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

• Published in: The FASEB journal 2023-07, Vol.37 (7), p.e23040-n/a
• Main Authors: Saw, Wuan‐Geok, Le, Khoa Cong Minh, Shin, Joon, Kwek, Jes Hui Min, Wong, Chui Fann, Ragunathan, Priya, Fong, Tuck Choy, Müller, Volker, Grüber, Gerhard
• Format: Article
• Language: English
• Subjects: ATP synthesis; bioenergetics; ESKAPE; F‐ATP synthase; ion channels; multidrug resistance; OXPHOS
• ISSN: 0892-6638; 1530-6860
• DOI: 10.1096/fj.202300175RR

The Acinetobacter baumannii F1FO‐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 AbF1FO‐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. The Acinetobacter baumannii F‐ATP synthase, composed of the subunits α3:β3:γ:ε:δ:a:b2:c10, is essential for growth of the pathogen by converting ADP and inorganic phosphate to the biological energy ATP. Similarly, the enzyme controls the homeostasis of ADP and ATP by regulating the reverse direction of ATP hydrolysis, resulting in a low ATPase activity of the enzyme. The C‐terminal domain of subunit ε (Abε) and its movements are critical for the regulation of latent ATP hydrolysis and favorable ATP synthesis.