ArsD Residues Cys12, Cys13, and Cys18Form an As(III)-binding Site Required for Arsenic Metallochaperone Activity
Journal of Biological Chemistry
The ArsA ATPase is the catalytic subunit of the ArsAB pump encoded by the arsRDABC operon of Escherichia coli plasmid R773. ArsD is a metallochaperone that delivers As(III) to ArsA, increasing its affinity for As(III), thus conferring resistance to environmental concentrations of arsenic. R773 ArsD is a homodimer with three vicinal cysteine pairs, Cys 12 -Cys 13 , Cys 112 -Cys 113 , and Cys 119 -Cys 120 , in each subunit. Each vicinal pair binds As(III) or Sb(III). Alignment of the primary
... nce of homologues of ArsD indicates that only the first vicinal cysteine pair, Cys 12 -Cys 13 , and an additional cysteine, Cys 18 , are conserved. The effect of cysteine-to-alanine substitutions and truncations were examined. By yeast two-hybrid analysis, nearly all of the ArsD mutants were able to interact with wild type ArsD, indicating that the mutations do not interfere with dimerization. ArsD mutants with alanines substituting for Cys 112 , Cys 113 , Cys 119 , or Cys 120 individually or in pairs or truncations lacking the vicinal pairs retained ability to interact with ArsA and to activate its ATPase activity. Cells expressing these mutants retained ArsD-enhanced As(III) efflux and resistance. In contrast, mutants with substitutions of conserved Cys 12 , Cys 13 , or Cys 18 , individually or in pairs, were unable to activate ArsA or to enhance the activity of the ArsAB pump. We propose that ArsD residues Cys 12 , Cys 13 , and Cys 18 , but not Cys 112 , Cys 113 , Cys 119 , or Cys 120 , are required for delivery of As(III) to and activation of the ArsAB pump.