A systematic assessment of mycobacterial F1-ATPase subunit ϵ’s role in latent ATPase hydrolysis
In contrast to most bacteria, the mycobacterial F1FO-ATP synthase (α3:β3:γ:δ:ϵ:a:b:b’:c9) does not perform ATP hydrolysis-driven proton translocation. Although subunits α, γ and ϵ of the catalytic F1-ATPase component α3:β3:γ:ϵ have all been implicated in the suppression of the enzyme’s ATPase ac...
| Main Authors: | , , , , , , , , , , , |
|---|---|
| Other Authors: | |
| Format: | Journal Article |
| Language: | English |
| Published: |
2021
|
| Subjects: | |
| Online Access: | https://hdl.handle.net/10356/149231 |
| _version_ | 1826117052478259200 |
|---|---|
| author | Wong, Chui-Fann Lau, Aik-Meng Harikishore, Amaravadhi Saw, Wuan-Geok Shin, Joon Ragunathan, Priya Bhushan, Shashi Ngan, So-Fong Cam Sze, Siu Kwan Bates, Roderick Wayland Dick, Thomas Grüber, Gerhard |
| author2 | School of Biological Sciences |
| author_facet | School of Biological Sciences Wong, Chui-Fann Lau, Aik-Meng Harikishore, Amaravadhi Saw, Wuan-Geok Shin, Joon Ragunathan, Priya Bhushan, Shashi Ngan, So-Fong Cam Sze, Siu Kwan Bates, Roderick Wayland Dick, Thomas Grüber, Gerhard |
| author_sort | Wong, Chui-Fann |
| collection | NTU |
| description | In contrast to most bacteria, the mycobacterial F1FO-ATP synthase
(α3:β3:γ:δ:ϵ:a:b:b’:c9) does not perform ATP hydrolysis-driven proton
translocation. Although subunits α, γ and ϵ of the catalytic F1-ATPase
component α3:β3:γ:ϵ have all been implicated in the suppression of the
enzyme’s ATPase activity, the mechanism remains poorly defined. Here, we
brought the central stalk subunit ϵ into focus by generating the recombinant
Mycobacterium smegmatis F1-ATPase (MsF1-ATPase), whose 3D
low-resolution structure is presented, and its ϵ-free form MsF1αβγ, which
showed an eightfold ATP hydrolysis increase and provided a defined system
to systematically study the segments of mycobacterial ϵ’s suppression
of ATPase activity. Deletion of four amino acids at ϵ’s N terminus, mutant
MsF1αβγϵΔ2-5, revealed similar ATP hydrolysis as MsF1αβγ. Together with
biochemical and NMR solution studies of a single, double, triple and
quadruple N-terminal ϵ-mutants, the importance of the first N-terminal
residues of mycobacterial ϵ in structure stability and latency is described.
