A novel method for detecting intramolecular coevolution: adding a further dimension to selective constraints analyses.

Protein evolution depends on intramolecular coevolutionary networks whose complexity is proportional to the underlying functional and structural interactions among sites. Here we present a novel approach that vastly improves the sensitivity of previous methods for detecting coevolution through a weighted comparison of divergence between amino acid sites. The analysis of the HIV-1 Gag protein detected convergent adaptive coevolutionary events responsible for the selective variability emerging between subtypes. Coevolution analysis and functional data for heat-shock proteins, Hsp90 and GroEL, highlight that almost all detected coevolving sites are functionally or structurally important. The results support previous suggestions pinpointing the complex interdomain functional interactions within these proteins and we propose new amino acid sites as important for interdomain functional communication. Three-dimensional information sheds light on the functional and structural constraints governing the coevolution between sites. Our covariation analyses propose two types of coevolving sites in agreement with previous reports: pairs of sites spatially proximal, where compensatory mutations could maintain the local structure stability, and clusters of distant sites located in functional domains, suggesting a functional dependency between them. All sites detected under adaptive evolution in these proteins belong to coevolution groups, further underlining the importance of testing for coevolution in selective constraints analyses.