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起核心作用。我们的长期目标是了解锌转运体的结构和机制，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，重点是来自阳离子扩散促进剂（CDF）家族的锌外排转运体。在当前预算期内，我们解决了来自大肠杆菌的原型CDF同源物YiiP的晶体结构。晶体结构显示出一个独特的Y形结构，在膜上有一个活性位点位于蛋白-脂质界面附近。尽管我们已有晶体学快照，但仍然缺乏基于结构的输运模型，并且YiiP晶体结构与其在膜中的天然结构的相关性尚不确定。此外，YiiP是一种双模块膜转运蛋白，其胞质结构域用于锌感应，而跨膜结构域则响应胞质锌浓度的波动而转运锌离子。锌转运的变构调节背后的构象变化尚未被探索。在下一个预算期内，我们将使用低分辨率生化技术和高分辨率晶体学分析的组合来解决三个问题：(i) YiiP如何将细胞质锌离子穿过膜屏障，（ii）活性位点如何被周围的脂质重塑，以及（iii） YiiP的构象变化如何使受调节的锌运输活性调节到细胞质锌浓度？因此，我们提出了三个具体目标：(i)建立锌运输的机制模型，（ii）评估膜中的YiiP构象，以及（iii）定义作用中的YiiP结构。在过去的两个预算周期中，我们开发了具有毫秒时间分辨率的锌运输功能分析，并在原子分辨率下获得了CDF同源物的结构信息。在下一个预算期内，我们将在原子细节（aim-1）、天然膜环境（aim-2）和行动（aim-3）中探索CDFs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).