Analysis of proteinprotein interactions by mutagenesis: direct versus indirect effects

Site-directed mutagenesis, including double-mutant cycles, isused routinely 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 knownto interact directly with subtilisin (Tyr42) and (ii) two CI2residues that do not have direct contacts with subtilisin (Arg46and Arg48). We find that there are similar changes in bindingenergy on mutation of these two sets of residues. It can thusbe difficult to interpret mutagenesis data in the absence ofstructural information.