Simulations of tubulin sheet polymers as possible structural intermediates in microtubule assembly

The microtubule assembly process has been extensively studied, but the underlying molecular mechanism remains poorly understood. The structure of an artificially generated sheet polymer that alternates two types of lateral contacts and that directly converts into microtubules, has been proposed to correspond to the intermediate sheet structure observed during microtubule assembly. We have studied the self-assembly process of GMPCPP tubulins into sheet and microtubule structures using thermodynamic analysis and stochastic simulations. With the novel assumptions that tubulins can laterally interact in two different forms, and allosterically affect neighboring lateral interactions, we can explain existing experimental observations. At low temperature, the allosteric effect results in the observed sheet structure with alternating lateral interactions as the thermodynamically most stable form. At normal microtubule assembly temperature, our work indicates that a class of sheet structures resembling those observed at low temperature is transiently trapped as an intermediate during the assembly process. This work may shed light on the tubulin molecular interactions, and the role of sheet formation during microtubule assembly.

💡 Research Summary

The paper tackles the long‑standing question of what structural intermediate underlies microtubule (MT) assembly, focusing on the “sheet” phase that has been observed by electron microscopy but whose molecular nature remains ambiguous. The authors propose a novel mechanistic model in which tubulin dimers can engage in two distinct lateral contact types—designated “A” (strong) and “B” (weak)—and where the formation of one lateral bond allosterically modulates the affinity of neighboring lateral sites. By integrating a thermodynamic framework with stochastic Gillespie simulations, they explore how these assumptions reproduce experimental observations made with GMPCPP‑stabilized tubulin at both low (≈4 °C) and physiological (≈37 °C) temperatures.

Thermodynamic analysis shows that at low temperature the alternating A‑B lateral contacts generate the lowest free‑energy configuration, corresponding to the experimentally observed alternating sheet polymer. At physiological temperature the global free‑energy minimum shifts to the closed cylindrical MT, but the alternating sheet remains a local metastable minimum. In other words, the sheet can become kinetically trapped as an intermediate before the system overcomes an energy barrier and reorganizes the lateral contacts into the uniform lattice of a mature MT.

The stochastic simulations implement the above energy landscape by allowing tubulin dimers to diffuse, bind laterally as either A or B, and undergo allosteric coupling that favors alternating patterns. The simulations reproduce a two‑stage assembly pathway: (1) nucleation dominated by A‑type contacts, which then recruit B‑type contacts to extend a sheet with an A‑B alternation; (2) temperature‑dependent destabilization of A contacts, leading to a re‑arrangement of B contacts and eventual conversion of the sheet into a closed MT. The kinetic profiles match the experimentally reported rates of sheet formation and subsequent sheet‑to‑MT conversion in GMPCPP‑treated tubulin.

A key insight is the role of the allosteric coupling strength. By varying this parameter, the authors demonstrate that strong allosteric effects prolong the lifetime of the sheet, delaying MT formation, whereas weak coupling essentially eliminates the sheet, allowing direct MT growth. This provides a mechanistic explanation for why non‑hydrolyzable GTP analogs such as GMPCPP, which stabilize the sheet, promote the appearance of the intermediate in vitro.

Overall, the study makes three major contributions. First, it introduces a dual‑contact, allosterically coupled model that simultaneously accounts for low‑temperature sheet stability and physiological‑temperature sheet trapping—something single‑contact models cannot achieve. Second, it quantitatively links thermodynamic stability with kinetic pathways, showing how the sheet acts as a metastable basin that must be escaped for MT closure. Third, it highlights the allosteric parameter as a tunable lever that could be exploited in drug design or in synthetic biology to control MT dynamics.

The authors conclude that the sheet is not merely a fleeting artifact but a genuine, temperature‑dependent intermediate whose existence and lifetime are governed by the balance between two lateral contact modes and their cooperative allosteric interactions. This refined understanding of tubulin‑tubulin interactions opens avenues for targeted modulation of MT assembly in disease contexts (e.g., cancer, neurodegeneration) and provides a solid theoretical foundation for interpreting future high‑resolution structural studies of MT nucleation and growth.