Energy Coupling and Regulation in the ATP Synthase of E. coli

大肠杆菌 ATP 合成酶的能量耦合和调控

• 批准号: 7931335
• 负责人: Thomas M Duncan
• 金额: $ 20万
• 依托单位: UPSTATE MEDICAL UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-30 至 2011-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7931335
• 关键词: ATP Hydrolysis; ATP Synthesis Pathway; Adopted; Animals; Anti-Bacterial Agents; Antibiotics; Bacteria; Binding; Binding Sites; Biochemical; Biological Models; C-terminal; Cardiac; Catalysis; Catalytic Domain; Cells; Chloroplasts; Complement; Complex; Coupled; Coupling; Cysteine; Defect; Disulfides; Enzymes; Escherichia coli; Event; Experimental Models; F1-ATPase; Family; Goals; Homologous Gene; Human; In Vitro; Infection; Ischemia; Lead; Membrane; Metabolic; Metabolism; Mitochondria; Mitochondrial Proton-Translocating ATPases; Molecular Conformation; Motor; Nature; Nucleotides; Organism; Peptides; Peripheral; Plants; Play; Protons; Regulation; Research; Resolution; Role; Structure; System; Water; Work; antibiotic design; base; crosslink; design; disulfide bond; fighting; human disease; insight; mutant; novel strategies; public health relevance; small molecule

DESCRIPTION (provided by applicant): The ATP synthase is a membrane-bound, energy-coupling rotary motor that is responsible for the synthesis of most cellular ATP in animals, plants and many bacteria. It consists of two sub-complexes with distinct, partial functions: the FO complex contains transmembrane subunits and functions in the transport of protons; the F1 is a peripheral complex, which contains the catalytic nucleotide binding sites for ATP synthesis. FO and F1 are coupled through two stalk-like connections of subunits: a central rotor shaft and a peripheral stator. In vitro, F1 can be dissociated from FO as a water-soluble enzyme that only catalyzes net hydrolysis of ATP, and this also serves as a simple sub-system for studying much of the enzymatic features of the enzyme. General features of catalysis by F1 and intact ATP synthase (FO F1) are shared among all types of the enzyme (mitochondrial, chloroplast and bacterial), but some factors that regulate its function appear to be adapted or unique to the metabolic demands of the specific organism. For example, mounting evidence indicates that in E.coli conformational changes in ?'s C-terminal domain (CTD) play one or more roles in regulating the activity and/or efficiency of energy coupling in the ATP synthase of bacteria and chloroplasts. The long-term goal of this project aims to gain a detailed understanding of the E. coli ATP synthase structure, function and regulation and thus provide a quantitative experimental model system to understand common structural/functional features of this important enzyme `family'. To achieve this goal we will focus our research in two directions: In Aim 1, we will carry out high-resolution crystallographic studies on the E. coli F1-ATPase. This structural work is aimed at providing atomic snapshots the EcF1-? with the ? subunit in an extended (open) and closed conformation. We hypothesize that these two structural conformations, respectively, best describe the inhibitory and activating conformations adopted by the ? subunit, which is a critical regulator of E. coli FOF1 ATP synthase. In Aim 2 we will study the dynamic nature of the ? subunit's inhibitory action on E.coli F1 and FOF1. Using ? mutants and disulfide crosslinking within ? we will restrict or bias the possible conformational states of ?'s CTD, in order to better define the regulatory roles of ? in EcF1 and EcFOF1, and which conformations of ?'s CTD are necessary for these roles. In addition, we will explore the possibility of using peptides spanning all or part of the ?-CTD to inhibit, in trans, the activity of F1 or FOF1. This will guide the rational basis for design of small molecules that selectively inhibit bacterial ATP synthases and not the mitochondrial enzyme. PUBLIC HEALTH RELEVANCE: The ATP synthase is a central enzyme in cellular metabolism - it is responsible for making most ATP, the primary energy currency used inside cells. Defects in its assembly or function can cause human diseases and aggravate harmful events such as cardiac ischemia. Understanding unique features of the bacterial ATP synthase may lead to new approaches to fight infections.

描述(申请人提供)：三磷酸腺苷合成酶是一种膜结合、能量耦合的旋转马达，负责动物、植物和许多细菌中大多数细胞三磷酸腺苷的合成。它由两个具有不同部分功能的亚复合体组成：FO复合体包含跨膜亚单位和质子运输功能；F1是外围复合体，包含ATP合成的催化核苷酸结合部位。F0和F1通过两个子单元的杆状连接连接：中央转子轴和外围定子。在体外，F1可以作为一种水溶酶从FO中解离出来，只催化ATP的净水解，这也可以作为一个简单的子系统来研究该酶的许多酶学特性。F1和完整的ATP合成酶(FO F1)催化的一般特征在所有类型的酶(线粒体、叶绿体和细菌)中都是相同的，但调节其功能的一些因素似乎是适应或独特于特定生物体的代谢需求。例如，越来越多的证据表明，在大肠杆菌中，S C末端结构域的构象变化在调节细菌和叶绿体三磷酸腺苷合成酶的能量偶联的活性和/或效率方面发挥着一个或多个作用。本项目的长期目标是详细了解大肠杆菌三磷酸腺苷合成酶的结构、功能和调控，从而提供一个定量的实验模型体系来理解这个重要的酶‘家族’的共同结构/功能特征。为了实现这一目标，我们将从两个方向进行研究：在目标1，我们将对大肠杆菌F1-ATPase进行高分辨率的结晶学研究。这项结构性工作旨在提供ECF1-？的原子快照。和那个？延伸的(开放的)和封闭的构象中的亚基。我们假设，这两种结构构象分别最好地描述了？亚单位，这是一个关键的调节大肠杆菌FOF1三磷酸腺苷合成酶。在目标2中，我们将研究？的动态性质？亚基对大肠杆菌F1和FOF1的抑制作用。使用？突变体和内部的二硫键交联物？我们将限制或偏向？‘S CTD的可能构象状态，以便更好地定义？在EcF1和EcFOF1中，这些角色需要哪些？‘S CTD构象。此外，我们将探索使用横跨全部或部分β-CTD的多肽来抑制反式F1或FOF1活性的可能性。这将指导设计选择性抑制细菌ATP合成酶而不是线粒体酶的小分子的合理基础。与公共健康相关：三磷酸腺苷合成酶是细胞新陈代谢的中心酶，它负责制造大多数三磷酸腺苷，细胞内使用的主要能量货币。它的组装或功能缺陷可能会导致人类疾病，并加剧心脏缺血等有害事件。了解细菌ATP合酶的独特功能可能会导致对抗感染的新方法。