Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

A conformational switch in complexin is required for synaptotagmin to trigger synaptic fusion

Nature Structural & Molecular Biology volume 18, pages 934–940 (2011)Cite this article

Subjects

Abstract

The crystal structure of complexin bound to a prefusion SNAREpin mimetic shows that the accessory helix extends away from the SNAREpin in an 'open' conformation, binding another SNAREpin and inhibiting its assembly, to clamp fusion. In contrast, the accessory helix in the postfusion complex parallels the SNARE complex in a 'closed' conformation. Here we use targeted mutations, FRET spectroscopy and a functional assay that reconstitutes Ca2+-triggered exocytosis to show that the conformational switch from open to closed in complexin is needed for synaptotagmin-Ca2+ to trigger fusion. Triggering fusion requires the zippering of three crucial aspartate residues in the switch region (residues 64–68) of v-SNARE. Conformational switching in complexin is integral to clamp release and is probably triggered when its accessory helix is released from its trans-binding to the neighboring SNAREpin, allowing the v-SNARE to complete zippering and open a fusion pore.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on SpringerLink
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Zippering of one turn of VAMP2 helix triggers the switch in CPX position.
Figure 2: Aspartate residues 64, 65 and 68 on VAMP2 mediate the switch in CPX position.
Figure 3: Interaction of CPXcen with aspartate residues 64, 65 and 68 on VAMP2 provides thermodynamic driving force for the switch.
Figure 4: The switch in CPXacc position is necessary for synaptotagmin-Ca2+ to trigger fusion.
Figure 5: Perturbation of a single SNARE complex in the zigzag array should be sufficient to rapidly disassemble the clamp in response to neuronal stimulus.

Similar content being viewed by others

References

  1. Söllner, T. et al. SNAP receptors implicated in vesicle targeting and fusion. Nature 362, 318–324 (1993).

    Article  Google Scholar 

  2. Hu, C. et al. Fusion of cells by flipped SNAREs. Science 300, 1745–1749 (2003).

    Article  CAS  Google Scholar 

  3. McNew, J.A. et al. Compartmental specificity of cellular membrane fusion encoded in SNARE proteins. Nature 407, 153–159 (2000).

    Article  CAS  Google Scholar 

  4. Weber, T. et al. SNAREpins: minimal machinery for membrane fusion. Cell 92, 759–772 (1998).

    Article  CAS  Google Scholar 

  5. Sutton, R.B., Fasshauer, D., Jahn, R. & Brunger, A.T. Crystal structure of a SNARE complex involved in synaptic exocytosis at 2.4 Å resolution. Nature 395, 347–353 (1998).

    Article  CAS  Google Scholar 

  6. Karatekin, E. et al. A fast, single-vesicle fusion assay mimics physiological SNARE requirements. Proc. Natl. Acad. Sci. USA 107, 3517–3521 (2010).

    Article  CAS  Google Scholar 

  7. Perin, M.S., Fried, V.A., Mignery, G.A., Jahn, R. & Sudhof, T.C. Phospholipid binding by a synaptic vesicle protein homologous to the regulatory region of protein kinase C. Nature 345, 260–263 (1990).

    Article  CAS  Google Scholar 

  8. Brose, N., Petrenko, A.G., Sudhof, T.C. & Jahn, R. Synaptotagmin: a calcium sensor on the synaptic vesicle surface. Science 256, 1021–1025 (1992).

    Article  CAS  Google Scholar 

  9. Fernández-Chacón, R. et al. Synaptotagmin I functions as a calcium regulator of release probability. Nature 410, 41–49 (2001).

    Article  Google Scholar 

  10. Geppert, M. et al. Synaptotagmin I: a major Ca2+ sensor for transmitter release at a central synapse. Cell 79, 717–727 (1994).

    Article  CAS  Google Scholar 

  11. Pang, Z.P., Shin, O.H., Meyer, A.C., Rosenmund, C. & Sudhof, T.C. A gain-of-function mutation in synaptotagmin-1 reveals a critical role of Ca2+-dependent soluble N-ethylmaleimide-sensitive factor attachment protein receptor complex binding in synaptic exocytosis. J. Neurosci. 26, 12556–12565 (2006).

    Article  CAS  Google Scholar 

  12. Ishizuka, T., Saisu, H., Odani, S. & Abe, T. Synaphin: a protein associated with the docking/fusion complex in presynaptic terminals. Biochem. Biophys. Res. Commun. 213, 1107–1114 (1995).

