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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.

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ST1710–promoter DNA interactions. (A) A ribbon diagram of ST1710–DNA complex colored as in Figure 4E. The full-length promoter DNA is shown in stick model. The T5-A27 and T5′-A27′ strands are in blue and yellow, respectively. (B) Schematic representation showing all ST1710–DNA contacts. The bases are shown in rectangles. The protein–DNA contacts in each of the ST1710 monomer are represented by the same color in (A). (C–F) The close-up view of the critical protein–DNA interactions in the complex is shown in A–D. The residues of nucleic acids and protein are shown in stick models. The hydrogen bonds are shown in broken lines.
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Figure 4: ST1710–promoter DNA interactions. (A) A ribbon diagram of ST1710–DNA complex colored as in Figure 4E. The full-length promoter DNA is shown in stick model. The T5-A27 and T5′-A27′ strands are in blue and yellow, respectively. (B) Schematic representation showing all ST1710–DNA contacts. The bases are shown in rectangles. The protein–DNA contacts in each of the ST1710 monomer are represented by the same color in (A). (C–F) The close-up view of the critical protein–DNA interactions in the complex is shown in A–D. The residues of nucleic acids and protein are shown in stick models. The hydrogen bonds are shown in broken lines.

Mentions: As explained above, Figure 4A represents the full length duplex-DNA bound to four monomers of the symmetry-related dimeric molecules. Although we used only a 30-mer duplexed DNA for our crystallization studies, we could see the duplexed-DNA consisting of T5 to A27 and T5′ to A27′ of the bases bound to the protein (Figures 4A and B). The 4 and 3 bases at the 5′- and 3′-end, respectively, were highly disordered in both of the DNA-strands and hence not modeled. The bound DNA adapted a B-form right handed structure, passing over the protein molecule by only contacting at the wHtH loop regions. The protein–DNA interactions seen in the asu of the complex (Figures 3B and C) were essentially same in all of the four symmetry-related molecules. Interestingly, as observed in the OhrR-ohrA operator complex, the –10 region (TAACAAT) of the promoter DNA (15–21) was recognized by the wHtH domains (Figures 4B–F). Of the bound 54 nucleotides, only 22 nucleotides make 36 contacts with six protein residues (Figures 4B–F). The side chain oxygen of S65 was bonded to the O5′ of Thy5′. Interestingly, the side chain (NH1) of residue R84 formed water-mediated hydrogen bonds to the N3 of bases G13′ and Ade17. In addition, side chain (NH1) of R89 hydrogen bonded to the backbone phosphate oxygen (O2P) of Thy14′. The residue R90 hydrogen bonded to the O4′ and O2 of base Cyt18 and the same residue made two salt-bridge contacts with D88. This salt bridge may assist in fixing the conformation of residue R90 in order to make contact with the nucleic acid base, Cyt18. Besides, the side chain atom (CD) bonded to the bases of Gua13′ (N2) and Thy14′ (O2). The side chain of K91 interacted with backbone phosphate of Ade19 and Ile91 to C5 of Ade20. Thus, the following residues S65, R84, D88, R89, R90, K91 and I92 interacted with the bound promoter DNA. To evaluate the protein–DNA interactions at the loop region, we prepared three mutant proteins (R89A, R90A and K91A) and analyzed the binding ability by gel-mobility shift assays. All three mutants failed to bind to DNA, suggesting that these three residues are important for protein–DNA interactions (Supplementary Figure S4). We also crystallized all three mutant proteins under the native protein conditions and solved their structures by molecular replacement method as explained previously (Table 1). All three mutant structures resembled the native ST1710, except very little changes were observed in the wHtH loop regions (Supplementary Figure S5). Furthermore, DNA-binding residues in ST1710 were highly conserved among the closely related proteins and OhrR (Figure 2E). The winged loop region connecting the strands β1 and β2 apparently plays a major role in modulating their conformation for binding to the DNA molecule, and this mode of recognition is anticipated for the proteins closely related to ST1710 as well as the family of MarR regulators. We observed Ca2+ ions in all of the mutant and native structures, but not in the salicylate and DNA-complex structures. Superimposition of the native, salicylate and DNA-complex structures suggested that the C-terminal helix (α6) in the DNA complex deviated greatly from the metal ion binding site and in the salicylate complex slight changes in side chain orientations were observed (Supplementary Figure S6). However, the functions of this metal ion observed in the native and mutant structures of ST1710 will need to be further investigated.Figure 4.


