Abstract

The mechanochemical protein dynamin is the prototype of the dynamin superfamily of large GTPases, which shape and remodel membranes in diverse cellular processes1. Dynamin forms predominantly tetramers in the cytosol, which oligomerize at the neck of clathrin-coated vesicles to mediate constriction and subsequent scission of the membrane1. Previous studies have described the architecture of dynamin dimers2,3, but the molecular determinants for dynamin assembly and its regulation have remained unclear. Here we present the crystal structure of the human dynamin tetramer in the nucleotide-free state. Combining structural data with mutational studies, oligomerization measurements and Markov state models of molecular dynamics simulations, we suggest a mechanism by which oligomerization of dynamin is linked to the release of intramolecular autoinhibitory interactions. We elucidate how mutations that interfere with tetramer formation and autoinhibition can lead to the congenital muscle disorders Charcot–Marie–Tooth neuropathy4 and centronuclear myopathy5, respectively. Notably, the bent shape of the tetramer explains how dynamin assembles into a right-handed helical oligomer of defined diameter, which has direct implications for its function in membrane constriction.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Accessions

Primary accessions

Protein Data Bank

Data deposits

The atomic coordinates and structure factors of human dynamin 3 have been deposited in the Protein Data Bank (PDB) with accession number 5A3F.

References

  1. 1.

    & Dynamin, a membrane-remodelling GTPase. Nature Rev. Mol. Cell Biol. 13, 75–88 (2012)

  2. 2.

    et al. Crystal structure of nucleotide-free dynamin. Nature 477, 556–560 (2011)

  3. 3.

    , & The crystal structure of dynamin. Nature 477, 561–566 (2011)

  4. 4.

    , , & Defective membrane remodeling in neuromuscular diseases: insights from animal models. PLoS Genet. 8, e1002595 (2012)

  5. 5.

    , , & Dynamin 2 and human diseases. J. Mol. Med. 88, 339–350 (2010)

  6. 6.

    , & BAR domain scaffolds in dynamin-mediated membrane fission. Cell 156, 882–892 (2014)

  7. 7.

    & Dynamin self-assembles into rings suggesting a mechanism for coated vesicle budding. Nature 374, 190–192 (1995)

  8. 8.

    , , & Tubular membrane invaginations coated by dynamin rings are induced by GTP-gamma S in nerve terminals. Nature 374, 186–190 (1995)

  9. 9.

    et al. A pseudoatomic model of the dynamin polymer identifies a hydrolysis-dependent powerstroke. Cell 147, 209–222 (2011)

  10. 10.

    et al. Structural insights into dynamin-mediated membrane fission. Structure 20, 1621–1628 (2012)

  11. 11.

    et al. The dynamin middle domain is critical for tetramerization and higher-order self-assembly. EMBO J. 26, 559–566 (2007)

  12. 12.

    et al. Structural insights into oligomerization and mitochondrial remodelling of dynamin 1-like protein. EMBO J. 32, 1280–1292 (2013)

  13. 13.

    et al. Structure of myxovirus resistance protein a reveals intra- and intermolecular domain interactions required for the antiviral function. Immunity 35, 514–525 (2011)

  14. 14.

    et al. Structural basis of oligomerization in the stalk region of dynamin-like MxA. Nature 465, 502–506 (2010)

  15. 15.

    , & Intrapolypeptide Interactions between the GTPase Effector Domain (GED) and the GTPase Domain Form the Bundle Signaling Element in Dynamin Dimers. Biochemistry 53, 5724–5726 (2014)

  16. 16.

    et al. Mild functional differences of dynamin 2 mutations associated to centronuclear myopathy and Charcot-Marie Tooth peripheral neuropathy. PLoS ONE 6, e27498 (2011)

  17. 17.

    et al. Dynamin 2 mutations in Charcot-Marie-Tooth neuropathy highlight the importance of clathrin-mediated endocytosis in myelination. Brain 135, 1395–1411 (2012)

  18. 18.

    et al. Differential curvature sensing and generating activities of dynamin isoforms provide opportunities for tissue-specific regulation. Proc. Natl Acad. Sci. USA 108, E234–E242 (2011)

  19. 19.

    et al. Importance of the pleckstrin homology domain of dynamin in clathrin-mediated endocytosis. Curr. Biol. 9, 257–263 (1999)

  20. 20.

    & Dynamin GTPase regulation is altered by PH domain mutations found in centronuclear myopathy patients. EMBO J. 29, 3054–3067 (2010)

  21. 21.

    , & (eds) An Introduction to Markov State Models and Their Application to Long Timescale Molecular Simulation. (Springer, 2014)

  22. 22.

