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 F1 FO -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. bauma...
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sg-ntu-dr.10356-1701252023-08-29T02:14:42Z 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 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 School of Biological Sciences NTU Institute of Structural Biology Science::Biological sciences ATP Synthesis Bioenergetics The Acinetobacter baumannii F1 FO -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 AbF1 FO -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. Ministry of Education (MOE) Nanyang Technological University Research reported in this publication is supported by the Singapore Ministry of Education Academic Research Fund Tier 1 (RG20/22) to G.G. The PhD scholarship of Khoa Le Cong Minh was supported by a Vingroup-NTU graduate scholarship. 2023-08-29T02:14:42Z 2023-08-29T02:14:42Z 2023 Journal Article Saw, W. G., Le, K. C. M., Shin, J., Kwek, J. H. M., Wong, C. F., Ragunathan, P., Fong, T. C., Müller, V. & Grüber, G. (2023). 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 FASEB Journal, 37(7), e23040-. https://dx.doi.org/10.1096/fj.202300175RR 0892-6638 https://hdl.handle.net/10356/170125 10.1096/fj.202300175RR 37318822 2-s2.0-85162161638 7 37 e23040 en RG20/22 The FASEB Journal © 2023 Federation of American Societies for Experimental Biology. All rights reserved. |
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Science::Biological sciences ATP Synthesis Bioenergetics 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 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 |
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The Acinetobacter baumannii F1 FO -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 AbF1 FO -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. |
author2 |
School of Biological Sciences |
author_facet |
School of Biological Sciences 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 |
author |
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 |
author_sort |
Saw, Wuan Geok |
title |
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 |
title_short |
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 |
title_full |
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 |
title_fullStr |
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 |
title_full_unstemmed |
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 |
title_sort |
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 |
publishDate |
2023 |
url |
https://hdl.handle.net/10356/170125 |
_version_ |
1779156431502573568 |