TRANSPORT ATPASE--ENERGY COUPLING

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The plasmid determined resistance to arsenic and antimony compounds in Escherichia coli is mediated by an anion-translocating ATPase. This ATPase does not fall into any of the known classes of the solute- translocating ATPases. It consists of an integral membrane protein ArsB that functions as a carrier, and a peripheral membrane protein ArsA that forms the catalytic subunit. Upon interaction, the ArsA and the ArsB proteins form a functional pump to extrude the anions out of the cell. The long term objective of the proposed research is to understand how the energy of ATP hydrolysis is coupled to the translocation of anions by the Ars ATPase. The catalytic component ArsA is an anion-stimulated ATPase and it undergoes dimerization in presence of the anion. Each monomer of ArsA has two homologous nucleotide binding domains, and it appears that an interaction of domains in trans between two polypeptide chains results in the formation of an interface. We will test the hypothesis that there are two active sites in the homodimer of the ArsA protein, and that each site is composed of residues from two polypeptide chains. The composition of the active sites will be examined by a hybridization approach, where mixing of two inactive point mutant proteins- each defective in one nucleotide binding domain- is expected to result in generation of one active site. Negative complementation between the wild type protein and a mutant defective in both domains will result in loss of ATPase activity if the active site is formed of shared residues between the two polypeptide chains. We will identify sites of contact between the subunits and alter residues in those regions by in vitro mutagenesis to determine the role of these interactions in the overall functioning of the pump. Analogs of ATP will be used to study the environment and the catalytic nature of the two ATP binding sites, which will be critical for understanding the mechanism of energy transduction. The possible interaction of nucleotide binding domains will have relevance to the F1 proton translocating ATPase, where rate of catalysis at the first site is increased several fold by binding of nucleotide to the second and the third site. This pump bears structural and functional similarity to members of the ABC type transporters such as the P-glycoprotein and the CFTR protein in the mammalian cells. Hence, these studies will give an insight into the molecular mechanism of energy coupling by Ars ATPase and by a variety of other ion and solute translocating pumps.