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A systematic assessment of mycobacterial F 1 ‐ATPase subunit ε’s role in latent ATPase hydrolysis

2020; Wiley; Volume: 288; Issue: 3 Linguagem: Inglês

10.1111/febs.15440

1742-4658

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

RNA and protein synthesis mechanisms

In contrast to most bacteria, the mycobacterial F1 FO -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 MsF1 αβγ, 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 MsF1 αβγεΔ2-5 , revealed similar ATP hydrolysis as MsF1 αβγ. 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 MsF1 αβγεΔ121 and MsF1 αβγεΔ103-121 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 α3 β3 -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.