Abstract
The Ndc80 complex is a key site of kinetochore-microtubule attachment during cell division. The human complex engages microtubules with a globular 'head' formed by tandem calponin-homology domains and an 80-amino-acid unstructured 'tail' that contains sites of phosphoregulation by the Aurora B kinase. Using biochemical, cell biological and electron microscopy analyses, we dissected the roles of the tail in binding of microtubules and mediation of cooperative interactions between Ndc80 complexes. Two segments of the tail that contain Aurora B phosphorylation sites become ordered at interfaces; one with tubulin and the second with an adjacent Ndc80 head on the microtubule surface, forming interactions that are disrupted by phosphorylation. We propose a model in which Ndc80's interaction with either growing or shrinking microtubule ends can be tuned by the phosphorylation state of its tail.
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Acknowledgements
We acknowledge G. Lander for assistance with image processing. GST tail expression constructs were generated by members of the QB3 Macrolab at University of California Berkeley. We thank T. Houweling, P. Grob and G. Kemalyan for computer and electron microscopy support. G.M.A. is partially supported by a US National Institutes of Health training grant. This work was funded by grants from the US National Institutes of Health (GM051487 to E.N. and GM081576 to P.T.S.). E.N. is also funded by the Howard Hughes Medical Institute.
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G.M.A. and E.N. designed research. G.M.A. and V.M. purified proteins and performed microtubule-binding assays. G.M.A. carried out electron microscopy experiments and image processing. D.M. and J.T. performed cell biology experiments and generated new constructs. G.M.A. and E.N. wrote the paper. G.M.A, V.M., D.M., J.T., P.T.S. and E.N. contributed to data analysis and editing of the manuscript.
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Supplementary Text and Figures
Supplementary Figures 1–6, Supplementary Table 1 (PDF 16158 kb)
Supplementary Video 1
Cryo-EM structure of the Ndc80–microtubule interface, which supplements Figure 5a. Crystal structures of two bonsai Δ1–80 molecules (PDB 2VE7) and tubulin (PDB 1JFF) docked into the improved cryo-EM density map, colored as in Figure 5a. Two densities not occupied by the crystal structures (magenta) were interpreted as corresponding to ordered regions of the N-terminal tail. (MOV 18038 kb)
Supplementary Video 2
Visualizing the Ndc80–E hook interface, which supplements Figure 5c. Same as Supplementary Movie 1, but with the cryo-EM map displayed at a lower threshold, where the tubulin E hooks (red) are visible. (MOV 8715 kb)
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Alushin, G., Musinipally, V., Matson, D. et al. Multimodal microtubule binding by the Ndc80 kinetochore complex. Nat Struct Mol Biol 19, 1161–1167 (2012). https://doi.org/10.1038/nsmb.2411
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DOI: https://doi.org/10.1038/nsmb.2411
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