Structure and Function of Copper Transporters

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)确定参与铜摄取的膜蛋白的结构，(2)获得有关其功能的机制见解。首先，我们的研究将集中在膜蛋白的铜转运蛋白家族（CTR）上。存在于除细菌外的所有生命形式中，cr -蛋白介导铜的摄取，出乎意料的是，它还参与化疗顺铂的摄取。虽然对细胞功能和治疗常见恶性肿瘤至关重要，但cr -蛋白的结构和铜/顺铂转运的分子机制尚未得到详细研究。因此，我们将集中努力实现三个具体目标：

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.