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.
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.
