title: Genetic mapping of the interface between the ArsD metallochaperone and the ArsA ATPase.
abstract: The ArsD metallochaperone delivers trivalent metalloids, As(III) or Sb(III), to the ArsA ATPase, the catalytic subunit of the ArsAB As(III) efflux pump. Transfer of As(III) increases the affinity of ArsA for As(III), allowing resistance to environmental arsenic concentrations. As(III) transfer is channelled from chaperone to ATPase, implying that ArsD and ArsA form an interface at their metal binding sites. A genetic approach was used to test this hypothesis. Thirteen ArsD mutants exhibiting either weaker or stronger interaction with ArsA were selected by either repressed transactivator yeast two-hybrid or reverse yeast two-hybrid assays. Additionally, Lys-37 and Lys-62 were identified as being involved in ArsD function by site-directed mutagenesis and chemical modification. Substitution at either position with arginine was tolerated, suggesting participation of a positive charge. By yeast two-hybrid analysis K37A and K62A mutants lost interaction with ArsA. All 15 mutations were mapped on the surface of the ArsD structure, and their locations are consistent with a structural model generated by in silico docking. Four are close to metalloid binding site residues Cys-12, Cys-13 and Cys-18, and seven are on the surface of helix 1. These results suggest that the interface involves one surface of helix 1 and the metalloid binding site.
authors.affiliation: Department of Biochemistry and Molecular Biology, Wayne State University, School of Medicine, Detroit, MI 48201, USA.
authors.forename: Jianbo
authors.initials: J
authors.lastname: Yang
authors.forename: Abdul Ajees Abdul
authors.initials: AA
authors.lastname: Salam
authors.forename: Barry P
authors.initials: BP
authors.lastname: Rosen
mesh_terms: D000107:Acetylation; D000251:Adenosine Triphosphatases / Q000378:metabolism*; D019943:Amino Acid Substitution; D001151:Arsenic / Q000378:metabolism; D001665:Binding Sites; D002874:Chromosome Mapping; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000235:genetics; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism*; D056511:Metallochaperones / Q000235:genetics / Q000378:metabolism*; D058955:Metalloids / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D017434:Protein Structure, Tertiary; D020798:Two-Hybrid System Techniques
chemical_list: D004269:DNA, Bacterial; D029968:Escherichia coli Proteins; D056511:Metallochaperones; D058955:Metalloids; D000251:Adenosine Triphosphatases; D001151:Arsenic