NMR Characterization of Copper-Binding Domains 4-6 of ATP7B
NMR Characterization of Copper-Binding Domains 4-6 of ATP7B
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DOI：
10.1021/bi1008535
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发表时间：
2010-10-05
期刊：
影响因子：
2.9
通讯作者：
Forman-Kay, Julie D.
中科院分区：
文献类型：
--
作者：
Fatemi, Negah;Korzhnev, Dmitry M.;Forman-Kay, Julie D.
肝豆状核变性蛋白（ATP 7 B）是P型ATP酶超家族的铜转运蛋白。在铜稳态中起着核心作用，并与铜伴侣Atox 1相互作用。ATP 7 B的N-末端由六个铜结合结构域（WCBD）组成，每个WCBD能够结合一个+1氧化态的铜原子。为了更好地理解铜与这些结构域结合的调节作用，我们对WCBD 4 -6（ATP 7 B的结构域4-6）进行了NMR表征。对载脂蛋白和Cu（I）结合的WCBD 4 -6的N-15弛豫测量表明，该三结构域构建物的动力学性质没有显著变化;结构域4和5之间的接头保持柔性，结构域5和6不形成完全刚性的二聚体而是相对于彼此具有一定的柔性，并且在这两种状态下畴的相对取向变化最小。我们还表明，与以前的报告相反，Atox 1和铜结合结构域之间的蛋白质-蛋白质相互作用发生，即使在没有铜。WCBD 1 -6的载脂蛋白和Cu（I）结合光谱的比较表明，Cu（I）的结合不会诱导形成作为单一实体翻滚的单元，这与我们对WCBD 4 -6的结果一致。我们建议，铜转移到威尔逊铜-ATP酶的N-末端结构域之间发生通过蛋白质相互作用，这是由连接器的灵活性和相对于彼此的结构域的运动自由促进。
The Wilson disease protein (ATP7B) is a copper-transporting member of the P-type ATPase superfamily, which. plays a central role in copper homeostasis and interacts with the copper chaperone Atox1. The N-terminus of ATP7B is comprised of six copper-binding domains (WCBDs), each capable of binding one copper atom in the +1 oxidation state. To better understand the regulatory effect of copper binding to these domains, we have performed NMR characterization of WCBD4-6 (domains 4-6 of ATP7B). N-15 relaxation measurements on the apo and Cu(I)-bound WCBD4-6 show that there is no dramatic change in the dynamic properties of this three-domain construct; the linker between domains 4 and 5 remains flexible, domains 5 and 6 do not form a completely rigid dimer but rather have some flexibility with respect to each other, and there is minimal change in the relative orientation of the domains in the two states. We also show that, contrary to previous reports, the protein-protein interaction between Atox1 and the copper-binding domains takes place even in the absence of copper. Comparison of apo and Cu(I)-bound spectra of WCBD1-6 shows that binding of Cu(I) does not induce the formation of a unit that tumbles as a single entity, consistent with our results for WCBD4-6. We propose that copper transfer to and between the N-terminal domains of the Wilson Cu-ATPase occurs via protein interactions that are facilitated by the flexibility of the linkers and the motional freedom of the domains with respect to each other.