How Phosphorylation of αβ-Tubulin Perturbs Microtubule Structure: A Computational Study

Microtubules are cytoskeletal structures composed of polymers of α/β-tubulin heterodimers. They play a central role in cell division and motility by a stochastic process of alternating polymerization and depolymerization episodes (dynamic instability) that can be modulated by phosphorylation. Protein kinase Cα and cyclin-dependent kinase 1 are known to phosphorylate Ser165 of α-tubulin (α:Ser165) and Ser172 of β-tubulin, (β:Ser172), respectively. Using all-atom molecular dynamics simulations of 6-mer αβ-tubulin systems modeled on the cryo-EM structure of a microtubule (PDB 3J6E), the impact of phosphorylation at each site is explored in terms of secondary structures (α:helix H8/loop T7 segment and β:loops T3/T5) that lie at the inter-dimer cleft near the E-site β:GTP. If properly aligned, α:Glu254 (helix H8) hydrolyzes β:GTP to GDP thereby triggering the transition from a polymerizing to a depolymerizing microtubule. α-Tubulin phosphorylated at α:Ser165 displaces helix H8 (α:Glu254/α:Gln256) and loop T5 towards the γ-phosphate of β:GTP. This movement coincides with a shift of the β:GTP nucleotide by 4.5-5.5 Å, stabilization of the γP of β:GTP by additional H-bonding and weakened inter-dimer interactions. In a phosphorylated β:Ser172 system, loop T5 is displaced toward β:GTP and coincides with stabilization of inter-dimer interactions. Therefore, phosphorylation of either α- or β-tubulin generates a distinct profile of intramolecular rearrangements that remodel the inter-dimer cleft and modulate dynamic instability. These profiles may provide a useful reference for screening mutations identified in tumor genomes.