Overexpression, purification, enzymatic and microscopic characterization of recombinant mycobacterial F-ATP synthase

Saw, Wuan‐Geok; Wong, Chui Fann; Dick, Thomas; Grüber, Gerhard

doi:10.1016/j.bbrc.2019.11.098

Cited by 9 publications

(7 citation statements)

References 33 publications

(55 reference statements)

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“…Recombinant M. smegmatis WT F-ATP synthase was purified following the protocol in Saw et al (2020) [27]. The purified enzyme was reconstituted into small unilamellar vesicles, which were generated from Phosphatidylcholine type II S soybeans (Sigma-Aldrich, Steinheim, Germany) as described recently [28].…”

Section: Reconstitution and Atp Synthesis Of Recombinant M Smegmatis F-atp Synthasementioning

confidence: 99%

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

et al. 2021

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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.

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Section: Reconstitution and Atp Synthesis Of Recombinant M Smegmatis F-atp Synthasementioning

confidence: 99%

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

et al. 2021

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“…To determine whether the NTD helix α5 residues R171, R177, and Q178 of the mycobacterial δ subunit are essential for ATP synthesis, the recombinant M. smegmatis (Ms) mc 2 155 F-ATP synthase mutants δR171K, δR171G, δR177Q, and δQ178R were engineered. The generated recombinant mutant enzymes were purified according to our developed protocols, 22 eluted at the same volume on the size exclusion columns, and showed similar subunit compositions like the wild type (WT) enzyme according to the sodium dodecyl sulfate−polyacrylamide (SDS) gel electrophoresis (Figure 1). WT MsF-ATP synthase or the respective mutant enzymes were reconstituted into proteoliposomes to assay ATP synthesis formation and to detect possible changes due to amino acid substitutions of the mutants generated.…”

Section: ■ Results and Discussionmentioning

confidence: 99%

“…12 Interestingly, mycobacteria species possess a more elongated δ subunit, including a canonical δ subunit consisting of 179 amino acids, which is similar in length to other prokaryotic δ subunits, and an N-terminal 111-amino acid extension, resulting in a 272-amino acid protein. 12 The recent low- 12,22 and atomic-resolution EM structures 7 of the M. smegmatis F-ATP synthase visualized subunit δ on top of the α 3 β 3 -headpiece, composed of the noncanonical NTD (helices α1−α6), the central α7−α12 helix domain (CD), and the Cterminal domain (CTD; β1−β4 and α13−α14). 7 The Nterminal helices of the ATP-(αTP) and ADP bound subunit α (αDP) interact with δ's NTD and CD domain, respectively (Supplementary Figure S1).…”

Section: ■ Introductionmentioning

confidence: 99%

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

et al. 2022

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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.

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“…This domain is connected by the central and rotating stalk subunits γ and ϵ [21], sitting on the rotating c 9 -ring [13] and enabling the coupling of ion transport from the a-and c-subunits to the α 3 :β 3 -headpiece. The peripheral stalk subunits, b and b', whose N termini are membrane-embedded, extend and become attached via subunit δ to the top of the α 3 :β 3 -headpiece [22,23]. Together, the peripheral and central stalks connect the F 1 -(α 3 :β 3 :γ:ϵ) and F O domains.…”

Section: Introductionmentioning

confidence: 99%

A systematic assessment of mycobacterial F₁‐ATPase subunit ε’s role in latent ATPase hydrolysis

et al. 2020

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In contrast to most bacteria, the mycobacterial F1FO‐ATP synthase (α3:β3:γ:δ:ε:a:b:b’:c9) does not perform ATP hydrolysis‐driven proton translocation. Although subunits α, γ and ε of the catalytic F1‐ATPase component α3:β3: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 F1‐ATPase (MsF1‐ATPase), whose 3D low‐resolution structure is presented, and its ε‐free form MsF1αβγ, 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 MsF1αβγεΔ2‐5, revealed similar ATP hydrolysis as MsF1αβγ. 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 MsF1αβγεΔ121 and MsF1αβγεΔ103‐121 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 α3β3‐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.

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Overexpression, purification, enzymatic and microscopic characterization of recombinant mycobacterial F-ATP synthase

Cited by 9 publications

References 33 publications

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

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

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

A systematic assessment of mycobacterial F₁‐ATPase subunit ε’s role in latent ATPase hydrolysis

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Overexpression, purification, enzymatic and microscopic characterization of recombinant mycobacterial F-ATP synthase

Cited by 9 publications

References 33 publications

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

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

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

A systematic assessment of mycobacterial F1‐ATPase subunit ε’s role in latent ATPase hydrolysis

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A systematic assessment of mycobacterial F₁‐ATPase subunit ε’s role in latent ATPase hydrolysis