Evolutionary Co-variance Analysis of DsrAB
Because many of the non-conserved residues in DsrAB are found at subunit interfaces, Evolutionary co-variance (EC) analysis on the EVCouplings server (as described in Materials and Methods) (39) was used to reveal how subunit contacts may have evolved with environment types or taxonomic groups that are distributed across the DsrAB phylogeny. We were particularly interested in False Positive Evolutionary Couplings (FPEC), as these often correspond to inter-subunit contacts within oligomers. They also can arise from long range allosteric pathways or dynamic structural heterogeneity (26), although these types of FPECs represent a small fraction of the total co-varying residues (48). Numerous FPECs were identified as evolutionarily co-varying residues that did not correspond to a contact in the 2D residue contact map of the monomer and were separated in space (within the monomers) by greater than 20 Å. Because the DsrA and DsrB subunits are highly homologous, both in sequence and in structure (Figure S4), both subunits were present in the multiple sequence alignments (MSAs) used in the EVCouplings analysis (38, 39). Moreover, because DsrAB complexes are hetero-tetramers, in principle, putative co-varying pairs of residues could correspond to eight different possible residue pairs (Res1A1-Res2A1, Res1B1-Res2B1, Res1A1-Res2B1, Res2A1-Res1B1, Res1A1-Res2A2, Res1A1-Res2B2, Res2A1-Res1B2, and Res1B1-Res2B2).