Journal
NUCLEIC ACIDS RESEARCH
Volume 44, Issue 2, Pages 595-607Publisher
OXFORD UNIV PRESS
DOI: 10.1093/nar/gkv955
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Funding
- Genome Canada Large-Scale Research Project [162MIC]
- Natural Science and Engineering Research Council Discovery grant [171359-13]
- Canada Research Chair
- National Institutes of Health [GM094585]
- US Department of Energy, Office of Biological and Environmental Research [DE-AC02-06CH11357]
- Midwest Center for Structural Genomics
- U.S. Department of Energy, Office of Biological and Environmental Research [DE-AC02-06CH11357]
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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 (K-d = 11 +/- 1 mu M). 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 (K-d = 89 +/- 6 mu M). 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.
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