How Phosphorylation of How Phosphorylation of alpha/beta-Tubulin Perturbs Microtubule Structure: A Computational Study

Microtubules are cytoskeletal structures composed of polymers of /{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 C and cyclin-dependent kinase 1 are known to phosphorylate Ser165 of -tubulin (:Ser165) and Ser172 of {beta}-tubulin, ({beta}:Ser172), respectively. Using all-atom molecular dynamics simulations of 6-mer {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 (: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, :Glu254 (helix H8) hydrolyzes {beta}:GTP to GDP thereby triggering the transition from a polymerizing to a depolymerizing microtubule. -Tubulin phosphorylated at :Ser165 displaces helix H8 (:Glu254/: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 - 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.