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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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Binding isotherms of MepR and selected ligands. (A through D) MepR-binding isotherms to various lengths of cognate DNA from the mepA promoter. (A) MepR binding to a 44-bp site encompassing the entire mepA promoter DNase I footprint. (B) MepR binding to a 26-bp site of the mepA promoter that has been truncated from the 3′-end. (C) MepR binding to a 22-bp site of the mepA promoter that has been truncated from the 3′-end. (D) MepR binding to a 31-bp site of the mepA promoter that has been truncated from the 5′-end. (E) MepR-binding isotherms to a 26-bp DNA binding site from the mepR promoter. (F) Nucleotide sequence of the mepA promoter. The –10 and –35 elements of the promoter are shown in bold and labelled and the transcription start site (TSS) is indicated as a bent arrow. The pseudo-inverted repeats of the promoter are shown by horizontal arrows and the signature GTTAG motifs are underlined. The boundaries of the 22, 26 and 31-bp sequences used in the binding studies are indicated by blue, red and purple rectangles, respectively. G–I, MepR-binding isotherms for three ‘drugs’. (G) The MepR-Et-binding isotherm. (H) The MepR-DAPI-binding isotherm. (I) The MepR-R6G-binding isotherm. The change in polarization, shown in red circles and indicated in millipolarization units (mP), was plotted against the MepR dimer concentration indicated inside each plot. The binding constants are shown in each plot. The chemical structure of each drug is shown below the respective binding curve.
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Figure 4: Binding isotherms of MepR and selected ligands. (A through D) MepR-binding isotherms to various lengths of cognate DNA from the mepA promoter. (A) MepR binding to a 44-bp site encompassing the entire mepA promoter DNase I footprint. (B) MepR binding to a 26-bp site of the mepA promoter that has been truncated from the 3′-end. (C) MepR binding to a 22-bp site of the mepA promoter that has been truncated from the 3′-end. (D) MepR binding to a 31-bp site of the mepA promoter that has been truncated from the 5′-end. (E) MepR-binding isotherms to a 26-bp DNA binding site from the mepR promoter. (F) Nucleotide sequence of the mepA promoter. The –10 and –35 elements of the promoter are shown in bold and labelled and the transcription start site (TSS) is indicated as a bent arrow. The pseudo-inverted repeats of the promoter are shown by horizontal arrows and the signature GTTAG motifs are underlined. The boundaries of the 22, 26 and 31-bp sequences used in the binding studies are indicated by blue, red and purple rectangles, respectively. G–I, MepR-binding isotherms for three ‘drugs’. (G) The MepR-Et-binding isotherm. (H) The MepR-DAPI-binding isotherm. (I) The MepR-R6G-binding isotherm. The change in polarization, shown in red circles and indicated in millipolarization units (mP), was plotted against the MepR dimer concentration indicated inside each plot. The binding constants are shown in each plot. The chemical structure of each drug is shown below the respective binding curve.

Mentions: In order to determine the minimal sequence required for MepR binding, fluorescent polarization-based DNA-binding assays were carried out with various lengths of oligodeoxy-nucleotides, which encompassed the proposed MepR-binding sites in the mepA and mepR promoters. Results from the titration experiments indicate that MepR binds to a 44 bp duplex covering the ‘full’ MepR footprint of the mepA promoter with a dissociation constant (Kd) of 6.3 nM (Figure 4A). Interestingly, truncation of downstream sequences had little effect on MepR binding whereby the dissociation constant for a 26 bp oligodeoxynucleotide, which does not include 18 bp from the 3′-end of the 44 bp site, was only ∼2.4-fold lower (Kd = 15.3 nM) than that of the complete mepA site (Figure 4B). Removal of four additional base pairs further impairs MepR binding to the mepA site another 3.3-fold (Kd = 46.4 nM) but binding remains relatively tight with only a cumulative ∼7-fold loss in affinity as compared to the 44 bp site (Figure 4C and F). By contrast, deletion of eight base pairs from the 5′-end of the 44mer causes a greater than 30-fold reduction (Kd = 186.9 nM) in the affinity of MepR for the mepA-binding site (Figure 4D). DNA-binding experiments with the 26 bp MepR-binding site from mepR promoter also showed high-affinity binding (Kd = 24.3 nM) that essentially matches the affinity of the tested 26-bp fragment from the mepA promoter and is only ∼4-fold lower than that of the full 44 bp mepA site (Figure 4B and E). Thus, the previously observed differential response to drugs, i.e. the slower release of MepR from mepR operator site, does not appear to correlate well with MepR–DNA-binding affinity and suggests that MepR binds to mepA and mepR-binding sites somewhat differently, whereby subsequent drug binding to MepR when bound to mepR promoter is less effective in induction than drug binding to MepR bound to mepA-binding site.Figure 4.


