Structural Basis of Selective Binding and Transport of Metal Ions in Membrane Tra

描述（由申请人提供）：锌转运蛋白在调节细胞内锌稳态中发挥核心作用。锌外流转运蛋白可以将 Zn2 与类似的细胞溶质阳离子区分开来，并通过针对陡峭的跨膜 Zn2 浓度梯度将过量的 Zn2+ 从细胞质中泵出，对其在稳态设定点附近的波动做出反应。目前，我们对任何锌转运蛋白的结构尚不了解。我们的长期目标是了解哺乳动物锌转运蛋白中锌离子选择性结合和赋能运动的结构基础。这项拟议的研究将重点关注阳离子扩散促进剂（CDF），它代表了从细菌到哺乳动物等多种生物体中锌外排泵的主要家族。具体来说，我们将使用 X 射线晶体学和生物化学方法来探索如何将锌亲和力、选择性和迁移率构建到 YiiP（一种来自大肠杆菌的原型 CDF 蛋白）的结构中。提出了两个具体目标； (1) 确定 YiiP 的晶体结构，以揭示 CDF 模型的分子结构，(2) 阐明 Zn2 结合和运输的结构基础。这将涉及 (a) 根据现有生化数据解释晶体结构，(b) 确定晶体结构中观察到的金属结合位点的功能作用，(c) 分析金属结合和 YiiP 去质子化之间的耦合，以及 (d) 表征 YiiP 运动响应金属结合的动力学。所提出的结构和生化分析将产生第一个锌转运蛋白结构的 X 射线晶体学解决方案，并为 YiiP 的构象变化提供详细的动力学模型，这些变化驱动 Zn2 的上坡泵送对抗 H 的相反的下坡流动。这些结果将为人类 CDF 结构的同源建模打开大门，从而为针对 ZnT-3（一种与阿尔茨海默病发病机制有关的同源人类 CDF）的基于结构的药物设计奠定基础。

DESCRIPTION (provided by applicant): Zinc transporters play a central role in regulating zinc homeostasis in cells. Zinc efflux transporters can discriminate Zn2+ from similar cytosolic cations and respond to its fluctuations around a homeostatic set point by pumping excess amounts out of the cytoplasm against steep transmembrane Zn2+ concentration gradients. At present, we have no knowledge of the structure of any zinc transporter. Our long-term goal is to understand the structural basis for selective binding and energized movement of zinc ions in mammalian zinc transporters. This proposed study will focus on cation diffusion facilitators (CDFs) that represent a major family of zinc efflux pumps in diverse organisms from bacteria to mammals. Specifically, we will use x-ray crystallographic- and biochemical-approaches to explore how the zinc affinity, selectivity and mobility are built into the structure of YiiP, a prototypic CDF protein from Escherichia coli. Two specific aims are proposed; (1) To determine the crystal structure of YiiP to reveal a molecular architecture for a model CDF, and, (2) To elucidate the structural basis for binding and transport of Zn2+. This will involve (a) interpreting the crystal structure based on existing biochemical data, (b) determining the functional roles of the observed metal-binding sites in the crystal structure, (c) analyzing the coupling between metal binding and YiiP deprotonation, and, (d) characterizing the kinetics of YiiP motions in response to metal binding. The proposed structural and biochemical analyses will lead to the first x-ray crystallographic solution of a zinc transporter structure and afford a detailed kinetic model for the conformational changes in YiiP that drive the uphill pumping of Zn2+ against an opposing downhill flow of H+. These results will open the door to homology modeling of human CDF structures, thereby setting the stage for structure-based drug design targeting ZnT- 3, a homologous human CDF that is implicated in the pathogenesis of Alzheimer's disease.