Structure-Function of Heavy Metal Transport CPx-ATPases

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The goal of this project is to understand the functional mechanism of heavy metal transport CPx-ATPases. These P-type ATPases transport heavy metals (ions of Cu, Ag, Zn, Co, Cd, Pb) across biomembranes. Found in all life kingdoms, these key enzymes are involved in micronutrient metal homeostasis. The transport stoichiometry, the determinants of metal ion specificity, and the regulatory mechanisms of CPx-ATPases are not known. This project aims to identify key structural features involved in ion selectivity and activity regulation. Simpler CPx-ATPases found in procaryotes will be used as models in these studies: Cu-ATPases from A. fulgidus, a Co-ATPase from A. pernix, and a Zn-ATPase from H. influenzae. A. fulgidus and A. pernix are extremophilic archaea. Experiments will be performed to test whether conserved sequences in the transmembrane region of CPx-ATPases determine ion specificity. CPx-ATPases have cytoplasmic N-terminal metal-binding domains, in addition to the transmembrane CPx site. The possible regulatory role of the metal-binding domain in enzyme kinetics will be studied. This project will also involve testing whether a metal chaperone molecule delivers the metal to the transport site. The fourth goal of this project is to determine the structure of a CPx-ATPase using X-ray crystallography. The structure of CPx-ATPases at atomic resolution would greatly enhance understanding the mechanism of heavy metal transport.Broader Impact: The project will contribute to an understanding of heavy metal homeostasis. Insight gained in metal selectivity might facilitate the design of metal bioremediation processes and the management of plant and animal mineral nutrition. This research will also contribute to ongoing educational activities (undergraduate teaching, curriculum development and summer research). Moreover, undergraduate and graduate students will perform major parts of this project.