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

Joel M. Walker, Dominik Huster, Martina Ralle, Clinton T. Morgan, Ninian Blackburn, Svetlana Lutsenko

Research output: Contribution to journalArticle

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Abstract

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.

Original languageEnglish (US)
Pages (from-to)15376-15384
Number of pages9
JournalJournal of Biological Chemistry
Volume279
Issue number15
DOIs
StatePublished - Apr 9 2004

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Copper
Metals
Binding Sites
Cysteine
Wilson disease protein
Metallochaperones
X-Ray Absorption Spectroscopy
Mutagenesis
Hepatolenticular Degeneration
X ray absorption spectroscopy
Chelating Agents
Site-Directed Mutagenesis
Liver
Labeling
Catalyst activity
Proteins
Mutation
Geometry

ASJC Scopus subject areas

  • Biochemistry

Cite this

The N-terminal Metal-binding Site 2 of the Wilson's Disease Protein Plays a Key Role in the Transfer of Copper from Atox1. / Walker, Joel M.; Huster, Dominik; Ralle, Martina; Morgan, Clinton T.; Blackburn, Ninian; Lutsenko, Svetlana.

In: Journal of Biological Chemistry, Vol. 279, No. 15, 09.04.2004, p. 15376-15384.

Research output: Contribution to journalArticle

Walker, Joel M. ; Huster, Dominik ; Ralle, Martina ; Morgan, Clinton T. ; Blackburn, Ninian ; Lutsenko, Svetlana. / The N-terminal Metal-binding Site 2 of the Wilson's Disease Protein Plays a Key Role in the Transfer of Copper from Atox1. In: Journal of Biological Chemistry. 2004 ; Vol. 279, No. 15. pp. 15376-15384.
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