Engineering ϵ’s C-terminal mutant MsF1αβγϵΔ121 and MsF1αβγϵΔ103-121
with deletion of the C-terminal residue D121 and the two C-terminal ɑ-helices,
respectively, revealed the requirement of the very C terminus for communication
with the catalytic α3β3-headpiece and its function in ATP
hydrolysis inhibition. Finally we applied the tools developed during the
study for an in silico screen to identify a novel subunit ϵ-targeting F-ATP
synthase inhibitor. |
| first_indexed | 2024-10-01T04:21:28Z |
| format | Journal Article |
| id | ntu-10356/149231 |
| institution | Nanyang Technological University |
| language | English |
| last_indexed | 2024-10-01T04:21:28Z |
| publishDate | 2021 |
| record_format | dspace |
| spelling | ntu-10356/1492312023-02-28T17:10:00Z A systematic assessment of mycobacterial F1-ATPase subunit ϵ’s role in latent ATPase hydrolysis Wong, Chui-Fann Lau, Aik-Meng Harikishore, Amaravadhi Saw, Wuan-Geok Shin, Joon Ragunathan, Priya Bhushan, Shashi Ngan, So-Fong Cam Sze, Siu Kwan Bates, Roderick Wayland Dick, Thomas Grüber, Gerhard School of Biological Sciences School of Physical and Mathematical Sciences Science::Biological sciences::Biochemistry Bioenergetics F-ATP Synthase In contrast to most bacteria, the mycobacterial F1FO-ATP synthase (α3:β3:γ:δ:ϵ:a:b:b’:c9) does not perform ATP hydrolysis-driven proton translocation. Although subunits α, γ and ϵ of the catalytic F1-ATPase component α3:β3:γ:ϵ have all been implicated in the suppression of the enzyme’s ATPase activity, the mechanism remains poorly defined. Here, we brought the central stalk subunit ϵ into focus by generating the recombinant Mycobacterium smegmatis F1-ATPase (MsF1-ATPase), whose 3D low-resolution structure is presented, and its ϵ-free form MsF1αβγ, which showed an eightfold ATP hydrolysis increase and provided a defined system to systematically study the segments of mycobacterial ϵ’s suppression of ATPase activity. Deletion of four amino acids at ϵ’s N terminus, mutant MsF1αβγϵΔ2-5, revealed similar ATP hydrolysis as MsF1αβγ. Together with biochemical and NMR solution studies of a single, double, triple and quadruple N-terminal ϵ-mutants, the importance of the first N-terminal residues of mycobacterial ϵ in structure stability and latency is described. Engineering ϵ’s C-terminal mutant MsF1αβγϵΔ121 and MsF1αβγϵΔ103-121 with deletion of the C-terminal residue D121 and the two C-terminal ɑ-helices, respectively, revealed the requirement of the very C terminus for communication with the catalytic α3β3-headpiece and its function in ATP hydrolysis inhibition. Finally we applied the tools developed during the study for an in silico screen to identify a novel subunit ϵ-targeting F-ATP synthase inhibitor. National Research Foundation (NRF) Accepted version This work as well as the research scholarship of Chui-Fann Wong was supported by the National Research Foundation (NRF) Singapore, NRF Competitive Research Programme (CRP), Grant Award Number NRF–CRP18–2017–01; Lead-PI G.G.). 2021-05-19T05:50:51Z 2021-05-19T05:50:51Z 2021 Journal Article Wong, C., Lau, A., Harikishore, A., Saw, W., Shin, J., Ragunathan, P., Bhushan, S., Ngan, S. C., Sze, S. K., Bates, R. W., Dick, T. & Grüber, G. (2021). A systematic assessment of mycobacterial F1-ATPase subunit ϵ’s role in latent ATPase hydrolysis. FEBS Journal, 288(3), 818-836. https://dx.doi.org/10.1111/febs.15440 1742-4658 https://hdl.handle.net/10356/149231 10.1111/febs.15440 3 288 818 836 en FEBS Journal © 2021 Federation of European Biochemical Societies. All rights reserved. This is the accepted version of the following article: Wong, C., Lau, A., Harikishore, A., Saw, W., Shin, J., Ragunathan, P., Bhushan, S., Ngan, S. C., Sze, S. K., Bates, R. W., Dick, T. & Grüber, G. (2020). A systematic assessment of mycobacterial F1-ATPase subunit ϵ’s role in latent ATPase hydrolysis. FEBS Journal, 288(3), 818-836. https://dx.doi.org/10.1111/febs.15440, which has been published in final form at https://doi.org/10.1111/febs.15440 application/pdf |
