Characterization of the Helicobacter pylori NikR-PureA DNA interaction:: Metal ion requirements and sequence specificity

HPNikR, a prokaryotic nickel binding transcription factor, is found in Helicobacter pylori where it functions as a regulator of multiple genes, including those involved in acid adaptation and nickel ion homeostasis. Particularly important is HPNikR's role in the regulation of the nickel-dependent enzyme urease which is critical for the organism's survival in the acidic environment of the gastric epithelium. The target operator sequences of the genes regulated by HPNikR do not contain any identifiable palindromes, and the exact mechanism(s) of the HPNikR-DNA recognition event is unknown. HPNikR was expressed and purified as a soluble protein containing mixed alpha/beta secondary structure with evidence of a tertiary fold. A direct and competitive fluorescence anisotropy (FA) assay to probe both the metal ion requirements and sequence specificity of HPNikR for P-ureA, the operator sequence for the urease gene, was developed. FA studies revealed that apo-HPNikR did not bind to P-ureA while Ni(II)HPNikR bound P-ureA with nanomolar affinity, but only in the presence of a second metal ion [magnesium, calcium, or manganese(II)], suggesting that HPNikR contains a second, low-affinity metal binding site. Cu(II)HPNikR also exhibited a requirement for a second metal ion to accomplish P-ureA binding. Removal of a loosely conserved "putative" palindrome sequence in the P-ureA operator abrogated HPNikR binding. Together, these results support a model of HPNikR-P-ureA binding in which specific metal ions must be coordinated to high- and low-affinity sites to modulate binding.