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Structural and mechanistic insights into Helicobacter pylori NikR activation.

Bahlawane C, Dian C, Muller C, Round A, Fauquant C, Schauer K, de Reuse H, Terradot L, Michaud-Soret I - Nucleic Acids Res. (2010)

Bottom Line: We show that a second metal is necessary for HpNikR/DNA binding, but only to some promoters.The crystal structures of selected mutants identify the effects of each mutation on HpNikR structure.This study unravels key structural features from which we derive a model for HpNikR activation where: (i) HA sites and an hydrogen bond network are required for DNA binding and (ii) metallation of a unique secondary external site (X) modulates HpNikR DNA binding to low-affinity promoters by disruption of a salt bridge.

View Article: PubMed Central - PubMed

Affiliation: CNRS UMR 5249 Laboratoire de Chimie et Biologie des Métaux, France.

ABSTRACT
NikR is a transcriptional metalloregulator central in the mandatory response to acidity of Helicobacter pylori that controls the expression of numerous genes by binding to specific promoter regions. NikR/DNA interactions were proposed to rely on protein activation by Ni(II) binding to high-affinity (HA) and possibly secondary external (X) sites. We describe a biochemical characterization of HpNikR mutants that shows that the HA sites are essential but not sufficient for DNA binding, while the secondary external (X) sites and residues from the HpNikR dimer-dimer interface are important for DNA binding. We show that a second metal is necessary for HpNikR/DNA binding, but only to some promoters. Small-angle X-ray scattering shows that HpNikR adopts a defined conformation in solution, resembling the cis-conformation and suggests that nickel does not trigger large conformational changes in HpNikR. The crystal structures of selected mutants identify the effects of each mutation on HpNikR structure. This study unravels key structural features from which we derive a model for HpNikR activation where: (i) HA sites and an hydrogen bond network are required for DNA binding and (ii) metallation of a unique secondary external site (X) modulates HpNikR DNA binding to low-affinity promoters by disruption of a salt bridge.

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Apo- and Ni-bound structures of M5 reveal an absence of X sites. (A) Structural comparison of apo-M5 (blue) with Ni-M5 structure (green). (B) Detailed comparison of Ni-M5 structure (green) with Ni1-HpNikR (grey). Ni-M5 structure was superimposed onto Ni1-HpNikR [r.m.s deviation of 0.98 Å (531 Cα)]. Nickel ions bound to HA, I and X sites are indicated as sphere and coloured as in Figure 1. Amino acids involved in nickel binding are shown as balls and stick and coloured by atom type (carbon as the ribbon, green; oxygen, red; nitrogen, blue; and sulphur, orange). Nickel coordination and hydrogen bonds are presented as full lines and green broken lines, respectively. Residues from M5 mutant are labelled in red.
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Figure 7: Apo- and Ni-bound structures of M5 reveal an absence of X sites. (A) Structural comparison of apo-M5 (blue) with Ni-M5 structure (green). (B) Detailed comparison of Ni-M5 structure (green) with Ni1-HpNikR (grey). Ni-M5 structure was superimposed onto Ni1-HpNikR [r.m.s deviation of 0.98 Å (531 Cα)]. Nickel ions bound to HA, I and X sites are indicated as sphere and coloured as in Figure 1. Amino acids involved in nickel binding are shown as balls and stick and coloured by atom type (carbon as the ribbon, green; oxygen, red; nitrogen, blue; and sulphur, orange). Nickel coordination and hydrogen bonds are presented as full lines and green broken lines, respectively. Residues from M5 mutant are labelled in red.

Mentions: The structure of M5 (H74G, H75G) was determined at 2.1 Å (Figure 7 and Supplementary Table S2). The residues H74 and H75 were previously identified in the Ni1-HpNikR structure (pdb code 2cad) as being involved in both the intermediary I (H74) and external X (H74, H75) sites. The structure of apo-M5 is almost identical to HpNikR apart from the mutated residues. Apo-M5 crystals were then soaked in a cryoprotectant containing 100 mM NiSO4 for 3 days resulting in Ni-M5 crystal structure (Figure 7A, Supplementary Table S2). Ni-M5 structure is almost identical to the Ni1-HpNikR structure (14) with four nickel-bound per tetramer, two at HA sites and two to modified I sites. The modified I site of Ni-M5 structure has a low occupancy (0.3) and presents an incomplete octahedral geometry in comparison with the Ni1-HpNikR structure.Figure 7.


