DESCRIPTION (provided by applicant): The mechanism of heavy-metal transporting P-type ATPases is crucial to the understanding of copper and iron homeostasis mechanisms. Wilson Disease is a disorder of copper metabolism, leading to a buildup of copper in the brain, liver, and kidneys, followed by subsequent neurological disorders, cirrhosis of the liver, and kidney failure. Wilson Protein and its homologues are essential for incorporation of copper into a multicopper oxidase that is vital for high affinity iron uptake. Thus copper and iron uptake are tightly coupled processes. The relationship between Wilson Disease and the mutations that cause the disease are not well understood. The N-terminal metal-binding domains of the Wilson protein are important for copper acquisition and subsequently delivery of copper into the post-Golgi vesicle that matures copper-containing multicopper oxidases. A number of different mutations give rise to Wilson disease, 3 of which occur in the N-terminal metal-binding domains. The molecular effect of these 3 mutations has not been elucidated. Wilson protein contains 6 N-terminal metal-binding domains. Intramolecular interactions between these domains facilitate the copper acquisition pathway. Characterization of these mutations and intradomain interactions is best accomplished by isolation of these metal-binding domains, followed by biochemical and structural studies. Experiments will be designed to test the transfer of copper between domains. Wild type vs. mutant forms will be compared by stability studies and assessment of the folding kinetics. Finally, it is likely that domains 5 and 6 of the Wilson protein act in concert with each other since no intervening sequence occurs between the putative consensus motifs. Expression of a construct containing domains 5 and 6 together will allow one to determine the role of two closely adjacent domains in homologous P-type ATPases. Finally, we are isolating the metal-binding domains of Arabidopsis thaliana RAN1 and the metallochaperone CCH, as a plant model for human disease.