, with HEL resid ue Gln121 proving the most critical for binding (Delta Delta G = 2.9 k cal/mol). These residues form a contiguous patch at the periphery of t he epitope recognized by D1.3. To understand how potentially disruptiv e mutations in the antigen are accommodated in the D1.3-HEL interface, we determined the crystal structure to 1.5 Angstrom resolution of the complex between D1.3 and HEL mutant Asp18 --> Ala. This mutation resu lts in a Delta Delta G of only 0.3 kcal/mol, despite the loss of a hyd rogen bond and seven van der Waals contacts to the Asp18 side chain. T he crystal structure reveals that three additional water molecules ape stably incorporated in the antigen-antibody interface at the site of the mutation. These waters help fill the cavity created by the mutatio n and form part of a rearranged solvent network linking the two protei ns. To further dissect the energetics of specific interactions in the D1.3-HEL interface, double mutant cycles were carried out to measure t he coupling of 14 amino acid pairs, 10 of which are in direct contact in the crystal structure. The highest coupling energies, 2.7 and 2.0 k cal/mol, were measured between HEL residue Gln121 and D1.3 residues V( L)Trp92 and V(L)Tyr32, respectively. The interaction between Gln121 an d V(L)Trp92 consists of three van der Waals contacts, while the intera ction of Gln121 with V(L)Tyr32 is mediated by a hydrogen bond. Surpris ingly, however, most cycles between interface residues in direct conta ct in the crystal structure showed no significant coupling. In particu lar, a number of hydrogen-bonded residue pairs were found to make no n et contribution to complex stabilization. We attribute these results t o accessibility of the mutation sites to water, such that the mutated residues exchange their interaction with each other to interact with w ater. This implies that the strength of the protein-protein hydrogen b onds in these particular cases is comparable to that of the protein-wa ter hydrogen bonds they replace. Thus, the simple fact that two residu es are in direct contact in a protein-protein interface cannot be take n as evidence that there necessarily exists a productive interaction b etween them. Rather, the majority of such contacts may be energeticall y neutral, as in the D1.3-HEL complex.