INOR 24 |
| HPNikR is a prokaryotic nickel binding transcription factor found in H. pylori where it regulates the expression of multiple genes including those involved in acid adaptation and nickel ion homeostasis. Of particular importance is HPNikR's role in the regulation of the nickel dependent enzyme urease which is critical for the organism's survival in its environmental niche - the acidic gastric epithelium. The target operator sequences of the genes regulated by HPNikR do not contain identifiable palindromes and the exact mechanism(s) by which HPNikR recognizes DNA is unknown. We have expressed and purified HPNikR as a soluble protein containing mixed alpha/beta secondary structure with evidence of tertiary fold. A direct and competitive fluorescence anisotropy (FA) assay has been developed to probe both the metal ion requirements and sequence specificity of HPNikR for PureA, the operator sequence for the urease gene. These studies have revealed that apo-HPNikR does not bind to PureA while Ni(II)HPNikR binds 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 exhibits a similar requirement for a second metal ion to accomplish PureA binding. In addition, removal of a loosely conserved ‘putative' palindrome sequence in the PureA operator abrogates HPNikR binding. Together, these findings support a model of HPNikR/PureA binding wherein specific metal ions must be coordinated to high and low affinity sites to modulate binding. These results as well as studies of wild type and mutant HPNikR constructs binding to additional operator sequences will be presented. |
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Bioinorganic Chemistry: Enzymes and Coenzymes
8:30 AM-12:10 PM, Sunday, August 19, 2007 BCEC -- 208, Oral
Division of Inorganic Chemistry |