The arsenical resistance (ars operon of the Escherichia coli plasmid R773 encodes a system for the active extrusion from cells of the toxic oxyanions arsenite (As(III)O21-) and antimonite (Sb(III)O21-) via an ATP-driven pump. The arsA and the arsB genes o the operon encode, respectively, the catalytic subunit (ATPase) and the membrane subunit of the pump. The arsC gene codes for a reductase that convert arsenate (As(V)O43) to arsenite and appears to channel it into the ArsA-ArsB pump, thus extending bacterial resistance also to the pentavalent state of arsenic. Crystals diffracting at high resolution (2.0 Angstrom) were obtained for both the catalytic subunit of the pump (ArsA) and for the reductase (ArsC) and native data sets have been collected. Structural studies are proposed to identify ArsA and ArsC binding sites for substrates and/or for allosteric effectors, and the regions of interaction with other proteins (e.g., ArsA with ArsB, ArsC and ArsA). Since expression of the arsB gene is highly toxic in E. coli, production of the protein in other hosts (Archaebacteria, yeast) will be pursued to obtain a large amount of pure protein for crystallization. Arsenical resistance is a useful model for the study of multiple drug resistance in both eukaryotic and prokaryotic cells. The ArsA-ArsB pump exhibits structural and functional similarity to the P-glycoprotein: both are efflux pumps for toxic compounds, have two nucleotide binding sites, are substrate-dependent ATPases, have 12 membrane spanning alpha-helices, and are each able to detoxify structurally distinct drugs. The latter point is illustrated for the ArsA-ArsB pump by the fact that while arsenate and arsenit are both oxyanions of arsenic, they are chemically dissimilar. The ArsA protein is also the only other ion-motive ATPase, besides the mitochondrial F1 ATPase, for which three-dimensional crystals have been obtained. Analysis of the similarities and differences between these two enzymes will further our understanding of how ions are transported across biological membranes.

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National Institute of Allergy and Infectious Diseases (NIAID)
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Physical Biochemistry Study Section (PB)
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Wayne State University
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