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J. Biol. Chem., Vol. 279, Issue 15, 15376-15384, April 9, 2004

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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 J. Blackburn||, and Svetlana Lutsenko**

From the Department of Biochemistry and Molecular Biology and ^||Department of Environmental and Biomolecular Systems, Oregon Health and Science University, Portland, Oregon 97239-3098

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.

Received for publication, January 5, 2004 , and in revised form, January 26, 2004.

^* This work was funded by National Science Foundation Grants MCB-00110057 (to S. L.) and PPG 1-P01-GM067166-01 (to S. L. and N. J. B.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

These authors contributed equally to this work.

^¶ Recipient of Deutsche Forschungsgemeinschaft postdoctoral fellowship HU 932/1-1.

^** To whom correspondence should be addressed. Tel.: 503-494-6953; Fax: 503-494-8393; E-mail: lutsenko{at}ohsu.edu.

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