Evidence for cooperativity between the four binding sites of dimeric ArsD, an As(III)-responsive transcriptional regulator.
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.
Li
Song
S
Department of Biochemistry and Molecular Biology, Wayne State University School of Medicine, Detroit, Michigan 48201, USA.
Rosen
Barry P
BP
Borges-Walmsley
M Ines
MI
Walmsley
Adrian R
AR
D000965:Antimony / Q000378:metabolism; D001151:Arsenic / Q000378:metabolism*; D001426:Bacterial Proteins; D001665:Binding Sites; D019281:Dimerization; D004229:Dithiothreitol / Q000494:pharmacology; D004926:Escherichia coli; D007700:Kinetics; D011487:Protein Conformation; D013050:Spectrometry, Fluorescence; D015534:Trans-Activators / Q000737:chemistry* / Q000378:metabolism
D001426:Bacterial Proteins; D015534:Trans-Activators; D000965:Antimony; D001151:Arsenic; D004229:Dithiothreitol