    & Metastability and Markov models in molecular dynamics: modeling, analysis, algorithmic approaches. In Courant Lecture Notes Vol. 24 (American Mathematical Society, 2013)

  23. 23.

    et al. Cloud-based simulations on Google Exacycle reveal ligand modulation of GPCR activation pathways. Nature Chem. 6, 15–21 (2014)

  24. 24.

    , , & The stalk region of dynamin drives the constriction of dynamin tubes. Nature Struct. Mol. Biol. 11, 574–575 (2004)

  25. 25.

    et al. Membrane curvature controls dynamin polymerization. Proc. Natl Acad. Sci. USA 107, 4141–4146 (2010)

  26. 26.

    et al. A dynamin mutant defines a super-constricted pre-fission state. Cell Rep. 8, 734–742 (2014)

  27. 27.

    , & Dynamin recruitment and membrane scission at the neck of a clathrin-coated pit. Mol. Biol. Cell 25, 3595–3609 (2014)

  28. 28.

    et al. Actin and dynamin2 dynamics and interplay during clathrin-mediated endocytosis. J. Cell Biol. 205, 721–735 (2014)

  29. 29.

    , , & Dynamin-dependent and dynamin-independent processes contribute to the regulation of single vesicle release kinetics and quantal size. Proc. Natl Acad. Sci. USA 99, 7124–7129 (2002)

  30. 30.

    & Integrating molecular and network biology to decode endocytosis. Nature 448, 883–888 (2007)

  31. 31.

    , & Differential distribution of dynamin isoforms in mammalian cells. Mol. Biol. Cell 9, 2595–2609 (1998)

  32. 32.

    XDS. Acta Crystallogr. D 66, 125–132 (2010)

  33. 33.

    et al. Phaser crystallographic software. J. Appl. Crystallogr. 40, 658–674 (2007)

  34. 34.

    , , & Features and development of Coot. Acta Crystallogr. D 66, 486–501 (2010)

  35. 35.

    et al. The Phenix software for automated determination of macromolecular structures. Methods 55, 94–106 (2011)

  36. 36.

    et al. MolProbity: all-atom structure validation for macromolecular crystallography. Acta Crystallogr. D 66, 12–21 (2010)

  37. 37.

    The PyMOL Molecular Graphics System. Version 1.7.0.1. (Schrödinger, LLC)

  38. 38.

    A solution for the best rotation to relate two sets of vectors. Acta Crystallogr. A 32, 922–923 (1976)

  39. 39.

    et al. Clustal W and Clustal X version 2.0. Bioinformatics 23, 2947–2948 (2007)

  40. 40.

    et al. UCSF Chimera–a visualization system for exploratory research and analysis. J. Comput. Chem. 25, 1605–1612 (2004)

  41. 41.

    Size-distribution analysis of macromolecules by sedimentation velocity ultracentrifugation and lamm equation modeling. Biophys. J. 78, 1606–1619 (2000)

  42. 42.

    & . Modern applications of analytical ultracentrifugation. Annu. Rev. Biophys. Biomol. Struct. 28, 75–100 (1999)

  43. 43.

    , , & in Analytical Ultracentrifugation in Biochemistry and Polymer Science (eds et al.) 90–125 (Royal Society of Chemistry, 1992)

  44. 44.

    et al. Computer control of microscopes using μManager. Curr. Protoc. Mol. Biol (2010)

  45. 45.

    , & VMD: visual molecular dynamics. J. Mol. Graph. 14, 33–38 (1996)

  46. 46.

    , & GROMACS 3.0: a package for molecular simulation and trajectory analysis. J. Mol. Model. 7, 306–317 (2001)

  47. 47.

    , & . Extending the treatment of backbone energetics in protein force fields: limitations of gas-phase quantum mechanics in reproducing protein conformational distributions in molecular dynamics simulations. J. Comput. Chem. 25, 1400–1415 (2004)

  48. 48.

    et al. Markov models of molecular kinetics: Generation and validation. J. Chem. Phys. 134, 174105 (2011)

  49. 49.

    , & Kinetic modulation of a disordered protein domain by phosphorylation. Nature Commun. 5, 5272 (2014)

  50. 50.

    et al. EMMA: A Software Package for Markov Model Building and Analysis. J. Chem. Theory Comput. 8, 2223–2238 (2012)

  51. 51.

    , & Describing protein folding kinetics by molecular dynamics simulations. 1. Theory. J. Phys. Chem. B 108, 6571–6581 (2004)

  52. 52.

    & Robust Perron cluster analysis in conformation dynamics. Linear Algebra Appl. 398, 161–184 (2005)

  53. 53.

