Limits...
ST1710-DNA complex crystal structure reveals the DNA binding mechanism of the MarR family of regulators.

Kumarevel T, Tanaka T, Umehara T, Yokoyama S - Nucleic Acids Res. (2009)

Bottom Line: Significantly large conformational changes occurred upon DNA binding and in each of the dimeric monomers in the asymmetric unit of the ST1710-DNA complex.Conserved wHtH loop residues interacting with the bound DNA and mutagenic analysis indicated that R89, R90 and K91 were important for DNA recognition.Significantly, the bound DNA exhibited a new binding mechanism.

View Article: PubMed Central - PubMed

Affiliation: RIKEN SPring-8 Center, Harima Institute, 1-1-1 Kouto, Sayo, Hyogo 679-5148, Japan. tskvel@spring8.or.jp

ABSTRACT
ST1710, a member of the multiple antibiotic resistance regulator (MarR) family of regulatory proteins in bacteria and archaea, plays important roles in development of antibiotic resistance, a global health problem. Here, we present the crystal structure of ST1710 from Sulfolobus tokodaii strain 7 complexed with salicylate, a well-known inhibitor of MarR proteins and the ST1710 complex with its promoter DNA, refined to 1.8 and 2.10 A resolutions, respectively. The ST1710-DNA complex shares the topology of apo-ST1710 and MarR proteins, with each subunit containing a winged helix-turn-helix (wHtH) DNA binding motif. Significantly large conformational changes occurred upon DNA binding and in each of the dimeric monomers in the asymmetric unit of the ST1710-DNA complex. Conserved wHtH loop residues interacting with the bound DNA and mutagenic analysis indicated that R89, R90 and K91 were important for DNA recognition. Significantly, the bound DNA exhibited a new binding mechanism.

Show MeSH

Related in: MedlinePlus

Salicylate binding and inhibition of ST1710–DNA complex assays. (A) DSC analysis of salicylate binding to ST1710. Typical excess heat capacity curves of ST1710 in the absence/presence of sodium salicylate ligand, at a scan rate of 90°C/hr. Salicylate concentration and peak temperature are noted on each curve. The binding constant was calculated using the non-linear regression fit, using the GraphPad Prism software. (B) Gel-mobility shift assay showing ST1710–DNA complex inhibition. All reactions were carried out in binding buffer containing 150 μM of protein and various amounts of sodium salicylate (to a final concentration of 0–250 mM; lanes 1–16 with 0, 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200 and 250 mM, respectively) were added and incubated at room temperature for 20 min. To this reaction mixture, 100 nM of 30-mer DNA added, and after for 20 min., the reactions were fractionated by 10% native PAGE and the DNA stained by SYBR Green (EMSA Kit, Invitrogen). The positions of the free and complex DNA are indicated by an arrow and an arrowhead, respectively.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2724296&req=5

Figure 1: Salicylate binding and inhibition of ST1710–DNA complex assays. (A) DSC analysis of salicylate binding to ST1710. Typical excess heat capacity curves of ST1710 in the absence/presence of sodium salicylate ligand, at a scan rate of 90°C/hr. Salicylate concentration and peak temperature are noted on each curve. The binding constant was calculated using the non-linear regression fit, using the GraphPad Prism software. (B) Gel-mobility shift assay showing ST1710–DNA complex inhibition. All reactions were carried out in binding buffer containing 150 μM of protein and various amounts of sodium salicylate (to a final concentration of 0–250 mM; lanes 1–16 with 0, 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200 and 250 mM, respectively) were added and incubated at room temperature for 20 min. To this reaction mixture, 100 nM of 30-mer DNA added, and after for 20 min., the reactions were fractionated by 10% native PAGE and the DNA stained by SYBR Green (EMSA Kit, Invitrogen). The positions of the free and complex DNA are indicated by an arrow and an arrowhead, respectively.

Mentions: Previous in vitro and in vivo analyses of MarR family of proteins suggested that salicylate is a broad inhibitor for MarR activity at the millimolar concentration levels (1). To investigate whether salicylate binds to ST1710, we used a differential scanning calorimetric method for the binding analysis. Various concentrations of sodium salicylate (0–300 mM) were mixed with the protein and the heat capacities (Cp) measured with the scan rate of 90°C/hr (Figure 1A). The binding constant (Kd) was 20 ± 4.9 mM for sodium salicylate as calculated by the GraphPad Prism software, while the Origin program produced similar values. This analysis clearly suggests that salicylate binds to ST1710, and the binding constants were comparable with other members of MarR family (21).Figure 1.


