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Structural and biochemical characterization of MepR, a multidrug binding transcription regulator of the Staphylococcus aureus multidrug efflux pump MepA.

Kumaraswami M, Schuman JT, Seo SM, Kaatz GW, Brennan RG - Nucleic Acids Res. (2009)

Bottom Line: 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 (K(d) = 2.6 and 4.5 microM, respectively), but with significantly lower affinity to the larger rhodamine 6G (K(d) = 62.6 microM).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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, University of Texas M.D. Anderson Cancer Center, Houston, TX 77030, USA.

ABSTRACT
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 A 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 (K(d) = 2.6 and 4.5 microM, respectively), but with significantly lower affinity to the larger rhodamine 6G (K(d) = 62.6 microM). 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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Related in: MedlinePlus

Structure of MepR. (A) Ribbon diagram of the MepR subunit. The secondary structural elements are labelled and rainbow colored from red (N-terminus) to blue (C-terminus). The N- and C-termini are indicated as N and C, respectively. With the exception of the loops between β1 and β2, all loops are colored grey. (B) Ribbon diagram of the MepR dimer. The secondary structure elements of one subunit is color coded as in Figure 2A. The dyadic mate is colored magenta. The dimerization interface and the winged helix-turn-helix motif (wHTH) are labelled. (C) Primary sequence alignment of MepR with other structurally characterized MarR family members [MepR, B. subtilis OhrR (OhrR-B), X. campestris OhrR (OhrR-X), E. coli MarR, P. aeruginosa MexR and Methanobacterium MarR, MTH313]. The multiple sequence alignment was made with ClustalW (45). The secondary structure elements of MepR are indicated above the alignment, α helices as rectangles and β strands as arrows, and colored as in Figure 2A. Identical residues are colored red and the chemically similar residues are blue. The conserved hydrophobic residues that are involved in dimerization are highlighted in shaded boxes. The magenta asterisks (*) indicate the positions of MepR mutations that had been identified from clinically isolated or in vitro selected multidrug-resistant strains of S. aureus.
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Figure 2: Structure of MepR. (A) Ribbon diagram of the MepR subunit. The secondary structural elements are labelled and rainbow colored from red (N-terminus) to blue (C-terminus). The N- and C-termini are indicated as N and C, respectively. With the exception of the loops between β1 and β2, all loops are colored grey. (B) Ribbon diagram of the MepR dimer. The secondary structure elements of one subunit is color coded as in Figure 2A. The dyadic mate is colored magenta. The dimerization interface and the winged helix-turn-helix motif (wHTH) are labelled. (C) Primary sequence alignment of MepR with other structurally characterized MarR family members [MepR, B. subtilis OhrR (OhrR-B), X. campestris OhrR (OhrR-X), E. coli MarR, P. aeruginosa MexR and Methanobacterium MarR, MTH313]. The multiple sequence alignment was made with ClustalW (45). The secondary structure elements of MepR are indicated above the alignment, α helices as rectangles and β strands as arrows, and colored as in Figure 2A. Identical residues are colored red and the chemically similar residues are blue. The conserved hydrophobic residues that are involved in dimerization are highlighted in shaded boxes. The magenta asterisks (*) indicate the positions of MepR mutations that had been identified from clinically isolated or in vitro selected multidrug-resistant strains of S. aureus.

Mentions: MepR is predominantly α helical with six α helices and a two-stranded antiparallel β hairpin and an overall topology of α1 (residues 5–26) -α2 (residues 31–42) -α3 (residues 50–57) -α4 (residues 61–74) -β1 (residues 77–81) - W1 (the tip of the Wing, residues 82–84)-β2 (residues 88–93) -α5 (residues 95–118) -α6 (residues 121–139) (Figure 2A and C). Each subunit is composed of two functional domains: a dimerization domain that includes helices α1, α5 and α6, and a DNA-binding domain with a winged helix-turn-helix (wHTH) motif (α3,α4,β1,W1, β2) in the middle of the polypeptide chain (Figure 2B). The DNA-binding domain is connected to the dimerization domain by helices α2 and α5. Unlike most MarR family members, the apo MepR structure lacks a third β strand typically located between α2 and α3. Residues located at the tip of the wing, between β1 and β2, are either missing completely or have had their side chains truncated to alanines. The disorder in this region is likely due to the inherent flexibility of the wing in the absence of DNA as observed in other MarR family members such as OhrR (36,37).Figure 2.