    Article  CAS  Google Scholar 

  13. McMahon, H.T., Missler, M., Li, C. & Sudhof, T.C. Complexins: cytosolic proteins that regulate SNAP receptor function. Cell 83, 111–119 (1995).

    Article  CAS  Google Scholar 

  14. Giraudo, C.G., Eng, W.S., Melia, T.J. & Rothman, J.E. A clamping mechanism involved in SNARE-dependent exocytosis. Science 313, 676–680 (2006).

    Article  CAS  Google Scholar 

  15. Giraudo, C.G. et al. Alternative zippering as an on-off switch for SNARE-mediated fusion. Science 323, 512–516 (2009).

    Article  CAS  Google Scholar 

  16. Maximov, A., Tang, J., Yang, X., Pang, Z.P. & Sudhof, T.C. Complexin controls the force transfer from SNARE complexes to membranes in fusion. Science 323, 516–521 (2009).

    Article  CAS  Google Scholar 

  17. Xue, M. et al. Distinct domains of complexin I differentially regulate neurotransmitter release. Nat. Struct. Mol. Biol. 14, 949–958 (2007).

    Article  CAS  Google Scholar 

  18. Xue, M. et al. Tilting the balance between facilitatory and inhibitory functions of mammalian and Drosophila Complexins orchestrates synaptic vesicle exocytosis. Neuron 64, 367–380 (2009).

    Article  CAS  Google Scholar 

  19. Südhof, T.C. & Rothman, J.E. Membrane fusion: grappling with SNARE and SM proteins. Science 323, 474–477 (2009).

    Article  Google Scholar 

  20. Li, F. et al. Complexin activates and clamps SNAREpins by a common mechanism involving an intermediate energetic state. Nat. Struct. Mol. Biol. doi:10.1038/nsmb.2102 (2011).

  21. Kümmel, D. et al. Complexin cross-links prefusion SNAREs into a zigzag array. Nat. Struct. Mol. Biol. doi:10.1038/nsmb.2101 (2011).

  22. Hua, S.Y. & Charlton, M.P. Activity-dependent changes in partial VAMP complexes during neurotransmitter release. Nat. Neurosci. 2, 1078–1083 (1999).

    Article  CAS  Google Scholar 

  23. Reim, K. et al. Complexins regulate a late step in Ca2+-dependent neurotransmitter release. Cell 104, 71–81 (2001).

    Article  CAS  Google Scholar 

  24. Tang, J. et al. A complexin/synaptotagmin 1 switch controls fast synaptic vesicle exocytosis. Cell 126, 1175–1187 (2006).

    Article  CAS  Google Scholar 

  25. Chen, X. et al. Three-dimensional structure of the complexin/SNARE complex. Neuron 33, 397–409 (2002).

    Article  CAS  Google Scholar 

  26. Bracher, A., Kadlec, J., Betz, H. & Weissenhorn, W. X-ray structure of a neuronal complexin-SNARE complex from squid. J. Biol. Chem. 277, 26517–26523 (2002).

    Article  CAS  Google Scholar 

  27. Yang, X., Kaeser-Woo, Y.J., Pang, Z.P., Xu, W. & Sudhof, T.C. Complexin clamps asynchronous release by blocking a secondary Ca2+ sensor via its accessory alpha helix. Neuron 68, 907–920 (2010).

    Article  CAS  Google Scholar 

  28. Shao, X., Fernandez, I., Sudhof, T.C. & Rizo, J. Solution structures of the Ca2+-free and Ca2+-bound C2A domain of synaptotagmin I: does Ca2+ induce a conformational change? Biochemistry 37, 16106–16115 (1998).

    Article  CAS  Google Scholar 

  29. Radhakrishnan, A., Stein, A., Jahn, R. & Fasshauer, D. The Ca2+ affinity of synaptotagmin 1 is markedly increased by a specific interaction of its C2B domain with phosphatidylinositol 4,5-bisphosphate. J. Biol. Chem. 284, 25749–25760 (2009).

    Article  CAS  Google Scholar 

  30. Vrljic, M. et al. Molecular mechanism of the synaptotagmin-SNARE interaction in Ca2+-triggered vesicle fusion. Nat. Struct. Mol. Biol. 17, 325–331 (2010).

    Article  CAS  Google Scholar 

  31. Ubach, J., Zhang, X., Shao, X., Sudhof, T.C. & Rizo, J. Ca2+ binding to synaptotagmin: how many Ca2+ ions bind to the tip of a C2-domain? EMBO J. 17, 3921–3930 (1998).

