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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-M7 structure suggests a weakening of β3–β′3 interactions and the disruption of the hydrogen bond network. (A) Detailed comparison of apo-HpNikR (grey) with apo-M7 (violet) in a view perpendicular to the 2-fold crystallographic axis showing the modifications induced by Q87F mutation (F87 side chain is indicated in red) at the tetramerization interface. The M7 structure was superimposed onto apo-HpNikR TD [r.m.s deviation of 1.76 Å (308 Cα)]. Accommodation of the F87 residue is accompanied by the displacement of α3 and α4 helices and the unzipping of β3–β′3 interactions at each side of the TD. These modifications unlock the TD interface and are echoed to the DBDs that adopt a modified closed trans-conformation. (B). Detailed comparison of the conserved hydrogen bond network linking two nickel ions at the HA sites within each ACT-like pair observed in Ni-EcNikR (pdb code 2HZV) and Ni1-HpNikR structures.
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Figure 8: Apo-M7 structure suggests a weakening of β3–β′3 interactions and the disruption of the hydrogen bond network. (A) Detailed comparison of apo-HpNikR (grey) with apo-M7 (violet) in a view perpendicular to the 2-fold crystallographic axis showing the modifications induced by Q87F mutation (F87 side chain is indicated in red) at the tetramerization interface. The M7 structure was superimposed onto apo-HpNikR TD [r.m.s deviation of 1.76 Å (308 Cα)]. Accommodation of the F87 residue is accompanied by the displacement of α3 and α4 helices and the unzipping of β3–β′3 interactions at each side of the TD. These modifications unlock the TD interface and are echoed to the DBDs that adopt a modified closed trans-conformation. (B). Detailed comparison of the conserved hydrogen bond network linking two nickel ions at the HA sites within each ACT-like pair observed in Ni-EcNikR (pdb code 2HZV) and Ni1-HpNikR structures.

Mentions: M7 (Q87F) crystal structure displayed a modified trans-conformation and TD compared with apo-HpNikR structure (Figure 8A). In the M7 crystal structure, the adjustment of the F87 side chain within the hydrophobic core formed between β3 and β4 strands and α3 and α4 helices results in the displacement of α3 helices away from the interface at both sides of the TD. Hence, the rearrangement of α3 opens the TD interface and modifies the interactions between β3 strands that are found much more distant in M7 structure than in HpNikR structure (Figure 8). When M7 crystals were soaked in nickel solutions, no structure could be obtained with HA sites. When Ni(II) are bound to HA sites, an important hydrogen bound network is formed between neighbouring residues, including Q87 (Figure 8B). This network is conserved in all NikR structures and considerably stabilizes the coordination of nickel ions (15). Therefore, the main effect of Q87 mutation is the disruption of the hydrogen bond network. M7 mutant showed a considerably reduced affinity for DNA and also less stable HA site in the nickel-binding study. Together with the crystal structure of M7, these results suggest that the hydrogen bond network plays a crucial role in securing Ni(II) binding and thus in positioning HpNikR in a conformation suitable for DNA binding.Figure 8.


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-M7 structure suggests a weakening of β3–β′3 interactions and the disruption of the hydrogen bond network. (A) Detailed comparison of apo-HpNikR (grey) with apo-M7 (violet) in a view perpendicular to the 2-fold crystallographic axis showing the modifications induced by Q87F mutation (F87 side chain is indicated in red) at the tetramerization interface. The M7 structure was superimposed onto apo-HpNikR TD [r.m.s deviation of 1.76 Å (308 Cα)]. Accommodation of the F87 residue is accompanied by the displacement of α3 and α4 helices and the unzipping of β3–β′3 interactions at each side of the TD. These modifications unlock the TD interface and are echoed to the DBDs that adopt a modified closed trans-conformation. (B). Detailed comparison of the conserved hydrogen bond network linking two nickel ions at the HA sites within each ACT-like pair observed in Ni-EcNikR (pdb code 2HZV) and Ni1-HpNikR structures.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2875016&req=5

Figure 8: Apo-M7 structure suggests a weakening of β3–β′3 interactions and the disruption of the hydrogen bond network. (A) Detailed comparison of apo-HpNikR (grey) with apo-M7 (violet) in a view perpendicular to the 2-fold crystallographic axis showing the modifications induced by Q87F mutation (F87 side chain is indicated in red) at the tetramerization interface. The M7 structure was superimposed onto apo-HpNikR TD [r.m.s deviation of 1.76 Å (308 Cα)]. Accommodation of the F87 residue is accompanied by the displacement of α3 and α4 helices and the unzipping of β3–β′3 interactions at each side of the TD. These modifications unlock the TD interface and are echoed to the DBDs that adopt a modified closed trans-conformation. (B). Detailed comparison of the conserved hydrogen bond network linking two nickel ions at the HA sites within each ACT-like pair observed in Ni-EcNikR (pdb code 2HZV) and Ni1-HpNikR structures.
Mentions: M7 (Q87F) crystal structure displayed a modified trans-conformation and TD compared with apo-HpNikR structure (Figure 8A). In the M7 crystal structure, the adjustment of the F87 side chain within the hydrophobic core formed between β3 and β4 strands and α3 and α4 helices results in the displacement of α3 helices away from the interface at both sides of the TD. Hence, the rearrangement of α3 opens the TD interface and modifies the interactions between β3 strands that are found much more distant in M7 structure than in HpNikR structure (Figure 8). When M7 crystals were soaked in nickel solutions, no structure could be obtained with HA sites. When Ni(II) are bound to HA sites, an important hydrogen bound network is formed between neighbouring residues, including Q87 (Figure 8B). This network is conserved in all NikR structures and considerably stabilizes the coordination of nickel ions (15). Therefore, the main effect of Q87 mutation is the disruption of the hydrogen bond network. M7 mutant showed a considerably reduced affinity for DNA and also less stable HA site in the nickel-binding study. Together with the crystal structure of M7, these results suggest that the hydrogen bond network plays a crucial role in securing Ni(II) binding and thus in positioning HpNikR in a conformation suitable for DNA binding.Figure 8.

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