A MUTATIONAL ANALYSIS OF THE BINDING OF 2 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 thro ugh 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 affinit y for HEL and for E5.2 were measured using surface plasmon resonance d etection, fluorescence quench titration, or sedimentation equilibrium, The energetics of the binding to HEL are dominated by only 3 of the 1 3 contact residues tested (Delta G(mutant) - Delta G(wild type) > 2.5 kcal/mol): V(L)W92, V(H)D1C0, and V(H)Y101. 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). This contrast s with the interaction of D1.3 with E5.2 in which most the contact res idues (11 of 15) were found to play a significant role in ligand bindi ng (> 1.5 kcal/mol). Furthermore, even though D1.3 contacts HEL and E5 .2 in very similar ways, the functionally important residues of D1.3 a re different for the two interactions, with only substitutions at D1.3 positions V(H)100 and V(H)101 greatly affecting binding to both ligan ds. Thus, the same protein may recognize different ligands in ways tha t are structurally similar yet energetically distinct.