Abstract

Synaptotagmin-1 and neuronal SNARE proteins have central roles in evoked synchronous neurotransmitter release; however, it is unknown how they cooperate to trigger synaptic vesicle fusion. Here we report atomic-resolution crystal structures of Ca2+- and Mg2+-bound complexes between synaptotagmin-1 and the neuronal SNARE complex, one of which was determined with diffraction data from an X-ray free-electron laser, leading to an atomic-resolution structure with accurate rotamer assignments for many side chains. The structures reveal several interfaces, including a large, specific, Ca2+-independent and conserved interface. Tests of this interface by mutagenesis suggest that it is essential for Ca2+-triggered neurotransmitter release in mouse hippocampal neuronal synapses and for Ca2+-triggered vesicle fusion in a reconstituted system. We propose that this interface forms before Ca2+ triggering, moves en bloc as Ca2+ influx promotes the interactions between synaptotagmin-1 and the plasma membrane, and consequently remodels the membrane to promote fusion, possibly in conjunction with other interfaces.

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

Data deposits

The coordinates of the atomic models and corresponding structure factors of the Syt1–SNARE complexes have been deposited in the Protein Data Bank under the accession codes 5CCG, 5CCH, 5CCI and 5CCJ.

References

  1. 1.

    & Membrane fusion. Nature Struct. Mol. Biol. 15, 658–664 (2008)

  2. 2.

    The Principle of Membrane Fusion in the Cell (Nobel Lecture). Angew. Chemie Int. Ed. 53, 12676–12694 (2014)

  3. 3.

    , , & Crystal structure of a SNARE complex involved in synaptic exocytosis at 2.4 Å resolution. Nature 395, 347–353 (1998)

  4. 4.

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

  5. 5.

    Neurotransmitter release: the last millisecond in the life of a synaptic vesicle. Neuron 80, 675–690 (2013)

  6. 6.

    & Cell biology of Ca2+-triggered exocytosis. Curr. Opin. Cell Biol. 22, 496–505 (2010)

  7. 7.

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

  8. 8.

    , & Synaptotagmin-1, -2, and -9: Ca2+ sensors for fast release that specify distinct presynaptic properties in subsets of neurons. Neuron 54, 567–581 (2007)

  9. 9.

    et al. Distinct roles for two synaptotagmin isoforms in synchronous and asynchronous transmitter release at zebrafish neuromuscular junction. Proc. Natl Acad. Sci. USA 107, 13906–13911 (2010)

  10. 10.

    et al. Synaptotagmin-1 and synaptotagmin-7 trigger synchronous and asynchronous phases of neurotransmitter release. Neuron 80, 947–959 (2013)

  11. 11.

    & Synaptotagmin I functions as a calcium sensor to synchronize neurotransmitter release. Neuron 36, 897–908 (2002)

  12. 12.

    & Autonomous function of synaptotagmin 1 in triggering synchronous release independent of asynchronous release. Neuron 48, 547–554 (2005)

  13. 13.

    & Synaptotagmin increases the dynamic range of synapses by driving Ca2+-evoked release and by clamping a near-linear remaining Ca2+ sensor. Neuron 69, 736–748 (2011)

  14. 14.

    & A single C2 domain from synaptotagmin I is sufficient for high affinity Ca2+/phospholipid binding. J. Biol. Chem. 268, 26386–26390 (1993)

  15. 15.

    , , & Synaptotagmin: a calcium sensor on the synaptic vesicle surface. Science 256, 1021–1025 (1992)

  16. 16.

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

  17. 17.

    et al. Post-translational modifications and lipid binding profile of insect cell-expressed full-length mammalian synaptotagmin 1. Biochemistry 50, 9998–10012 (2011)

  18. 18.

    et al. Augmenting neurotransmitter release by enhancing the apparent Ca2+ affinity of synaptotagmin 1. Proc. Natl Acad. Sci. USA 102, 18664–18669 (2005)

  19. 19.

    et al. Close membrane-membrane proximity induced by Ca2+-dependent multivalent binding of synaptotagmin-1 to phospholipids. Nature Struct. Mol. Biol. 13, 209–217 (2006)

  20. 20.

    , , , & Synaptotagmin-mediated bending of the target membrane is a critical step in Ca2+-regulated fusion. Cell 138, 709–721 (2009)

  21. 21.

    , & How synaptotagmin promotes membrane fusion. Science 316, 1205–1208 (2007)

  22. 22.

    , & Mechanism of phospholipid binding by the C2A-domain of synaptotagmin I. Biochemistry 37, 12395–12403 (1998)

  23. 23.

    , , , & Membrane-bound orientation and position of the synaptotagmin C2B domain determined by site-directed spin labeling. Biochemistry 44, 18–28 (2005)

  24. 24.

