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

Microtubules are cytoskeletal structures composed of polymers of alpha/beta-tubulin heterodimers that enable cell division and motility by a process of alternating episodes of polymerization and depolymerization (dynamic instability). Transition from a polymerizing to a depolymerizing microtubule is triggered at the interdimer interface by Glu254 in alpha-tubulin (alpha:Glu254), which hydrolyzes GTP bound to beta-tubulin (beta:GTP). The process is regulated by phosphorylation of alpha-tubulin (Ser165) or beta-tubulin (Ser172) via signaling protein kinases (PKC, CDK1). All-atom molecular dynamics simulations of alpha/beta tubulin 6-mer systems are used to screen the cryo-EM structure of a microtubule (PDB 3J6E) for structural responses to phosphorylation of each tubulin subunit. In terms of global structure, microtubules with phosphorylated alpha-tubulin have a straight conformation attributed to a growing microtubule, whereas MTs with phosphorylated beta-tubulin are curved, characteristic of a disassembling MT. Phospho--alpha-tubulin initiates displacement of key secondary structures (helix H8, loop T5) at the inter-dimer interface, shifts the beta:GTP nucleotide by 5 A, and immobilizes the gamma-phosphate of beta:GTP through increased H-bonding with beta-tubulin. Phospho-beta-tubulin produces fewer structural effects and has a more flexible beta:GTP For beta:GTP hydrolysis, the phospho-beta-tubulin system displays an extensive network of water molecules between alpha:Glu254 and the gamma-phosphate of beta:GTP, facilitating its hydrolysis. In contrast, phospho-alpha-tubulin displays a discontinuous network of water molecules that predicts a diminished capacity for beta:GTP hydrolysis. These findings provide a detailed framework for understanding how phosphorylation of each tubulin subunit restructures the inter-dimer interface and modulates beta:GTP hydrolysis, global structure, and dynamic instability in response to key signaling protein kinases.