    & Building a fission machine–structural insights into dynamin assembly and activation. J. Cell Sci. 126, 2773–2784 (2013)

Download references

Acknowledgements

This project was supported by grants from the Deutsche Forschungsgemeinschaft (MA1081/8-2 to D.J.M.; SFB740/D7 and SFB958/A04 to F.N.; SFB740/C8 and SFB 958/A7 to V.H.; SFB 740/C7 and SFB958/A12 to O.D.; and ES410/2-1 to S.E.), an ERC consolidator grant (ERC-2013-CoG-616024 to O.D.), an ERC starting grant (pcCell to F.N.) and a grant from the Einstein Foundation Berlin (SOoPiC to N.P.). T.F.R. acknowledges partial financial support by the Cluster of Excellence REBIRTH (DFG EXC 62/1). We thank B. Purfürst for help with electron microscopy; S. Hertel, L. Litz, P. Straub and S. Wohlgemuth for experimental assistance; and the staff at beamlines X06SA (PXI) and X06DA (PXIII) at the Swiss Light Source (Villigen, Switzerland) for help during data collection. We thank Y.-W. Liu for discussions, and A. Wittinghofer for his support and discussions in the initial stages of the project.

Author information

Author notes

    • Thomas F. Reubold
    •  & Katja Faelber

    These authors contributed equally to this work.

    • York Posor

    Present address: Cancer Institute, University College London, 72 Huntley Street, London WC1E 6DD, UK.

Affiliations

  1. Institut für Biophysikalische Chemie, Medizinische Hochschule Hannover, Carl-Neuberg-Str. 1, 30625 Hannover, Germany

    • Thomas F. Reubold
    • , Ute Curth
    • , Roopsee Anand
    • , Dietmar J. Manstein
    •  & Susanne Eschenburg
  2. Max-Delbrück-Centrum für Molekulare Medizin, Kristallographie, Robert-Rössle-Straße 10, 13125 Berlin, Germany

    • Katja Faelber
    • , Jeanette Schlegel
    •  & Oliver Daumke
  3. Institut für Mathematik, Freie Universität Berlin, Arnimallee 6, 14195 Berlin, Germany

    • Nuria Plattner
    •  & Frank Noé
  4. Leibniz-Institut für Molekulare Pharmakologie, Robert-Rössle-Straße 10, 13125 Berlin, Germany

    • York Posor
    • , Katharina Ketel
    •  & Volker Haucke
  5. Forschungseinrichtung Strukturanalyse, Medizinische Hochschule Hannover, Carl-Neuberg-Str. 1, 30625 Hannover, Germany

    • Ute Curth
    •  & Dietmar J. Manstein
  6. Institut für Chemie und Biochemie, Freie Universität Berlin, Takustraße 6, 14195 Berlin, Germany

    • Volker Haucke
    •  & Oliver Daumke

Authors

  1. Search for Thomas F. Reubold in:

  2. Search for Katja Faelber in:

  3. Search for Nuria Plattner in:

  4. Search for York Posor in:

  5. Search for Katharina Ketel in:

  6. Search for Ute Curth in:

  7. Search for Jeanette Schlegel in:

  8. Search for Roopsee Anand in:

  9. Search for Dietmar J. Manstein in:

  10. Search for Frank Noé in:

  11. Search for Volker Haucke in:

  12. Search for Oliver Daumke in:

  13. Search for Susanne Eschenburg in:

Contributions

T.F.R. grew the crystals and collected data; K.F. solved the structure; T.F.R., K.F. and S.E. refined the structure; T.F.R. and R.A. purified protein for crystallization and monomeric dynamin; K.F. and J.S. purified all other proteins, performed liposome co-sedimentation, EM and GTPase assays; U.C. performed and analysed analytical ultracentrifugation experiments; Y.P. and K.K. performed transferrin uptake assays; N.P. and. F.N. conducted and analysed molecular modelling and molecular dynamics simulations. N.P. and Y.P. contributed equally to this work. T.F.R., K.F., F.N., V.H., O.D. and S.E. interpreted structural data. T.F.R., K.F., Y.P., N.P., U.C., D.J.M., F.N., V.H., O.D. and S.E. designed the research. T.F.R., K.F., F.N., O.D. and S.E. wrote the manuscript.

Competing interests

The authors declare no competing financial interests.

Corresponding authors

Correspondence to Katja Faelber or Oliver Daumke or Susanne Eschenburg.

Extended data

Supplementary information

PDF files

  1. 1.

    Supplementary Information

    This file contains Supplementary Figure 1, sequence alignment of the dynamin superfamily. The figure contains a structure-based alignment of protein sequences of important members of the dynamin superfamily. Amino acid residues mutated in this study are highlighted. It also contains gel images for Figure 2 and Extended Data Figures 5 and 6.

About this article

Publication history

Received

Accepted

Published

DOI

https://doi.org/10.1038/nature14880

Further reading

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.