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.
Zambelli
Barbara
B
Laboratory of Bioinorganic Chemistry, Department of Agro-Environmental Science and Technology, University of Bologna, Bologna, Italy.
Danielli
Alberto
A
Romagnoli
Simona
S
Neyroz
Paolo
P
Ciurli
Stefano
S
Scarlato
Vincenzo
V
D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism*; D001665:Binding Sites; D002151:Calorimetry; D002413:Cations, Divalent / Q000378:metabolism; D003035:Cobalt / Q000378:metabolism; D018983:DNA Footprinting; D004269:DNA, Bacterial / Q000378:metabolism; D024202:Electrophoretic Mobility Shift Assay; D029968:Escherichia coli Proteins / Q000378:metabolism; D016480:Helicobacter pylori / Q000201:enzymology*; D009532:Nickel / Q000378:metabolism*; D009875:Operator Regions, Genetic; D011401:Promoter Regions, Genetic / Q000235:genetics*; D011485:Protein Binding; D017433:Protein Structure, Secondary; D012097:Repressor Proteins / Q000737:chemistry / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D017186:Titrimetry; D014510:Urease / Q000235:genetics*; D015032:Zinc / Q000378:metabolism
D001426:Bacterial Proteins; D002413:Cations, Divalent; D004269:DNA, Bacterial; D029968:Escherichia coli Proteins; C402409:NikR protein, E coli; C523724:NikR protein, Helicobacter pylori; D012097:Repressor Proteins; D003035:Cobalt; D009532:Nickel; D014510:Urease; D015032:Zinc