Interaction of ATP binding sites in the ArsA ATPase, the catalytic subunit of the Ars pump.
The ArsA ATPase is the catalytic subunit of the Ars pump that catalyzes arsenical extrusion in Escherichia coli, thus providing resistance. The active form of ArsA is a homodimer with four nucleotide binding sites, two from each monomer. The codons for Gly-15 in the N-terminal consensus nucleotide binding sequence and Gly-334 in the C-terminal sequence were individually mutated to cysteine codons. Cells expressing an arsAG334C mutation retained arsenite resistance, while an arsAG15C mutation resulted in substantial reductions in arsenite resistance, transport, and ATPase activity. Selection for suppression of the G15C mutation that restored arsenite resistance yielded an A344V substitution. Ala-344 is located adjacent to the C-terminal nucleotide binding sequence. The second site mutation did not suppress the loss of resistance resulting from G18D, G20S, or T22I substitutions in the N-terminal nucleotide binding site. Cells expressing the G15C/A344V double mutant regained arsenite extrusion. These results suggest a spatial proximity of Gly-15 and Ala-344 and support a model for interaction of the nucleotide binding sites in ArsA.
Li
J
J
Department of Biochemistry and Molecular Biology, Wayne State University, School of Medicine, Detroit, Michigan 48201, USA.
Liu
S
S
Rosen
B P
BP
D000251:Adenosine Triphosphatases / Q000737:chemistry / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism*; D000595:Amino Acid Sequence; D053501:Arsenite Transporting ATPases; D018053:Arsenites / Q000494:pharmacology; D001665:Binding Sites; D003062:Codon; D016384:Consensus Sequence; D003545:Cysteine; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005798:Genes, Bacterial; D005998:Glycine; D016623:Ion Pumps; D046911:Macromolecular Substances; D008565:Membrane Proteins / Q000378:metabolism; D008961:Models, Structural; D009097:Multienzyme Complexes; D016297:Mutagenesis, Site-Directed; D017354:Point Mutation; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism
D018053:Arsenites; D003062:Codon; D016623:Ion Pumps; D046911:Macromolecular Substances; D008565:Membrane Proteins; D009097:Multienzyme Complexes; D011994:Recombinant Proteins; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases; D053501:Arsenite Transporting ATPases; D003545:Cysteine; D005998:Glycine