The family of P-type ATPases is responsible for maintaining the ionic homeostasis of cells. In eukaryotes, individual pumps use ATP to pump housekeeping cations: Ca, Na, K, H. The resulting gradients are coupled to signal transduction, nervous impulses, pH balance and nutrient uptake. A related group of pumps handles transition and heavy metal ions: Cu, Zn, Ag, Cd, Pb. In the case of Cu and Zn, import pumps ensure sufficient concentrations for assembly of metalloproteins. However, export pumps prevent accumulation of these potentially toxic metal ions, providing established resistance mechanisms in many bacteria. Ca-ATPase represents an archetype for the family and its recent x-ray crystal structure provides a framework on which to address mechanisms employed by various members of the family. In the current application, we propose structural studies aimed at understanding the basic reaction cycle as well as functional specializations of several other P-type ATPases. In particular, studies of Ca-ATPase, will reveal the conformational dynamics that drive its reaction cycle. Studies of Na,K-ATPase and Kdp will address their oligomeric assemblies. Studies of plasma membrane Ca-ATPase and phospholamban are aimed at understanding their mechanisms of regulation. Studies of bacterial pumps will address the role of their specialized N-terminal, metal-binding domains either in transport or in regulation. Structures will be determined either by cryoelectron microscopy of 2D crystals or by x-ray crystallography of 3D crystals. In some cases, 2D crystals will be produced within native microscomes; in others, purified, detergent-solubilized pumps will be reconstituted prior to 2D crystallization. Structures by cryoelectron microscopy at 6-10 Angstroms resolution will be modeled using the recent x-ray structure for Ca-ATPase, thus characterizing conformational changes and the location of structural specializations. 3D crystallization trials will be conducted in hanging drops and structures solved by x-ray crystallography. Relevance to human health comes from the importance of Ca-ATPase, phospholamban and Na/K-ATPase to cardiac function, of PMCA to signal transduction in the hair cells of the inner ear and the similarity of bacterial pumps to those defective in Menkes and Wilson's diseases involving Cu imbalances.
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