Structural basis for transmembrane Mg2+ transport

摘要Mg2+在多种细胞功能中发挥着重要作用，如酶促辅助因子、脂质衍生的第二信使的调节剂和基因组稳定性的促进剂等。在本论文中，我们将重点研究CorA Mg2+转运体/通道，它是真细菌和古细菌的主要Mg2+摄取系统。最近在中等分辨率下测定了海洋热藓CorA同源物的结构，揭示了一个漏斗状的同构体，具有2个跨膜（TM）螺旋和一个大的，主要是螺旋的细胞外区域。该项目的总体和长期目标是了解Mg2+在原核机械敏感通道中的转运和调控的分子机制。尽管最近对CorA晶体结构的测定大大提高了我们对这类分子的认识，但仍有许多机理问题有待解决。对于通道/传输门控的分子事件尤其如此。在这方面，我们计划通过实验解决几个基本问题：CorA是离子通道的耦合转运体吗？什么区域的CorA形成门(s)和他们如何移动产生门？从Mg2+结合开始到蛋白质运动结束的能量转导步骤的物理基础是什么？关键功能态的结构是什么？我们计划采用的方法将报告群光谱技术（自旋标记/EPR，荧光）x射线晶体学和电生理方法与经典的生化，遗传和分子生物学方法相结合。功能研究将以了解CorA能量转导的物理基础为目标。自旋标记突变体的EPR分析将获得有关CorA的拓扑结构、二级和三级结构以及结构相似的同源物的信息。这些数据将被解释为在每种类型的通道中产生门控通路的不同阶段的主干模型。这一建议开辟了一条新的实验途径，将有助于理解原核生物中Mg2+的动态平衡，特别是离子转运和门控以及信号转导的机制。公共卫生相关性：了解CorA的结构和功能直接关系到健康和疾病，不仅是细胞功能最基本方面的关键因素，而且由于其与真核细胞线粒体Mg++稳态机制的关系。这是相关的，因为已知线粒体在细胞凋亡中的作用。CorA在原核生物中也是一种毒力因子，因此是一种重要的潜在抗生素靶点。

DESCRIPTION (provided by applicant): Structural basis for transmembrane Mg2+ transport Abstract Mg2+ plays an essential role in a variety of cellular functions, as enzymatic cofactor, regulator of lipid-derived second messengers and promoter of genomic stability, among other functions. In this proposal we will focus on the CorA Mg2+ transporter/channel, which functions as the primary Mg2+ uptake system for Eubacteria and Archaea. The structure of the CorA ortholog from Thermotoga maritima has been recently determined at medium resolution, revealing a funnel-shaped homopentamer with 2 transmembrane (TM) helices and a large, mostly helical extracellular region. The overall, long-term goal of this project is to understand the molecular mechanism of Mg2+ transport and regulation in prokaryotic mechanosensitive channels. Although the recent determination of the CorA crystal structures has dramatically improved our knowledge of this class of molecules, a number of mechanistic questions remain to be solved. This is particularly true for the molecular events underlying channel/transport gating. In this respect, we plan to experimentally address several fundamental questions: Is CorA a coupled transporter of an ion channel? What regions of CorA form the gate(s) and how do they move to produce gating? What is the physical basis of the energy transduction steps, starting with Mg2+ binding and culminating in protein motion? What are the structures of the key functional states? The approach we plan to pursue combines reporter-group spectroscopic techniques (spin labeling/EPR, Fluorescence) X-ray crystallography and electrophysiological methods with classical biochemical, genetic and molecular biological procedures. Functional studies will be targeted to understand the physical basis of energy transduction in CorA. Information on the topology, secondary, and tertiary structure of CorA and structurally-similar orthologs will be obtained from EPR analysis of spin labeled mutants. The data will be interpreted to generate backbone models of the different stages of the gating pathway in each type of channel. This proposal opens up a new experimental avenue that will contribute to the understanding of Mg2+ homeostasis in prokaryotes with particular emphasis o the mechanisms of ion translocation and gating, and signal transduction. PUBLIC HEALTH RELEVANCE: Understanding of CorA structure and function relates directly to health and disease, not only as key element in the most basic aspect of cellular function but due to its relationship to the mechanism of mitochondrial Mg++ homeostasis in eukaryotic cells. This is relevant because of the known role of mitochondria in apoptosis. CorA is also a virulence factor in prokaryotes and thus an important potential antibiotic target.