Role of signature lysines in the deviant walker a motifs of the ArsA ATPase

Hsueh Liang Fu, A. Abdul Ajees, Barry P. Rosen, Hiranmoy Bhattacharjee

Research output: Contribution to journalArticlepeer-review

5 Citations (Scopus)

Abstract

(Figure Presented) The ArsA ATPase belongs to the P-loop GTPase subgroup within the GTPase superfamily of proteins. Members of this subgroup have a deviant Walker A motif which contains a signature lysine that is predicted to make intermonomer contact with the bound nucleotides and to play a role in ATP hydrolysis. ArsA has two signature lysines located at positions 16 and 335. The role of Lys16 in the A1 half and Lys335 in the A2 half was investigated by altering the lysines individually to alanine, arginine, leucine, methionine, glutamate, and glutamine by site-directed mutagenesis. While Lys16 mutants show similar resistance phenotypes as the wild type, the Lys335 mutants are sensitive to higher concentrations of arsenite. K16Q ArsA shows 70% of wild-type ATPase activity while K335Q ArsA is inactive. ArsA is activated by binding of Sb(III), and both wild-type and mutant ArsAs bind Sb(III) with a 1:1 stoichiometry. Although each ArsA binds nucleotide, the binding affinity decreases in the order wild type > K16Q > K335Q. The results of limited trypsin digestion analysis indicate that both wild type and K16Q adopt a similar conformation during activated catalysis, whereas K335Q adopts a conformation that is resistant to trypsin cleavage. These biochemical data along with structural modeling suggest that, although Lys16 is not critical for ATPase activity, Lys335 is involved in intersubunit interaction and activation of ATPase activity in both halves of the protein. Taken together, the results indicate that Lys16 and Lys335, located in the A1 and A2 halves of the protein, have different roles in ArsA catalysis, consistent with our proposal that the nucleotide binding domains in these two halves are functionally nonequivalent. The ArsAB pump in Escherichia coli, encoded by the ars operon of plasmid R773, confers resistance to arsenicals and antimonials. ArsA is the catalytic subunit of the pump that hydrolyzes ATP in the presence of arsenite [As(III)] or antimonite [Sb(III)]. ATP hydrolysis is coupled to extrusion of As(III) or Sb(III) through ArsB, which serves both as a membrane anchor for ArsA and as the substrate-conducting pathway (1). When expressed from a multicopy plasmid, the majority of ArsA is soluble in the cytosol, which allows large-scale purification and characterization. Purified ArsA exhibits low, basal level ATPase activity in the absence of metalloid and is activated by As(III) or Sb(III) (2). ArsA is composed of homologous N-terminal (A1)1 and C-terminal (A2) halves that are most likely the result of an ancestral gene duplication and fusion (3). Each half has a consensus Walker A motif or P-loop (4, 5), which interacts with the phosphate moiety of ATP (6). In ArsA, the P-loop sequences are G15KGGVGKT and G334KGGVGKT in A1 and A2, respectively. This sequence differs from the classical Walker A motif in that it contains a "signature" lysine residue (Lys16 and Lys335) near the N-terminal end, and ArsA has been classified in the "deviant" Walker A motif subgroup (7).

Original languageEnglish
Pages (from-to)356-364
Number of pages9
JournalBiochemistry
Volume49
Issue number2
DOIs
Publication statusPublished - 19-01-2010

All Science Journal Classification (ASJC) codes

  • Biochemistry

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