Oncogenic β-tubulin mutations disrupt nucleotide-dependent allostery and free energy landscape of tubulin dimer.

Dynamic instability of microtubules arises from nucleotide-dependent conformational changes within the tubulin dimers; however, little is known about the molecular mechanisms linking specific mutations to microtubule dysfunction. Here, we combined molecular-dynamics simulations with multi-parametric analysis to investigate wild-type and four lung cancer-associated β-tubulin mutations: Q134L, D177H, G269S, and Q426E. GTP-bound tubulin dimers exhibited enhanced flexibility in the H1-S2, T5, M-loop, and H7 regions, and strong correlated motions across longitudinal interfaces were observed consistent with an assembly-competent tubulin dimer conformation. Our analyses show that each mutation perturbs tubulin heterodimer stability through distinct mechanisms. Mutations such as Q134L and Q426E mutations loosened tubulin dimer inter-subunit packing and shifted the H7 helix toward open conformations, producing fragmented shallow free energy basins. D177H mutation preserved global stability but the tubulin dimer skewed toward a compact closed state. G269S mutation promoted tighter packing with heterogeneous conformers. These findings identify the core helix H7 as a central pivot linking nucleotide state, local perturbations, and global conformational equilibria. Principal component and free energy analyses reveal that these mutations shift the conformational equilibrium toward flexible, energetically unfavorable states incompatible with stable microtubule formation. Thus, our results provide atomistic insights into how these mutations remodel long-range allosteric communication within the tubulin dimer, offering a structural framework for comprehending the regulation of microtubule dynamics.