Mutations of an epitope hot-spot residue alter rate limiting steps of antigen-antibody protein-protein associations.

The antibodies, HyHEL-10 and HyHEL-26 (H10 and H26, respectively), share over 90% sequence homology and recognize with high affinity the same epitope on hen egg white lysozyme (HEL) but differ in degree of cross-reactivity with mutant lysozymes. The binding kinetics, as measured by BIAcore surface plasmon resonance, of monovalent Fab from both Abs (Fab10 and Fab26) to HEL and mutant lysozymes are best described by a two-step association model consistent with an encounter followed by docking that may include conformational changes. In their complexes with HEL, both Abs make the transition to the docked phase rapidly. For H10, the encounter step is rate limiting, whereas docking is also partially rate limiting for H26. The forward rate constants of H10 are higher than those of H26. The docking equilibrium as well as the overall equilibrium constant are also higher for H10 than for H26. Most of the free energy change of association (Delta G degrees) occurs during the encounter phase (Delta G1) of both Abs. H10 derives a greater amount and proportion of free energy change from the docking phase (Delta G2) than does H26. In the H10--HEL(R21Q) complex, a significant slowing of docking results in lowered affinity, a loss of most of Delta G2, and apparently faster dissociation. Slower encounter and docking cause lowered affinity and a loss of free energy change primarily in the encounter step (Delta G1) of H26 with mutant HEL(R21Q). Overall, in the process of complex formation with lysozyme, the mutations HEL(R21X) affect primarily the docking phase of H10 association and both phases of H26. Our results are consistent with the interpretation that the free energy barriers to conformational rearrangement are highest in H26, especially with mutant antigen.