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bioRxiv · 10.64898/2025.12.30.697104

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

Taheri, A.·Ashaduzzaman, M.·Gill, V.·Al-Bassam, J.
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Abstract

Microtubules polymerize from cytoplasmic pools of soluble {beta}-tubulin heterodimers that support diverse cellular functions. The tubulin cofactors, TBCC, TBCD, TBCE, and the Arl2 GTPase, form TBC-DEG assemblies that regulate {beta}-tubulin assembly and disassembly from - and {beta}-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 {beta}-tubulin intermediates and determine their cryo-EM structures. The structures reveal that TBC-DEG disassembles {beta}-tubulin by releasing -tubulin through a lever-arm-like rotation in TBCE coupled to major conformational change in Arl2 upon its nucleotide release, while TBCD tightly holds {beta}-tubulin. TBCD dissociates -tubulin by refolding the {beta}-tubulin H10-S8 loop at its intradimer interface. The TBC-DEG-{beta}-tubulin or TBC-DE-{beta}-tubulin assemblies undergo extensive back-to-back dimerization mediated by {beta}-{beta}-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 -tubulin or removes it from beneath the TBCD-bound {beta}-tubulin and is directionally regulated by TBCC stabilizing {beta}-tubulin interfaces. Our findings suggest that TBC-DEG/TBCC catalyzing heterodimerization of -tubulin with {beta}-tubulin may have evolved to counteract the {beta}-tubulin intrinsic tendency to form off-pathway toxic homodimers through its exposed -tubulin-binding intradimer interface.

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Taheri, A., Ashaduzzaman, M., Gill, V., Al-Bassam, J.. 2025-12-31. The Structural Basis of alpha/beta-tubulin Assembly and Disassembly by Tubulin Cofactors. https://doi.org/10.64898/2025.12.30.697104

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