Evolutionary Co-variance and Anisotropic Network Analysis
Evolutionary covariance (EC) analysis (25, 26, 38) was performed using the EV Couplings server (https://evcouplings.org/ (39)) to identify instances of False Positive Evolutionary Covariance (FPEC), defined as significant covariance between residues that are too distant (>20 Å) to be in direct contact within the monomeric subunits. Given the strong homology between DsrA and DsrB subunits that are suspected to have originated from gene duplication, it was not possible to use the EV Complex Couplings option to identify co-varying residues at subunit interfaces in the oligomers. Instead, EC analysis was carried out using the monomeric sequences of the DsrA and DsrB subunits of A. fulgidus , D. vulgaris, and MV2-Eury separately as input. The values of Neff (number of non-redundant sequences in the alignment normalized to the number of residues) was between 1.16 and 2.84, depending upon the query, with the number of non-redundant sequences was between 481 and 2786. Queries were made by separately submitting the A and B subunits from the A. fulgidus and MV2-Eury sequences. Recovered coupling probabilities were between 80 and 94%. To model the possible dynamic fluctuations that might be associated with the putative allosteric pathway that was identified through EC analysis, we carried out Anisotropic Network Modeling (ANM) (40) using the webserver from the Bahar group (http://anm.csb.pitt.edu/cgi-bin/anm2/anm2.cgi; (41)).