ArsD residues Cys12, Cys13, and Cys18 form an As(III)-binding site required for arsenic metallochaperone activity.
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 sequence 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.
Lin
Yung-Feng
YF
Department of Biochemistry and Molecular Biology, Wayne State University, School of Medicine, Detroit, Michigan 48201, USA.
Yang
Jianbo
J
Rosen
Barry P
BP
D000595:Amino Acid Sequence; D001151:Arsenic / Q000378:metabolism*; D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism*; D001665:Binding Sites; D001692:Biological Transport; D001643:Bridged Bicyclo Compounds / Q000737:chemistry; D003545:Cysteine / Q000378:metabolism*; D004926:Escherichia coli / Q000378:metabolism; D018832:Molecular Chaperones / Q000737:chemistry / Q000378:metabolism*; D008969:Molecular Sequence Data; D017386:Sequence Homology, Amino Acid; D015534:Trans-Activators / Q000737:chemistry / Q000378:metabolism*; D020798:Two-Hybrid System Techniques
D001426:Bacterial Proteins; D001643:Bridged Bicyclo Compounds; D018832:Molecular Chaperones; D015534:Trans-Activators; C037094:dibromobimane; D003545:Cysteine; D001151:Arsenic