title: Novobiocin and peptide analogs of α-factor are positive allosteric modulators of the yeast G protein-coupled receptor Ste2p.
abstract: G protein-coupled receptors (GPCRs) are the target of many drugs prescribed for human medicine and are therefore the subject of intense study. It has been recognized that compounds called allosteric modulators can regulate GPCR activity by binding to the receptor at sites distinct from, or overlapping with, that occupied by the orthosteric ligand. The purpose of this study was to investigate the nature of the interaction between putative allosteric modulators and Ste2p, a model GPCR expressed in the yeast Saccharomyces cerevisiae that binds the tridecapeptide mating pheromone α-factor. Biological assays demonstrated that an eleven amino acid α-factor analog and the antibiotic novobiocin were positive allosteric modulators of Ste2p. Both compounds enhanced the biological activity of α-factor, but did not compete with α-factor binding to Ste2p. To determine if novobiocin and the 11-mer shared a common allosteric binding site, a biologically-active analog of the 11-mer peptide ([Bio-DOPA]11-mer) was chemically cross-linked to Ste2p in the presence and absence of novobiocin. Immunoblots probing for the Ste2p-[Bio-DOPA]11-mer complex revealed that novobiocin markedly decreased cross-linking of the [Bio-DOPA]11-mer to the receptor, but cross-linking of the α-factor analog [Bio-DOPA]13-mer, which interacts with the orthosteric binding site of the receptor, was minimally altered. This finding suggests that both novobiocin and [Bio-DOPA]11-mer compete for an allosteric binding site on the receptor. These results indicate that Ste2p may provide an excellent model system for studying allostery in a GPCR.
authors.affiliation: Department of Microbiology, University of Tennessee, Knoxville, TN 37996, United States.
authors.forename: Jeffrey K
authors.initials: JK
authors.lastname: Rymer
authors.affiliation: Department of Microbiology, University of Tennessee, Knoxville, TN 37996, United States.
authors.forename: Melinda
authors.initials: M
authors.lastname: Hauser
authors.affiliation: Department of Chemistry, University of Tennessee, Knoxville, TN 37996, United States.
authors.forename: Allen K
authors.initials: AK
authors.lastname: Bourdon
authors.affiliation: Department of Chemistry, University of Tennessee, Knoxville, TN 37996, United States.
authors.forename: Shawn R
authors.initials: SR
authors.lastname: Campagna
authors.affiliation: Department of Chemistry and Macromolecular Assemblies Institute, College of Staten Island, CUNY, New York, NY 10314, United States; Graduate School and University Center, CUNY, New York, NY 10314, United States.
authors.forename: Fred
authors.initials: F
authors.lastname: Naider
authors.affiliation: Department of Microbiology, University of Tennessee, Knoxville, TN 37996, United States. Electronic address: jbecker@utk.edu.
authors.forename: Jeffrey M
authors.initials: JM
authors.lastname: Becker
mesh_terms: D000494:Allosteric Regulation / Q000187:drug effects*; D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D001665:Binding Sites; D001667:Binding, Competitive; D003432:Cross-Linking Reagents; D006801:Humans; D015151:Immunoblotting; D008024:Ligands; D000072235:Mating Factor; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism; D009675:Novobiocin / Q000494:pharmacology*; D010446:Peptide Fragments / Q000494:pharmacology*; D010455:Peptides / Q000494:pharmacology*; D011485:Protein Binding; D051964:Receptors, Mating Factor / Q000235:genetics / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000187:drug effects / Q000254:growth & development / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
chemical_list: D000900:Anti-Bacterial Agents; D003432:Cross-Linking Reagents; C490024:FUS1 protein, S cerevisiae; D008024:Ligands; D008565:Membrane Proteins; D010446:Peptide Fragments; D010455:Peptides; D051964:Receptors, Mating Factor; C498031:STE2 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D009675:Novobiocin; D000072235:Mating Factor
keywords: Allostery; DOPA; G protein-coupled receptor (GPCR); Novobiocin; Ste2p; Yeast