Browsing Theses and Dissertations School of Pharmacy by Author "West, Abby L."
H. pylori NikR: a new nickel regulatory proteinWest, Abby L.; Michel, Sarah L. J. (2012)My research has focused on understanding the mechanism of DNA recognition by the nickel regulatory protein NikR from Helicobacter pylori (HpNikR). H. pylori colonize the highly acidic gastric epithelium of the human stomach. One feature that enables H. pylori to survive under acidic conditions is the ability to release large quantities of ammonia, produced by the NikR regulated enzyme urease, to neutralize its immediate environment. HpNikR also regulates the expression of multiple other genes as either an activator or repressor including those involved in nickel ion homeostasis, acid adaptation and iron uptake. The genes for which direct regulation have been established contain variable recognition sequences; and the biophysical basis for DNA recognition and discrimination by HpNikR is currently unresolved. Crystallographic studies produced an unanticipated structure of Holo-HpNikR in which nickel ions are coordinated to two distinct binding sites: a 4-coordinate, square planar site (called the 4-site) and a 5/6-coordinate square-pyramidal/octahedral site (called the 5/6-site) in contrast to previous NikR structures where all four ions are coordinated to square planar sites. A mutant of HpNikR called H74A was designed to force all four nickels to the 4-sites, and the crystal structure confirmed singular coordination. DNA binding studies revealed that when Ni(II) is restricted from the 5/6 sites, DNA binding properties are abrogated compared to Holo-HpNikR. These data support a mechanism in which nickel coordination to the 5/6 site of HpNikR is critical for function. A reporter assay was also developed to monitor transcription of urease as a function of nickel concentration and promoter sequence directly within H. pylori. Unexpectedly, initial findings from this study directed us towards a novel cooperative effect in vivo where the presence of active HpArsR-P, from the ArsRS two component system, in conjunction with HpNikR leads to maximum Ni(II) dependent induction of urease. HpArsR is also able to bind the HpNikR PureA mutant operator sites in vitro as determined via fluorescence anisotropy at neutral pH with a high nanomolar to low micromolar affinity. Through the use of both in vitro and in vivo approaches, a novel model has been proposed for DNA recognition by HpNikR.