Abstract
Wilson's disease, an autosomal disorder associated with vast accumulation of copper in tissues, is caused by mutations in a gene encoding a copper-transporting ATPase (Wilson's disease protein, WNDP). Numerous mutations have been identified throughout the WNDP sequence, particularly in the Lys(1010)-Lys(1325) segment; however, the biochemical properties and molecular mechanism of WNDP remain poorly characterized. Here, the Lys(1010)-Lys(1325) fragment of WNDP was overexpressed, purified, and shown to form an independently folded ATP-binding domain (ATP-BD). ATP-BD binds the fluorescent ATP analogue trinitrophenyl-ATP with high affinity, and ATP competes with trinitrophenyl-ATP for the binding site; ADP and AMP appear to bind to ATP-BD at the site separate from ATP. Purified ATP-BD hydrolyzes ATP and interacts specifically with the N-terminal copper-binding domain of WNDP (N-WNDP). Strikingly, copper binding to N-WNDP diminishes these interactions, suggesting that the copper-dependent change in domain-domain contact may represent the mechanism of WNDP regulation. In agreement with this hypothesis, N-WNDP induces conformational changes in ATP-BD as evidenced by the altered nucleotide binding properties of ATP-BD in the presence of N-WNDP. Significantly, the effects of copper-free and copper-bound N-WNDP on ATP-BD are not identical. The implications of these results for the WNDP function are discussed.
Highlights
Copper overload is as deleterious to cells as copper deficiency, presumably because of the ability of copper to participate in reactions that generate highly reactive oxygen species [3]
The maximum emission wavelength (max) for ATP-binding domain (ATP-BD) is 330 nm in contrast to max for free L-Trp (348 nm), indicating that the purified ATP-BD is folded and that the tryptophan residue in folded ATP-BD is buried. In agreement with this conclusion, unfolding of ATP-BD with 6 M guanidine hydrochloride (GuHCl) is accompanied by a shift of max to 348 nm, which is identical to the maximum of the solvent-exposed tryptophan (Fig. 3)
ATP-BD is relatively resistant to treatment with denaturing reagents: at least 2.0 M GuHCl was required to observe some red shift in the max, and in the presence of 6 M GuHCl the maximum was fully shifted to 348 nm
Summary
Copper overload is as deleterious to cells as copper deficiency, presumably because of the ability of copper to participate in reactions that generate highly reactive oxygen species [3]. The specific activity (67.6 Ϯ 15.7 nmol Pi/mg/min) was reproducible for protein batches with different ATP-BD concentrations, and essentially no ATPase activity was detected in control eluates from the Ni2ϩ affinity resin, containing a similar amount of unrelated His tag protein or just a buffer (Fig. 6).
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