| spellingShingle | Science::Biological sciences::Biochemistry Bioenergetics F-ATP Synthase Wong, Chui-Fann Lau, Aik-Meng Harikishore, Amaravadhi Saw, Wuan-Geok Shin, Joon Ragunathan, Priya Bhushan, Shashi Ngan, So-Fong Cam Sze, Siu Kwan Bates, Roderick Wayland Dick, Thomas Grüber, Gerhard A systematic assessment of mycobacterial F1-ATPase subunit ϵ’s role in latent ATPase hydrolysis |
| title | A systematic assessment of mycobacterial F1-ATPase subunit ϵ’s role in latent ATPase hydrolysis |
| title_full | A systematic assessment of mycobacterial F1-ATPase subunit ϵ’s role in latent ATPase hydrolysis |
| title_fullStr | A systematic assessment of mycobacterial F1-ATPase subunit ϵ’s role in latent ATPase hydrolysis |
| title_full_unstemmed | A systematic assessment of mycobacterial F1-ATPase subunit ϵ’s role in latent ATPase hydrolysis |
| title_short | A systematic assessment of mycobacterial F1-ATPase subunit ϵ’s role in latent ATPase hydrolysis |
| title_sort | systematic assessment of mycobacterial f1 atpase subunit ϵ s role in latent atpase hydrolysis |
| topic | Science::Biological sciences::Biochemistry Bioenergetics F-ATP Synthase |
| url | https://hdl.handle.net/10356/149231 |
| work_keys_str_mv | AT wongchuifann asystematicassessmentofmycobacterialf1atpasesubunitesroleinlatentatpasehydrolysis AT lauaikmeng asystematicassessmentofmycobacterialf1atpasesubunitesroleinlatentatpasehydrolysis AT harikishoreamaravadhi asystematicassessmentofmycobacterialf1atpasesubunitesroleinlatentatpasehydrolysis AT sawwuangeok asystematicassessmentofmycobacterialf1atpasesubunitesroleinlatentatpasehydrolysis AT shinjoon asystematicassessmentofmycobacterialf1atpasesubunitesroleinlatentatpasehydrolysis AT ragunathanpriya asystematicassessmentofmycobacterialf1atpasesubunitesroleinlatentatpasehydrolysis AT bhushanshashi asystematicassessmentofmycobacterialf1atpasesubunitesroleinlatentatpasehydrolysis AT ngansofongcam asystematicassessmentofmycobacterialf1atpasesubunitesroleinlatentatpasehydrolysis AT szesiukwan asystematicassessmentofmycobacterialf1atpasesubunitesroleinlatentatpasehydrolysis AT batesroderickwayland asystematicassessmentofmycobacterialf1atpasesubunitesroleinlatentatpasehydrolysis AT dickthomas asystematicassessmentofmycobacterialf1atpasesubunitesroleinlatentatpasehydrolysis AT grubergerhard asystematicassessmentofmycobacterialf1atpasesubunitesroleinlatentatpasehydrolysis AT wongchuifann systematicassessmentofmycobacterialf1atpasesubunitesroleinlatentatpasehydrolysis AT lauaikmeng systematicassessmentofmycobacterialf1atpasesubunitesroleinlatentatpasehydrolysis AT harikishoreamaravadhi systematicassessmentofmycobacterialf1atpasesubunitesroleinlatentatpasehydrolysis AT sawwuangeok systematicassessmentofmycobacterialf1atpasesubunitesroleinlatentatpasehydrolysis AT shinjoon systematicassessmentofmycobacterialf1atpasesubunitesroleinlatentatpasehydrolysis AT ragunathanpriya systematicassessmentofmycobacterialf1atpasesubunitesroleinlatentatpasehydrolysis AT bhushanshashi systematicassessmentofmycobacterialf1atpasesubunitesroleinlatentatpasehydrolysis AT ngansofongcam systematicassessmentofmycobacterialf1atpasesubunitesroleinlatentatpasehydrolysis AT szesiukwan systematicassessmentofmycobacterialf1atpasesubunitesroleinlatentatpasehydrolysis AT batesroderickwayland systematicassessmentofmycobacterialf1atpasesubunitesroleinlatentatpasehydrolysis AT dickthomas systematicassessmentofmycobacterialf1atpasesubunitesroleinlatentatpasehydrolysis AT grubergerhard systematicassessmentofmycobacterialf1atpasesubunitesroleinlatentatpasehydrolysis |