Exploring the Effect of Mechanical Anisotropy of Protein Structures in the Unfoldase Mechanism of AAA+ Molecular Machines

Abstract

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.

Microtubule severing enzymes oligomerization and allostery: a tale of two domains

“…12,14,16,17 Several proposed mechanisms are: unfoldase, which is similar to the hand-over-hand action, wedge-like, or a combined unfoldase and wedge named death-spiral. 16,18–20 Our previous studies showed that, while the unfoldase action is important, 21,22 the wedge mechanism is most likely making a large contribution to the disassembly of a microtubule by severing proteins. 23–25…”

“…27 Interestingly, the disassembly of the hexameric state into lower order oligomers also occurred in our recent simulations of the action of spastin on microtubule filaments, especially when the interactions between the severing machine and the microtubule surface reach levels comparable to the interactions between kinesin-1 motors and microtubules. 22…”

“…12,14,16,17 Several proposed mechanisms are: unfoldase, which is similar to the hand-over-hand action, wedge-like, or a combined unfoldase and wedge named death-spiral. 16,[18][19][20] Our previous studies showed that, while the unfoldase action is important, 21,22 the wedge mechanism is most likely making a large contribution to the disassembly of a microtubule by severing proteins. [23][24][25] An important functional aspect of severing proteins found in experimental studies is the instability in the hexamers upon ATP hydrolysis and/or disengagement from the substrate protein.…”

“…27 Interestingly, the disassembly of the hexameric state into lower order oligomers also occurred in our recent simulations of the action of spastin on microtubule filaments, especially when the interactions between the severing machine and the microtubule surface reach levels comparable to the interactions between kinesin-1 motors and microtubules. 22 Another fascinating aspect of microtubule severing enzymes is that, unlike other machines from the AAA+ family, which require only ATP binding to reach their oligomeric functional configuration, they are believed to form higher-order oligomers (hexamers) only if their substrate, a MT filament or, at the minimum, the C-terminal tail (CTT) from a tubulin subunit, is present as well. 28 For example, for ClpB sedimentation studies found that, in the absence of nucleotides, ClpB from E.coli undergoes reversible self-association that involves protein concentration-dependent populations of monomers, higher-order oligomers (heptamers or hexamers) and intermediate-size oligomers, most likely dimers.…”

“…Adding to these findings, in our previous studies of microtubule severing AAA+ nanomachines the hexamer in its spiral state showed a propensity for dissociation into trimers or dimers, especially in the absence of binding partners. 22,33 In the present study, we addressed a number of questions regarding the mechanism of oligomerization/disassembly of severing proteins: (1) which oligomeric species is likely to be stable depending on conditions (ligands bound or just the APO state)? ; (2) what drives the disassembly of some oligomeric species, and what keeps other oligomers intact?…”