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 Ni2+ enzymes and other proteins involved in Ni2+ trafficking. In the human pathogen Helicobacter pylori, NikR controls transcription of the Ni2+ 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 PureA, the promoter of the urease operon, were the object of this study. Electrophoretic mobility shift and DNase I footprinting assays indicated that Ni2+ is necessary and sufficient to promote HpNikR binding to PureA, while the effect of other metal ions in identical conditions is significantly lower (Zn2+ and Co2+) or absent (Ca2+ and Mg2+). Isothermal titration calorimetry (ITC) demonstrated the absence of specific Ca2+ and Mg2+ binding to the protein. ITC also established the binding of Zn2+ and Co2+ to two sets of high-affinity sites on HpNikR, differing in stoichiometry (n1=2, n2=4) and dissociation constant (Kd1=6 nM, Kd2=90 nM for Zn2+; Kd1=0.3 microM, Kd2=2.7 microM for Co2+). Additional low-affinity binding sites were observed for Zn2+ (n=8, Kd=1.6 microM). Mobility shift assays and ITC proved that binding of stoichiometric Ni2+ (but not Zn2+ or Co2+) 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 Kd=56 nM. A protein conformational rearrangement is selectively induced by Ni2+ and not by Zn2+, as indicated by fluorescence spectroscopy and microcalorimetry. Accordingly, competition experiments showed that stoichiometric Ni2+ outperforms Zn2+, as well as Co2+, in functionally activating HpNikR toward high affinity binding to PureA. A general scheme for the nickel-selective HpNikR-DNA interaction is proposed.