Song, L. and Rosen, B. P. and Borges-Walmsley, M. I. and Walmsley, A. (2002) 'Evidence for cooperativity between the four binding sites of dimeric ArsD, an As (III)-responsive transcriptional regulator.', Journal of biological chemistry., 277 (29). pp. 25992-26002.
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, Cys12-Cys13 and Cys112-Cys113, 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 Cys112-Cys113 site by mutagenesis of arsD, but not the Cys12-Cys13 site, largely abolished this cooperativity, indicative of interactions between adjacent Cys112-Cys113 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.
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|Publisher Web site:||http://dx.doi.org/10.1074/jbc.M201619200|
|Record Created:||26 Aug 2008|
|Last Modified:||08 Apr 2009 16:31|
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