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

Abstract

Microtubules are cytoskeletal structures composed of polymers of /{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 -tubulin (:Glu254), which hydrolyzes GTP bound to {beta}-tubulin ({beta}:GTP). The process is regulated by phosphorylation of -tubulin (Ser165) or {beta}-tubulin (Ser172) via signaling protein kinases (PKC, CDK1). All-atom molecular dynamics simulations of /{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 -tubulin have a straight conformation attributed to a growing microtubule, whereas MTs with phosphorylated {beta}-tubulin are curved, characteristic of a disassembling MT. Phospho--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 :Glu254 and the {gamma}-phosphate of {beta}:GTP, facilitating its hydrolysis. In contrast, phospho--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 to modulate {beta}:GTP hydrolysis, global structure, and dynamic instability in response to key signaling protein kinases.