Alternative Sigma Factor of Staphylococcus aureus Interacts with the Cognate Antisigma Factor Primarily Using Its...

σ^B, an alternative sigma factor, is usually employed to tackle the general stress response in Staphylococcus aureus and other Gram-positive bacteria. This protein, involved in S. aureus-mediated pathogenesis, is typically blocked by RsbW, an antisigma factor having serine kinase activity. σ^B, a σ⁷⁰-like sigma factor, harbors three conserved domains designated σ^B2, σ^B3, and σ^B4. To better understand the interaction between RsbW and σ^B or its domains, we have studied their recombinant forms, rRsbW, rσ^B, rσ^B2, rσ^B3, and rσ^B4, using different probes. The results show that none of the rσ^B domains, unlike rσ^B, showed binding to a cognate DNA in the presence of a core RNA polymerase. However, both rσ^B2 and rσ^B3, like rσ^B, interacted with rRsbW, and the order of their rRsbW binding affinity looks like rσ^B > rσ^B3 > rσ^B2. Furthermore, the reaction between rRsbW and rσ^B or rσ^B3 was exothermic and occurred spontaneously. rRsbW and rσ^B3 also associate with each other at a stoichiometry of 2:1, and different types of noncovalent bonds might be responsible for their interaction. A structural model of the RsbW-σ^B3 complex that has supported our experimental results indicated the binding of rσ^B3 at the putative dimeric interface of RsbW. A genetic study shows that the tentative dimer-forming region of RsbW is crucial for preserving its rσ^B binding ability, serine kinase activity, and dimerization ability. Additionally, a urea-induced equilibrium unfolding study indicated a notable thermodynamic stabilization of σ^B3 in the presence of RsbW. Possible implications of the stabilization data in drug discovery were discussed at length.