    , & Syntaxin: a synaptic protein implicated in docking of synaptic vesicles at presynaptic active zones. Science 257, 255–259 (1992)

  25. 25.

    et al. Ca2+-dependent and -independent activities of neural and non-neural synaptotagmins. Nature 375, 594–599 (1995)

  26. 26.

    , , , & 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)

  27. 27.

    et al. Conserved prefusion protein assembly in regulated exocytosis. Mol. Biol. Cell 17, 283–294 (2006)

  28. 28.

    et al. Single-molecule FRET-derived model of the synaptotagmin 1-SNARE fusion complex. Nature Struct. Mol. Biol. 17, 318–324 (2010)

  29. 29.

    , , & A quaternary SNARE-synaptotagmin-Ca2+-phospholipid complex in neurotransmitter release. J. Mol. Biol. 367, 848–863 (2007)

  30. 30.

    et al. Dynamic binding mode of a Synaptotagmin-1–SNARE complex in solution. Nature Struct. Mol. Biol. 22, 555–564 (2015)

  31. 31.

    , , , & Structure of the first C2 domain of synaptotagmin I: a novel Ca2+/phospholipid-binding fold. Cell 80, 929–938 (1995)

  32. 32.

    et al. Three-dimensional structure of the synaptotagmin 1 C2B-domain: synaptotagmin 1 as a phospholipid binding machine. Neuron 32, 1057–1069 (2001)

  33. 33.

    , , & Structure of human synaptotagmin 1 C2AB in the absence of Ca2+ reveals a novel domain association. Biochemistry 46, 13041–13048 (2007)

  34. 34.

    et al. Natively inhibited Trypanosoma brucei cathepsin B structure determined by using an X-ray laser. Science 339, 227–230 (2013)

  35. 35.

    , , , & Synaptotagmin C2B domain regulates Ca2+-triggered fusion in vitro: critical residues revealed by scanning alanine mutagenesis. J. Biol. Chem. 283, 31763–31775 (2008)

  36. 36.

    , , , & The Janus-faced nature of the C2B domain is fundamental for synaptotagmin-1 function. Nature Struct. Mol. Biol. 15, 1160–1168 (2008)

  37. 37.

    et al. Synaptic proteins promote calcium-triggered fast transition from point contact to full fusion. Elife 1, e00109 (2012)

  38. 38.

    et al. Complexin inhibits spontaneous release and synchronizes Ca2+-triggered synaptic vesicle fusion by distinct mechanisms. Elife 3, 1–14 (2014)

  39. 39.

    & Electrostatic interaction of internal Mg2+ with membrane PIP2 Seen with KCNQ K+ channels. J. Gen. Physiol. 130, 241–256 (2007)

  40. 40.

    et al. Controlling synaptotagmin activity by electrostatic screening. Nature Struct. Mol. Biol. 19, 991–997 (2012)

  41. 41.

    , , & Synaptotagmin-1 functions as a Ca2+ sensor for spontaneous release. Nature Neurosci. 12, 759–766 (2009)

  42. 42.

    et al. Conformational dynamics of calcium-triggered activation of fusion by synaptotagmin. Biophys. J. 105, 2507–2516 (2013)

  43. 43.

    et al. Calcium sensitive ring-like oligomers formed by synaptotagmin. Proc. Natl Acad. Sci. USA 111, 13966–13971 (2014)

  44. 44.

    et al. Ca2+-independent syntaxin binding to the C2B effector region of synaptotagmin. Mol. Cell. Neurosci. 49, 1–8 (2012)

  45. 45.

    & Mutations in the second C2 domain of synaptotagmin disrupt synaptic transmission at Drosophila neuromuscular junctions. J. Comp. Neurol. 436, 4–16 (2001)

  46. 46.

    et al. Single reconstituted neuronal SNARE complexes zipper in three distinct stages. Science 337, 1340–1343 (2012)

  47. 47.

    , , , & Lipid-anchored SNAREs lacking transmembrane regions fully support membrane fusion during neurotransmitter release. Neuron 80, 470–483 (2013)

  48. 48.

    , , & Energetics of stalk intermediates in membrane fusion are controlled by lipid composition. Proc. Natl Acad. Sci. USA 109, E1609–E1618 (2012)

  49. 49.

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

  50. 50.

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

  51. 51.

    , , , & Linker length and composition influence the flexibility of Oct-1 DNA binding. EMBO J. 16, 2043–2053 (1997)

  52. 52.

    , , & The structure of an Arf-ArfGAP complex reveals a Ca2+ regulatory mechanism. Cell 141, 812–821 (2010)

  53. 53.

    & High resolution structure, stability, and synaptotagmin binding of a truncated neuronal SNARE complex. J. Biol. Chem. 278, 8630–8636 (2003)

  54. 54.

    et al. Processive ATP-driven disassembly of SNARE complexes by the N-ethylmaleimide sensitive factor molecular machine. J. Biol. Chem. (2013)

  55. 55.

