Structure and Function of Copper Transporters

描述（由申请人提供）：本项目的长期目标是了解细胞获得必需微量金属铜的分子机制。为了实现这一目标，我们提出了一个综合的方法，结合电子晶体学，扫描诱变，和生化方法（1）确定参与铜吸收的膜蛋白的结构，和（2）获得有关其功能的机械见解。最初，我们的研究将集中在铜转运蛋白家族（CTR）的膜蛋白。除了细菌之外，CTR蛋白存在于所有形式的生命中，介导铜的摄取，并且出乎意料地，还参与化疗顺铂的摄取。虽然对细胞功能至关重要并用于治疗常见的恶性肿瘤，但CTR蛋白的结构和铜/顺铂转运的分子机制尚未详细研究。因此，我们将集中努力实现三个具体目标： 目的1：确定人CTR 1的结构。 我们已经生成了人类CTR 1的二维晶体。这些晶体将以大约6埃的分辨率产生hCTR 1的第一张图片，这将揭示膜嵌入结构域内α-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，大多数结构和功能上重要的氨基酸残基的膜嵌入域的yCTR 3是不保守的家庭。这表明，在一个共同的整体结构，CTR-蛋白质已经演变出多种解决方案，以允许紧密包装，寡聚化和铜运输。我们将测试这一假设，通过确定螺旋-螺旋包装的相互作用，在两个遥远的相关CTR通过诱变方法的组合。目的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-蛋白质有什么可能是一个铜渗透孔，将允许铜吸收的直接机制。使用取代的半胱氨酸可及性方法和丙氨酸扫描诱变，我们将测试这一假设，确定如何不同的跨膜螺旋有助于这样的铜吸收。

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.