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Activating mutations in quorum-sensing regulator Rgg2 and its conformational flexibility in the absence of an intermolecular disulfide bond.
- Source :
-
The Journal of biological chemistry [J Biol Chem] 2017 Dec 15; Vol. 292 (50), pp. 20544-20557. Date of Electronic Publication: 2017 Oct 13. - Publication Year :
- 2017
-
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 <subscript>Sd</subscript> ). Here, we compared two Rgg2 <subscript>Sd</subscript> (C45S) X-ray crystal structures with that of wild-type Rgg2 <subscript>Sd</subscript> and found that in the absence of the intermolecular disulfide, the Rgg2 <subscript>Sd</subscript> dimer interface is destabilized and Rgg2 <subscript>Sd</subscript> can adopt multiple conformations. One conformation closely resembled the "disulfide-locked" Rgg2 <subscript>Sd</subscript> 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 <subscript>Sp</subscript> ) that conferred pheromone-independent transcriptional activation of an Rgg2-stimulated promoter. Eight mutations yielding constitutive Rgg2 activity, designated Rgg2 <subscript>Sp</subscript> *, were identified, and five of them clustered in or near an Rgg2 region that underwent conformational changes in one of the Rgg2 <subscript>Sd</subscript> (C45S) crystal structures. The Rgg2 <subscript>Sp</subscript> * mutations increased Rgg2 <subscript>Sp</subscript> sensitivity to pheromone and pheromone variants while displaying decreased sensitivity to the Rgg2 antagonist cyclosporine A. We propose that Rgg2 <subscript>Sp</subscript> * 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 <subscript>Sp</subscript> .<br /> (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)
- Subjects :
- Amino Acid Substitution
Bacterial Proteins antagonists & inhibitors
Bacterial Proteins chemistry
Bacterial Proteins genetics
Binding Sites
Crystallography, X-Ray
Cyclosporine pharmacology
Dimerization
Drug Resistance, Bacterial
Kinetics
Mutagenesis, Site-Directed
Pheromones chemistry
Pheromones metabolism
Pheromones pharmacology
Protein Conformation
Protein Interaction Domains and Motifs
Protein Isoforms antagonists & inhibitors
Protein Isoforms chemistry
Protein Isoforms genetics
Protein Isoforms metabolism
Protein Stability drug effects
Recombinant Proteins chemistry
Recombinant Proteins metabolism
Static Electricity
Streptococcus pyogenes drug effects
Trans-Activators antagonists & inhibitors
Trans-Activators chemistry
Trans-Activators genetics
Transcription Factors antagonists & inhibitors
Transcription Factors chemistry
Transcription Factors genetics
Bacterial Proteins metabolism
Cysteine chemistry
Models, Molecular
Point Mutation
Streptococcus pyogenes metabolism
Trans-Activators metabolism
Transcription Factors metabolism
Subjects
Details
- Language :
- English
- ISSN :
- 1083-351X
- Volume :
- 292
- Issue :
- 50
- Database :
- MEDLINE
- Journal :
- The Journal of biological chemistry
- Publication Type :
- Academic Journal
- Accession number :
- 29030429
- Full Text :
- https://doi.org/10.1074/jbc.M117.801670