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

Microtubules are cytoskeletal structures composed of polymers of alpha-beta-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 Calpha and cyclin-dependent kinase 1 are known to phosphorylate Ser165 of alpha-tubulin (alpha:Ser165) and Ser172 of beta-tubulin, (alpha:Ser172), respectively. Using all-atom molecular dynamics simulations of 6-mer alpha/beta-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 (alpha:helix H8/loop T7 segment and beta:loops T3/T5) that lie at the inter-dimer cleft near the E-site beta:GTP. If properly aligned, alpha:Glu254 (helix H8) hydrolyzes beta:GTP to GDP thereby triggering the transition from a polymerizing to a depolymerizing microtubule. alpha-Tubulin phosphorylated at alpha:Ser165 displaces helix H8 (alpha:Glu254/alpha:Gln256) and loop T5 towards the gamma-phosphate of beta:GTP. This movement coincides with a shift of the beta:GTP nucleotide by 4.5-5.5 A, stabilization of the gamma-P of beta-GTP by additional H-bonding and weakened inter-dimer interactions. In a phosphorylated beta:Ser172 system, loop T5 is displaced toward beta:GTP and coincides with stabilization of inter-dimer interactions. Therefore, phosphorylation of either alpha- or beta-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.