TY - JOUR
T1 - Structural and biochemical characterization of MepR, a multidrug binding transcription regulator of the Staphylococcus aureus multidrug efflux pump MepA
AU - Kumaraswami, Muthiah
AU - Schuman, Jason T.
AU - Seo, Susan M.
AU - Kaatz, Glenn W.
AU - Brennan, Richard G.
N1 - Funding Information:
This work was supported in part by funds from the National Institutes of Health grant (AI048593 to R.G.B.), the Robert A. Welch Foundation grant (G-0040 to R.G.B.) and Veteran Administration Research Funds (to G.W.K.). Funding for open access charge: R.A. Welch Foundation grant G-0040 (to R.G.B).
Funding Information:
We thank the beamline scientists at ALS BL 8.3.1 for their great help with MAD data collection. We also acknowledge the Advanced Light Source supported by the Director, Office of science, Office of Basic Energy Sciences, Material Sciences Division, of the US Department of Energy under contract No. DE-AC03-76SF00098 at Lawrence Berkeley National Laboratory. We thank Dr H. P. Bächinger at Shriners Hospital for Sick Children at Portland, Oregon for determining the absolute values of our protein concentrations by amino acid analysis.
PY - 2009
Y1 - 2009
N2 - MepR is a multidrug binding transcription regulator that represses expression of the Staphylococcus aureus multidrug efflux pump gene, mepA, as well as its own gene. MepR is induced by multiple cationic toxins, which are also substrates of MepA. In order to understand the gene regulatory and drug-binding mechanisms of MepR, we carried out biochemical, in vivo and structural studies. The 2.40 Å resolution structure of drug-free MepR reveals the most open MarR family protein conformation to date, which will require a huge conformational change to bind cognate DNA. DNA-binding data show that MepR uses a dual regulatory binding mode as the repressor binds the mepA operator as a dimer of dimers, but binds the mepR operator as a single dimer. Alignment of the six half sites reveals the consensus MepR binding site, 5′-GTTAGAT-3′. 'Drug' binding studies show that MepR binds to ethidium and DAPI with comparable affinities (Kd = 2.6 and 4.5 μM, respectively), but with significantly lower affinity to the larger rhodamine 6G (Kd = 62.6 μM). Mapping clinically relevant or in vitro selected MepR mutants onto the MepR structure suggests that their defective repressor phenotypes are due to structural and allosteric defects.
AB - MepR is a multidrug binding transcription regulator that represses expression of the Staphylococcus aureus multidrug efflux pump gene, mepA, as well as its own gene. MepR is induced by multiple cationic toxins, which are also substrates of MepA. In order to understand the gene regulatory and drug-binding mechanisms of MepR, we carried out biochemical, in vivo and structural studies. The 2.40 Å resolution structure of drug-free MepR reveals the most open MarR family protein conformation to date, which will require a huge conformational change to bind cognate DNA. DNA-binding data show that MepR uses a dual regulatory binding mode as the repressor binds the mepA operator as a dimer of dimers, but binds the mepR operator as a single dimer. Alignment of the six half sites reveals the consensus MepR binding site, 5′-GTTAGAT-3′. 'Drug' binding studies show that MepR binds to ethidium and DAPI with comparable affinities (Kd = 2.6 and 4.5 μM, respectively), but with significantly lower affinity to the larger rhodamine 6G (Kd = 62.6 μM). Mapping clinically relevant or in vitro selected MepR mutants onto the MepR structure suggests that their defective repressor phenotypes are due to structural and allosteric defects.
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U2 - 10.1093/nar/gkn1046
DO - 10.1093/nar/gkn1046
M3 - Article
C2 - 19129225
AN - SCOPUS:62549114521
VL - 37
SP - 1211
EP - 1224
JO - Nucleic Acids Research
JF - Nucleic Acids Research
SN - 0305-1048
IS - 4
ER -