STRUCTURAL BIOLOGY OF COPPER HOMEOSTASIS

遗传和生化研究有力地支持了CTR家族膜蛋白的观点 调节细胞的铜摄取。类似地，铜转运ATPase和细胞内铜的作用 铜分泌中的伴侣和铜离子向铜酶活性部位的转移已被研究 站稳了脚跟。仍然难以捉摸的是，这些不同的成分是如何在分子水平上整合的 这在很大程度上是因为参与这些过程的膜蛋白的结构未知。 这个项目的长期目标是在结构层面上了解铜 转运膜蛋白与细胞内铜伴侣相互作用完成 细胞内铜的定向流动。 目标1的工作利用了我们在人类高亲和力结构确定方面的进展 铜转运蛋白hCTR1。该结构表明hCTR1三聚体中的C-末端聚集 为铜离子和铜伴侣创建结合部位，这将允许直接转移 铜从hCTR1转移到伴侣。值得注意的是，形成这个位点的氨基酸残基在 哺乳动物的CTR2，提出了一个问题，是否所有的CTR-蛋白都使用相同的机制来分配 铜到下游接受者。我们建议用电子结晶学的方法解决hCTR2的结构问题 研究hCTR2中另一组金属结合残基可以在功能上取代的假设 在hCTR1中观察到的C末端伴侣对接位置进一步追求了直接 CTR：伴侣相互作用，目标2的目标是捕获CTR：伴侣复合体，并可视化其 用电子显微镜单粒子法测定其结构。最后，目标3的长期目标是解决 铜转运ATPase 7B的结构，它参与铜的分泌。的关键方面 这些研究将描绘包埋膜和胞浆之间的空间关系 对于ATPase复杂的运输行为的功能和调节至关重要的结构域。

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.