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

The Acinetobacter baumannii F₁F_O-ATP synthase (α₃:β₃:γ:δ:ε:a:b₂:c₁₀), 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 F₁-ATPase (AbF₁–ATPase) composed of subunits α₃:β₃:γ:ε, showing latent ATP hydrolysis. A 3.0 Å cryo-electron microscopy structure visualizes the architecture and regulatory element of this enzyme, in which the C-terminal domain of subunit ε (Abε) is present in an extended position. An ε-free AbF₁-ɑβγ complex generated showed a 21.5-fold ATP hydrolysis increase, demonstrating that Abε is the major regulator of AbF₁-ATPase's latent ATP hydrolysis. The recombinant system enabled mutational studies of single amino acid substitutions within Abε or its interacting subunits β and γ, respectively, as well as C-terminal truncated mutants of Abε, providing a detailed picture of Abε's main element for the self-inhibition mechanism of ATP hydrolysis. Using a heterologous expression system, the importance of Abε's C-terminus in ATP synthesis of inverted membrane vesicles, including AbF₁F_O-ATP synthases, has been explored. In addition, we are presenting the first NMR solution structure of the compact form of Abε, revealing interaction of its N-terminal β-barrel and C-terminal ɑ-hairpin domain. A double mutant of Abε highlights critical residues for Abε's domain–domain formation which is important also for AbF₁–ATPase's stability. Abε 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 F₁-ATPases in bacteria, chloroplasts, and mitochondria to prevent wasting of ATP.