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

In contrast to most bacteria, the mycobacterial F 1 F O ‐ATP synthase (α 3 :β 3 :γ:δ:ε: a : b : b’ : c 9 ) does not perform ATP hydrolysis‐driven proton translocation. Although subunits α, γ and ε of the catalytic F 1 ‐ATPase component α 3 :β 3 :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 F 1 ‐ATPase ( Ms F 1 ‐ATPase), whose 3D low‐resolution structure is presented, and its ε‐free form Ms F 1 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 Ms F 1 αβγε Δ2‐5 , revealed similar ATP hydrolysis as Ms F 1 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 Ms F 1 αβγε Δ121 and Ms F 1 αβγε Δ103‐121 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 α 3 β 3 ‐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.