Structure of human Wilson protein domains 5 and 6 and their interplay with domain 4 and the copper chaperone HAM in copper uptake
Structure of human Wilson protein domains 5 and 6 and their interplay with domain 4 and the copper chaperone HAM in copper uptake
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DOI：
10.1073/pnas.0504472103
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发表时间：
2006-04-11
影响因子：
11.1
通讯作者：
Huffman, DL
中科院分区：
文献类型：
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作者：
Achila, D;Banci, L;Huffman, DL
人Wilson蛋白是一种位于分泌途径的铜转运ATP酶，N端有6个金属结合结构域。在这里，我们专注于最接近铜泵的囊泡部分的金属结合域的功能，即，结构域4（WLN 4）和结构域5和6的构建体（WLN 5 -6）。为了比较的目的，还用结构域2（WLN 2）进行了一些实验。apoWLN 5 -6的溶液结构由两个通过短连接子连接的铁氧还蛋白折叠组成，N-15弛豫速率测量表明它在溶液中表现为一个单元。用金属伴侣Cu（I）HAH 1对apoWLN 5 -6进行NMR滴定显示两种蛋白质之间没有形成络合物并且没有铜交换，而用WLN 4对Cu（I）HAH 1进行滴定显示形成加合物，该加合物在NMR时间尺度上与分离的蛋白质种类快速交换，如通过15 N弛豫数据所证实的。在Cu（I）HAH 1和WLN 2之间也观察到类似的相互作用;然而，蛋白质混合物中加合物的相对量较低。用Cu（I）WLN 4对apoWLN 5 -6进行NMR滴定显示铜转移，首先转移到WLN 6，然后转移到WLN 5，而没有形成加合物。因此，我们认为WLN 4和WLN 2是来自HAH 1的Cu（I）的两个受体，然后在ATP驱动的铜跨囊泡膜转运之前，以某种方式将铜路由到WLN 5 -6。
Human Wilson protein is a copper-transporting ATPase located in the secretory pathway possessing six N-terminal metal-binding domains. Here we focus on the function of the metal-binding domains closest to the vesicular portion of the copper pump, i.e., domain 4 (WLN4), and a construct of domains 5 and 6 (WLN5-6). For comparison purposes, some experiments were also performed with domain 2 (WLN2). The solution structure of apoWLN5-6 consists of two ferredoxin folds connected by a short linker, and N-15 relaxation rate measurements show that it behaves as a unit in solution. An NMR titration of apoWLN5-6 with the metallochaperone Cu(I)HAH1 reveals no complex formation and no copper exchange between the two proteins, whereas titration of Cu(I)HAH1 with WLN4 shows the formation of an adduct that is in fast exchange on the NMR time scale with the isolated protein species as confirmed by 15N relaxation data. A similar interaction is also observed between Cu(I)HAH1 and WLN2; however, the relative amount of the adduct in the protein mixture is lower. An NMR titration of apoWLN5-6 with Cu(I)WLN4 shows copper transfer, first to WLN6 then to WLN5, without the formation of an adduct. Therefore, we suggest that WLN4 and WLN2 are two acceptors of Cu(I) from HAH1, which then somehow route copper to WLN5-6, before the ATP-driven transport of copper across the vesicular membrane.