    Protein production by auto-induction in high-density shaking cultures. Protein Expr. Purif. 41, 207–234 (2005)

  56. 56.

    , & Collective instabilities and high-gain regime in a free electron laser. Opt. Commun. 50, 373–378 (1984)

  57. 57.

    & Generation of coherent radiation by a relativistic electron beam in an undulator. Part. Accel. 10, 207–216 (1980)

  58. 58.

    , , , & Potential for biomolecular imaging with femtosecond X-ray pulses. Nature 406, 752–757 (2000)

  59. 59.

    Imaging biological specimens with high-intensity soft x rays. J. Opt. Soc. Am. B 3, 1551 (1986)

  60. 60.

    et al. Goniometer-based femtosecond crystallography with X-ray free electron lasers. Proc. Natl Acad. Sci. USA 111, 17122–17127 (2014)

  61. 61.

    et al. Data exploration toolkit for serial diffraction experiments. Acta Crystallogr. D 71, 352–356 (2015)

  62. 62.

    et al. Accurate macromolecular structures using minimal measurements from X-ray free-electron lasers. Nature Methods 11, 545–548 (2014)

  63. 63.

    , , & New Python-based methods for data processing. Acta Crystallogr. D 69, 1274–1282 (2013)

  64. 64.

    et al. Improved crystal orientation and physical properties from single-shot XFEL stills. Acta Crystallogr. D 70, 3299–3309 (2014)

  65. 65.

    et al. Enabling X-ray free electron laser crystallography for challenging biological systems from a limited number of crystals. Elife 4, e05421 (2015)

  66. 66.

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

  67. 67.

    et al. Overview of the CCP4 suite and current developments. Acta Crystallogr. D 67, 235–242 (2011)

  68. 68.

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

  69. 69.

    & Coot: Model-building tools for molecular graphics. Acta Crystallogr. D 60, 2126–2132 (2004)

  70. 70.

    Version 1.2 of the Crystallography and NMR system. Nature Protocols 2, 2728–2733 (2007)

  71. 71.

    et al. Crystallography & NMR system: A new software suite for macromolecular structure determination. Acta Crystallogr. D 54, 905–921 (1998)

  72. 72.

    et al. PHENIX: building new software for automated crystallographic structure determination. Acta Crystallogr. D 58, 1948–1954 (2002)

  73. 73.

    , & Super-resolution biomolecular crystallography with low-resolution data. Nature 464, 1218–1222 (2010)

  74. 74.

    , , & Crystallographic model quality at a glance. Acta Crystallogr. D 65, 297–300 (2009)

  75. 75.

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

  76. 76.

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

  77. 77.

    & Inference of macromolecular assemblies from crystalline state. J. Mol. Biol. 372, 774–797 (2007)

  78. 78.

    & Synaptotagmin has an essential function in synaptic vesicle positioning for synchronous release in addition to its role as a calcium sensor. Neuron 63, 482–496 (2009)

  79. 79.

    et al. Synaptotagmin-1 docks secretory vesicles to syntaxin-1/SNAP-25 acceptor complexes. Cell 138, 935–946 (2009)

  80. 80.

    et al. Synaptotagmin interaction with SNAP-25 governs vesicle docking, priming, and fusion triggering. J. Neurosci. 33, 14417–14430 (2013)

  81. 81.

    et al. In vitro system capable of differentiating fast Ca2+-triggered content mixing from lipid exchange for mechanistic studies of neurotransmitter release. Proc. Natl Acad. Sci. USA 108, E304–E313 (2011)

  82. 82.

    , , , & Studying calcium-triggered vesicle fusion in a single vesicle-vesicle content and lipid-mixing system. Nature Protocols 8, 1–16 (2013)

  83. 83.

    et al. Dynamic Ca2+-dependent stimulation of vesicle fusion by membrane-anchored synaptotagmin 1. Science 328, 760–763 (2010)

  84. 84.

    , , , & The synaptotagmin 1 linker may function as an electrostatic zipper that opens for docking but closes for fusion pore opening. Biochem. J. 456, 25–33 (2013)

  85. 85.

    , , & Reconstituted synaptotagmin I mediates vesicle docking, priming, and fusion. J. Cell Biol. 195, 1159–1170 (2011)

  86. 86.

    & A distinct tethering step is vital for vacuole membrane fusion. Elife 3, e03251 (2014)

  87. 87.

