Structure and Function of Copper Transporters

铜转运蛋白的结构和功能

• 批准号: 7924962
• 负责人: VINZENZ UNGER
• 金额: $ 9.87万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-30 至 2010-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7924962
• 关键词: Ablation; Affinity; Alanine; Alzheimer' s Disease; Amino Acids; Bacteria; Binding; Biochemical; Carbon; Cell physiology; Cells; Cisplatin; Complement; Copper; Creutzfeldt-Jakob Syndrome; Cysteine; Data; Disease; Embryo; Family; Future; Generations; Goals; Homeostasis; Human; Impairment; Knowledge; Life; Malignant Neoplasms; Measures; Mediating; Membrane; Membrane Proteins; Metabolism; Methods; Molecular; Mus; Mutagenesis; Phenotype; Positioning Attribute; Protein Family; Proteins; Research Personnel; Resistance; Resolution; Role; Sampling; Scanning; Site; Solutions; Source; Structural Models; Structure; Suppressor Mutations; Testing; Trace metal; Tryptophan; Vestibule; Visual; Work; alpha helix; base; common treatment; electron crystallography; in vivo; insight; membrane model; monomer; mutant; orexin A receptor; programs; research study; tumor; two-dimensional; uptake

DESCRIPTION (provided by applicant): The long-term goal of this project is to understand the molecular mechanism by which cells acquire the essential trace metal copper. Towards this goal, we propose an integrated approach that combines electron crystallography, scanning mutagenesis, and biochemical methods to (1) determine the structure of the membrane proteins that are involved in copper uptake, and (2) to gain mechanistic insights about their function. Initially, our studies will focus on the copper transporter family (CTR) of membrane proteins. Being present in all forms of life, except bacteria, CTR-proteins mediate uptake of copper and, unexpectedly, also participate in the uptake of the chemotherapeutic cisplatin. While essential for cellular function and exploited for treating common malignancies, the structure of CTR-proteins and the molecular mechanism of copper/cisplatin transport have not been studied in detail. We therefore will focus our efforts towards three specific aims: Aim 1: Determine the Structure of human CTR1. We have generated two-dimensional crystals of human CTR 1. These crystals will yield a first picture of hCTR1 at approximately 6 Angstrom resolution, which will reveal the detailed arrangement of alpha-helices within the membrane embedded domain. Moreover, we will determine conformational changes that occur upon binding of copper and cisplatin, which will be an important step towards understanding the molecular mechanism underlying the uptake of copper and cisplatin. Aim 2: Determine whether all CTR proteins share the same structure. Our preliminary studies show that most structurally and functionally important amino acid residues in the membrane embedded domain of yCTR3 are not conserved in the family. This suggests that within a shared overall structure, CTR-proteins have evolved multiple solutions to allow tight packing, oligomerization and copper transport. We will test this hypothesis by determining helix-helix packing interaction in two distantly related CTRs through a combination of mutagenesis approaches. Aim 3: Determine the Role of the Transmembrane Helices in Copper Uptake. Our preliminary data suggest that CTR-proteins have what may be a copper permeable pore that would allow copper uptake by a direct mechanism. Using the substituted cysteine accessibility method and alanine scanning mutagenesis, we will test this hypothesis by determining how the different transmembrane helices contribute to such a copper uptake.

描述（由申请人提供）：该项目的长期目标是了解细胞获取必需微量金属铜的分子机制。为了实现这一目标，我们提出了一种结合电子晶体学、扫描诱变和生化方法的综合方法，以（1）确定参与铜吸收的膜蛋白的结构，以及（2）获得有关其功能的机制见解。最初，我们的研究将集中在膜蛋白的铜转运蛋白家族（CTR）上。 CTR 蛋白存在于除细菌之外的所有生命形式中，介导铜的摄取，并且出人意料地还参与化疗顺铂的摄取。虽然 CTR 蛋白对于细胞功能至关重要并用于治疗常见恶性肿瘤，但其结构和铜/顺铂转运的分子机制尚未得到详细研究。因此，我们将集中精力实现三个具体目标： 目标 1：确定人类 CTR1 的结构。 我们已经生成了人类 CTR 1 的二维晶体。这些晶体将产生约 6 埃分辨率的 hCTR1 的第一张图片，这将揭示膜嵌入域内 α 螺旋的详细排列。此外，我们将确定铜和顺铂结合时发生的构象变化，这将是理解铜和顺铂吸收分子机制的重要一步。目标 2：确定所有 CTR 蛋白是否具有相同的结构。我们的初步研究表明，yCTR3 膜嵌入域中大多数结构和功能重要的氨基酸残基在该家族中并不保守。这表明，在共享的整体结构中，CTR 蛋白已经进化出多种解决方案，以实现紧密堆积、寡聚化和铜运输。我们将通过结合诱变方法确定两个远缘相关 CTR 中的螺旋-螺旋堆积相互作用来检验这一假设。目标 3：确定跨膜螺旋在铜吸收中的作用。我们的初步数据表明，CTR 蛋白可能具有铜可渗透孔，可以通过直接机制吸收铜。使用取代的半胱氨酸可及性方法和丙氨酸扫描诱变，我们将通过确定不同的跨膜螺旋如何促进这种铜的吸收来检验这一假设。