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Abstract Microtubule (MT)‐associated proteins regulate the dynamic behavior of MTs during cellular processes. MT severing enzymes are the associated proteins which destabilize MTs by removing subunits from the lattice. One model for how severing enzymes remove tubulin dimers from the MT lattice is by unfolding its subunits through pulling on the carboxy‐terminal tails of tubulin dimers. This model stems from the fact that severing enzymes are AAA+ unfoldases. To test this mechanism, we apply pulling forces on the carboxy‐terminal regions of MT subunits using coarse grained molecular simulations. In our simulations, we used different MT lattices and concentrations of severing enzymes. We compare our simulation results with data from in vitro severing assays and find that the experimental data is best fit by a model of cooperative removal of protofilament fragments by severing enzymes, which depends on the severing enzyme concentration and placement on the MT lattice.
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Exploring the Effect of Mechanical Anisotropy of Protein Structures in the Unfoldase Mechanism of AAA+ Molecular Machines
Essential cellular processes of microtubule disassembly and protein degradation, which span lengths from tens of μm to nm, are mediated by specialized molecular machines with similar hexameric structure and function. Our molecular simulations at atomistic and coarse-grained scales show that both the microtubule-severing protein spastin and the caseinolytic protease ClpY, accomplish spectacular unfolding of their diverse substrates, a microtubule lattice and dihydrofolate reductase (DHFR), by taking advantage of mechanical anisotropy in these proteins. Unfolding of wild-type DHFR requires disruption of mechanically strong β-sheet interfaces near each terminal, which yields branched pathways associated with unzipping along soft directions and shearing along strong directions. By contrast, unfolding of circular permutant DHFR variants involves single pathways due to softer mechanical interfaces near terminals, but translocation hindrance can arise from mechanical resistance of partially unfolded intermediates stabilized by β-sheets. For spastin, optimal severing action initiated by pulling on a tubulin subunit is achieved through specific orientation of the machine versus the substrate (microtubule lattice). Moreover, changes in the strength of the interactions between spastin and a microtubule filament, which can be driven by the tubulin code, lead to drastically different outcomes for the integrity of the hexameric structure of the machine.
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- PAR ID:
- 10329143
- Date Published:
- Journal Name:
- Nanomaterials
- Volume:
- 12
- Issue:
- 11
- ISSN:
- 2079-4991
- Page Range / eLocation ID:
- 1849
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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- Free Publicly Accessible Full Text
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Accepted Manuscript1.0
- Journal Article:
- https://doi.org/10.3390/nano12111849
