Spatial proximity of Cys(113), Cys(172), and Cys(422) in the metalloactivation domain of the ArsA ATPase

ArsA ATPase activity is allosterically activated by salts of the semimetal arsenic or antimony. Activation is associated with the presence of three cysteine residues in ArsA: Cys(113), Cys(172), and Cys(422). To determine the distance between cysteine residues, wild type ArsA and ArsA proteins with cysteine to serine substitutions were treated with the bifunctional alkylating agent dibromobimane, which reacts with thiol pairs within 3-6 Angstrom of each other to form a fluorescent adduct. ArsA proteins in which single cysteine residues were altered by site-directed mutagenesis still formed fluorescent adducts. Proteins in which two of the three cysteine residues were substituted did not form fluorescent adducts, These results demonstrate that Cys(113), Cys(172), and Cys(422) are in close proximity of each other. We propose a model in which As(III) or Sb(III) interacts with these three cysteines in a trigonal pyramidal geometry, forming a novel soft metal-thiol cage.