| Title: | Activating mutations in quorum-sensing regulator Rgg2 and its conformational flexibility in the absence of an intermolecular disulfide bond |
| Author(s): | Wilkening RV; Capodagli GC; Khataokar A; Tylor KM; Neiditch MB; Federle MJ; |
| Address: | "From the Department of Microbiology and Immunology, University of Illinois at Chicago, Chicago, Illinois 60607. the Department of Microbiology, Biochemistry, and Molecular Genetics, New Jersey Medical School, Rutgers, State University of New Jersey, Newark, New Jersey 07103, and. the Department of Microbiology, Biochemistry, and Molecular Genetics, New Jersey Medical School, Rutgers, State University of New Jersey, Newark, New Jersey 07103, and matthew.neiditch@rutgers.edu. From the Department of Microbiology and Immunology, University of Illinois at Chicago, Chicago, Illinois 60607, mfederle@uic.edu. the Department of Medicinal Chemistry and Pharmacognosy, Center for Biomolecular Sciences, University of Illinois at Chicago, Chicago, Illinois 60607" |
| ISSN/ISBN: | 1083-351X (Electronic) 0021-9258 (Print) 0021-9258 (Linking) |
| 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)" |
| Keywords: | "Amino Acid Substitution Bacterial Proteins/antagonists & inhibitors/chemistry/genetics/*metabolism Binding Sites Crystallography, X-Ray Cyclosporine/pharmacology Cysteine/*chemistry Dimerization Drug Resistance, Bacterial Kinetics *Models, Molecular Mutag;" |
| Notes: | "MedlineWilkening, Reid V Capodagli, Glenn C Khataokar, Atul Tylor, Kaitlyn M Neiditch, Matthew B Federle, Michael J eng R01 AI091779/AI/NIAID NIH HHS/ R01 AI125452/AI/NIAID NIH HHS/ Comparative Study Research Support, N.I.H., Extramural Research Support, Non-U.S. Gov't 2017/10/17 J Biol Chem. 2017 Dec 15; 292(50):20544-20557. doi: 10.1074/jbc.M117.801670. Epub 2017 Oct 13" |
