The overall objective of the proposal is to determine the 3D structure of a P-type ATPase, specifically the Kdp-ATPase from E. coli. P-type ATPases are membrane bound active transporters involved in ion homeostasis in all forms of life. Although, extensive work has been reported on the biochemistry and molecular biology of P-type ATPases, the molecular mechanisms of ion translocation and energy transduction remain unknown due to the lack of atomic resolution structures. This proposal is directed towards filling this gap in knowledge. X-ray crystallographic methods will be used to determine the structure of Kdp-ATPase, a high affinity K+ transporter. Kdp-ATPase from E. coli was chosen for structural studies as large quantities of protein can be expressed and purified easily as a multi-subunit complex. In addition, its biochemical properties are well characterized and a number of mutants are available. However, to overcome the difficulties associated with crystallography of membrane proteins, a multi-pronged approach involving crystallization of Kdp-ATPase with detergents, complexed to antibody fragments, bound to inhibitors and in lipidic cubic phases, are proposed. Besides enhancing our understanding of the Kdp complex, the 3D structure will elucidate the functioning of related eukaryotic ATPases including the human sarcoplasmic Ca2+ ATPase. Further, homologs of Kdp-ATPase are found only among eubacteria including many pathogenic bacteria. The availability of a high-resolution structure will be a start point for rational drug design efforts.
