Evidence for cooperativity between the four binding sites of dimeric ArsD, an As(III)-responsive transcriptional regulator

Li, S., Rosen, B.P., Borges-Walmsley, M.I. and Walmsley, A.R. (2002) Evidence for cooperativity between the four binding sites of dimeric ArsD, an As(III)-responsive transcriptional regulator. Journal of Biological Chemistry, 277, pp. 25992-26002. (doi: 10.1074/jbc.M201619200)

Abstract

rsD 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-Cys113site 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.

• Item Type: Articles
• Status: Published
• Refereed: Yes
• Glasgow Author(s) Enlighten ID: UNSPECIFIED
• Authors: Li, S., Rosen, B.P., Borges-Walmsley, M.I., and Walmsley, A.R.
• College/School: College of Medical Veterinary and Life Sciences
• Journal Name: Journal of Biological Chemistry
• Journal Abbr.: J Biol Chem.
• Publisher: American Society for Biochemistry and Molecular Biology, Inc.
• ISSN: 0021-9258
• ISSN (Online): 1083-351X