Targeting Mycobacterial F-ATP Synthase C-Terminal α Subunit Interaction Motif on Rotary Subunit γ

Abstract

Mycobacteria regulate their energy (ATP) levels to sustain their survival even in stringent living conditions. Recent studies have shown that mycobacteria not only slow down their respiratory rate but also block ATP hydrolysis of the F-ATP synthase (α3:β3:γ:δ:ε:a:b:b’:c9) to maintain ATP homeostasis in situations not amenable for growth. The mycobacteria-specific α C-terminus (α533-545) has unraveled to be the major regulative of latent ATP hydrolysis. Its deletion stimulates ATPase activity while reducing ATP synthesis. In one of the six rotational states of F-ATP synthase, α533-545 has been visualized to dock deep into subunit γ, thereby blocking rotation of γ within the engine. The functional role(s) of this C-terminus in the other rotational states are not clarified yet and are being still pursued in structural studies. Based on the interaction pattern of the docked α533-545 region with subunit γ, we attempted to study the druggability of the α533-545 motif. In this direction, our computational work has led to the development of an eight-featured α533-545 peptide pharmacophore, followed by database screening, molecular docking, and pose selection, resulting in eleven hit molecules. ATP synthesis inhibition assays using recombinant ATP synthase as well as mycobacterial inverted membrane vesicles show that one of the hits, AlMF1, inhibited the mycobacterial F-ATP synthase in a micromolar range. The successful targeting of the α533-545-γ interaction motif demonstrates the potential to develop inhibitors targeting the α site to interrupt rotary coupling with ATP synthesis.

Molecular mechanism on forcible ejection of ATPase inhibitory factor 1 from mitochondrial ATP synthase

“…The ζ subunit of α-proteobacteria inhibits hydrolysis by binding to the interface of the αβ DP pair in a manner similar to IF 1 61 – 63 . Mycobacteria have a specific insertion sequence at the C-terminal of the α subunit called the α-extension loop 64 , 65 . Recent cryo-EM analysis showed that the α-extension loop binds to a specific loop on the γ subunit, which is considered to block rotation in a counterclockwise direction 26 , 66 .…”

Evolution of the Inhibitory and Non-Inhibitory ε, ζ, and IF1 Subunits of the F1FO-ATPase as Related to the Endosymbiotic Origin of Mitochondria

“…( 6) Another inhibitory protein named AtpΘ has been recently found in photosynthetic cyanobacteria; however, it is only a partial F O inhibitor of the cyanobacterial ATP synthase [18,19]. (7) A different inhibitory mechanism of latent ATP hydrolysis has also been described in mycobacteria, in which an extra C-terminus of the α subunit of about 3.5 kDa interacts with subunit γ [20,21] and leads to a novel anti-tuberculosis compound [22,23]. In summary, nature evolutively designed several F 1 F O -ATPase inhibitory proteins or domains to complement the insufficient kinetic and partial inhibitory MgADP regulation.…”

Mutational Analysis of Mycobacterial F-ATP Synthase Subunit δ Leads to a Potent δ Enzyme Inhibitor

“…Reconstitution of MsF-ATP synthase was carried out as described previously. 38 ATP Synthesis Assay. ATP synthesis was performed as described previously.…”

“…ATP synthesis was performed as described previously. 38 Fluorescence Quenching Assay. A Varian Cary Eclipse spectrofluorometer was used, and all experiments were carried out at 20 °C.…”