Genetic mapping of the interface between the ArsD metallochaperone and the ArsA ATPase
Open Access
- 7 December 2010
- journal article
- Published by Wiley in Molecular Microbiology
- Vol. 79 (4), 872-881
- https://doi.org/10.1111/j.1365-2958.2010.07494.x
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.Keywords
This publication has 30 references indexed in Scilit:
- The 1.4 Å Crystal Structure of the ArsD Arsenic Metallochaperone Provides Insights into Its Interaction with the ArsA ATPaseBiochemistry, 2010
- Arsenic Binding and Transfer by the ArsD As(III) MetallochaperoneBiochemistry, 2010
- Mapping of Protein–Protein Interaction Sites by the ‘Absence of Interference’ ApproachJournal of Molecular Biology, 2008
- ArsD Residues Cys12, Cys13, and Cys18 Form an As(III)-binding Site Required for Arsenic Metallochaperone ActivityPublished by Elsevier BV ,2007
- Identification of protein interaction antagonists using the repressed transactivator two-hybrid systemBioTechniques, 2007
- An arsenic metallochaperone for an arsenic detoxification pumpProceedings of the National Academy of Sciences, 2006
- MetallochaperonesCell Chemical Biology, 2002
- Randomization of genes by PCR mutagenesis.Genome Research, 1992
- A novel genetic system to detect protein–protein interactionsNature, 1989
- A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye bindingAnalytical Biochemistry, 1976