The distinct roles of the N-terminal copper-binding sites in regulation of catalytic activity of the Wilson's disease protein.
Wilson's disease protein (WNDP) is a copper-transporting ATPase essential for normal distribution of copper in human cells. Recent studies demonstrate that copper regulates WNDP through several mechanisms. Six metal-binding sites (MBS) at the N terminus of WNDP are predicted to be involved in copper-dependent regulation of WNDP; however, specific roles of MBS remain poorly understood. To address this issue, we generated WNDP variants with mutations or truncation in the N-terminal region and characterized their functional properties. We show that copper cooperatively stimulates catalytic activity of WNDP and that this effect requires the presence of both MBS5 and MBS6. Mutations of MBS6 or MBS1-5 result in non-cooperative activation of the enzyme by copper, whereas the deletion of MBS1-4 does not abolish cooperativity. Our data further suggest that MBS5 and MBS6 together regulate the affinity of the intramembrane-binding site(s) for copper. Analysis of the copper-dependent stimulation of catalytic phosphorylation demonstrate that the MBS6 and MBS1-5 mutants have a 7-8-fold lower EC50 for copper activation, suggesting that their affinity for copper is increased. This conclusion is confirmed by a markedly decreased inhibition of these mutants by a copper chelator bathocuproine disulphonate. In contrast, deletion of MBS1-4 does not affect the affinity of sites important for catalytic phosphorylation. Rather, the MBS1-4 region appears to control access of copper to the functionally important metal-binding sites. The implications of these findings for intracellular regulation of WNDP are discussed.
Huster
Dominik
D
Department of Biochemistry and Molecular Biology, Oregon Health and Science University, Portland, Oregon, 97239-3098, USA.
Lutsenko
Svetlana
S
D000251:Adenosine Triphosphatases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D001483:Base Sequence; D001665:Binding Sites; D002384:Catalysis; D027682:Cation Transport Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D003300:Copper / Q000378:metabolism*; D000073840:Copper-Transporting ATPases; D017931:DNA Primers; D010766:Phosphorylation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D027682:Cation Transport Proteins; D017931:DNA Primers; D011994:Recombinant Proteins; D003300:Copper; D000251:Adenosine Triphosphatases; D000073840:Copper-Transporting ATPases