Department of Biochemistry, University of Saskatchewan, Saskatoon, Saskatchewan, Canada.
J Biol Chem. 2011 May 6;286(18):16355-62. doi: 10.1074/jbc.M110.198101. Epub 2011 Mar 11.
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.
威尔逊病（WD）是一种由铜转运 ATP 酶 ATP7B 基因突变引起的铜代谢紊乱。WD 的表型变异很大，其分子基础知之甚少。先前的研究表明，ATP7B N 结构域中的 E1064A 突变会破坏 ATP 结合。我们通过 NMR 确定了包含该突变的 N 结构域的结构，并比较了 E1064A 和另一种 ATP 结合受损的突变体 H1069Q 的性质。E1064A 突变不会改变 N 结构域的整体折叠。然而，包含 Glu（1064）和 His（1069）的α1,α2-螺旋发夹（α-HH）的位置发生了变化。与野生型 ATP 结合形式相比，α-HH 的移动产生了更开放的结构，并且使 ATP 配位残基失准，从而完全解释了 ATP 结合的丧失。在细胞中，ATP7B-E1064A 向反式高尔基体网络的稳定性和靶向性与野生型相比没有显著差异。这与同一α-HH 内的 H1069Q 突变形成对比，该突变在体外和细胞内都大大降低了蛋白的稳定性。两种突变体之间的差异可以归因于在生理温度下 H1069Q 变体中α-HH 的稳定性较低。我们得出结论，N 结构域的结构稳定性而不是 ATP 结合的丧失在 ATP7B 到达反式高尔基体网络的能力中起着决定性作用，从而导致 WD 的表型变异。
