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

描述(由申请人提供)：锌转运体在调节细胞内锌稳态方面起着核心作用。锌外流转运体可以将锌离子与类似的胞内阳离子区分开来，并通过将过量的锌离子从细胞质中抽出，对抗陡峭的跨膜锌离子浓度梯度，对其在动态平衡设定点附近的波动做出反应。目前，我们对任何锌转运体的结构一无所知。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带电运动的结构基础。这项拟议的研究将集中在阳离子扩散促进剂(CDF)，它们代表了从细菌到哺乳动物的不同生物体中锌外排泵的一个主要家族。具体地说，我们将使用X射线结晶学和生化方法来探索锌的亲和力、选择性和流动性是如何构建在Escherichia Coli的CDF原型蛋白YIIP的结构中的。提出了两个具体的目标：(1)确定YIIP的晶体结构，以揭示模型CDF的分子结构；(2)阐明锌结合和运输的结构基础。这将涉及(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部位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YIIP运动响应于金属结合的动力学。提出的结构和生化分析将导致第一个锌转运体结构的X射线结晶学解决方案，并提供一个详细的动力学模型，说明驱动向上泵送锌2以对抗相反的下坡H流的Y1iP的构象变化。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的基于结构的药物设计奠定基础。ZNT-3是一种与阿尔茨海默病的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.