The N-terminal Metal-binding Site 2 of the Wilson's Disease Protein Plays a Key Role in the Transfer of Copper from Atox1

• Published in: The Journal of biological chemistry 2004-04, Vol.279 (15), p.15376-15384
• Main Authors: Walker, Joel M., Huster, Dominik, Ralle, Martina, Morgan, Clinton T., Blackburn, Ninian J., Lutsenko, Svetlana
• Format: Article
• Language: English
• Subjects: Adenosine Triphosphatases - chemistry; Amino Acid Motifs; Amino Acid Sequence; Animals; Binding Sites; Binding, Competitive; Biological Transport; Catalysis; Cation Transport Proteins - chemistry; Cell Line; Cell Membrane - metabolism; Copper - chemistry; Copper - metabolism; Copper Transport Proteins; Copper-Transporting ATPases; Cysteine - chemistry; DNA, Complementary - metabolism; Dose-Response Relationship, Drug; Escherichia coli - metabolism; Humans; Insecta; Ions; Metallochaperones; Models, Molecular; Models, Statistical; Molecular Chaperones - chemistry; Molecular Chaperones - metabolism; Molecular Sequence Data; Phosphorylation; Protein Binding; Protein Structure, Tertiary; Recombinant Proteins - chemistry; Scattering, Radiation; Sequence Homology, Amino Acid; Spectrometry, X-Ray Emission
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M400053200

The Wilson's disease protein (WNDP) is a copper-transporting ATPase regulating distribution of copper in the liver. Mutations in WNDP lead to a severe metabolic disorder, Wilson's disease. The function of WNDP depends on Atox1, a cytosolic metallochaperone that delivers copper to WNDP. We demonstrate that the metal-binding site 2 (MBS2) in the N-terminal domain of WNDP (N-WNDP) plays an important role in this process. The transfer of one copper from Atox1 to N-WNDP results in selective protection of the metal-coordinating cysteines in MBS2 against labeling with a cysteine-directed probe. Such selectivity is not observed when free copper is added to N-WNDP. Similarly, site-directed mutagenesis of MBS2 eliminates stimulation of the catalytic activity of WNDP by the copper-Atox1 complex but not by free copper. The Atox1 preference toward MBS2 is likely due to specific protein-protein interactions and is not due to unique surface exposure of the metal-coordinating residues or higher copper binding affinity of MBS2 compared with other sites. Competition experiments using a copper chelator revealed that MBS2 retained copper much better than Atox1, and this may facilitate the metal transfer process. X-ray absorption spectroscopy of the isolated recombinant MBS2 demonstrated that this sub-domain coordinates copper with a linear biscysteinate geometry, very similar to that of Atox1. Therefore, non-coordinating residues in the vicinity of the metal-binding sites are responsible for the difference in the copper binding properties of MBS2 and Atox1. The intramolecular changes that accompany transfer of a single copper to N-WNDP are discussed.