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起核心作用。锌外排转运蛋白可以将Zn2+与类似的细胞质阳离子区分开来，并通过将过量的Zn2+泵出细胞质以抵抗陡峭的跨膜Zn2+浓度梯度来响应其在稳态设定点附近的波动。目前，我们还不知道锌转运体的结构。我们的长期目标是了解哺乳动物锌转运体中锌离子选择性结合和通电运动的结构基础。本研究将重点关注从细菌到哺乳动物等多种生物中锌外排泵的主要家族——阳离子扩散促进剂（CDFs）。具体来说，我们将使用x射线晶体学和生化方法来探索锌的亲和力，选择性和迁移性如何构建到YiiP的结构中，YiiP是一种来自大肠杆菌的原型CDF蛋白。提出了两个具体目标；(1)确定YiiP的晶体结构，揭示模型CDF的分子结构；(2)阐明Zn2+结合和转运的结构基础。这将包括(a)根据现有的生化数据解释晶体结构，(b)确定观察到的金属结合位点在晶体结构中的功能作用，(c)分析金属结合和YiiP去质子化之间的耦合，以及(d)表征响应金属结合的YiiP运动的动力学。所提出的结构和生化分析将导致第一个锌转运体结构的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，这些构象变化驱动Zn2+向上泵送而H+相反向下流动。这些结果将为人类CDF结构的同源性建模打开大门，从而为基于结构的药物设计奠定基础，靶向ZnT- 3，这是一种与阿尔茨海默病发病机制有关的同源人类CDF。

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.