Identification of ATP synthase α subunit as a new maternal factor capable of protecting zebrafish embryos from bacterial infection

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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Structure of the ATP synthase from Mycobacterium smegmatis provides targets for treating tuberculosis

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.2111899118 ◽

2021 ◽

Vol 118 (47) ◽

pp. e2111899118

Author(s):

Martin G. Montgomery ◽

Jessica Petri ◽

Tobias E. Spikes ◽

John E. Walker

Keyword(s):

Atp Synthase ◽

Mycobacterium Smegmatis ◽

Atp Hydrolysis ◽

Terminal Region ◽

Catalytic Cycle ◽

Α Subunit ◽

Antitubercular Drugs ◽

Safe Mechanism ◽

Fail Safe ◽

Α Subunits

The structure has been determined by electron cryomicroscopy of the adenosine triphosphate (ATP) synthase from Mycobacterium smegmatis. This analysis confirms features in a prior description of the structure of the enzyme, but it also describes other highly significant attributes not recognized before that are crucial for understanding the mechanism and regulation of the mycobacterial enzyme. First, we resolved not only the three main states in the catalytic cycle described before but also eight substates that portray structural and mechanistic changes occurring during a 360° catalytic cycle. Second, a mechanism of auto-inhibition of ATP hydrolysis involves not only the engagement of the C-terminal region of an α-subunit in a loop in the γ-subunit, as proposed before, but also a “fail-safe” mechanism involving the b′-subunit in the peripheral stalk that enhances engagement. A third unreported characteristic is that the fused bδ-subunit contains a duplicated domain in its N-terminal region where the two copies of the domain participate in similar modes of attachment of the two of three N-terminal regions of the α-subunits. The auto-inhibitory plus the associated “fail-safe” mechanisms and the modes of attachment of the α-subunits provide targets for development of innovative antitubercular drugs. The structure also provides support for an observation made in the bovine ATP synthase that the transmembrane proton-motive force that provides the energy to drive the rotary mechanism is delivered directly and tangentially to the rotor via a Grotthuss water chain in a polar L-shaped tunnel.

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Removal of dystroglycan causes severe muscular dystrophy in zebrafish embryos

Development ◽

10.1242/dev.129.14.3505 ◽

2002 ◽

Vol 129 (14) ◽

pp. 3505-3512 ◽

Cited By ~ 4

Author(s):

Michael J. Parsons ◽

Isabel Campos ◽

Elizabeth M. A. Hirst ◽

Derek L. Stemple

Keyword(s):

Sarcoplasmic Reticulum ◽

Muscular Dystrophy ◽

Muscle Cells ◽

Laminin Receptor ◽

Central Component ◽

Zebrafish Embryos ◽

Term Survival ◽

Α Subunit ◽

Glycoprotein Complex ◽

Dystrophin Glycoprotein Complex

Muscular dystrophy is frequently caused by disruption of the dystrophin-glycoprotein complex (DGC), which links muscle cells to the extracellular matrix. Dystroglycan, a central component of the DGC, serves as a laminin receptor via its extracellular α subunit, and interacts with dystrophin (and thus the actin cytoskeleton) through its integral membrane β subunit. We have removed the function of dystroglycan in zebrafish embryos. In contrast to mouse, where dystroglycan mutations lead to peri-implantation lethality, dystroglycan is dispensable for basement membrane formation during early zebrafish development. At later stages, however, loss of dystroglycan leads to a disruption of the DGC, concurrent with loss of muscle integrity and necrosis. In addition, we find that loss of the DGC leads to loss of sarcomere and sarcoplasmic reticulum organisation. The DGC is required for long-term survival of muscle cells in zebrafish, but is dispensable for muscle formation. Dystroglycan or the DGC is also required for normal sarcomere and sarcoplasmic reticulum organisation. Because zebrafish embryos lacking dystroglycan share several characteristics with human muscular dystrophy, they should serve as a useful model for the disease. In addition, knowing the dystroglycan null phenotype in zebrafish will facilitate the isolation of other molecules involved in muscular dystrophy pathogenesis.

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