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

Abstract

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.