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

Abstract

While many bacteria are able to bypass the requirement for oxidative phosphorylation when grown on carbohydrates, Mycobacterium tuberculosis is unable to do so. Differences of amino acid composition and structural features of the mycobacterial F-ATP synthase (α 3 :β 3 :γ:δ:ε:a:b:b′:c 9 ) compared to its prokaryotic or human counterparts were recently elucidated and paved avenues for the discovery of molecules interfering with various regulative mechanisms of this essential energy converter. In this context, the mycobacterial peripheral stalk subunit δ came into focus, which displays a unique Nterminal 111-amino acid extension. Here, mutants of recombinant mycobacterial subunit δ were characterized, revealing significant reduction in ATP synthesis and demonstrating essentiality of this subunit for effective catalysis. These results provided the basis for the generation of a four-feature model forming a δ receptor-based pharmacophore and to identify a potent subunit δ inhibitor DeMF1 via in silico screening. The successful targeting of the δ subunit demonstrates the potential to advance δ's flexible coupling as a new area for the development of F-ATP synthase inhibitors.

Binding properties of the anti-TB drugs bedaquiline and TBAJ-876 to a mycobacterial F-ATP synthase

“…The revolution of the c -ring also drives rotation of the central stalk subunits γ and ε, which causes conformational changes in the catalytic sites of the α 3 β 3 hexamer leading to ATP formation ( Guo et al., 2021 ; Montgomery et al., 2021 ). The peripheral stalk subunits b : b’: δ provide the flexibility to smoothen the transmission of power between the rotary c -ring and the α 3 :β 3 :γ:ε domain ( Harikishore et al., 2022 ; Montgomery et al., 2021 ).

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GaMF1.39’s antibiotic efficacy and its enhanced antitubercular activity in combination with clofazimine, Telacebec, ND-011992, or TBAJ-876

“…Mycobacterial-specific modifications of the F-ATP synthase subunits α, δ, and γ, including a C-terminal elongation ( 14 , 20 , 21 ), an inserted domain ( 18 ), or a 12–14 amino acid extra loop ( 13 ), respectively, have been described as regulative ( 14 , 20 , 21 ) or essential features for catalysis ( 13 , 14 , 22 ). For example, the mycobacterial extra loop of subunit γ, absent in the human homolog and other prokaryotes, is important for ATP synthesis as well as ATP hydrolysis and ATP-driven proton pumping regulation ( 19 ) and has been identified as a new drug target ( 9 , 13 , 23 ).…”