title: The activity of CouR, a MarR family transcriptional regulator, is modulated through a novel molecular mechanism.
abstract: CouR, a MarR-type transcriptional repressor, regulates the cou genes, encoding p-hydroxycinnamate catabolism in the soil bacterium Rhodococcus jostii RHA1. The CouR dimer bound two molecules of the catabolite p-coumaroyl-CoA (Kd = 11 ± 1 μM). The presence of p-coumaroyl-CoA, but neither p-coumarate nor CoASH, abrogated CouR's binding to its operator DNA in vitro. The crystal structures of ligand-free CouR and its p-coumaroyl-CoA-bound form showed no significant conformational differences, in contrast to other MarR regulators. The CouR-p-coumaroyl-CoA structure revealed two ligand molecules bound to the CouR dimer with their phenolic moieties occupying equivalent hydrophobic pockets in each protomer and their CoA moieties adopting non-equivalent positions to mask the regulator's predicted DNA-binding surface. More specifically, the CoA phosphates formed salt bridges with predicted DNA-binding residues Arg36 and Arg38, changing the overall charge of the DNA-binding surface. The substitution of either arginine with alanine completely abrogated the ability of CouR to bind DNA. By contrast, the R36A/R38A double variant retained a relatively high affinity for p-coumaroyl-CoA (Kd = 89 ± 6 μM). Together, our data point to a novel mechanism of action in which the ligand abrogates the repressor's ability to bind DNA by steric occlusion of key DNA-binding residues and charge repulsion of the DNA backbone.
authors.affiliation: Department of Microbiology and Immunology, Life Sciences Institute, The University of British Columbia, Vancouver, British Columbia V6T 1Z3, Canada.
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Otani
authors.affiliation: Department of Chemical Engineering and Applied Chemistry, University of Toronto, Toronto, Ontario M5S 3E5, Canada.
authors.forename: Peter J
authors.initials: PJ
authors.lastname: Stogios
authors.affiliation: Department of Chemical Engineering and Applied Chemistry, University of Toronto, Toronto, Ontario M5S 3E5, Canada.
authors.forename: Xiaohui
authors.initials: X
authors.lastname: Xu
authors.affiliation: Structural Biology Center, Biosciences Division, Argonne National Laboratory and the Midwest Center for Structural Genomics, Lemont, IL 60439, USA.
authors.forename: Boguslaw
authors.initials: B
authors.lastname: Nocek
authors.affiliation: Department of Microbiology and Immunology, Life Sciences Institute, The University of British Columbia, Vancouver, British Columbia V6T 1Z3, Canada.
authors.forename: Shu-Nan
authors.initials: SN
authors.lastname: Li
authors.affiliation: Department of Chemical Engineering and Applied Chemistry, University of Toronto, Toronto, Ontario M5S 3E5, Canada.
authors.forename: Alexei
authors.initials: A
authors.lastname: Savchenko
authors.affiliation: Department of Microbiology and Immunology, Life Sciences Institute, The University of British Columbia, Vancouver, British Columbia V6T 1Z3, Canada leltis@mail.ubc.ca.
authors.forename: Lindsay D
authors.initials: LD
authors.lastname: Eltis
mesh_terms: D000214:Acyl Coenzyme A / Q000737:chemistry* / Q000378:metabolism; D000409:Alanine / Q000737:chemistry / Q000378:metabolism; D019943:Amino Acid Substitution; D001120:Arginine / Q000737:chemistry / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D001665:Binding Sites; D003065:Coenzyme A / Q000737:chemistry / Q000378:metabolism; D003373:Coumaric Acids / Q000737:chemistry / Q000378:metabolism; D018360:Crystallography, X-Ray; D004247:DNA / Q000737:chemistry* / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial; D057927:Hydrophobic and Hydrophilic Interactions; D008024:Ligands; D008958:Models, Molecular; D011401:Promoter Regions, Genetic; D011422:Propionates; D011485:Protein Binding; D054730:Protein Interaction Domains and Motifs; D055503:Protein Multimerization; D012097:Repressor Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D012240:Rhodococcus; D055672:Static Electricity; D014158:Transcription, Genetic
chemical_list: D000214:Acyl Coenzyme A; D001426:Bacterial Proteins; D003373:Coumaric Acids; D008024:Ligands; D011422:Propionates; D012097:Repressor Proteins; C058644:4-coumaroyl-coenzyme A; D004247:DNA; D001120:Arginine; C495469:p-coumaric acid; D000409:Alanine; D003065:Coenzyme A