The N-terminal metal-binding site 2 of the Wilson's Disease Protein plays a key role in the transfer of copper from Atox1.
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.
Walker
Joel M
JM
Department of Biochemistry and Molecular Biology, Biomolecular Systems, Oregon Health and Science University, Portland, Oregon 97239-3098, USA.
Huster
Dominik
D
Ralle
Martina
M
Morgan
Clinton T
CT
Blackburn
Ninian J
NJ
Lutsenko
Svetlana
S
D000251:Adenosine Triphosphatases / Q000737:chemistry*; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D001667:Binding, Competitive; D001692:Biological Transport; D002384:Catalysis; D027682:Cation Transport Proteins / Q000737:chemistry*; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D003300:Copper / Q000737:chemistry* / Q000378:metabolism; D000079922:Copper Transport Proteins; D000073840:Copper-Transporting ATPases; D003545:Cysteine / Q000737:chemistry; D018076:DNA, Complementary / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004926:Escherichia coli / Q000378:metabolism; D006801:Humans; D007313:Insecta; D007477:Ions; D056511:Metallochaperones; D008958:Models, Molecular; D015233:Models, Statistical; D018832:Molecular Chaperones / Q000737:chemistry* / Q000378:metabolism; D008969:Molecular Sequence Data; D010766:Phosphorylation; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry; D012542:Scattering, Radiation; D017386:Sequence Homology, Amino Acid; D013052:Spectrometry, X-Ray Emission
C474500:ATOX1 protein, human; D027682:Cation Transport Proteins; D000079922:Copper Transport Proteins; D018076:DNA, Complementary; D007477:Ions; D056511:Metallochaperones; D018832:Molecular Chaperones; D011994:Recombinant Proteins; D003300:Copper; D000251:Adenosine Triphosphatases; D000073840:Copper-Transporting ATPases; D003545:Cysteine