Structure and Function of Copper Transporters

描述(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结晶学、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，(2)从机制上了解它们的功能。首先，我们的研究将集中在膜蛋白的铜转运蛋白家族(CTR)上。CTR-蛋白存在于除细菌以外的所有生命形式中，介导铜的吸收，并且出人意料地也参与了化疗顺铂的吸收。虽然CTR-蛋白对细胞功能至关重要，并被用于治疗常见的恶性肿瘤，但其结构和铜/顺铂转运的分子机制尚未被详细研究。因此，我们将集中努力实现三个具体目标： 目的1：确定人CTR1的结构。我们已经产生了人CTR-1的二维晶体。这些晶体将产生大约6埃分辨率的hCTR1的第一张图片，这将揭示膜嵌入结构域中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，这将是了解铜和顺铂吸收的分子机制的重要一步。目的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膜嵌入区的大多数结构和功能上重要的氨基酸残基在该家族中并不保守。这表明在一个共享的整体结构中，CTR-蛋白质已经进化出多种解决方案，以允许紧密堆积、齐聚和铜的运输。我们将通过结合突变方法确定两个远缘CTR中的螺旋-螺旋堆积相互作用来检验这一假设。目的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-蛋白质可能有一个铜渗透孔，这将允许通过直接机制吸收铜。使用替代半胱氨酸可及性方法和丙氨酸扫描突变，我们将通过确定不同的跨膜螺旋如何促进这种铜摄取来检验这一假说。

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.