ST1710-DNA complex crystal structure reveals the DNA binding mechanism of the MarR family of regulators.

Kumarevel T, Tanaka T, Umehara T, Yokoyama S - Nucleic Acids Res. (2009)

Salicylate binding and inhibition of ST1710–DNA complex assays. (A) DSC analysis of salicylate binding to ST1710. Typical excess heat capacity curves of ST1710 in the absence/presence of sodium salicylate ligand, at a scan rate of 90°C/hr. Salicylate concentration and peak temperature are noted on each curve. The binding constant was calculated using the non-linear regression fit, using the GraphPad Prism software. (B) Gel-mobility shift assay showing ST1710–DNA complex inhibition. All reactions were carried out in binding buffer containing 150 μM of protein and various amounts of sodium salicylate (to a final concentration of 0–250 mM; lanes 1–16 with 0, 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200 and 250 mM, respectively) were added and incubated at room temperature for 20 min. To this reaction mixture, 100 nM of 30-mer DNA added, and after for 20 min., the reactions were fractionated by 10% native PAGE and the DNA stained by SYBR Green (EMSA Kit, Invitrogen). The positions of the free and complex DNA are indicated by an arrow and an arrowhead, respectively.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2724296&req=5

Figure 1: Salicylate binding and inhibition of ST1710–DNA complex assays. (A) DSC analysis of salicylate binding to ST1710. Typical excess heat capacity curves of ST1710 in the absence/presence of sodium salicylate ligand, at a scan rate of 90°C/hr. Salicylate concentration and peak temperature are noted on each curve. The binding constant was calculated using the non-linear regression fit, using the GraphPad Prism software. (B) Gel-mobility shift assay showing ST1710–DNA complex inhibition. All reactions were carried out in binding buffer containing 150 μM of protein and various amounts of sodium salicylate (to a final concentration of 0–250 mM; lanes 1–16 with 0, 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200 and 250 mM, respectively) were added and incubated at room temperature for 20 min. To this reaction mixture, 100 nM of 30-mer DNA added, and after for 20 min., the reactions were fractionated by 10% native PAGE and the DNA stained by SYBR Green (EMSA Kit, Invitrogen). The positions of the free and complex DNA are indicated by an arrow and an arrowhead, respectively.
Mentions: Previous in vitro and in vivo analyses of MarR family of proteins suggested that salicylate is a broad inhibitor for MarR activity at the millimolar concentration levels (1). To investigate whether salicylate binds to ST1710, we used a differential scanning calorimetric method for the binding analysis. Various concentrations of sodium salicylate (0–300 mM) were mixed with the protein and the heat capacities (Cp) measured with the scan rate of 90°C/hr (Figure 1A). The binding constant (Kd) was 20 ± 4.9 mM for sodium salicylate as calculated by the GraphPad Prism software, while the Origin program produced similar values. This analysis clearly suggests that salicylate binds to ST1710, and the binding constants were comparable with other members of MarR family (21).Figure 1.

Bottom Line: Significantly large conformational changes occurred upon DNA binding and in each of the dimeric monomers in the asymmetric unit of the ST1710-DNA complex.Conserved wHtH loop residues interacting with the bound DNA and mutagenic analysis indicated that R89, R90 and K91 were important for DNA recognition.Significantly, the bound DNA exhibited a new binding mechanism.

View Article: PubMed Central - PubMed

Affiliation: RIKEN SPring-8 Center, Harima Institute, 1-1-1 Kouto, Sayo, Hyogo 679-5148, Japan. tskvel@spring8.or.jp

ABSTRACT
ST1710, a member of the multiple antibiotic resistance regulator (MarR) family of regulatory proteins in bacteria and archaea, plays important roles in development of antibiotic resistance, a global health problem. Here, we present the crystal structure of ST1710 from Sulfolobus tokodaii strain 7 complexed with salicylate, a well-known inhibitor of MarR proteins and the ST1710 complex with its promoter DNA, refined to 1.8 and 2.10 A resolutions, respectively. The ST1710-DNA complex shares the topology of apo-ST1710 and MarR proteins, with each subunit containing a winged helix-turn-helix (wHtH) DNA binding motif. Significantly large conformational changes occurred upon DNA binding and in each of the dimeric monomers in the asymmetric unit of the ST1710-DNA complex. Conserved wHtH loop residues interacting with the bound DNA and mutagenic analysis indicated that R89, R90 and K91 were important for DNA recognition. Significantly, the bound DNA exhibited a new binding mechanism.

Show MeSH
Related in: MedlinePlus