Mechanism of an ATP-coupled arsenical pump

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Resistance to arsenic and antimony is widely spread in both gram-positive and gram-negative bacteria. The best- characterized system encoded by the clinically isolated resistance factor plasmid R773 in E. coli confers resistance against oxyanions of arsenic (arsenite and arsenate) and antimony (antimonite). The ars operon codes for two regulatory (ArsR and ArsD) and three structural (ArsA, ArsB and ArsC) proteins. Resistance correlates with active extrusion of arsenite from the cell by a primary pump. The arsA gene product is an ATPase that serves as the catalytic subunit of the pump. ArsA is a 63-kDa peripheral membrane protein that catalyzes As(III)/Sb(III)- stimulated ATP hydrolysis. ArsA has two homologous halves, A1 and A2, connected by a short linker. Each has a consensus nucleotide binding site (NBS), and both NBS are required for activity. ArsA binds to ArsB, which s the ion-conducting subunit of the pump. ArsB is a 45-kDa integral membrane protein that spans the inner membrane 12 times. ArsB has a novel dual mode of energy coupling depending on its association with ArsA. Arsenic efflux bacteria can be catalyzed by either ArsB alone functioning as a secondary transporter or by the ArsAB complex, functioning as a transport ATPase. Our overall goal is elucidation of the molecular mechanism of the ArsAB pump. Analysis includes the following specific aims: 1. Structure and function of the ArsA ATPase: The function and properties of the metal binding site, nucleotide binding sites and signal transduction sites of ArsA will be examined. The composition and function of residues that form the interface between A1 and A2 will be determined. 2. Catalytic mechanism of the ArsA ATPase will be examined using single tryptophan ArsA mutants as spectroscopic probes, by isotope trapping and by vanadate trapping and cleavage experiments. 3. Structure of the ArsAB pump: Residues that form the sites of interaction of the ArsA and ArsB subunits will be identified. The stoichiometry of the ArsA and ArsB subunits in the pump will be determined.