High-Affinity Ni2+ Binding Selectively Promotes Binding of Helicobacter pylori NikR to Its Target Urease Promoter

NikR is a prokaryotic transcription factor that regulates the expression of Ni(2+) enzymes and other proteins involved in Ni(2+) trafficking. In the human pathogen Helicobacter pylori, NikR controls transcription of the Ni(2+) enzyme Urease, which allows survival of the bacterium in the acidic gastric niche. The in vitro affinity of NikR from H. pylori (HpNikR) for different metal ions and the metal-ion-dependent capability of HpNikR to bind P(ureA), the promoter of the urease operon, were the object of this study. Electrophoretic mobility shift and DNase I footprinting assays indicated that Ni(2+) is necessary and sufficient to promote HpNikR binding to P(ureA), while the effect of other metal ions in identical conditions is significantly lower (Zn(2+) and Co(2+)) or absent (Ca(2+) and Mg(2+)). Isothermal titration calorimetry (ITC) demonstrated the absence of specific Ca(2+) and Mg(2+) binding to the protein. ITC also established the binding of Zn(2+) and Co(2+) to two sets of high- affinity sites on HpNikR, differing in stoichiometry (n(1)=2, n(2)=4) and dissociation constant (K(d1) = 6 nM, K(d2) = 90 nM for Zn(2+); K(d1) = 0.3 mu M, K(d2)=2.7 mu M for Co(2+)). Additional low-affinity binding sites were observed for Zn(2+) (n=8, K(d)=1.6 mu M). Mobility shift assays and ITC proved that binding of stoichiometric Ni(2+) (but not Zn(2+) or Co(2+)) to the high-affinity sites (but not to the low-affinity sites) selectively activates HpNikR to bind its target operator with. 1:1 stoichiometry and K(d)=56 nM. A protein conformational rearrangement is selectively induced by Ni2+ and not by Zn(2+), as indicated by fluorescence spectroscopy and microcalorimetry. Accordingly, competition experiments showed that stoichiometric Ni(2+) outperforms Zn(2+) as well as Co(2+) in functionally activating HpNikR toward high affinity binding to P(ureA). A general scheme for the nickel-selective HpNikR-DNA interaction is proposed. (c) 2008 Elsevier Ltd. All rights reserved.