Role of the recognition helix of response regulator WalR from Bacillus anthracis in DNA binding and specificity.

WalRK two-component system of Bacillus anthracis potentially regulates multiple genes spanning diverse cellular functions. Its constituent response regulator (RR), WalR belongs to the OmpR/PhoB family which possesses a winged helix-turn-helix motif for DNA binding. An in silico knowledge based model of WalR C-terminal DNA binding domain in complex with its ftsE promoter region binding motif was used to identify specific residues of the recognition helix important for DNA binding. The model was validated by mutagenesis in conjunction with in vitro DNA binding analysis. The ftsE promoter region DNA binding motif was also varied. Optimal binding of WalR to DNA required the presence of both half-sites in its binding motif. Substitution of invariant bases of WalR DNA binding motif abrogated the binding whereas changes at variable motif positions governed affinity. D199 was not in direct contact with the DNA but its substitution modified the WalR-DNA specificity indicating the importance of contact avoidance by this residue for DNA specificity. This represents the first in-depth study of RR-DNA interaction from B. anthracis.