Analysis of protein-protein interactions by mutagenesis: direct versus indirect effects

Site-directed mutagenesis, including double-mutant cycles, is used routinel y for studying protein-protein interactions. We now present a case analysis of chymotrypsin inhibitor 2 (CI2) and subtilisin BPN' using (i) a residue in CI2 that is known to interact directly with subtilisin (Tyr42) and (ii) two CI2 residues that do not have direct contacts with subtilisin (Arg46 an d Arg48). We find that there are similar changes in binding energy on mutat ion of these two sets of residues. It can thus be difficult to interpret mu tagenesis data in the absence of structural information.