Mechanistic implications of urate interaction with transcriptional regulator HucR from Deinococcus radiodurans

Abstract

The hypothetical uricase regulator (HucR) is a ligand‐responsive MarR family transcriptional regulator from D. radiodurans that regulates uricase expression. Regulation of uric acid levels may contribute to the bacterium's extreme resistance to reactive oxygen species. Molecular docking was used to predict the urate binding site in HucR. A site was predicted above the DNA recognition helices, and mutation of residues predicted to contact uric acid, R80, D73, and W20, shows that all three residues are required for DNA binding antagonism in presence of urate. Although DNA binding is not antagonized by urate, the R80 mutant shows Trp fluorescence quenching at 338 nm on urate binding, indicating ligand binding to the predicted site. In contrast, mutation of D73 abolishes fluorescence quenching with increasing urate concentrations. Other purines with the exception of xanthine have no effect on DNA binding affinity of HucR and display no concentration‐dependent fluorescence quenching. Based on these observations we propose that urate‐dependent perturbation of DNA binding by HucR may be due to the binding of N3‐deprotonated urate changing the orientation of D73 which in turn affects the disposition of the recognition helix due to altered interaction with R106. Due to high DNA and ligand binding affinity, HucR serves as a model to study mechanistic implications of ligand binding to MarR type transcriptional regulators.