Crystallization and preliminary X-ray studies of the N-domain of the Wilson disease associated protein

Department of Biochemistry, University of Saskatchewan, Saskatoon, Saskatchewan, S7N 5E5, Canada.
Acta Crystallographica Section F Structural Biology and Crystallization Communications (Impact Factor: 0.53). 07/2009; 65(Pt 6):621-4. DOI: 10.1107/S1744309109017023
Source: PubMed


Wilson disease associated protein (ATP7B) is essential for copper transport in human cells. Mutations that affect ATP7B function result in Wilson's disease, a chronic copper toxicosis. Disease-causing mutations within the N-domain of ATP7B (WND) are known to disrupt ATP binding, but a high-resolution X-ray structure of the ATP-binding site has not been reported. The N-domain was modified to delete the disordered loop comprising residues His1115-Asp1138 (WNDDelta(1115-1138)). Unlike the wild-type N-domain, WNDDelta(1115-1138) formed good-quality crystals. Synchrotron diffraction data have been collected from WNDDelta(1115-1138) at the Canadian Light Source. A native WNDDelta(1115-1138) crystal diffracted to 1.7 A resolution and belonged to space group P4(2)2(1)2, with unit-cell parameters a = 39.2, b = 39.2, c = 168.9 A. MAD data were collected to 2.7 A resolution from a SeMet-derivative crystal with unit-cell parameters a = 38.4, b = 38.4, c = 166.7 A. The WNDDelta(1115-1138) structure is likely to be solved by phasing from multiwavelength anomalous diffraction (MAD) experiments.

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    ABSTRACT: Wilson disease (WD) is a disorder of copper metabolism caused by mutations in the Cu-transporting ATPase ATP7B. WD is characterized by significant phenotypic variability, the molecular basis of which is poorly understood. The E1064A mutation in the N-domain of ATP7B was previously shown to disrupt ATP binding. We have now determined, by NMR, the structure of the N-domain containing this mutation and compared properties of E1064A and H1069Q, another mutant with impaired ATP binding. The E1064A mutation does not change the overall fold of the N-domain. However, the position of the α1,α2-helical hairpin (α-HH) that houses Glu(1064) and His(1069) is altered. The α-HH movement produces a more open structure compared with the wild-type ATP-bound form and misaligns ATP coordinating residues, thus explaining complete loss of ATP binding. In the cell, neither the stability nor targeting of ATP7B-E1064A to the trans-Golgi network differs significantly from the wild type. This is in a contrast to the H1069Q mutation within the same α-HH, which greatly destabilizes protein both in vitro and in cells. The difference between two mutants can be linked to a lower stability of the α-HH in the H1069Q variant at the physiological temperature. We conclude that the structural stability of the N-domain rather than the loss of ATP binding plays a defining role in the ability of ATP7B to reach the trans-Golgi network, thus contributing to phenotypic variability in WD.
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