) The superfamily of P-type ATPases catalyzes the energy-dependent transport of charged substrates across membranes. These ubiquitous pumps transport different cations and carry out a wide variety of functions, for example, they generate the transmembrane ion gradients necessary for nutrient uptake, signal transduction, and maintenance of suitable pH and ion concentrations as well as maintain the asymmetric distribution of phospholipids across plasma membranes of animal cells. Soft-metal P-type ATPases maintain homeostasis of the essential metals, Cu(I), Zn (II) and Co(II) and also mediate resistance to Ag(I), Pb(II), Cd(II), and other highly toxic metal cations. The human Cu(I)-transporting ATPases associated with Menkes and Wilson diseases, are examples of monovalent soft-metal ATPases whereas ZntA from Escherichia coli, the focus of this application, is a divalent soft-metal ATPase. The objective of this proposal is to use ZntA as a prototype to study the mechanism by which soft-metal ATPases recognize and transport specific metal ions. ZntA is ideal for this study because it has been cloned, overexpressed, solubilized and purified. An ATP-dependent transport assay has been developed for ZntA. A soft-metal dependent ATP hydrolysis assay has been optimized; ZntA is the first soft-metal ATPase for which this activity has been demonstrated.
The specific aims i nclude biochemical characterization of ZntA and analysis of the contributions of conserved residues and different domains, including the cysteine-rich amino-terminal domain of soft-metal ATPases, towards metal recognition and transport. The interaction between the metal-binding and transport domains with the ATP hydrolysis domain will be examined. The coordination and geometry adopted by different metal ions will be studied. Efforts to address these specific aims will include transport, ATP hydrolysis and phosphoenzyme formation assays, site-specific mutagenesis, and fluorescence EXAFS and x-ray structural studies.