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)

Binding isotherms of MepR and selected ligands. (A through D) MepR-binding isotherms to various lengths of cognate DNA from the mepA promoter. (A) MepR binding to a 44-bp site encompassing the entire mepA promoter DNase I footprint. (B) MepR binding to a 26-bp site of the mepA promoter that has been truncated from the 3′-end. (C) MepR binding to a 22-bp site of the mepA promoter that has been truncated from the 3′-end. (D) MepR binding to a 31-bp site of the mepA promoter that has been truncated from the 5′-end. (E) MepR-binding isotherms to a 26-bp DNA binding site from the mepR promoter. (F) Nucleotide sequence of the mepA promoter. The –10 and –35 elements of the promoter are shown in bold and labelled and the transcription start site (TSS) is indicated as a bent arrow. The pseudo-inverted repeats of the promoter are shown by horizontal arrows and the signature GTTAG motifs are underlined. The boundaries of the 22, 26 and 31-bp sequences used in the binding studies are indicated by blue, red and purple rectangles, respectively. G–I, MepR-binding isotherms for three ‘drugs’. (G) The MepR-Et-binding isotherm. (H) The MepR-DAPI-binding isotherm. (I) The MepR-R6G-binding isotherm. The change in polarization, shown in red circles and indicated in millipolarization units (mP), was plotted against the MepR dimer concentration indicated inside each plot. The binding constants are shown in each plot. The chemical structure of each drug is shown below the respective binding curve.
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Figure 4: Binding isotherms of MepR and selected ligands. (A through D) MepR-binding isotherms to various lengths of cognate DNA from the mepA promoter. (A) MepR binding to a 44-bp site encompassing the entire mepA promoter DNase I footprint. (B) MepR binding to a 26-bp site of the mepA promoter that has been truncated from the 3′-end. (C) MepR binding to a 22-bp site of the mepA promoter that has been truncated from the 3′-end. (D) MepR binding to a 31-bp site of the mepA promoter that has been truncated from the 5′-end. (E) MepR-binding isotherms to a 26-bp DNA binding site from the mepR promoter. (F) Nucleotide sequence of the mepA promoter. The –10 and –35 elements of the promoter are shown in bold and labelled and the transcription start site (TSS) is indicated as a bent arrow. The pseudo-inverted repeats of the promoter are shown by horizontal arrows and the signature GTTAG motifs are underlined. The boundaries of the 22, 26 and 31-bp sequences used in the binding studies are indicated by blue, red and purple rectangles, respectively. G–I, MepR-binding isotherms for three ‘drugs’. (G) The MepR-Et-binding isotherm. (H) The MepR-DAPI-binding isotherm. (I) The MepR-R6G-binding isotherm. The change in polarization, shown in red circles and indicated in millipolarization units (mP), was plotted against the MepR dimer concentration indicated inside each plot. The binding constants are shown in each plot. The chemical structure of each drug is shown below the respective binding curve.
Mentions: In order to determine the minimal sequence required for MepR binding, fluorescent polarization-based DNA-binding assays were carried out with various lengths of oligodeoxy-nucleotides, which encompassed the proposed MepR-binding sites in the mepA and mepR promoters. Results from the titration experiments indicate that MepR binds to a 44 bp duplex covering the ‘full’ MepR footprint of the mepA promoter with a dissociation constant (Kd) of 6.3 nM (Figure 4A). Interestingly, truncation of downstream sequences had little effect on MepR binding whereby the dissociation constant for a 26 bp oligodeoxynucleotide, which does not include 18 bp from the 3′-end of the 44 bp site, was only ∼2.4-fold lower (Kd = 15.3 nM) than that of the complete mepA site (Figure 4B). Removal of four additional base pairs further impairs MepR binding to the mepA site another 3.3-fold (Kd = 46.4 nM) but binding remains relatively tight with only a cumulative ∼7-fold loss in affinity as compared to the 44 bp site (Figure 4C and F). By contrast, deletion of eight base pairs from the 5′-end of the 44mer causes a greater than 30-fold reduction (Kd = 186.9 nM) in the affinity of MepR for the mepA-binding site (Figure 4D). DNA-binding experiments with the 26 bp MepR-binding site from mepR promoter also showed high-affinity binding (Kd = 24.3 nM) that essentially matches the affinity of the tested 26-bp fragment from the mepA promoter and is only ∼4-fold lower than that of the full 44 bp mepA site (Figure 4B and E). Thus, the previously observed differential response to drugs, i.e. the slower release of MepR from mepR operator site, does not appear to correlate well with MepR–DNA-binding affinity and suggests that MepR binds to mepA and mepR-binding sites somewhat differently, whereby subsequent drug binding to MepR when bound to mepR promoter is less effective in induction than drug binding to MepR bound to mepA-binding site.Figure 4.

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