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: 7743023
• 负责人: Thomas M Duncan
• 金额: $ 31.86万
• 依托单位: UPSTATE MEDICAL UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-12-01 至 2012-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7743023
• 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（由申请人提供）：ATP合酶是一种膜结合的能量耦合旋转马达，负责动物、植物和许多细菌中大多数细胞ATP的合成。它由两个具有不同部分功能的子复合体组成：FO 复合体包含跨膜亚基，具有质子运输功能； F1 是一种外周复合物，包含 ATP 合成的催化核苷酸结合位点。 FO 和 F1 通过子单元的两个茎状连接进行耦合：中心转子轴和外围定子。在体外，F1 可以作为水溶性酶从 FO 中解离出来，仅催化 ATP 的净水解，并且这也可以作为研究该酶的许多酶学特征的简单子系统。 F1 和完整 ATP 合酶 (FO F1) 催化的一般特征是所有类型的酶（线粒体、叶绿体和细菌）共有的，但调节其功能的一些因素似乎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，α的C端结构域(CTD)的构象变化在调节细菌和叶绿体的ATP合酶中的能量耦合的活性和/或效率中发挥一种或多种作用。该项目的长期目标是详细了解大肠杆菌ATP合酶的结构、功能和调控，从而提供定量实验模型系统来了解这一重要酶“家族”的共同结构/功能特征。为了实现这一目标，我们将把研究重点放在两个方向：目标1，我们将对大肠杆菌F1-ATP酶进行高分辨率晶体学研究。这项结构工作旨在提供 EcF1-? 的原子快照。与？处于延伸（开放）和闭合构象的亚基。我们假设这两种结构构象分别最好地描述了 ? 采用的抑制构象和激活构象。亚基，是大肠杆菌 FOF1 ATP 合酶的关键调节因子。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 ？突变体和二硫键交联？我们将限制或偏向?的CTD可能的构象状态，以便更好地定义?的调节作用。在 EcF1 和 EcFOF1 中，以及 ? 的 CTD 的哪些构象对于这些作用是必需的。此外，我们将探索使用跨越全部或部分β-CTD的肽来反式抑制F1或FOF1活性的可能性。这将为设计选择性抑制细菌 ATP 合酶而非线粒体酶的小分子提供合理基础。公共健康相关性：ATP 合酶是细胞代谢中的一种核心酶 - 它负责制造大多数 ATP，而 ATP 是细胞内使用的主要能量货币。其组装或功能的缺陷会导致人类疾病并加剧心脏缺血等有害事件。了解细菌 ATP 合酶的独特特征可能会带来对抗感染的新方法。