Nucleotide-dependent Conformational Dynamics and Energetics of Tubulin

Abstract

Microtubules (MTs) are key components of the cytoskeleton and play a central role in cell division and development. They undergo stochastic switching between phases of growth and shrinkage driven by GTP hydrolysis in αβ-tubulin dimers. MT assembly requires that tubulin adopts a straight conformation in the lattice, while during disassembly it adopts a kinked conformation that is incompatible with the MT geometry. The mechanism by which GTP binding renders individual tubulin dimers polymerization-competent is still poorly understood. In particular, two models of MT assembly are being controversially discussed: in the allosteric model, GTP binding directly promotes tubulin straightening, whereas in the lattice model tubulin is kinked regardless of its nucleotide state, and it is the assembling MT itself that forces tubulin into a straight conformation. In order to distinguish between these two hypotheses, we have characterized the conformational dynamics and energetics of free tubulin in solution using atomistic molecular dynamics simulations and free energy calculations. Unexpectedly, and contradictory to the above models, we find that GTP-tubulin samples a broad range of intrinsic curvatures all of which are almost isoenergetic. In contrast, GDP-tubulin is confined to low-curvature regions of the conformational space. Furthermore, an additional energy barrier is seen in all simulated tubulin ensembles that is associated with collective rearrangements in the β-subunit of tubulin but not with the nucleotide state. This barrier separates different bending modi and may be related to the inhibitory mechanism of MT-associated proteins such as stathmin which suppresses tubulin polymerization in a nucleotide-independent manner. Overall, our results suggest a new combined conformational selection / induced fit model in which GTP binding renders tubulin more flexible at the intadimer interface and allows a specific type of tubulin bending that facilitates MT assembly.