Atomic insights of an up and down conformation of the Acinetobacter baumannii F₁-ATPase subunit e and deciphering the residues critical for ATP hydrolysis inhibition and ATP synthesis

The Acinetobacter baumannii F1FO-ATP synthase (alpha(3):beta(3):gamma:delta:epsilon:a:b(2):c(10)), which is essential for this strictly respiratory opportunistic human pathogen, is incapable of ATP-driven proton translocation due to its latent ATPase activity. Here, we generated and purified the first recombinant A. baumannii F-1-ATPase (AbF(1)-ATPase) composed of subunits alpha(3):beta(3):gamma:epsilon, showing latent ATP hydrolysis. A 3.0 angstrom cryo-electron microscopy structure visualizes the architecture and regulatory element of this enzyme, in which the C-terminal domain of subunit epsilon (Ab epsilon) is present in an extended position. An epsilon-free AbF(1)-alpha beta gamma complex generated showed a 21.5-fold ATP hydrolysis increase, demonstrating that Ab epsilon is the major regulator of AbF(1)-ATPase's latent ATP hydrolysis. The recombinant system enabled mutational studies of single amino acid substitutions within Ab epsilon or its interacting subunits beta and gamma, respectively, as well as C-terminal truncated mutants of Ab epsilon, providing a detailed picture of Ab epsilon's main element for the self-inhibition mechanism of ATP hydrolysis. Using a heterologous expression system, the importance of Ab epsilon's C-terminus in ATP synthesis of inverted membrane vesicles, including AbF(1)FO-ATP synthases, has been explored. In addition, we are presenting the first NMR solution structure of the compact form of Ab epsilon, revealing interaction of its N-terminal beta-barrel and C-terminal alpha-hairpin domain. A double mutant of Ab epsilon highlights critical residues for Ab epsilon's domain-domain formation which is important also for AbF(1)-ATPase's stability. Ab epsilon does not bind MgATP, which is described to regulate the up and down movements in other bacterial counterparts. The data are compared to regulatory elements of F-1-ATPases in bacteria, chloroplasts, and mitochondria to prevent wasting of ATP.