Evidence for Cooperativity between the Four Binding Sites of Dimeric ArsD, an As (III)-responsive Transcriptional Regulator

Song, L.; Rosen, B.P.; Borges-Walmsley, M.I.; Walmsley, A.

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, 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.

Citation

Song, L., Rosen, B., Borges-Walmsley, M., & 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), 25992-26002. https://doi.org/10.1074/jbc.m201619200

• Journal Article Type: Article
• Publication Date: Jul 19, 2002
• Deposit Date: Aug 26, 2008
• Publicly Available Date: Aug 29, 2017
• Journal: Journal of Biological Chemistry
• Print ISSN: 0021-9258
• Electronic ISSN: 1083-351X
• Publisher: American Society for Biochemistry and Molecular Biology
• Peer Reviewed: Peer Reviewed
• Volume: 277
• Issue: 29
• Pages: 25992-26002
• DOI: https://doi.org/10.1074/jbc.m201619200
• Public URL: https://durham-repository.worktribe.com/output/1566907