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内的金属代谢和动态平衡中起着核心作用。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。这是一个持续研究项目的第二次竞争性更新，该项目专注于阳离子扩散促进剂(CDF)家族的锌外流转运蛋白。在本预算期间，我们解决了来自E.Coli的CDF同源原型蛋白YIIP的晶体结构。晶体结构显示出在膜中唯一定向的Y形结构，其活性中心位于蛋白质-脂质界面附近。尽管我们现有的晶体快照，仍然缺乏一个基于结构的运输模型，并且YIIP晶体结构与其在膜中的天然结构的关联性是不确定的。此外，YIIP是一种双模膜转运蛋白，具有锌敏感的细胞质结构域和响应细胞质锌浓度波动而转运锌离子的跨膜结构域。锌转运的变构调控背后的构象变化还有待研究。在下一个预算期间，我们将结合低分辨率生化技术和高分辨率结晶学分析来解决三个问题：(I)YiiP如何将细胞质锌离子移动到膜屏障上，(Ii)活性部位如何被周围的脂类重塑，以及(Iii)YiiP的构象变化如何使调节的锌转运活性可调节到细胞质锌浓度？因此，我们提出了三个具体的目标：(I)建立锌转运的机制模型，(Ii)评估膜中的YIIP构象，以及(Iii)确定作用中的YIIP结构。在过去的两个预算周期中，我们开发了毫秒时间分辨率的锌转运功能分析，并获得了原子分辨率的CDF直系物的结构信息。在下一个预算期，我们将探索CDF在原子细节(AIM-1)、自然膜环境中(AIM-2)和行动中(AIM-3)的内部工作原理。

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).