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得必需痕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）确定参与铜摄取的膜蛋白的结构，以及（2）获得有关其功能的机械洞察力。最初，我们的研究将集中于膜蛋白的铜转运蛋白家族（CTR）。除了细菌外，CTR蛋白以外的各种生命形式存在介导铜的吸收，并且出乎意料地参与了化学治疗的顺铂的摄取。虽然尚未详细研究CTR蛋白质的结构和铜/顺铂转运的分子机制，但尚未详细研究CTR蛋白质的结构和分子机制。因此，我们将把精力集中在三个具体目标上： 目标1：确定人CTR1的结构。 我们已经生成了人类CTR 1的二维晶体。这些晶体将在大约6个Angstrom分辨率下产生HCTR1的第一张图片，这将揭示膜嵌入域中alpha螺旋的详细排列。此外，我们将确定铜和顺铂结合后发生的构象变化，这将是理解铜和顺铂摄取的分子机制的重要步骤。目标2：确定所有CTR蛋白是否共享相同的结构。我们的初步研究表明，在YCTR3的膜嵌入结构域中，大多数结构和功能上重要的氨基酸残基在家族中都不保守。这表明在共享的整体结构中，CTR蛋白质已经进化了多种溶液，以允许堆积，低聚和铜运输。我们将通过通过诱变方法组合在两个远距离相关的CTR中确定螺旋 - 螺旋填料相互作用来检验这一假设。目标3：确定跨膜螺旋在铜摄取中的作用。我们的初步数据表明，CTR蛋白具有可能是铜渗透孔，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有助于这种铜摄取来检验这一假设。