    Article  CAS  Google Scholar 

  32. Bai, J., Tucker, W.C. & Chapman, E.R. PIP2 increases the speed of response of synaptotagmin and steers its membrane-penetration activity toward the plasma membrane. Nat. Struct. Mol. Biol. 11, 36–44 (2004).

    Article  CAS  Google Scholar 

  33. Chapman, E.R. & Davis, A.F. Direct interaction of a Ca2+-binding loop of synaptotagmin with lipid bilayers. J. Biol. Chem. 273, 13995–14001 (1998).

    Article  CAS  Google Scholar 

  34. McMahon, H.T., Kozlov, M.M. & Martens, S. Membrane curvature in synaptic vesicle fusion and beyond. Cell 140, 601–605 (2010).

    Article  CAS  Google Scholar 

  35. Xue, M. et al. Binding of the complexin N terminus to the SNARE complex potentiates synaptic-vesicle fusogenicity. Nat. Struct. Mol. Biol. 17, 568–575 (2010).

    Article  CAS  Google Scholar 

  36. Yoon, T.Y. et al. Complexin and Ca2+ stimulate SNARE-mediated membrane fusion. Nat. Struct. Mol. Biol. 15, 707–713 (2008).

    Article  CAS  Google Scholar 

  37. Sabatini, B.L. & Regehr, W.G. Timing of neurotransmission at fast synapses in the mammalian brain. Nature 384, 170–172 (1996).

    Article  CAS  Google Scholar 

  38. Evans, E. Probing the relation between force–lifetime–and chemistry in single molecular bonds. Annu. Rev. Biophys. Biomol. Struct. 30, 105–128 (2001).

    Article  CAS  Google Scholar 

  39. Scott, B.L. et al. Liposome fusion assay to monitor intracellular membrane fusion machines. Methods Enzymol. 372, 274–300 (2003).

    Article  CAS  Google Scholar 

  40. Ji, H. et al. Protein determinants of SNARE-mediated lipid mixing. Biophys. J. 99, 553–560 (2010).

    Article  CAS  Google Scholar 

  41. Giraudo, C.G. et al. Distinct domains of complexins bind SNARE complexes and clamp fusion in vitro. J. Biol. Chem. 283, 21211–21219 (2008).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We wish to thank T. Melia (Yale University) for critical reading of the manuscript. This work was supported by US National Institutes of Health grants to J.E.R. and K.M.R., an Agence Nationale de la Recherche (ANR) Physique et Chimie du Vivant (PCV) grant to F.P., and a grant from the Deutsche Forschungsgemeinschaft to D.K.

Author information

Author notes

  1. Shyam S Krishnakumar and Daniel T Radoff: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Cell Biology, Yale University School of Medicine, New Haven, Connecticut, USA

    Shyam S Krishnakumar, Daniel T Radoff, Daniel Kümmel, Claudio G Giraudo, Feng Li, Lavan Khandan, Stephanie Wood Baguley, Jeff Coleman, Karin M Reinisch, Frederic Pincet & James E Rothman

  2. Department of Biochemistry and Molecular Biophysics, Columbia University, New York, New York, USA

    Daniel T Radoff

  3. Laboratoire de Physique Statistique, Unité Mixte de Recherche 8550, Centre National de la Recherche Scientifique associée aux Université Paris VI et Paris VII, Ecole Normale Supérieure, Paris, France

    Frederic Pincet

Authors
  1. Shyam S Krishnakumar
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Daniel T Radoff
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Daniel Kümmel
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Claudio G Giraudo
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Feng Li
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Lavan Khandan
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Stephanie Wood Baguley
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Jeff Coleman
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Karin M Reinisch
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Frederic Pincet
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. James E Rothman
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

S.S.K., D.T.R. and D.K. did the mutagenesis and protein purification, S.S.K. and D.T.R. carried out the fluorescence measurements, F.L. took the ITC measurements and C.G.G. did the cell-cell fusion assay. L.K., S.B. and J.C. provided technical assistance. Results were analyzed and discussed by all authors. The manuscript was prepared by S.S.K., D.T.R., K.M.R., F.P. and J.E.R.

Corresponding author

Correspondence to James E Rothman.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–4 (PDF 846 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Krishnakumar, S., Radoff, D., Kümmel, D. et al. A conformational switch in complexin is required for synaptotagmin to trigger synaptic fusion. Nat Struct Mol Biol 18, 934–940 (2011). https://doi.org/10.1038/nsmb.2103

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nsmb.2103

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing