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

ATP合成酶是一种膜结合的、能量耦合的旋转马达，负责 在动物、植物和许多细菌中合成大多数细胞的三磷酸腺苷。它由两个子复合体组成 具有不同的部分功能：FO复合体包含跨膜亚单位和在 质子的运输；F1是一个外围复合体，它包含催化核苷酸结合 三磷酸腺苷合成部位。F0和F1通过亚基的两个茎状连接连接：A 中心转子轴和外围定子。在体外，F1可以作为水溶性的FO从FO解离出来 仅催化ATP净水解的酶，这也是一个简单的子系统，用于 研究了该酶的许多酶学特性。F1和INTERNAL催化作用的一般特征 三磷酸腺苷合成酶(FOF1)在所有类型的酶(线粒体、叶绿体和 细菌)，但一些调节其功能的因子似乎适应或独特于新陈代谢 特定生物体的需求。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胞周期中起着一个或多个作用 细菌和叶绿体三磷酸腺苷合成酶中能量偶联的活性和/或效率。 这个项目的长期目标是对大肠杆菌三磷酸腺苷合成酶有一个详细的了解。 结构、功能和调控，从而提供了定量的实验模型系统 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，我们将从两个方向进行研究： 在目标1中，我们将对大肠杆菌F1-ATPase进行高分辨率的结晶学研究。这 结构工作的目的是提供原子快照ECF1-？在扩展的 (开放)和闭合构象。我们假设这两种结构构象， 分别最好地描述了？亚基所采用的抑制构象和激活构象 是大肠杆菌FOF1三磷酸腺苷合成酶的关键调控因子。 在目标2中，我们将研究该亚基对E.ColiF1和FOF1的抑制作用的动态性质。 使用突变体和内部的二硫键交联，我们将限制或偏向可能的构象 S CTD的状态，以便更好地界定ECF1和ECFOF1中的调节作用，并且 S的CTD构象是这些角色所必需的。此外，我们还将探索 使用横跨全部或部分CTD的多肽，以反式抑制F1或FOF1的活性。这将是 指导选择性抑制细菌ATP合成酶的小分子设计的合理基础 而不是线粒体酶。

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 (FOF1) 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.