How phosphorylation of α/β-tubulin perturbs microtubule structure: A computational study.

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