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Crystal structure of γ-tubulin complex protein GCP4 provides insight into microtubule nucleation

Nature Structural & Molecular Biology volume 18, pages 915–919 (2011)Cite this article

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Abstract

Microtubule nucleation in all eukaryotes involves γ-tubulin small complexes (γTuSCs) that comprise two molecules of γ-tubulin bound to γ-tubulin complex proteins (GCPs) GCP2 and GCP3. In many eukaryotes, multiple γTuSCs associate with GCP4, GCP5 and GCP6 into large γ-tubulin ring complexes (γTuRCs). Recent cryo-EM studies indicate that a scaffold similar to γTuRCs is formed by lateral association of γTuSCs, with the C-terminal regions of GCP2 and GCP3 binding γ-tubulin molecules. However, the exact role of GCPs in microtubule nucleation remains unknown. Here we report the crystal structure of human GCP4 and show that its C-terminal domain binds directly to γ-tubulin. The human GCP4 structure is the prototype for all GCPs, as it can be precisely positioned within the γTuSC envelope, revealing the nature of protein-protein interactions and conformational changes regulating nucleation activity.

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Figure 1: The crystal structure of GCP4 reveals a previously undescribed fold.
Figure 2: GCP4 binds to γ-tubulin through its C-terminal domain.
Figure 3: A molecular model of γTuSC.

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Acknowledgements

We thank M. Wright and J.-E. Gairin (Centre de Recherche en Pharmacologie-Santé, Centre National de la Recherche Scientifique Pierre Fabre, Toulouse) for their advice and support; the staff of synchrotron beamlines PROXIMA 1 at SOLEIL Synchrotron, ID14-1, ID14-2, ID23-2 and ID29 at the European Synchrotron Radiation Facility; P. Legrand from the PROXIMA 1 beamline for his help during data collection and preliminary calculation of experimental phases; and F. Viala for her help in preparing the figures. This project was financed in part with grant 08-BLAN-0281 from the French 'Agence Nationale de la Recherche' (A.M. and L.M.) and by the Howard Hughes Medical Institute and the US National Institutes of Health grant GM31627 (J.M.K. and D.A.A.).

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Author notes

  1. Valérie Guillet, Martine Knibiehler and Lynn Gregory-Pauron: These authors contributed equally to this work.

Authors and Affiliations

  1. Institut de Pharmacologie et de Biologie Structurale, Centre National de la Recherche Scientifique, Toulouse, France

    Valérie Guillet, Lynn Gregory-Pauron, Cécile Bon & Lionel Mourey

  2. Université de Toulouse, Université Paul Sabatier, Institut de Pharmacologie et de Biologie Structurale, Toulouse, France

    Valérie Guillet, Lynn Gregory-Pauron, Cécile Bon & Lionel Mourey

  3. Centre de Recherche en Pharmacologie-Santé, Centre National de la Recherche Scientifique–Pierre Fabre, Toulouse, France

    Martine Knibiehler, Marie-Hélène Remy, Cécile Chemin, Brigitte Raynaud-Messina & Andreas Merdes

  4. Department of Biochemistry and Biophysics, Howard Hughes Medical Institute, University of California, San Francisco, California, USA

    Justin M Kollman & David A Agard

  5. Keck Advanced Microscopy Center, University of California, San Francisco, California, USA

    Justin M Kollman & David A Agard

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  1. Valérie Guillet
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Contributions

V.G. helped optimize protein production and purification, grew the crystals and conducted diffraction data collection, analyzed the structure, prepared figures and participated in manuscript writing. M.K. optimized native and SeMet-labeled protein production and purification. L.G.-P. participated in data processing and did structure determination and refinement, analyzed the structure and helped prepare tables and figures. M.-H.R. made the constructs, produced and purified the proteins, carried out Flag pulldown experiments and prepared figures. C.C. made the constructs and carried out Flag pulldown experiments. B.R.-M. did initial purification studies. C.B. participated in protein characterization. J.M.K. analyzed structure-function relationships, carried out the fitting into the cryo-EM map of γTuSC and prepared figures. D.A.A. analyzed the data and revised the manuscript. A.M. devised the experiments, designed figures and wrote the manuscript. L.M. devised the experiments, participated in diffraction data collection, analyzed the structure, designed tables and figures and wrote the manuscript.

Corresponding authors

Correspondence to Andreas Merdes or Lionel Mourey.

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Supplementary Figures 1–7, Supplementary Table 1 and Supplementary Discussion (PDF 18948 kb)

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Guillet, V., Knibiehler, M., Gregory-Pauron, L. et al. Crystal structure of γ-tubulin complex protein GCP4 provides insight into microtubule nucleation. Nat Struct Mol Biol 18, 915–919 (2011). https://doi.org/10.1038/nsmb.2083

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