Structure and function of the F-type ATP synthase from Fusobacterium nucleatum
ATP synthases are multi-subunit membrane-bound enzymes that utilise the energy stored in a transmembrane electrochemical ion gradient to produce ATP, the major energy currency of all living cells. The F-type ATP synthase consists of two domains: FO, a membrane-embedded complex that is responsible for the translocation of H+/Na+ ions across the membrane, and F1, a water-soluble complex responsible for the synthesis or hydrolysis of ATP. The goal of this study was to carry out a biochemical and structural analysis of the F-type (F1Fo) sodium-dependent ATP synthase from the anaerobic opportunistic oral pathogen Fusobacterium nucleatum. An expression system in Escherichia coli was developed to overproduce and purify the F1 complex from F . nucleatum. Pure F1 sub-complexes (i.e. F1∆δ and F1∆δ(ε∆c)) were purified via a His10-linker-TEV-linker that was attached to the N-terminal domain of subunit ε. The pure F1 sub-complexes had a final specific activity of 4-7 U/mg with an overall purification of 11-fold. Biochemical characterisation of the pure F1 sub-complexes revealed apparent Km values for ATP and Mg2+ of 0.1 mM and 0.2 mM, respectively. The pH profile of the F1 sub-complexes was broad with an optimum between pH 8-8.5. A striking feature of the F1 complex was its extreme temperature optimum (44 U/mg protein at 65 ̊C) relative to the growth temperature of the bacterium (i.e. 37°C). Truncation of subunit ε was without significant effect on the ATP hydrolysis activity of the enzyme. Mg-ADP inhibited the ATP hydrolysis activity of both F1∆δ and F1∆δ(ε∆c) suggesting regulation of enzyme activity through Mg-ADP binding to the catalytic sites. The F1∆δ sub-complex was initially crystallised under 22 conditions. Further analysis identified a buffer system (0.1 M magnesium acetate tetrahydrate, 0.1 M tri-sodium citrate, 15.5% (w/v) PEG5000 MME; pH 6) resulting in crystals that diffracted to a resolution of 9 AÅ. This condition was further optimised to obtain crystals that diffracted to 4.2 Å. Diffraction data obtained from protein crystallised in a gel matrix was used for structural determination of the F1∆δ complex. The structure was solved by molecular replacement to a resolution of 3.45 Å and the unit cell contained two molecules of F1∆δ with a space group of P21. Comparison of the F. nucleatum complex to the F1 complexes of other organisms revealed that the overall structure of the enzyme was very similar. A unique feature of the F. nucleatum F1-complex was that Mg2+-ATP was bound to all non-catalytic α sites and Mg2+-ADP bound to two catalytic sites (βDP and βTP). Furthermore, the C-terminal domain of subunit ε, which is proposed to play a role in regulation of ATP synthesis and hydrolysis, was in a down-state position. This work reports the first structural determination of an F1 complex from a sodium-dependent ATP synthase and will form an important platform for rotational analysis of the enzyme and studies aimed at determining the entire structure of this class of enzyme.
Advisor: Cook, Gregory M.
Degree Name: Doctor of Philosophy
Degree Discipline: Microbiology and Immunology
Publisher: University of Otago
Keywords: ATP synthase; Fusobacterium nucleatum; Structure; Regulation
Research Type: Thesis