A mutational analysis of binding interactions in an antigen-antibody protein-protein complex.

Alanine scanning mutagenesis, double mutant cycles, and X-ray crystallography were used to characterize the interface between the anti-hen egg white lysozyme (HEL) antibody D1.3 and HEL. Twelve out of the 13 nonglycine contact residues on HEL, as determined by the high-resolution crystal structure of the D1.3−HEL complex, were individually truncated to alanine. Only four positions showed a ΔΔG (ΔGmutant − ΔGwild-type) of greater than 1.0 kcal/mol, with HEL residue Gln121 proving the most critical for binding (ΔΔG = 2.9 kcal/mol). These residues form a contiguous patch at the periphery of the epitope recognized by D1.3. To understand how potentially disruptive mutations in the antigen are accommodated in the D1.3−HEL interface, we determined the crystal structure to 1.5 A resolution of the complex between D1.3 and HEL mutant Asp18 → Ala. This mutation results in a ΔΔG of only 0.3 kcal/mol, despite the loss of a hydrogen bond and seven van der Waals contacts to the Asp18 side chain. The crystal structure re...