Structural and biochemical characterization of MepR, a multidrug binding transcription regulator of the Staphylococcus aureus multidrug efflux pump MepA.

Kumaraswami M, Schuman JT, Seo SM, Kaatz GW, Brennan RG - Nucleic Acids Res. (2009)

Structure of MepR. (A) Ribbon diagram of the MepR subunit. The secondary structural elements are labelled and rainbow colored from red (N-terminus) to blue (C-terminus). The N- and C-termini are indicated as N and C, respectively. With the exception of the loops between β1 and β2, all loops are colored grey. (B) Ribbon diagram of the MepR dimer. The secondary structure elements of one subunit is color coded as in Figure 2A. The dyadic mate is colored magenta. The dimerization interface and the winged helix-turn-helix motif (wHTH) are labelled. (C) Primary sequence alignment of MepR with other structurally characterized MarR family members [MepR, B. subtilis OhrR (OhrR-B), X. campestris OhrR (OhrR-X), E. coli MarR, P. aeruginosa MexR and Methanobacterium MarR, MTH313]. The multiple sequence alignment was made with ClustalW (45). The secondary structure elements of MepR are indicated above the alignment, α helices as rectangles and β strands as arrows, and colored as in Figure 2A. Identical residues are colored red and the chemically similar residues are blue. The conserved hydrophobic residues that are involved in dimerization are highlighted in shaded boxes. The magenta asterisks (*) indicate the positions of MepR mutations that had been identified from clinically isolated or in vitro selected multidrug-resistant strains of S. aureus.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC2651776&req=5

Figure 2: Structure of MepR. (A) Ribbon diagram of the MepR subunit. The secondary structural elements are labelled and rainbow colored from red (N-terminus) to blue (C-terminus). The N- and C-termini are indicated as N and C, respectively. With the exception of the loops between β1 and β2, all loops are colored grey. (B) Ribbon diagram of the MepR dimer. The secondary structure elements of one subunit is color coded as in Figure 2A. The dyadic mate is colored magenta. The dimerization interface and the winged helix-turn-helix motif (wHTH) are labelled. (C) Primary sequence alignment of MepR with other structurally characterized MarR family members [MepR, B. subtilis OhrR (OhrR-B), X. campestris OhrR (OhrR-X), E. coli MarR, P. aeruginosa MexR and Methanobacterium MarR, MTH313]. The multiple sequence alignment was made with ClustalW (45). The secondary structure elements of MepR are indicated above the alignment, α helices as rectangles and β strands as arrows, and colored as in Figure 2A. Identical residues are colored red and the chemically similar residues are blue. The conserved hydrophobic residues that are involved in dimerization are highlighted in shaded boxes. The magenta asterisks (*) indicate the positions of MepR mutations that had been identified from clinically isolated or in vitro selected multidrug-resistant strains of S. aureus.
Mentions: MepR is predominantly α helical with six α helices and a two-stranded antiparallel β hairpin and an overall topology of α1 (residues 5–26) -α2 (residues 31–42) -α3 (residues 50–57) -α4 (residues 61–74) -β1 (residues 77–81) - W1 (the tip of the Wing, residues 82–84)-β2 (residues 88–93) -α5 (residues 95–118) -α6 (residues 121–139) (Figure 2A and C). Each subunit is composed of two functional domains: a dimerization domain that includes helices α1, α5 and α6, and a DNA-binding domain with a winged helix-turn-helix (wHTH) motif (α3,α4,β1,W1, β2) in the middle of the polypeptide chain (Figure 2B). The DNA-binding domain is connected to the dimerization domain by helices α2 and α5. Unlike most MarR family members, the apo MepR structure lacks a third β strand typically located between α2 and α3. Residues located at the tip of the wing, between β1 and β2, are either missing completely or have had their side chains truncated to alanines. The disorder in this region is likely due to the inherent flexibility of the wing in the absence of DNA as observed in other MarR family members such as OhrR (36,37).Figure 2.

Bottom Line: 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 (K(d) = 2.6 and 4.5 microM, respectively), but with significantly lower affinity to the larger rhodamine 6G (K(d) = 62.6 microM).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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, University of Texas M.D. Anderson Cancer Center, Houston, TX 77030, USA.

ABSTRACT
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 A 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 (K(d) = 2.6 and 4.5 microM, respectively), but with significantly lower affinity to the larger rhodamine 6G (K(d) = 62.6 microM). 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.

Show MeSH
Related in: MedlinePlus