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

• Main Authors: 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
• Other Authors: School of Biological Sciences
• Format: Article
• Language: English
• Published: 2021
• Subjects: Science::Biological sciences::Biochemistry; Bioenergetics; F-ATP Synthase
• Online Access: https://hdl.handle.net/10356/149231
• Institution: Nanyang Technological University

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.