Estévez-Gallego, JuanBlum, Thorsten B.Ruhnow, FelixGili, MariaSperoni, SilviaGarcia-Castellanos, RaquelSteinmetz, Michel O.Surrey, Thomas2025-05-152025-05-152025Estévez-Gallego J, Blum TB, Ruhnow F, Gili M, Speroni S, García-Castellanos R, et al. Hydrolysis-deficient mosaic microtubules as faithful mimics of the GTP cap. Nat Commun. 2025 Mar 10;16(1):2396. DOI: 10.1038/s41467-025-57555-62041-1723http://hdl.handle.net/10230/70397A critical feature of microtubules is their GTP cap, a stabilizing GTP-tubulin rich region at growing microtubule ends. Microtubules polymerized in the presence of GTP analogs or from GTP hydrolysis-deficient tubulin mutants have been used as GTP-cap mimics for structural and biochemical studies. However, these analogs and mutants generate microtubules with diverse biochemical properties and lattice structures, leaving it unclear what is the most faithful GTP mimic and hence the structure of the GTP cap. Here, we generate a hydrolysis-deficient human tubulin mutant, αE254Q, with the smallest possible modification. We show that αE254Q-microtubules are stable, but still exhibit mild mutation-induced growth abnormalities. However, mixing two GTP hydrolysis-deficient tubulin mutants, αE254Q and αE254N, at an optimized ratio eliminates growth and lattice abnormalities, indicating that these 'mosaic microtubules' are faithful GTP cap mimics. Their cryo-electron microscopy structure reveals that longitudinal lattice expansion, but not protofilament twist, is the primary structural feature distinguishing the GTP-tubulin containing cap from the GDP-tubulin containing microtubule shaft. However, alterations in protofilament twist may be transiently needed to allow lattice compaction and GTP hydrolysis. Together, our results provide insights into the structural origin of GTP cap stability, the pathway of GTP hydrolysis and hence microtubule dynamic instability.application/pdfeng© The Author(s) 2025. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.info:eu-repo/semantics/articlehttp://dx.doi.org/10.1038/s41467-025-57555-6Cytoskeletal proteinsStructural biologyinfo:eu-repo/semantics/openAccess