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)

ST1710–promoter DNA interactions. (A) A ribbon diagram of ST1710–DNA complex colored as in Figure 4E. The full-length promoter DNA is shown in stick model. The T5-A27 and T5′-A27′ strands are in blue and yellow, respectively. (B) Schematic representation showing all ST1710–DNA contacts. The bases are shown in rectangles. The protein–DNA contacts in each of the ST1710 monomer are represented by the same color in (A). (C–F) The close-up view of the critical protein–DNA interactions in the complex is shown in A–D. The residues of nucleic acids and protein are shown in stick models. The hydrogen bonds are shown in broken lines.
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Figure 4: ST1710–promoter DNA interactions. (A) A ribbon diagram of ST1710–DNA complex colored as in Figure 4E. The full-length promoter DNA is shown in stick model. The T5-A27 and T5′-A27′ strands are in blue and yellow, respectively. (B) Schematic representation showing all ST1710–DNA contacts. The bases are shown in rectangles. The protein–DNA contacts in each of the ST1710 monomer are represented by the same color in (A). (C–F) The close-up view of the critical protein–DNA interactions in the complex is shown in A–D. The residues of nucleic acids and protein are shown in stick models. The hydrogen bonds are shown in broken lines.
Mentions: As explained above, Figure 4A represents the full length duplex-DNA bound to four monomers of the symmetry-related dimeric molecules. Although we used only a 30-mer duplexed DNA for our crystallization studies, we could see the duplexed-DNA consisting of T5 to A27 and T5′ to A27′ of the bases bound to the protein (Figures 4A and B). The 4 and 3 bases at the 5′- and 3′-end, respectively, were highly disordered in both of the DNA-strands and hence not modeled. The bound DNA adapted a B-form right handed structure, passing over the protein molecule by only contacting at the wHtH loop regions. The protein–DNA interactions seen in the asu of the complex (Figures 3B and C) were essentially same in all of the four symmetry-related molecules. Interestingly, as observed in the OhrR-ohrA operator complex, the –10 region (TAACAAT) of the promoter DNA (15–21) was recognized by the wHtH domains (Figures 4B–F). Of the bound 54 nucleotides, only 22 nucleotides make 36 contacts with six protein residues (Figures 4B–F). The side chain oxygen of S65 was bonded to the O5′ of Thy5′. Interestingly, the side chain (NH1) of residue R84 formed water-mediated hydrogen bonds to the N3 of bases G13′ and Ade17. In addition, side chain (NH1) of R89 hydrogen bonded to the backbone phosphate oxygen (O2P) of Thy14′. The residue R90 hydrogen bonded to the O4′ and O2 of base Cyt18 and the same residue made two salt-bridge contacts with D88. This salt bridge may assist in fixing the conformation of residue R90 in order to make contact with the nucleic acid base, Cyt18. Besides, the side chain atom (CD) bonded to the bases of Gua13′ (N2) and Thy14′ (O2). The side chain of K91 interacted with backbone phosphate of Ade19 and Ile91 to C5 of Ade20. Thus, the following residues S65, R84, D88, R89, R90, K91 and I92 interacted with the bound promoter DNA. To evaluate the protein–DNA interactions at the loop region, we prepared three mutant proteins (R89A, R90A and K91A) and analyzed the binding ability by gel-mobility shift assays. All three mutants failed to bind to DNA, suggesting that these three residues are important for protein–DNA interactions (Supplementary Figure S4). We also crystallized all three mutant proteins under the native protein conditions and solved their structures by molecular replacement method as explained previously (Table 1). All three mutant structures resembled the native ST1710, except very little changes were observed in the wHtH loop regions (Supplementary Figure S5). Furthermore, DNA-binding residues in ST1710 were highly conserved among the closely related proteins and OhrR (Figure 2E). The winged loop region connecting the strands β1 and β2 apparently plays a major role in modulating their conformation for binding to the DNA molecule, and this mode of recognition is anticipated for the proteins closely related to ST1710 as well as the family of MarR regulators. We observed Ca2+ ions in all of the mutant and native structures, but not in the salicylate and DNA-complex structures. Superimposition of the native, salicylate and DNA-complex structures suggested that the C-terminal helix (α6) in the DNA complex deviated greatly from the metal ion binding site and in the salicylate complex slight changes in side chain orientations were observed (Supplementary Figure S6). However, the functions of this metal ion observed in the native and mutant structures of ST1710 will need to be further investigated.Figure 4.

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