Analysis of functional domains of Wilson's disease protein (ATP7B).

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ATP7B gene, which encodes a putative copper transporting P-type ATPase (ATP7B protein), is defective in the patients with Wilson's disease, resulting in the excessive accumulation of copper in the liver. We have recently revealed the functional importance of ATP7B protein hepatic copper metabolism. Based on the amino acid sequence, ATP7B protein is assumed to have several functional domains in its molecule, however, how these domains correlate with the manifestation of ATP7B function is not known yet. To address this, we introduced various mutations, such as point mutation or deletion, into ATP7B cDNA, transfected these mutants into a yeast strain lacking the CCC2 gene (Δccc2), the yeast homologue of ATP7B, and examined the function of mutated ATP7B protein by the complementation test.Among the mutants bearing the deletion within the copper binding domains at the N terminus of ATP7B, the mutant containing only the sixth copper binding domain could rescue the growth of Δccc2, indicating the sixth copper binding domain is sufficient for the manifestation of ATP7B function. In addition, ATP7B protein lacking the 88 amino acids at its C terminus was found to lose its function. Similarly, the N1270S mutant, located in the hinge domain of ATP7B protein and found in patients showing the fulminant type of the disease, could not rescue the Δccc2 mutant, implying the N1270S mutation highly associates with the occurrence of Wilson's disease. In conclusion, the sixth copper binding domain, the hinge domain, and the 88 amino acids at the C terminus play important roles in ATP7B function.