Activating mutations in quorum-sensing regulator Rgg2 and its conformational flexibility in the absence of an intermolecular disulfide bond

Abstract

Rap/Rgg/NprR/PlcR/PrgX (RRNPP) quorum-sensing systems use extracellular peptide pheromones that are detected by cytoplasmic receptors to regulate gene expression in firmicute bacteria. Rgg-type receptors are allosterically regulated through direct pheromone binding to control transcriptional activity; however, the receptor activation mechanism remains poorly understood. Previous work has identified a disulfide bond between Cys-45 residues within the homodimer interface of Rgg2 from Streptococcus dysgalactiae (Rgg2Sd). Here, we compared two Rgg2Sd(C45S) X-ray crystal structures with that of wild-type Rgg2Sd and found that in the absence of the intermolecular disulfide, the Rgg2Sd dimer interface is destabilized and Rgg2Sd can adopt multiple conformations. One conformation closely resembled the "disulfide-locked" Rgg2Sd secondary and tertiary structures, but another displayed more extensive rigidbody shifts as well as dramatic secondary structure changes. In parallel experiments, agenetic screen was usedtoidentify mutations in rgg2 of Streptococcus pyogenes (rgg2Sp) that conferred pheromone-independent transcriptional activation of an Rgg2-stimulated promoter. Eight mutations yielding constitutive Rgg2 activity, designated Rgg2Sp∗, were identified, and five of them clustered in or near an Rgg2 region that underwent conformational changes in one of the Rgg2Sd(C45S) crystal structures. The Rgg2Sp∗ mutations increased Rgg2Sp sensitivity to pheromone and pheromone variants while displaying decreased sensitivity to the Rgg2 antagonist cyclosporine A. We propose that Rgg2Sp∗ mutations invoke shifts in free-energy bias to favor the active stateofthe protein. Finally, wepresent evidence for an electrostatic interaction between an N-terminal Asp of the pheromone and Arg-153 within the proposed pheromone-binding pocket of Rgg2Sp.