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Nucleotide-dependent bending flexibility of tubulin regulates microtubule assembly

Abstract

The atomic structure of tubulin in a polymerized, straight protofilament is clearly distinct from that in a curved conformation bound to a cellular depolymerizer. The nucleotide contents are identical, and in both cases the conformation of the GTP-containing, intra-dimer interface is indistinguishable from the GDP-containing, inter-dimer contact. Here we present two structures corresponding to the start and end points in the microtubule polymerization and hydrolysis cycles that illustrate the consequences of nucleotide state on longitudinal and lateral assembly. In the absence of depolymerizers, GDP-bound tubulin shows distinctive intra-dimer and inter-dimer interactions and thus distinguishes the GTP and GDP interfaces. A cold-stable tubulin polymer with the non-hydrolysable GTP analogue GMPCPP, containing semi-conserved lateral interactions, supports a model in which the straightening of longitudinal interfaces happens sequentially, starting with a conformational change after GTP binding that straightens the dimer enough for the formation of lateral contacts into a non-tubular intermediate. Closure into a microtubule does not require GTP hydrolysis.

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Figure 1: Cryo-EM reconstruction of double-layered tubes of GDP-bound tubulin and docking of crystallographic models.
Figure 2: Intra-dimer and inter-dimer bends in different tubulin polymers.
Figure 3: Cryo-EM reconstruction of GMPCPP-tubulin tubes and docking of the crystallographic model.
Figure 4: GMPCPP tubes: comparison with and conversion into microtubules.

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Acknowledgements

We thank G. Barnes for fluorescent tubulin; S. Westermann for help with the optical microscopy; K. Downing, P. Grob and A. Leschziner for their comments on the manuscript; and W. Wriggers for generating the energy-minimized atomic models after the addition of missing loops. This work was funded by a grant from NIHGMS to E.N. and by the Office of Biological and Environmental Research of the US Department of Energy. E.N. is a Howard Hughes Medical Investigator.

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Correspondence to Eva Nogales.

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The density maps have been deposited at the Macromolecular Structure Database under accession numbers EMD-1129 (GDP-tube, inner layer), EMD-1130 (GDP-tube, outer layer), and EMD-1131 (GMPCPP-tube). Reprints and permissions information is available at npg.nature.com/reprintsandpermissions. The authors declare no competing financial interests.

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Supplementary Notes

This contains the Supplementary Methods, Supplementary Figure Legends and Supplementary Tables S1 and S2. (DOC 95 kb)

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Wang, HW., Nogales, E. Nucleotide-dependent bending flexibility of tubulin regulates microtubule assembly. Nature 435, 911–915 (2005). https://doi.org/10.1038/nature03606

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