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Atomic insights of an up and down conformation of the Acinetobacter baumannii F 1 ‐ATPase subunit ε and deciphering the residues critical for ATP hydrolysis inhibition and ATP synthesis

2023; Wiley; Volume: 37; Issue: 7 Linguagem: Inglês

10.1096/fj.202300175rr

1530-6860

Wuan Geok Saw, Khoa C. M. Le, Joon Shin, Jes Hui Min Kwek, Chui Fann Wong, Priya Ragunathan, Tuck Choy Fong, Volker Müller, Gerhard Grüber,

RNA modifications and cancer

Abstract The Acinetobacter baumannii F 1 F O ‐ATP synthase (α 3 :β 3 :γ:δ:ε: 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 ( Ab F 1 –ATPase) composed of subunits α 3 :β 3 :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 ( Ab ε) is present in an extended position. An ε‐free Ab F 1 ‐ɑβγ complex generated showed a 21.5‐fold ATP hydrolysis increase, demonstrating that Ab ε is the major regulator of Ab F 1 ‐ATPase's latent ATP hydrolysis. The recombinant system enabled mutational studies of single amino acid substitutions within Ab ε or its interacting subunits β and γ, respectively, as well as C‐terminal truncated mutants of Ab ε, providing a detailed picture of Ab ε's main element for the self‐inhibition mechanism of ATP hydrolysis. Using a heterologous expression system, the importance of Ab ε's C‐terminus in ATP synthesis of inverted membrane vesicles, including Ab F 1 F O ‐ATP synthases, has been explored. In addition, we are presenting the first NMR solution structure of the compact form of Ab ε, revealing interaction of its N‐terminal β‐barrel and C‐terminal ɑ‐hairpin domain. A double mutant of Ab ε highlights critical residues for Ab ε's domain–domain formation which is important also for Ab F 1 –ATPase's stability. Ab ε 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.