Structural model for differential cap maturation at growing microtubule ends

Abstract

Microtubules (MTs) are hollow cylinders made of tubulin, a GTPase responsible for essential functions during cell growth and division, and thus, key target for anti-tumor drugs. In MTs, GTP hydrolysis triggers structural changes in the lattice, which are responsible for interaction with regulatory factors. The stabilizing GTP-cap is a hallmark of MTs and the mechanism of the chemical-structural link between the GTP hydrolysis site and the MT lattice is a matter of debate. We have analyzed the structure of tubulin and MTs assembled in the presence of fluoride salts that mimic the GTP-bound and GDP•Pi transition states. Our results challenge current models because tubulin does not change axial length upon GTP hydrolysis. Moreover, analysis of the structure of MTs assembled in the presence of several nucleotide analogues and of taxol allows us to propose that previously described lattice expansion could be a post-hydrolysis stage involved in Pi release.

Microtubule instability driven by longitudinal and lateral strain propagation

“…Rather, our results are consistent with a weakening of longitudinal bonds upon GTP hydrolysis, based on the stronger response of single GDP-PFs to mechanical stretching (Fig 2C), which is indicative of more fragile longitudinal bonds between GDP dimers. Further, our results do not support the most recent model by Estévez-Gallego et al [24] postulating that both pre-and post-hydrolysis MT segments are equally compact, while the lattice undergoes a transient, hydrolysis-energy-consuming expansion (approximated by the GMPCPP lattice) to release the γ-phosphate. Our combined evidence (Figs 2-5) rather suggests: (a) whatever the GMPCPP state corresponds to in the GTPase cycle of tubulin, it most likely precedes the GDP state; and (b) a pure GDP lattice is higher in free energy than a pure GMPCPP one because the transition to the GDP lattice can only be achieved by investing a per-dimer energy on the order of 11 k B T. Thus, the conformational cycle proposed by Estévez-Gallego et al would involve around twice the energy available from GTP hydrolysis to first expand and then compact the lattice, which is energetically implausible according to our estimates.…”

“…Recent high- and low-resolution structural studies have revealed, in line with the early finding [ 19 ], that the use of a non-hydrolyzable GTP analog, GMPCPP, for MT assembly results in a more expanded MT lattice compared to a fully hydrolyzed GDP-lattice [ 20 – 24 ], which is commonly interpreted as the lattice response to GTP hydrolysis ( Fig 1B ). Because by itself this global rearrangement does not fully indicate how and whether at all it is linked to GTP hydrolysis and strain accumulation at the single-dimer level, several competing models of MT cap maturation and MT disassembly have been proposed.…”

“…Also here, recent atomistic simulations of full MT lattices suggest that lateral interactions in GDP-MTs might be weaker than those in GTP-MTs [25]. Finally, the most recent 'no expansion' model [24] provides an alternative view of the cap maturation process in which both pure GTP-and pure GDP-MTs have equally compacted lattices, while the higher-energy expanded lattice induced by GTP hydrolysis (mimicked by GMPCPP) corresponds to an intermediate, phosphate-releasing state. This model is partially supported by the observation that the extent of lattice compaction differs across different eukaryotic species [28].…”

“…Our computational findings provide quantitative insights into tubulin mechanochemistry and, therefore, the structural and energetic basis of MT dynamic instability. The results presented here enable us to test the three major models of MT cap maturation and MT catastrophe: (i) the strain model proposed by Nogales, Alushin, Zhang and colleagues [ 20 – 22 ], (ii) the bond model proposed by the Moores lab [ 23 , 27 ], and (iii) the most recent ‘no expansion’ model by Estévez-Gallego et al [ 24 ]. Our results do not support the bond model because we do not find a statistically significant effect of the nucleotide state on the lateral bond stability, which is key to the bond model.…”

The Mechanism of Tubulin Assembly into Microtubules: Insights from Structural Studies

“…Another level of complexity comes from the fact that most structures of the microtubule have been determined with a marker (e.g., a kinesin) used to distinguish between the α and β subunits during image analysis, a protein which itself may influence microtubule structure ( Peet et al., 2018 ). As a consequence, the effect of GTP hydrolysis remains a matter of debate ( Alushin et al., 2014 ; Estévez-Gallego et al., 2020 ; Manka and Moores, 2018b ). The recent characterization of a tubulin mutant unable to hydrolyze GTP ( Roostalu et al., 2020 ) and the determination of the structure of undecorated microtubules ( Zhang et al., 2018 ) open the possibility to establish the structure of bona fide GTP-microtubules, although these would be constituted of a mutated tubulin.…”