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Architecture of the Dam1 kinetochore ring complex and implications for microtubule-driven assembly and force-coupling mechanisms

Nature Structural & Molecular Biology volume 14, pages 721–726 (2007)Cite this article

Abstract

The Dam1 kinetochore complex is essential for chromosome segregation in budding yeast. This ten-protein complex self-assembles around microtubules, forming ring-like structures that move with depolymerizing microtubule ends, a mechanism with implications for cellular function. Here we used EM-based single-particle and helical analyses to define the architecture of the Dam1 complex at 30-Å resolution and the self-assembly mechanism. Ring oligomerization seems to be facilitated by a conformational change upon binding to microtubules, suggesting that the Dam1 ring is not preformed, but self-assembles around kinetochore microtubules. The C terminus of the Dam1p protein, where most of the Aurora kinase Ipl1 phosphorylation sites reside, is in a strategic location to affect oligomerization and interactions with the microtubule. One of Ipl1's roles might be to fine-tune the coupling of the microtubule interaction with the conformational change required for oligomerization, with phosphorylation resulting in ring breakdown.

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Figure 1: Negative-stain EM of unassembled Dam1 complexes.
Figure 2: Single-particle three-dimensional reconstructions of wild-type and ΔC Dam1 complexes.
Figure 3: End-on views of wild-type and ΔC Dam1 ring complexes.
Figure 4: Helical reconstruction of wild-type Dam1 complex helical assembly around microtubules.
Figure 5: Modeled conformational changes in the Dam1 complex between the unassembled and assembled states, viewed looking out from the microtubule.
Figure 6: Docking of the single-particle Dam1 complex reconstruction into the helical assembly around the microtubule.
Figure 7: Model of microtubule-induced self-assembly of the Dam1 ring complex at the kinetochore.

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Acknowledgements

We thank D. DeRosier for invaluable help with helical reconstruction of the assembled Dam1 complex and comments on the manuscript, J. Kuriyan and M. Lamers for help in performing the light-scattering experiments, E. Zelenova for help in preparing the helical crystal of Dam1 with microtubules, and J. Fang for Dam1 complex purification. This work was supported by grants from the National Institute of General Medical Sciences of the US National Institutes of Health (G.B. and E.N), from the Office of Biological and Environmental Research of the US Department of Energy (E.N.) and from Philip Morris USA of Philip Morris International (D.G.D.); by a postdoctoral fellowship of the Deutsche Forschungsgemeinschaft (S.W.); and by a predoctoral training grant from the US National Institutes of Health (V.H.R.). E.N. is a Howard Hughes Medical Institute Investigator.

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  1. Stefan Westermann

    Present address: Present address: Research Institute of Molecular Pathology, Dr. Bohr-Gasse 7, Vienna, Austria.,

Authors and Affiliations

  1. Life Sciences Division, Lawrence Berkeley National Laboratory, 1 Cyclotron Rd., Berkeley, 94720, California, USA

    Hong-Wei Wang, Vincent H Ramey, Julie P I Welburn & Eva Nogales

  2. Department of Molecular and Cell Biology, University of California, Berkeley, 94720, California, USA

    Vincent H Ramey, Stefan Westermann, Andres E Leschziner, Yuko Nakajima, David G Drubin, Georjana Barnes & Eva Nogales

  3. Howard Hughes Medical Institute, University of California, Berkeley, 94720, California, USA

    Eva Nogales

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Contributions

H.-W.W., S.W., D.G.D., G.B. and E.N. designed the research. H.-W.W. performed the electron microscopy and the helical reconstruction of the helical Dam1-microtubule assemblies. H.-W.W. and J.P.I.W. determined the handedness of the helical assembly. V.H.R. and A.E.L. performed the single-particle analysis of free Dam1 complexes in solution. S.W. prepared and purified the wild-type and mutant complexes. Y.N. built the GFP-labeled Dam1 construct and J.P.I.W. purified it. D.G.D. and G.B. supervised the biochemical experiments. E.N. supervised the electron microscopy and data analysis. H.-W.W., V.H.R. and E.N. performed the model docking and prepared the manuscript. All authors contributed to scientific discussions.

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Correspondence to Eva Nogales.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–8, Supplementary Table 1 and Supplementary Methods (PDF 1585 kb)

Supplementary Video 1

Two monomers fit back in the dimer reconstruction of wild-type Dam1. The colors are coded the same as in Figure 2b. (MOV 2335 kb)

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Wang, HW., Ramey, V., Westermann, S. et al. Architecture of the Dam1 kinetochore ring complex and implications for microtubule-driven assembly and force-coupling mechanisms. Nat Struct Mol Biol 14, 721–726 (2007). https://doi.org/10.1038/nsmb1274

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