Activating mutations in quorum-sensing regulator Rgg2 and its conformational flexibility in the absence of an intermolecular disulfide bond.
Wilkening, R.V., Capodagli, G.C., Khataokar, A., Tylor, K.M., Neiditch, M.B., Federle, M.J.(2017) J Biological Chem 292: 20544-20557
- PubMed: 29030429
- DOI: https://doi.org/10.1074/jbc.M117.801670
- Primary Citation of Related Structures:
5W4M, 5W4N - PubMed 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 (Rgg2 Sd ). Here, we compared two Rgg2 Sd (C45S) X-ray crystal structures with that of wild-type Rgg2 Sd and found that in the absence of the intermolecular disulfide, the Rgg2 Sd dimer interface is destabilized and Rgg2 Sd can adopt multiple conformations. One conformation closely resembled the "disulfide-locked" Rgg2 Sd 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 ( rgg2 Sp ) that conferred pheromone-independent transcriptional activation of an Rgg2-stimulated promoter. Eight mutations yielding constitutive Rgg2 activity, designated Rgg2 Sp *, were identified, and five of them clustered in or near an Rgg2 region that underwent conformational changes in one of the Rgg2 Sd (C45S) crystal structures. The Rgg2 Sp * mutations increased Rgg2 Sp sensitivity to pheromone and pheromone variants while displaying decreased sensitivity to the Rgg2 antagonist cyclosporine A. We propose that Rgg2 Sp * 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 Sp .
Organizational Affiliation:
From the Department of Microbiology and Immunology, University of Illinois at Chicago, Chicago, Illinois 60607.