The state of the guanosine nucleotide allosterically affects the interfaces of tubulin in protofilament

Abstract

The dynamics of microtubules is essential for many microtubule-dependent cellular functions such as the mitosis. It has been recognized for a long time that GTP hydrolysis in αβ-tubulin polymers plays a critical role in this dynamics. However, the effects of the changes in the nature of the guanosine nucleotide at the E-site in β-tubulin on microtubule structure and stability are still not well understood. In the present work, we performed all-atom molecular dynamics simulations of a αβα-tubulin heterotrimer harboring a guanosine nucleotide in three different states at the E-site: GTP, GDP-Pi and GDP. We found that changes in the nucleotide state is associated with significant conformational variations at the α-tubulin N- and β-tubulin M-loops which impact the interactions between tubulin protofilaments. The results also show that GTP hydrolysis reduces αβ-tubulin interdimer contacts in favor of intradimer interface. From an atomistic point view, we propose a role for α-tubulin glutamate residue 254 in catalytic magnesium coordination and identified a water molecule in the nucleotide binding pocket which is most probably required for nucleotide hydrolysis. Finally, the results are discussed with reference to the role of taxol in microtubule stability and the recent tubulin-sT2R crystal structures.

Multi-scale Computational Modeling of Tubulin-Tubulin Lateral Interaction

“…To investigate the nature and strength of the lateral interaction between two tubulin dimers using our multi-scale approach, we first built a molecular dynamics model of two tubulin dimers laterally paired with one of three nucleotide-states: GDP-, GMPCPP-, or GTP-tubulin. Similar to earlier molecular dynamic studies of tubulin heterodimers (32,33,61), our simulations (~200ns vs. 10-100ns equilibration in previous studies) indicated that αβ-tubulin structure is stable and consistent in aqueous environment regardless of the nucleotide state or lateral neighbor (see Movie S1-S3 in the Supporting Material). Mean root-mean-squared displacement (RMSD) of backbone atoms was measured from the starting coordinates obtained from the cryo-EM structure as the protein structure stability for three nucleotide states of the dimer.…”

“…Hence, by performing molecular dynamics (MD) simulations of tubulin structures, we are able to study the dynamic evolution of this protein in solution and sample the thermal fluctuations through time. For example, MD simulations of curved structures of tubulin have been used to confirm that they preserve their curvature in solution, with no significant difference at intra-vs interdimer bending angles (31,32). However, using the right time scale and sampling of the ensemble is an important factor in drawing conclusions from the simulation results.…”

Microtubule assembly governed by tubulin allosteric gain in flexibility and lattice induced fit

“…The intrinsic conformational dynamics of both GTP- and GDP-tubulin in solution have also been simulated for several tens of nanoseconds using all-atom molecular dynamics (MD) and both were found to be kinked ( Gebremichael et al, 2008 ). Longer MD simulations (up to 100 ns) have not revealed GTP-induced tubulin straightening either ( Bennett et al, 2009 ; Grafmüller and Voth, 2011 ; André et al, 2012 ; Grafmüller et al, 2013 ). In another computational study, lateral binding of two GDP-tubulin dimers has been shown to shift the conformations of individual dimers toward the straight state; the GTP-state was not analyzed though ( Peng et al, 2014 ).…”

Microtubule Simulations Provide Insight into the Molecular Mechanism Underlying Dynamic Instability

“…Solution x-ray scattering results and the structures of free ab-tubulin dimers solved in different nucleotide states also did not reveal any significant structural differences, thus favoring the lattice model (24,(31)(32)(33)(34)(35). In addition, computer simulations of tubulin dimers, as well as PFs in both the GTP-and GDP-bound states, have confirmed that they adopt an intrinsic bent conformation (36)(37)(38). More recently, a combination of the two limiting models was proposed (39), in which GTP binding is thought to induce the increased flexibility of tubulin dimers, thereby lowering the free energy barrier of the transition between the curved and straight conformations, which in turn facilitates the recruitment of GTP-tubulin dimers to the MT growing end.…”

“…The lateral contacts in all three states are reported to be very similar, dominated by the interaction of the M-loop (S7-H9 loop) on one side and sandwiched by the H2-S3 loop and the H1-S2 loop on the other side. Molecular dynamics (MD) simulations have also been widely used to study the effects of nucleotide states to MT systems from the scale of tubulin dimers to MT lattice patches (36)(37)(38)(39)(47)(48)(49)(50)(51)(52)(53)(54)(55)(56)(57)(58)(59)(60). For example, all atomic simulations by Grafm€ uller et al (48) have suggested that both the lateral and longitudinal contacts in the GDP MT lattice are weakened and more likely to disassemble when exposed at the growing end.…”