Alternative Sigma Factor Of Staphylococcus Aureus Interacts With The Cognate Antisigma Factor Primarily Using Its Domain 3

sigma(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. sigma(B), a sigma(70)-like sigma factor, harbors three conserved domains designated sigma(B2), sigma(B3), and sigma(B4). To better understand the interaction between RsbW and sigma(B) or its domains, we have studied their recombinant forms, rRsbW, r sigma(B), r sigma(B2), r sigma(B3), and r sigma(B4), using different probes. The results show that none of the r sigma B domains, unlike r sigma(B), showed binding to a cognate DNA in the presence of a core RNA polymerase. However, both r sigma(B2) and r sigma(B3), like r sigma(B), interacted with rRsbW, and the order of their rRsbW binding affinity looks like r sigma(B) > r sigma(B3) > r sigma(B2). Furthermore, the reaction between rRsbW and r sigma(B) or r sigma(B3) was exothermic and occurred spontaneously. rRsbW and r sigma(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-sigma(B3) complex that has supported our experimental results indicated the binding of r sigma(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 r sigma(B) binding ability, serine kinase activity, and dimerization ability. Additionally, a urea-induced equilibrium unfolding study indicated a notable thermodynamic stabilization of sigma(B3) in the presence of RsbW. Possible implications of the stabilization data in drug discovery were discussed at length.