Characterization of the Helicobacter pylori NikR-P(ureA) 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 PureA, the operator sequence for the urease gene, was developed. FA studies revealed that apo-HPNikR did not bind to PureA while Ni(II)HPNikR bound PureA 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 PureA binding. Removal of a loosely conserved "putative" palindrome sequence in the PureA operator abrogated HPNikR binding. Together, these results support a model of HPNikR-PureA binding in which specific metal ions must be coordinated to high- and low-affinity sites to modulate binding.
Dosanjh
Nuvjeevan S
NS
Department of Pharmaceutical Sciences, School of Pharmacy, University of Maryland, Baltimore, Maryland 21201-1180, USA.
Hammerbacher
Nicole A
NA
Michel
Sarah L J
SL
D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism*; D001483:Base Sequence; D002942:Circular Dichroism; D004247:DNA / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D005454:Fluorescence Polarization; D016480:Helicobacter pylori / Q000378:metabolism*; D008670:Metals / Q000378:metabolism*; D011485:Protein Binding; D017433:Protein Structure, Secondary; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013056:Spectrophotometry, Ultraviolet
D001426:Bacterial Proteins; D008670:Metals; D004247:DNA