Structural and mechanistic insights into Helicobacter pylori NikR activation.
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.
Bahlawane
C
C
CNRS UMR 5249 Laboratoire de Chimie et Biologie des Métaux, France.
Dian
C
C
Muller
C
C
Round
A
A
Fauquant
C
C
Schauer
K
K
de Reuse
H
H
Terradot
L
L
Michaud-Soret
I
I
D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism; D008958:Models, Molecular; D009154:Mutation; D009532:Nickel / Q000378:metabolism; D011485:Protein Binding; D012097:Repressor Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D053838:Scattering, Small Angle; D014961:X-Ray Diffraction
D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; C523724:NikR protein, Helicobacter pylori; D012097:Repressor Proteins; D009532:Nickel