Abstract
Microtubule polarity and dynamic polymerization arise from the self-association properties of the αβ-tubulin heterodimer. For decades, it has remained unclear how the tubulin cofactors TBCD, TBCE, TBCC, and the Arl2 GTPase mediate the biogenesis of αβ-tubulin from individual α- and β-tubulins. Here, we use cryo-electron microscopy to determine structures of tubulin cofactors bound to αβ-tubulin. TBCD, TBCE, and Arl2 form a heterotrimeric cage-like assembly, we term TBC-DEG, around the αβ-tubulin heterodimer. The TBC-DEG-αβ-tubulin structures show that TBC-DEG wraps around β-tubulin while TBCE extends along α-tubulin. The TBC-DEG/TBCC-αβ-tubulin structures reveal that TBCC forms multi-domain interactions with Arl2 and TBCD to engage the αβ-tubulin intradimer-interface, promoting TBCE rotation while TBCD holds β-tubulin. TBCC engages the GTP-bound Arl2, multiple sites of TBCD, and the native αβ-tubulin intradimer interface near the α-tubulin N-site GTP. Together, these structures uncover transition states for αβ-tubulin biogenesis and degradation, suggesting a vise-like, GTP-hydrolysis-dependent mechanism in which TBCC binding to TBC-DEG modulates αβ-tubulin interfaces. Our studies provide structural evidence that tubulin cofactors act as enzymatic regulators that assemble the invariant αβ-tubulin architecture. By catalyzing α- and β-tubulin biogenesis and degradation, the TBC-DEG and TBCC assemblies regulate the polymerization competency of αβ-tubulin for microtubule formation.
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Data availability
Cryo-EM maps and models are available in the Electron Microscopy Database (EMDB) with the EMBD-IDs: EMD-47949, EMD-47954, EMD-47947, EMD-47948. The corresponding atomic coordinates for models are available at the Protein Data Bank (PDB) with the accession numbers, PDB-ID: 9EDT, 9EEB, 9EDR, 9EDS, respectively. The work also utilized the following coordinates for model building and comparisons: 1FFX, 4DRX, and 6GWD. Source data are provided with this paper.
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Acknowledgements
We thank the Bay Area Cryo-EM consortium led by Prof. Eva Nogales (Molecular Cell Biology, UC-Berkeley) and the UC-Davis BioEM facility for Cryo-EM data collection support. Large Scale Cryo-EM data were collected at UC-Davis and the Cryo-EM facility at UC-San Francisco with support from Dr Alexander Mysanikov (Biochemistry and Biophysics, UC-San Francisco). We thank Dr. Camille Scott and the UC-Davis High-Performance Computing Facility (HPCCF) for computational HPC infrastructure building and support. We thank Dr Stanley Nithianantham (Molecular Cellular Biology, UC-Davis) for the preliminary biochemical studies. We thank Prof Jeffrey K Moore (Cell Developmental Biology, University of Colorado, Anschutz Medical Campus) for advice and suggestions and for the critical reading of this manuscript. We thank Prof Richard McKenney, Prof Jonathan Scholey (Molecular Cellular Biology, UC-Davis), and Prof Ahmet Yildiz (Molecular Cell Biology, UC-Berkeley) for comments on the manuscript. J.A.B. acknowledges funding support from the National Institutes of Health (GM110283 and GM158334).
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A.T. purified and assembled TBC-DEG/TBCC-αβ-tubulin complexes, collected cryo-EM data, determined and refined all single particle cryo-EM structures, built, refined, and validated all models for assemblies, prepared figures, wrote and edited the manuscript. Z.W. purified and assembled TBC-DEG-αβ-tubulin complexes, collected cryo-EM data, and determined initial single particle cryo-EM structures. B.S. built and refined initial models. F.G. supported cryo-EM grid preparation, cryo-EM screening, and large-scale cryo-EM data collection. J.A.B. planned and managed the project, trained scientists, obtained funding for the project, prepared assemblies, prepared figures, and wrote and edited the manuscript.
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Taheri, A., Wang, Z., Singal, B. et al. Cryo-EM structures of the tubulin cofactors reveal the molecular basis of alpha/beta-tubulin biogenesis. Nat Commun (2025). https://doi.org/10.1038/s41467-025-68142-0
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DOI: https://doi.org/10.1038/s41467-025-68142-0
