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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.

 

In cells, microtubule location, length, and dynamics are regulated by a host of microtubule-associated proteins and enzymes that read where to bind and act based on the microtubule “tubulin code,” which is predominantly encoded in the tubulin carboxy-terminal tail (CTT). Katanin is a highly conserved AAA ATPase enzyme that binds to the tubulin CTTs to remove dimers and sever microtubules. We have previously demonstrated that short CTT peptides are able to inhibit katanin severing. Here, we examine the effects of CTT sequences on this inhibition activity. Specifically, we examine CTT sequences found in nature, alpha1A (TUBA1A), detyrosinated alpha1A, Δ2 alpha1A, beta5 (TUBB/TUBB5), beta2a (TUBB2A), beta3 (TUBB3), and beta4b (TUBB4b). We find that these natural CTTs have distinct abilities to inhibit, most noticeably beta3 CTT cannot inhibit katanin. Two non-native CTT tail constructs are also unable to inhibit, despite having 94% sequence identity with alpha1 or beta5 sequences. Surprisingly, we demonstrate that poly-E and poly-D peptides are capable of inhibiting katanin significantly. An analysis of the hydrophobicity of the CTT constructs indicates that more hydrophobic polypeptides are less inhibitory than more polar polypeptides. These experiments not only demonstrate inhibition, but also likely interaction and targeting of katanin to these various CTTs when they are part of a polymerized microtubule filament. 

The nanomachine from the ATPases associated with various cellular activities superfamily, called spastin, severs microtubules during cellular processes. To characterize the functionally important allostery in spastin, we employed methods from evolutionary information, to graph-based networks, to machine learning applied to atomistic molecular dynamics simulations of spastin in its monomeric and the functional hexameric forms, in the presence or absence of ligands. Feature selection, using machine learning approaches, for transitions between spastin states recognizes all the regions that have been proposed as allosteric or functional in the literature. The analysis of the composition of the Markov State Model macrostates in the spastin monomer, and the analysis of the direction of change in the top machine learning features for the transitions, indicate that the monomer favors the binding of ATP, which primes the regions involved in the formation of the inter-protomer interfaces for binding to other protomer(s). Allosteric path analysis of graph networks, built based on the cross-correlations between residues in simulations, shows that perturbations to a hub specific for the pre-hydrolysis hexamer propagate throughout the structure by passing through two obligatory regions: the ATP binding pocket, and pore loop 3, which connects the substrate binding site to the ATP binding site. Our findings support a model where the changes in the terminal protomers due to the binding of ligands play an active role in the force generation in spastin. The secondary structures in spastin, which are found to be highly degenerative within the network paths, are also critical for feature transitions of the classification models, which can guide the design of allosteric effectors to enhance or block allosteric signaling. 

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.