PMID:11980902
| Citation |
Li, S, Rosen, BP, Borges-Walmsley, MI and Walmsley, AR (2002) Evidence for cooperativity between the four binding sites of dimeric ArsD, an As(III)-responsive transcriptional regulator. J. Biol. Chem. 277:25992-6002 |
|---|---|
| Abstract |
ArsD is a trans-acting repressor of the arsRDABC operon that confers resistance to arsenicals and antimonials in Escherichia coli. It possesses two-pairs of vicinal cysteine residues, Cys(12)-Cys(13) and Cys(112)-Cys(113), that potentially form separate binding sites for the metalloids that trigger dissociation of ArsD from the operon. However, as a homodimer it has four vicinal cysteine pairs. Titration of the steady-state fluorescence of ArsD with metalloids revealed positive cooperativity, with a Hill coefficient of 2, between these sites. Disruption of the Cys(112)-Cys(113) site by mutagenesis of arsD, but not the Cys(12)-Cys(13) site, largely abolished this cooperativity, indicative of interactions between adjacent Cys(112)-Cys(113) sites within the dimer. The kinetics of metalloid binding were determined by stopped flow spectroscopy; the rate increased in a sigmoidal manner, with a Hill coefficient of 4, indicating that the pre-steady-state measurements reported cooperativity between all four sites of the dimer rather than just the intermolecular interactions reported by the steady-state measurements. The kinetics of Sb(III) displacement by As(III) revealed that the metalloid-binding sites behave differentially, with the rapid exchange of As(III) for Sb(III) at one site retarding the release of Sb(III) from the other sites. We propose a model involving the sequential binding and release of metalloids by the four binding sites of dimeric ArsD, with only one site releasing free metalloids. |
| Links |
PubMed Online version:10.1074/jbc.M201619200 |
| Keywords |
Antimony/metabolism; Arsenic/metabolism; Bacterial Proteins; Binding Sites; Dimerization; Dithiothreitol/pharmacology; Escherichia coli; Kinetics; Protein Conformation; Spectrometry, Fluorescence; Trans-Activators/chemistry; Trans-Activators/metabolism |
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