Characterizing the role of ensemble modulation in mutation-induced changes in binding affinity.

Protein conformational fluctuations are key contributors to biological function, mediating important processes such as enzyme catalysis, molecular recognition, and allosteric signaling. To better understand the role of conformational fluctuations in substrate/ligand recognition, we analyzed, experimentally and computationally, the binding reaction between an SH3 domain and the recognition peptide of its partner protein. The fluctuations in this SH3 domain were enumerated by using an algorithm based on the hard sphere collision model, and the binding energetics resulting from these fluctuations were calculated using a structure-based energy function parametrized to solvent accessible surface areas. Surprisingly, this simple model reproduced the effects of mutations on the experimentally determined SH3 binding energetics, within the uncertainties of the measurements, indicating that conformational fluctuations in SH3, and in particular the RT loop region, are structurally diverse and are well-approximated by the randomly configured states. The mutated positions in SH3 were distant to the binding site and involved Ala and Gly substitutions of solvent exposed positions in the RT loop. To characterize these fluctuations, we applied principal coordinate analysis to the computed ensembles, uncovering the principal modes of conformational variation. It is shown that the observed differences in binding affinity between each mutant, and thus the apparent coupling between the mutated sites, can be described in terms of the changes in these principal modes. These results indicate that dynamic loops in proteins can populate a broad conformational ensemble and that a quantitative understanding of molecular recognition requires consideration of the entire distribution of states.