STRUCTURAL BIOLOGY OF COPPER HOMEOSTASIS

遗传和生物化学研究有力地支持了膜蛋白CTR家族 介导细胞铜摄取。同样，铜转运ATP酶和细胞内铜的作用 在铜分泌和铜离子转移到铜酶的活性位点中的分子伴侣已经被 牢固确立。仍然难以捉摸的是这些不同的成分如何在分子水平上整合 水平，主要是因为参与这些过程的膜蛋白的结构是未知的。 该项目的长期目标是在结构层面上了解铜 转运膜蛋白和细胞铜分子伴侣相互作用， 细胞内铜的定向流动。 实现目标1的工作利用了我们在人高亲和力多肽的结构测定方面的进展。 铜转运蛋白hCTR 1。该结构表明hCTR 1三聚体中C-末端的聚集 为铜离子和铜分子伴侣创造了一个结合位点，这将允许直接转移 铜从hCTR 1转移到分子伴侣。值得注意的是，形成该位点的氨基酸残基在哺乳动物中不存在。 哺乳动物的CTR 2，提出了一个问题，是否所有的CTR蛋白质使用相同的机制来分配 铜到下游受体。我们建议通过电子晶体学解析hCTR 2的结构， 研究hCTR 2中一组替代的金属结合残基可以在功能上取代 在hCTR 1中观察到的C-末端分子伴侣对接位点 CTR：分子伴侣相互作用，目的2的目标是捕获CTR：分子伴侣复合物，并可视化它们的相互作用。 用电子显微镜单粒子方法进行结构化。最后，目标3的长期目标是 铜转运ATP酶7 B的结构，其参与铜分泌。的关键方面 这些研究将描述包埋在细胞膜和细胞质之间的空间关系。 这些结构域对于ATP酶的复杂运输行为的功能和调节至关重要。

Genetic and biochemical studies lend strong support to the idea that the CTR-family of membrane proteins mediate cellular copper uptake. Similarly, the roles of copper transporting ATPases and intracellular copper chaperones in copper secretion and transfer of copper ions to the active sites of cuproenzymes have been firmly established. What remains elusive, is how these different components are integrated at the molecular level, largely because the structures of the membrane proteins involved in these processes are unknown. The longterm goal of this project is to understand at a structural level the mechanism by which copper transporting membrane proteins and cellular copper chaperones interact with each other to accomplish directed flow of copper within cells. Work towards Aim 1 capitalizes on our progress in the structure determination of the human high affinity copper transporter hCTR1. The structure suggests that clustering of the C-termini in the hCTR1 trimer creates a binding site for both copper ions and copper chaperones, which would allow for a direct transfer of copper from hCTR1 to the chaperones. Notably, the amino acid residues forming this site are absent in mammalian CTR2, raising the question whether all CTR-proteins use the same mechanism to distribute copper to downstream acceptors. We propose to solve the structure of hCTR2 by electron crystallography to investigate the hypothesis that in hCTR2 an alternate set of metal binding residues can functionally replace the C-terminal chaperone docking site observed in hCTRl Further pursuing the idea of direct CTR:chaperone interactions, the goal of Aim 2 is to trap CTR:chaperone complexes, and to visualize their structure by electron microscopic single particle methods. Finally, the longterm goal for Aim 3 is to solve the structure of the copper transporting ATPase 7B, which is involved in copper secretion. The key aspect of these studies will be to delineate the spatial relationships between the membrane embedded and cytosolic domains that are critical for function and the regulation of the ATPase's complex trafficking behavior.