Structural basis for transmembrane Mg2+ transport

跨膜 Mg2 运输的结构基础

• 批准号: 8136457
• 负责人: Eduardo A Perozo
• 金额: $ 32.11万
• 依托单位: UNIVERSITY OF CHICAGO
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-01 至 2013-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8136457
• 关键词: Address; Alkalies; Antibiotics; Antibodies; Apoptosis; Archaea; Binding; Biochemical Genetics; Biological; Biological Assay; Biological Models; Cations; Cell physiology; Chemistry; Computing Methodologies; Coupled; Crystallography; DNA Sequence Rearrangement; Data; Disease; Electrodes; Eubacterium; Eukaryotic Cell; Event; Fluorescence; Generations; Genome Stability; Goals; Health; Homeostasis; Hydration status; In Vitro; Indium; Ion Channel; Ion Channel Gating; Ions; Knowledge; Lead; Link; Lipids; Magnesium; Measures; Membrane; Membrane Proteins; Metals; Methods; Mitochondria; Modeling; Molecular; Molecular Chaperones; Monitor; Motion; Mutagenesis; Nature; Oocytes; Orthologous Gene; Pathway interactions; Play; Procedures; Process; Prokaryotic Cells; Proteins; Regulation; Reporter; Resolution; Role; Second Messenger Systems; Shapes; Signal Transduction; Solutions; Solvents; Specificity; Spin Labels; Staging; Structure; System; Techniques; Thermotoga maritima; Transition Elements; Vertebral column; Virulence Factors; X-Ray Crystallography; abstracting; base; cofactor; extracellular; improved; in vivo; loss of function; member; mutant; promoter; protein folding; second messenger; three-dimensional modeling; tool; uptake; voltage clamp

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.

描述（由申请人提供）：跨膜Mg 2+转运的结构基础摘要Mg 2+在多种细胞功能中起重要作用，作为酶辅因子、脂质衍生的第二信使的调节剂和基因组稳定性的启动子，以及其它功能。在这个建议中，我们将集中在CorA Mg 2+转运蛋白/通道，其功能作为真细菌和假单胞菌的主要Mg 2+摄取系统。最近在中等分辨率下确定了来自海栖热袍菌的CorA直向同源物的结构，揭示了具有2个跨膜（TM）螺旋和大的、主要是螺旋的胞外区的漏斗形同源五聚体。该项目的总体长期目标是了解原核机械敏感通道中Mg 2+转运和调控的分子机制。虽然最近确定的CorA晶体结构，大大提高了我们的知识，这类分子，一些机械的问题仍然有待解决。这对于通道/转运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蛋白吗？CorA的哪些区域形成门控，它们如何移动以产生门控？从Mg 2+结合到蛋白质运动的能量转换步骤的物理基础是什么？关键功能状态的结构是什么？我们计划采用的方法结合了荧光基团光谱技术（自旋标记/EPR，荧光）X射线晶体学和电生理学方法与经典的生物化学，遗传学和分子生物学程序。功能研究的目标是了解CorA中能量转导的物理基础。从自旋标记突变体的EPR分析中获得关于CorA和结构相似的直系同源物的拓扑结构、二级和三级结构的信息。将对数据进行解释，以生成每种类型通道中门控途径不同阶段的主干模型。这一提议开辟了一条新的实验途径，将有助于理解镁离子在原核生物中的稳态，特别强调离子易位和门控，以及信号转导的机制。公共卫生关系：对CorA结构和功能的理解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中线粒体Mg++稳态机制的关系。这是相关的，因为线粒体在细胞凋亡中的已知作用。CorA也是原核生物中的毒力因子，因此是重要的潜在抗生素靶标。