STRUCTURAL BIOLOGY OF COPPER HOMEOSTASIS

铜稳态的结构生物学

基本信息

批准号：
7690563
负责人：
VINZENZ UNGER
金额：
$ 27.75万
依托单位：
JOHNS HOPKINS UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2009
资助国家：
美国
起止时间：
2009-09-15 至 2014-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7690563
关键词：
ATP phosphohydrolase Active Sites Address Affinity Alzheimer&apos s Disease Amino Acids Antineoplastic Agents Area Behavior Binding Binding Sites Biochemical Biology C-terminal Cell physiology Cell secretion Cells Cisplatin Complex Copper Creutzfeldt-Jakob Syndrome Crystallization Cues Data Disease Docking Electrons Engineering Family Genetic Glycophorin A Goals Homeostasis Human Ions Letters Light Link Mediating Membrane Membrane Proteins Membrane Transport Proteins Menkes Kinky Hair Syndrome Metals Methods Microscopic Molecular Molecular Chaperones Mus Parkinson Disease Physiological Process Proteins Regulation Resolution Role Sampling Site Structure Testing Wilson disease protein Work Yeasts base copper-transporting ATPase crosslink electron crystallography genetic regulatory protein in vivo insight metalloenzyme neuropathology novel orexin A receptor orexin B receptor particle reconstruction response spatial relationship structural biology tool trafficking uptake

项目摘要

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.

遗传和生化研究有力地支持了膜蛋白 CTR 家族的观点介导细胞铜的吸收。同样，铜转运 ATP 酶和细胞内铜的作用铜分泌和铜离子转移到铜酶活性位点的分子伴侣已被牢固地建立起来。仍然难以捉摸的是这些不同的成分如何在分子上整合水平，很大程度上是因为参与这些过程的膜蛋白的结构尚不清楚。该项目的长期目标是从结构层面了解铜转运膜蛋白和细胞铜伴侣相互作用以完成铜在细胞内的定向流动。实现目标 1 的工作利用了我们在人类高亲和力结构确定方面取得的进展铜转运蛋白 hCTR1。该结构表明 hCTR1 三聚体中 C 末端的聚集为铜离子和铜伴侣创建一个结合位点，这将允许直接转移铜从 hCTR1 到伴侣。值得注意的是，形成该位点的氨基酸残基不存在于哺乳动物 CTR2，提出了一个问题：是否所有 CTR 蛋白都使用相同的机制来分布铜到下游受体。我们建议通过电子晶体学解析 hCTR2 的结构研究以下假设：在 hCTR2 中，一组替代的金属结合残基可以在功能上替代在 hCTRl 中观察到的 C 末端伴侣对接位点进一步追求直接的想法 CTR：伴侣相互作用，目标 2 的目标是捕获 CTR：伴侣复合物，并可视化它们通过电子显微镜单粒子方法进行结构。最后，目标 3 的长期目标是解决铜转运 ATP 酶 7B 的结构，参与铜分泌。关键方面这些研究将描绘膜嵌入和胞质之间的空间关系这些结构域对于 ATP 酶复杂运输行为的功能和调节至关重要。