A Mutational Analysis of the Binding of Two Different Proteins to the Same Antibody

The crystal structures of the complexes between the anti-hen egg white lysozyme (HEL) antibody D1.3 and HEL and between D1.3 and the anti-D1.3 antibody E5.2 have shown that D1.3 contacts these two proteins through essentially the same set of combining site residues [Fields, B. A., Goldbaum, F. A., Ysern, X., Poljak, R. J., & Mariuzza, R. A. (1995) Nature 374, 739−742]. To probe the relative contribution of individual residues to complex stabilization, single alanine substitutions were introduced in the combining site of D1.3, and their effects on affinity for HEL and for E5.2 were measured using surface plasmon resonance detection, fluorescence quench titration, or sedimentation equilibrium. The energetics of the binding to HEL are dominated by only 3 of the 13 contact residues tested (ΔGmutant − ΔGwild type > 2.5 kcal/mol):  VLW92, VHD100, and VHY101. These form a patch at the center of the interface and are surrounded by residues whose apparent contributions are much less pronounced (<1.5 kcal/mol). Thi...