The Lys(1010)-Lys(1325) fragment of the Wilson's disease protein binds nucleotides and interacts with the N-terminal domain of this protein in a copper-dependent manner

Wilson's disease, an autosomal disorder associated with vast accumulation o f copper in tissues, is caused by mutations in a gene encoding a copper-tra nsporting ATPase (Wilson's disease protein, WNDP), Numerous mutations have been identified throughout the WNDP sequence, particularly in the Lys(1010) -Lys(1325) segment; however, the biochemical properties and molecular mecha nism of WNDP remain poorly characterized. Here, the Lys(1010)-Lys(1325) fra gment of WNDP was overexpressed, purified, and shown to form an independent ly folded ATP-binding domain (ATP-BD). ATP-BD binds the fluorescent ATP ana logue trinitrophenyl-ATP with high affinity, and ATP competes with trinitro phenyl-ATP for the binding site; ADP and AMP appear to bind to ATP-BD at th e site separate from ATP. Purified ATP-BD hydrolyzes ATP and interacts spec ifically with the N-terminal copper-binding domain of WNDP (N-WNDP). Striki ngly, copper binding to N-WNDP diminishes these interactions, suggesting th at the copper-dependent change in domain-domain contact may represent the m echanism of WNDP regulation. In agreement with this hypothesis, N-WNDP indu ces conformational changes in ATP-BD as evidenced by the altered nucleotide binding properties of ATP-BD in the presence of N-WNDP, Significantly, the effects of copper-free and copper-bound N-WNDP on ATP-BD are not identical . The implications of these results for the WNDP function are discussed.