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 rigid-body shifts as well as dramatic secondary structure changes. In parallel experiments, a genetic screen was used to identify 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 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 Rgg2Sp.
Highlights
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
By these structural findings, combined with previous in vivo and in vitro characterization of Rgg2, we propose that the protein possesses four primary structural features that define its function: a peptide-binding groove within the repeat domain establishing an allosteric regulatory site; an XRE family helix-turn-helix DNA-binding domain (DBD); a dimerization interface that includes the Cys-45–Cys-45 cysteine pair allowing for intermolecular disulfide bond formation; and an undefined region that provides the ability to promote transcription, likely by recruiting RNA polymerase when Rgg2 is bound to SHP
We propose that the Rgg2 from Streptococcus dysgalactiae (Rgg2Sd)(C45S)x2 rigid body and secondary structure changes are enabled by structural flexibility otherwise absent upon formation of the Cys-45–Cys-45 intermolecular disulfide bond that connects the wild-type Rgg2 DBDs
Summary
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. The structures indicated several residues likely to be important for SHP-Rgg2Sd interaction, including Arg-153 in a putative peptide-binding pocket, here proposed to form electrostatic interactions with SHP necessary for full activation [20] By these structural findings, combined with previous in vivo and in vitro characterization of Rgg, we propose that the protein possesses four primary structural features that define its function: a peptide-binding groove within the repeat domain establishing an allosteric regulatory site; an XRE family helix-turn-helix DBD; a dimerization interface that includes the Cys-45–Cys-45 cysteine pair allowing for intermolecular disulfide bond formation; and an undefined region that provides the ability to promote transcription, likely by recruiting RNA polymerase when Rgg is bound to SHP. Results of site-directed mutational analysis of Arg-153 provides evidence that electrostatic interactions between this residue and the SHP pheromone are central in activation of Rgg2Sp
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