The Structural Basis of alpha/beta-tubulin Assembly and Disassembly by Tubulin Cofactors

Microtubules polymerize from cytoplasmic pools of soluble αβ-tubulin heterodimers that support diverse cellular functions. The tubulin cofactors, TBCC, TBCD, TBCE, and the Arl2 GTPase, form TBC-DEG assemblies that regulate αβ-tubulin assembly and disassembly from α- and β-tubulins, yet their underlying mechanisms remain incompletely understood. Here, we reconstitute the human TBC-DE and TBC-DEG assemblies from eukaryotic cells co-purified with monomeric β-tubulin intermediates and determine their cryo-EM structures. The structures reveal that TBC-DEG disassembles αβ-tubulin by releasing α-tubulin through a lever-arm-like rotation in TBCE coupled to major conformational change in Arl2 upon its nucleotide release, while TBCD tightly holds β-tubulin. TBCD dissociates α-tubulin by refolding the β-tubulin H10-S8 loop at its intradimer interface. The TBC-DEG-β-tubulin or TBC-DE-β-tubulin assemblies undergo extensive back-to-back dimerization mediated by β-β-tubulin homodimers, formed through their dissociated H8 helices at unoccupied intradimer interfaces. Structural comparisons demonstrate that the TBCE mechanical rotation, driven by the Arl2 GTPase cycle, either delivers α-tubulin or removes it from beneath the TBCD-bound β-tubulin and is directionally regulated by TBCC stabilizing αβ-tubulin interfaces. Our findings suggest that TBC-DEG/TBCC catalyzing heterodimerization of α-tubulin with β-tubulin may have evolved to counteract the β-tubulin intrinsic tendency to form off-pathway toxic homodimers through its exposed α-tubulin-binding intradimer interface.