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Disrupting coupling within mycobacterial F-ATP synthases subunit ? causes dysregulated energy production and cell wall biosynthesis.

ABSTRACT: The dynamic interaction of the N- and C-terminal domains of mycobacterial F-ATP synthase subunit ? is proposed to contribute to efficient coupling of H+-translocation and ATP synthesis. Here, we investigate crosstalk between both subunit ? domains by introducing chromosomal atpC missense mutations in the C-terminal helix 2 of ? predicted to disrupt inter domain and subunit ?-? crosstalk and therefore coupling. The ? mutant ?R105A,R111A,R113A,R115A (?4A) showed decreased intracellular ATP, slower growth rates and lower molar growth yields on non-fermentable carbon sources. Cellular respiration and metabolism were all accelerated in the mutant strain indicative of dysregulated oxidative phosphorylation. The ?4A mutant exhibited an altered colony morphology and was hypersusceptible to cell wall-acting antimicrobials suggesting defective cell wall biosynthesis. In silico screening identified a novel mycobacterial F-ATP synthase inhibitor disrupting ?'s coupling activity demonstrating the potential to advance this regulation as a new area for mycobacterial F-ATP synthase inhibitor development.

SUBMITTER: Saw WG

PROVIDER: S-EPMC6856130 | biostudies-literature | 2019 Nov

REPOSITORIES: biostudies-literature

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Disrupting coupling within mycobacterial F-ATP synthases subunit ε causes dysregulated energy production and cell wall biosynthesis.

Saw Wuan-Geok WG Wu Mu-Lu ML Ragunathan Priya P Biuković Goran G Lau Aik-Meng AM Shin Joon J Harikishore Amaravadhi A Cheung Chen-Yi CY Hards Kiel K Sarathy Jickky Palmae JP Bates Roderick W RW Cook Gregory M GM Dick Thomas T Grüber Gerhard G

Scientific reports 20191114 1

The dynamic interaction of the N- and C-terminal domains of mycobacterial F-ATP synthase subunit ε is proposed to contribute to efficient coupling of H<sup>+</sup>-translocation and ATP synthesis. Here, we investigate crosstalk between both subunit ε domains by introducing chromosomal atpC missense mutations in the C-terminal helix 2 of ε predicted to disrupt inter domain and subunit ε-α crosstalk and therefore coupling. The ε mutant εR105A,R111A,R113A,R115A (ε<sup>4A</sup>) showed decreased int ...[more]

PMID: 31727946

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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

Disrupting coupling within mycobacterial F-ATP synthases subunit ? causes dysregulated energy production and cell wall biosynthesis.

Publications

Disrupting coupling within mycobacterial F-ATP synthases subunit ε causes dysregulated energy production and cell wall biosynthesis.

Similar Datasets

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