Abstract
The MarR family transcriptional regulator CouR, from the soil bacterium Rhodopseudomonas palustris CGA009, has recently been shown to negatively regulate a p-coumarate catabolic operon. Unlike most characterized MarR repressors that respond to small metabolites at concentrations in the millimolar range, repression by CouR is alleviated by the 800-Da ligand p-coumaroyl-CoA with high affinity and specificity. Here we report the crystal structures of ligand-free CouR as well as the complex with p-coumaroyl-CoA, each to 2.1-Å resolution, and the 2.85-Å resolution cocrystal structure of CouR bound to an oligonucleotide bearing the cognate DNA operator sequence. In combination with binding experiments that uncover specific residues important for ligand and DNA recognition, these structures provide glimpses of a MarR family repressor in all possible states, providing an understanding of the molecular basis of DNA binding and the conformation alterations that accompany ligand-induced dissociation for activation of the operon.
Highlights
The multiple antibiotic resistance regulator (MarR) family transcriptional regulator CouR, from the soil bacterium Rhodopseudomonas palustris CGA009, has recently been shown to negatively regulate a p-coumarate catabolic operon
Unlike most characterized MarR repressors that respond to small metabolites at concentrations in the millimolar range, repression by CouR is alleviated by the 800-Da ligand p-coumaroyl–CoA with high affinity and specificity
Because of ease of reproducibility, structure determination focused on using crystals of the CouR–DNA complex, and crystallographic phases were determined by the single-wavelength anomalous diffraction method from data collected on crystals of selenomethionine-labeled CouR
Summary
Cocrystallization efforts with CouR and various synthetic oligonucleotides bearing the inverted repeat DNA recognition sequence yielded several candidates, but most of these crystals did not diffract beyond 3.5-Å resolution. Structures of ligand-free CouR and a complex with synthetic pCC each produced crystals that diffracted to 2.1-Å resolution. Each monomer consists of six ␣-helices and two -strands containing a wHTH motif, where helices ␣3-␣4 and strands 1-2 define the DNA binding elements (Fig. 1, A and D). Dimerization between the two monomers is mediated by extensive burial of numerous hydrophobic residues to form the structural core. Residues at this interface include Leu-51, Leu-55, Val-62, and Phe-66 from helix ␣1; Val-152, Leu-160, and Leu-164 from helix ␣5; and Leu-172, Leu-176, and Ile-179 from helix ␣6. The closest homologs include a putative regulator of unknown function from Pseudomonas aeruginosa (PDB code 2NNN, Z score of 17.5, RMSD of 1.4 Å over 133 aligned C␣ atoms), the Streptomyces coelicolor -ketoadipate regulator PcaV (PDB code 4FHT, Z score of 17.1, RMSD of 2.7 Å over 141 aligned C␣ atoms), E. coli MarR (PDB code 3VOE, Z score of 17.1, RMSD of 2.7 Å over 136 aligned C␣ atoms), and the multidrug efflux regulator MexR from P. aeruginosa (PDB code 1LNW, Z score of 17.1, RMSD of 1.7 Å over 140 aligned C␣ atoms)
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