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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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Ni(II) binding to NikR and M5, M6, M7 and M10 derivatives and kinetics of metal release for HpNikR and derivatives. (A) Comparison of the binding profiles of HpNikR, M5, M6, M7 and M10 determined by the changes in absorbance at 305 nm upon the addition of up to 3 equivalents of NiSO4. The presented data correspond to the difference in the absorption at 305 nm between the absorption spectra of the holo and apo forms for each protein. Data were collected at the equilibrium, 30 min after addition of NiSO4 to 200 µM of protein solution in HEPES 20 mM, pH 7.4. The results are homogenous (variation is <5%) and were reproduced at least three times with two different protein batches. (B) Comparison of the kinetics of Ni(II) release from HpNikR, M5, M6, M7 and M10. UV-visible absorption spectra were continuously measured for 1 h after EDTA addition. The changes in absorbance at 305 nm are presented as function of time after addition of 10 mM EDTA to a Ni(II)–protein complex with a 1 : 1 stoichiometry. Experiments were reproduced three times giving similar results.
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Figure 2: Ni(II) binding to NikR and M5, M6, M7 and M10 derivatives and kinetics of metal release for HpNikR and derivatives. (A) Comparison of the binding profiles of HpNikR, M5, M6, M7 and M10 determined by the changes in absorbance at 305 nm upon the addition of up to 3 equivalents of NiSO4. The presented data correspond to the difference in the absorption at 305 nm between the absorption spectra of the holo and apo forms for each protein. Data were collected at the equilibrium, 30 min after addition of NiSO4 to 200 µM of protein solution in HEPES 20 mM, pH 7.4. The results are homogenous (variation is <5%) and were reproduced at least three times with two different protein batches. (B) Comparison of the kinetics of Ni(II) release from HpNikR, M5, M6, M7 and M10. UV-visible absorption spectra were continuously measured for 1 h after EDTA addition. The changes in absorbance at 305 nm are presented as function of time after addition of 10 mM EDTA to a Ni(II)–protein complex with a 1 : 1 stoichiometry. Experiments were reproduced three times giving similar results.

Mentions: The ability of the purified HpNikR mutated proteins to bind Ni(II) at the final HA sites was measured by UV/vis spectroscopy at 305 nm corresponding to a Cys 107 Sγ→Ni ligand to metal charge transfer (LMCT) band (18,32). The difference between the absorption spectra of the apo and holo proteins upon Ni(II) addition shows the appearance of peaks at 305 and 475 nm (Supplementary Figure S1A), except for mutant M1. The binding profiles of HpNikRs are shown in Figure 2A by the changes in absorbance at 305 nm upon the addition of up to 3 equivalents of NiSO4 per subunit. The intensity at 305 nm increased linearly up to one equivalent nickel per monomer similarly for HpNikR, M5, M6, M7 and M10 mutants and saturation was observed at a stoichiometry of 1 Ni(II) per monomer. This indicated that HpNikR mutants accommodate one nickel per monomer i.e. four HA sites per HpNikR tetramer.Figure 2.


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)

Ni(II) binding to NikR and M5, M6, M7 and M10 derivatives and kinetics of metal release for HpNikR and derivatives. (A) Comparison of the binding profiles of HpNikR, M5, M6, M7 and M10 determined by the changes in absorbance at 305 nm upon the addition of up to 3 equivalents of NiSO4. The presented data correspond to the difference in the absorption at 305 nm between the absorption spectra of the holo and apo forms for each protein. Data were collected at the equilibrium, 30 min after addition of NiSO4 to 200 µM of protein solution in HEPES 20 mM, pH 7.4. The results are homogenous (variation is <5%) and were reproduced at least three times with two different protein batches. (B) Comparison of the kinetics of Ni(II) release from HpNikR, M5, M6, M7 and M10. UV-visible absorption spectra were continuously measured for 1 h after EDTA addition. The changes in absorbance at 305 nm are presented as function of time after addition of 10 mM EDTA to a Ni(II)–protein complex with a 1 : 1 stoichiometry. Experiments were reproduced three times giving similar results.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 2: Ni(II) binding to NikR and M5, M6, M7 and M10 derivatives and kinetics of metal release for HpNikR and derivatives. (A) Comparison of the binding profiles of HpNikR, M5, M6, M7 and M10 determined by the changes in absorbance at 305 nm upon the addition of up to 3 equivalents of NiSO4. The presented data correspond to the difference in the absorption at 305 nm between the absorption spectra of the holo and apo forms for each protein. Data were collected at the equilibrium, 30 min after addition of NiSO4 to 200 µM of protein solution in HEPES 20 mM, pH 7.4. The results are homogenous (variation is <5%) and were reproduced at least three times with two different protein batches. (B) Comparison of the kinetics of Ni(II) release from HpNikR, M5, M6, M7 and M10. UV-visible absorption spectra were continuously measured for 1 h after EDTA addition. The changes in absorbance at 305 nm are presented as function of time after addition of 10 mM EDTA to a Ni(II)–protein complex with a 1 : 1 stoichiometry. Experiments were reproduced three times giving similar results.
Mentions: The ability of the purified HpNikR mutated proteins to bind Ni(II) at the final HA sites was measured by UV/vis spectroscopy at 305 nm corresponding to a Cys 107 Sγ→Ni ligand to metal charge transfer (LMCT) band (18,32). The difference between the absorption spectra of the apo and holo proteins upon Ni(II) addition shows the appearance of peaks at 305 and 475 nm (Supplementary Figure S1A), except for mutant M1. The binding profiles of HpNikRs are shown in Figure 2A by the changes in absorbance at 305 nm upon the addition of up to 3 equivalents of NiSO4 per subunit. The intensity at 305 nm increased linearly up to one equivalent nickel per monomer similarly for HpNikR, M5, M6, M7 and M10 mutants and saturation was observed at a stoichiometry of 1 Ni(II) per monomer. This indicated that HpNikR mutants accommodate one nickel per monomer i.e. four HA sites per HpNikR tetramer.Figure 2.

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