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

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 (Rgg2). Here, we compared two Rgg2(C45S) X-ray crystal structures with that of wild-type Rgg2 and found that in the absence of the intermolecular disulfide, the Rgg2 dimer interface is destabilized and Rgg2 can adopt multiple conformations. One conformation closely resembled the "disulfide-locked" Rgg2 secondary and tertiary structures, but another displayed more extensive rigid-body shifts as well as dramatic secondary structure changes. In parallel experiments, a genetic screen was used to identify mutations in of ( ) that conferred pheromone-independent transcriptional activation of an Rgg2-stimulated promoter. Eight mutations yielding constitutive Rgg2 activity, designated Rgg2*, were identified, and five of them clustered in or near an Rgg2 region that underwent conformational changes in one of the Rgg2(C45S) crystal structures. The Rgg2* mutations increased Rgg2 sensitivity to pheromone and pheromone variants while displaying decreased sensitivity to the Rgg2 antagonist cyclosporine A. We propose that Rgg2* mutations invoke shifts in free-energy bias to favor the active state of the protein. Finally, we present evidence for an electrostatic interaction between an N-terminal Asp of the pheromone and Arg-153 within the proposed pheromone-binding pocket of Rgg2.