A systematic assessment of mycobacterial F1-ATPase subunit ε’s role in latent ATPase hydrolysis

Chui Fann Wong, Aik Meng Lau, Amaravadhi Harikishore, Wuan Geok Saw, Joon Shin, Priya Ragunathan, Shashi Bhushan, So Fong Cam Ngan, Siu Kwan Sze, Roderick W. Bates, Thomas Dick, Gerhard Grüber

Research output: Contribution to journalArticlepeer-review

5 Scopus citations


In contrast to most bacteria, the mycobacterial F1FO-ATP synthase (α33: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 α33: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.

Original languageEnglish (US)
Pages (from-to)818-836
Number of pages19
JournalFEBS Journal
Issue number3
StatePublished - Feb 2021

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology
  • Cell Biology


  • F-ATP synthase
  • Mycobacterium
  • bioenergetics
  • subunit ε
  • tuberculosis


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