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.
Highlights
Copper is essential for cell growth and development as a cofactor of cytochrome c oxidase, copper,zinc-dependent superoxide dismutase, ceruloplasmin, lysyl oxidase, and other important metabolic enzymes
The Atox1-mediated Transfer of Copper to N-Wilson’s disease protein (WNDP) Selectively Protects Cysteines in metal-binding site 2 (MBS2) against Labeling with CPM— Previously, we demonstrated that Atox1 transferred copper to N-WNDP, stimulating the catalytic activity of WNDP [9]
Copper binding to N-WNDP protects the metal-coordinating cysteines in N-WNDP from labeling with CPM [14]; the difference in the intensity of fluorescent labeling can be utilized for identification of the copper-bound metal binding subdomains (MBS)
Summary
Recombinant Proteins—Expression and purification of Atox were carried out using a published protocol [9]. The copper-bound form of Atox (Cuϩ-Atox1) was generated by incubation of apoAtox with equimolar amount of a copper-glutathione complex as described [9] and dialyzed into buffer A. After a wash with 3 resin volumes of buffer A, apoAtox or Cuϩ-Atox was added and incubated with N-WNDP for 10 min. For copper binding in the absence of Atox, N-WNDP was incubated with increasing concentrations of CuCl2 (Sigma) dissolved in buffer A containing freshly prepared 200 M ascorbate (Fisher). Under these conditions all copper is present in the reduced form (our data). The membranes were resuspended in phosphorylation buffer containing 100 M ascorbate, 100 M Tris(2-carboxyethyl)phosphine hydrochloride followed by the addition of increasing concentrations of CuCl2 or Cuϩ-Atox. The incorporation of 32P into WNDP was normalized to the WNDP protein levels
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