Structural and mechanistic insights into Helicobacter pylori NikR activation.

Bahlawane C, Dian C, Muller C, Round A, Fauquant C, Schauer K, de Reuse H, Terradot L, Michaud-Soret I - Nucleic Acids Res. (2010)

Apo- and Ni-bound structures of M5 reveal an absence of X sites. (A) Structural comparison of apo-M5 (blue) with Ni-M5 structure (green). (B) Detailed comparison of Ni-M5 structure (green) with Ni1-HpNikR (grey). Ni-M5 structure was superimposed onto Ni1-HpNikR [r.m.s deviation of 0.98 Å (531 Cα)]. Nickel ions bound to HA, I and X sites are indicated as sphere and coloured as in Figure 1. Amino acids involved in nickel binding are shown as balls and stick and coloured by atom type (carbon as the ribbon, green; oxygen, red; nitrogen, blue; and sulphur, orange). Nickel coordination and hydrogen bonds are presented as full lines and green broken lines, respectively. Residues from M5 mutant are labelled in red.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Figure 7: Apo- and Ni-bound structures of M5 reveal an absence of X sites. (A) Structural comparison of apo-M5 (blue) with Ni-M5 structure (green). (B) Detailed comparison of Ni-M5 structure (green) with Ni1-HpNikR (grey). Ni-M5 structure was superimposed onto Ni1-HpNikR [r.m.s deviation of 0.98 Å (531 Cα)]. Nickel ions bound to HA, I and X sites are indicated as sphere and coloured as in Figure 1. Amino acids involved in nickel binding are shown as balls and stick and coloured by atom type (carbon as the ribbon, green; oxygen, red; nitrogen, blue; and sulphur, orange). Nickel coordination and hydrogen bonds are presented as full lines and green broken lines, respectively. Residues from M5 mutant are labelled in red.
Mentions: The structure of M5 (H74G, H75G) was determined at 2.1 Å (Figure 7 and Supplementary Table S2). The residues H74 and H75 were previously identified in the Ni1-HpNikR structure (pdb code 2cad) as being involved in both the intermediary I (H74) and external X (H74, H75) sites. The structure of apo-M5 is almost identical to HpNikR apart from the mutated residues. Apo-M5 crystals were then soaked in a cryoprotectant containing 100 mM NiSO4 for 3 days resulting in Ni-M5 crystal structure (Figure 7A, Supplementary Table S2). Ni-M5 structure is almost identical to the Ni1-HpNikR structure (14) with four nickel-bound per tetramer, two at HA sites and two to modified I sites. The modified I site of Ni-M5 structure has a low occupancy (0.3) and presents an incomplete octahedral geometry in comparison with the Ni1-HpNikR structure.Figure 7.

Bottom Line: We show that a second metal is necessary for HpNikR/DNA binding, but only to some promoters.The crystal structures of selected mutants identify the effects of each mutation on HpNikR structure.This study unravels key structural features from which we derive a model for HpNikR activation where: (i) HA sites and an hydrogen bond network are required for DNA binding and (ii) metallation of a unique secondary external site (X) modulates HpNikR DNA binding to low-affinity promoters by disruption of a salt bridge.

View Article: PubMed Central - PubMed

Affiliation: CNRS UMR 5249 Laboratoire de Chimie et Biologie des Métaux, France.

ABSTRACT
NikR is a transcriptional metalloregulator central in the mandatory response to acidity of Helicobacter pylori that controls the expression of numerous genes by binding to specific promoter regions. NikR/DNA interactions were proposed to rely on protein activation by Ni(II) binding to high-affinity (HA) and possibly secondary external (X) sites. We describe a biochemical characterization of HpNikR mutants that shows that the HA sites are essential but not sufficient for DNA binding, while the secondary external (X) sites and residues from the HpNikR dimer-dimer interface are important for DNA binding. We show that a second metal is necessary for HpNikR/DNA binding, but only to some promoters. Small-angle X-ray scattering shows that HpNikR adopts a defined conformation in solution, resembling the cis-conformation and suggests that nickel does not trigger large conformational changes in HpNikR. The crystal structures of selected mutants identify the effects of each mutation on HpNikR structure. This study unravels key structural features from which we derive a model for HpNikR activation where: (i) HA sites and an hydrogen bond network are required for DNA binding and (ii) metallation of a unique secondary external site (X) modulates HpNikR DNA binding to low-affinity promoters by disruption of a salt bridge.

Show MeSH
Related in: MedlinePlus