Analysis of binding interactions in an idiotope-antiidiotope protein-protein complex by double mutant cycles.

The idiotope−antiidiotope complex between the anti-hen egg whitelysozyme antibody D1.3 and the anti-D1.3 antibody E5.2 provides a useful model for studying protein−protein interactions. A high-resolution crystal structure of the complex is available [Fields, B. A., Goldbaum, F. A., Ysern, X., Poljak, R. J., & Mariuzza, R. A. (1995) Nature 374, 739−742], and both components are easily produced and manipulated in Escherichia coli. We previously analyzed the relative contributions of individual residues of D1.3 to complex stabilization by site-directed mutagenesis[Dall'Acqua, W., Goldman, E. R., Eisenstein, E., & Mariuzza, R. A. (1996) Biochemistry 35, 9667−9676]. In the current work, we introduced single alanine substitutions in 9 out of 21 positions in the combining site of E5.2 involved in contacts with D1.3 and found that 8 of them play a significant role in ligand binding (ΔGmutant − ΔGwild type > 1.5 kcal/mol). Furthermore, energetically important E5.2 and D1.3 residues tend to be juxtaposed in the c...