Alternative Sigma Factor of Staphylococcus aureus Interacts with the Cognate Antisigma Factor Primarily Using Its Domain 3

Abstract

Metrics & MoreArticle Recommendations * sı Supporting Information ABSTRACT: 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 σ 70 -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.