Genetic mapping of the interface between the ArsD metallochaperone and the ArsA ATPase

Jianbo Yang, Abdul Ajees Abdul Salam, Barry P. Rosen

Research output: Contribution to journalArticle

16 Citations (Scopus)

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.

Original languageEnglish
Pages (from-to)872-881
Number of pages10
JournalMolecular Microbiology
Volume79
Issue number4
DOIs
Publication statusPublished - 01-02-2011

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Metallochaperones
Metalloids
Adenosine Triphosphatases
Binding Sites
Yeasts
Two-Hybrid System Techniques
Trans-Activators
Structural Models
Arsenic
Site-Directed Mutagenesis
Computer Simulation
Arginine
Catalytic Domain
Metals
Mutation

All Science Journal Classification (ASJC) codes

  • Microbiology
  • Molecular Biology

Cite this

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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.",
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Genetic mapping of the interface between the ArsD metallochaperone and the ArsA ATPase. / Yang, Jianbo; Abdul Salam, Abdul Ajees; Rosen, Barry P.

In: Molecular Microbiology, Vol. 79, No. 4, 01.02.2011, p. 872-881.

Research output: Contribution to journalArticle

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