The protein-water H-bond network for bacterial response regulator REC domain activation

The bacterial response regulator (RR) superfamily is activated by aspartyl phosphorylation of the receiver (REC) domain for diverse functions. Crystal structures of the enteric CheY chemotaxis RRs (CheY_es) have revealed water-residue hydrogen bonds (H-bonds) essential for activation. Here, we compared the H-bond networks, computed from MD simulations, of the basal and phosphorylated conformational landscapes in silico. Representative REC domains were selected by filtering the sequence and structure RR databases based on sequence information and fold variability. Residues bonded with water included the aspartate triad for divalent metal ion coordination and phosphoryl transfer, plus K109. The water-residue H-bond propensity of these residues was balanced by the impaired stability of adjacent residue contacts maintained by evolution. Upon phosphorylation, the CheY_es aspartyl phosphate (D57-PO₃ ^-) became the major node, connected via the conserved switch residues K109, T87 to the distant target binding surface with H-bond and associated long-range couplings measured by mutual information. The communication was mediated by transient H-bonded chains that included internal water in the hydrophobic core. Study of two other RR representatives indicates that an increased D57-PO₃ ^- node strength in the REC protein-water H-bond network upon phosphorylation is a generic feature of RR allostery. There are subtle differences in the connectivity between D57 and an aromatic residue (CheY_es Y106 or equivalent), diagnostic of interfacial dynamics. These differences, influenced by the water contribution, may reflect function-related spatiotemporal constraints useful for the design of RR subfamily-specific, allosteric inhibitors.