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Atomic solution structure of Mycobacterium abscessus F-ATP synthase subunit ε and identification of Ep1MabF1 as a targeted inhibitor.

ABSTRACT: Mycobacterium abscessus (Mab) is a nontuberculous mycobacterium of increasing clinical relevance. The rapidly growing opportunistic pathogen is intrinsically multi-drug-resistant and causes difficult-to-cure lung disease. Adenosine triphosphate, generated by the essential F₁ F_O ATP synthase, is the major energy currency of the pathogen, bringing this enzyme complex into focus for the discovery of novel antimycobacterial compounds. Coupling of proton translocation through the membrane-embedded F_O sector and ATP formation in the F₁ headpiece of the bipartite F₁ F_O ATP synthase occurs via the central stalk subunits γ and ε. Here, we used solution NMR spectroscopy to resolve the first atomic structure of the Mab subunit ε (Mabε), showing that it consists of an N-terminal β-barrel domain (NTD) and a helix-loop-helix motif in its C-terminal domain (CTD). NMR relaxation measurements of Mabε shed light on dynamic epitopes and amino acids relevant for coupling processes within the protein. We describe structural differences between other mycobacterial ε subunits and Mabε's lack of ATP binding. Based on the structural insights, we conducted an in silico inhibitor screen. One hit, Ep1MabF1, was shown to inhibit the growth of Mab and bacterial ATP synthesis. NMR titration experiments and docking studies described the binding epitopes of Ep1MabF1 on Mabε. Together, our data demonstrate the potential to develop inhibitors targeting the ε subunit of Mab F₁ F_O ATP synthase to interrupt the coupling process.

SUBMITTER: Shin J

PROVIDER: S-EPMC10609657 | biostudies-literature | 2022 Oct

REPOSITORIES: biostudies-literature

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Atomic solution structure of Mycobacterium abscessus F-ATP synthase subunit ε and identification of Ep1MabF1 as a targeted inhibitor.

Shin Joon J Harikishore Amaravadhi A Wong Chui Fann CF Ragunathan Priya P Dick Thomas T Grüber Gerhard G

The FEBS journal 20220605 20

Mycobacterium abscessus (Mab) is a nontuberculous mycobacterium of increasing clinical relevance. The rapidly growing opportunistic pathogen is intrinsically multi-drug-resistant and causes difficult-to-cure lung disease. Adenosine triphosphate, generated by the essential F<sub>1</sub> F<sub>O</sub> ATP synthase, is the major energy currency of the pathogen, bringing this enzyme complex into focus for the discovery of novel antimycobacterial compounds. Coupling of proton translocation through th ...[more]

PMID: 35612822

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Project description:The F(1)F(o)-ATP synthases of alkaliphilic bacteria exhibit latent ATPase activity, and for the thermoalkaliphile Bacillus sp. strain TA2.A1, this activity is intrinsic to the F(1) moiety. To study the mechanism of ATPase inhibition, we developed a heterologous expression system in Escherichia coli to produce TA2F(1) complexes from this thermoalkaliphile. Like the native F(1)F(o)-ATP synthase, the recombinant TA2F(1) was blocked in ATP hydrolysis activity, and this activity was stimulated by the detergent lauryldimethylamine oxide. To determine if the C-terminal domain of the epsilon subunit acts as an inhibitor of ATPase activity and if an electrostatic interaction plays a role, a TA2F(1) mutant with either a truncated epsilon subunit [i.e., TA2F(1)(epsilon(DeltaC))] or substitution of basic residues in the second alpha-helix of epsilon with nonpolar alanines [i.e., TA2F(1)(epsilon(6A))] was constructed. Both mutants showed ATP hydrolysis activity at low and high concentrations of ATP. Treatment of the purified F(1)F(o)-ATP synthase and TA2F(1)(epsilon(WT)) complex with proteases revealed that the epsilon subunit was resistant to proteolytic digestion. In contrast, the epsilon subunit of TA2F(1)(epsilon(6A)) was completely degraded by trypsin, indicating that the C-terminal arm was in a conformation where it was no longer protected from proteolytic digestion. In addition, ATPase activity was not further activated by protease treatment when compared to the untreated control, supporting the observation that epsilon was responsible for inhibition of ATPase activity. To study the effect of the alanine substitutions in the epsilon subunit in the entire holoenzyme, we reconstituted recombinant TA2F(1) complexes with F(1)-stripped native membranes of strain TA2.A1. The reconstituted TA2F(o)F(1)(epsilon(WT)) was blocked in ATP hydrolysis and exhibited low levels of ATP-driven proton pumping consistent with the F(1)F(o)-ATP synthase in native membranes. Reconstituted TA2F(o)F(1)(epsilon(6A)) exhibited ATPase activity that correlated with increased ATP-driven proton pumping, confirming that the epsilon subunit also inhibits ATPase activity of TA2F(o)F(1).

Dataset Information

Atomic solution structure of Mycobacterium abscessus F-ATP synthase subunit ε and identification of Ep1MabF1 as a targeted inhibitor.

Publications

Atomic solution structure of Mycobacterium abscessus F-ATP synthase subunit ε and identification of Ep1MabF1 as a targeted inhibitor.

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