Structure and mechanism of zinc efflux transporters

描述（由申请人提供）：锌转运蛋白在所有生物体的金属代谢和体内平衡中发挥核心作用。我们的长期目标是了解锌转运蛋白的结构和机制以及金属离子、脂质和药物对其功能的调节。这是一个持续的研究项目的第二个竞争性更新，重点是从阳离子扩散促进剂（CDF）家庭锌外排转运。在本预算期间，我们解析了YiiP的晶体结构，YiiP是来自大肠杆菌的原型CDF直向同源物。杆菌晶体结构揭示了Y形结构，其在膜中独特地取向，具有位于蛋白质-脂质界面附近的活性位点。尽管我们现有的晶体学快照，仍然缺乏基于结构的传输模型，并且YiiP晶体结构与其在膜中的天然结构的相关性是不确定的。此外，YiiP是具有用于锌传感的胞质结构域和响应于胞质锌浓度的波动而转运锌离子的跨膜结构域的双模块膜转运蛋白。锌转运的变构调节的构象变化还有待探讨。在下一个预算期间，我们将使用低分辨率的生化技术和高分辨率的晶体学分析相结合，以解决三个问题：（i）YiiP如何移动细胞质锌离子穿过膜屏障，（ii）如何通过周围的脂质重塑活性位点，以及（iii）如何在YiiP构象变化使调节锌转运活动可调的细胞质锌浓度？因此，我们提出了三个具体的目标：（i）开发一个机制模型锌运输，（ii）评估YiiP构象的膜，和（iii）定义的YiiP结构的行动。在过去的两个预算周期中，我们已经开发了功能测定与毫秒时间分辨率的锌运输，并获得了原子分辨率的CDF直系同源物的结构信息。在下一个预算期间，我们将探索CDF在原子细节（aim-1），天然膜环境（aim-2）和行动（aim-3）中的内部运作。 公共卫生相关性：锌外排转运蛋白是阳离子扩散促进剂（CDF）家族的重要分子标志物，也是糖尿病和阿尔茨海默病的重要药物靶点。通过拟议的结构-功能研究获得的基础知识将阐明人类CDF多态性的功能后果，从而为通过遗传筛查进行疾病风险评估提供基础。此外，人CDF直系同源物的同源性建模将有助于结构指导的药物发现工作。

DESCRIPTION (provided by applicant): Zinc transporters play a central role in metal metabolism and homeostasis in all living organisms. Our long- term goal is to understand the structure and mechanism of zinc transporters and their functional regulation by metal ions, lipids, and drugs. This is the second competitive renewal of a continuing research project focusing on zinc-efflux transporters from the cation diffusion facilitator (CDF) family. In the current budget period, we solved the crystal structure of YiiP, a prototypic CDF ortholog from E. coli. The crystal structure reveals a Y- shaped architecture that is uniquely oriented in the membrane with an active-site situated near the protein-lipid interface. Despite our existing crystallographic snapshots, a structure-based transport model still is lacking, and the relevance of the YiiP crystal structure to its native structure in the membrane is uncertain. Further, YiiP is a two-modular membrane transporter with a cytoplasmic domain for zinc sensing, and a transmembrane domain that transports zinc ions in response to fluctuation of cytoplasmic zinc concentrations. The conformational changes underlying the allosteric regulation of zinc transport are yet to be explored. In the next budget period, we will use a combination of low-resolution biochemical techniques and high-resolution crystallographic analysis to address three questions: (i) how does YiiP move a cytoplasmic zinc ion across the membrane barrier, (ii) how is the active-site reshaped by surrounding lipids, and, (iii) how do conformational changes in YiiP enable a regulated zinc-transport activity tunable to the cytoplasmic zinc concentration? Accordingly, we propose three specific aims: (i) developing a mechanistic model for zinc transport, (ii) evaluating YiiP conformations in the membrane, and, (iii) defining the YiiP structure in action. During the past two budget cycles, we have developed functional assays for zinc transport with millisecond time-resolution, and obtained structural information on a CDF ortholog at atomic resolution. In the next budget period, we will explore the inner workings of CDFs in atomic detail (aim-1), in native membrane environments (aim-2), and in action (aim-3). PUBLIC HEALTH RELEVANCE: Zinc efflux transporters in the cation diffusion facilitator (CDF) family are important molecular markers and active drug targets for diabetes and Alzheimer's disease. The fundamental knowledge gained through the proposed structure-function study will illuminate the functional consequences of human CDF polymorphisms, thereby providing the basis for disease-risk assessment by genetic screening. Further, homology modeling of human CDF orthologs will be instrumental to structure-guided drug discovery efforts.