    , , & Monitoring synaptic transmission in primary neuronal cultures using local extracellular stimulation. J. Neurosci. Methods 161, 75–87 (2007)

Download references

Acknowledgements

We thank M. S. Padolina for help with protein purification, the Northeastern Collaborative Access Team (supported by NIH P41 GM103403) at Advanced Photon Source for X-ray data collection, and the SSRL/LCLS scientists E. L. Baxter, P. Ehrensberger, T. I. Eriksson, Y. Feng, M. Hollenbeck, E. G. Kovaleva, S. E. McPhillips, S. Nelson, J. Song, Y. Tsai, V. Vinetsky and D. Zhu for their invaluable assistance with data collection at the LCLS XPP facility. Use of the Stanford Synchrotron Radiation Lightsource (SSRL) and Linac Coherent Light Source (LCLS), SLAC National Accelerator Laboratory, is supported by the US Department of Energy, Office of Science, Office of Basic Energy Sciences under contract no. DE-AC02-76SF00515. The SSRL Structural Molecular Biology Program is supported by the DOE Office of Biological and Environmental Research, and by the National Institutes of Health, National Institute of General Medical Sciences (including P41GM103393). Portions of this research were carried out at the Linac Coherent Light Source (LCLS) at the SLAC National Accelerator Laboratory. LCLS is an Office of Science User Facility operated for the US Department of Energy Office of Science by Stanford University. This research was supported in part by the National Institutes of Health (R37MH63105 to A.T.B.; MH086403 to T.C.S.; GM095887 and GM102520 to N.K.S and A.S.B); and by a HHMI Collaborative Innovation Award (HCIA) to A.T.B. and W.I.W.

Author information

Author notes

    • Ying Lai
    • , Taulant Bacaj
    •  & Minglei Zhao

    These authors contributed equally to this work.

Affiliations

  1. Department of Molecular and Cellular Physiology, Howard Hughes Medical Institute, Stanford University, Stanford, California 94305, USA

    • Qiangjun Zhou
    • , Ying Lai
    • , Taulant Bacaj
    • , Minglei Zhao
    • , Artem Y. Lyubimov
    • , Monarin Uervirojnangkoorn
    • , Oliver B. Zeldin
    • , Richard A. Pfuetzner
    • , Ucheor B. Choi
    • , Jiajie Diao
    • , Thomas C. Südhof
    •  & Axel T. Brunger
  2. Departments of Neurology and Neurological Sciences, Photon Science, and Structural Biology, Stanford University, Stanford, California 94305, USA

    • Qiangjun Zhou
    • , Ying Lai
    • , Minglei Zhao
    • , Artem Y. Lyubimov
    • , Monarin Uervirojnangkoorn
    • , Oliver B. Zeldin
    • , Richard A. Pfuetzner
    • , Ucheor B. Choi
    • , Jiajie Diao
    •  & Axel T. Brunger
  3. Physical Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley, California 94720, USA

    • Aaron S. Brewster
    •  & Nicholas K. Sauter
  4. SLAC National Accelerator Laboratory, Stanford, California 94305, USA

    • Aina E. Cohen
    • , S. Michael Soltis
    • , Roberto Alonso-Mori
    • , Matthieu Chollet
    •  & Henrik T. Lemke
  5. Departments of Structural Biology, Molecular and Cellular Physiology, and Photon Science, Stanford University, Stanford, California 94305, USA

    • William I. Weis

Authors

  1. Search for Qiangjun Zhou in:

  2. Search for Ying Lai in:

  3. Search for Taulant Bacaj in:

  4. Search for Minglei Zhao in:

  5. Search for Artem Y. Lyubimov in:

  6. Search for Monarin Uervirojnangkoorn in:

  7. Search for Oliver B. Zeldin in:

  8. Search for Aaron S. Brewster in:

  9. Search for Nicholas K. Sauter in:

  10. Search for Aina E. Cohen in:

  11. Search for S. Michael Soltis in:

  12. Search for Roberto Alonso-Mori in:

  13. Search for Matthieu Chollet in:

  14. Search for Henrik T. Lemke in:

  15. Search for Richard A. Pfuetzner in:

  16. Search for Ucheor B. Choi in:

  17. Search for William I. Weis in:

  18. Search for Jiajie Diao in:

  19. Search for Thomas C. Südhof in:

  20. Search for Axel T. Brunger in:

Contributions

Q.Z. designed, expressed, purified and crystallized the Syt1–SNARE complexes and the Syt1 C2B quintuple mutant, performed CD experiments, and designed the mutants to disrupt the primary interface. Q.Z. and M.Z. collected all diffraction data, determined and refined the crystal structures. Y.L. and J.D. performed the reconstituted single vesicle–vesicle experiments. T.B. performed the co-immunoprecipitation and electrophysiological experiments of neuronal cultures. A.Y.L., M.U., O.B.Z., N.K.S., A.S.B., W.I.W. and A.T.B. analysed and processed the LCLS diffraction data. A.E.C. and S.M.S. designed the goniometer-based setup at LCLS-XPP and helped with data collection. R.A.-M., M.C. and H.T.L. helped with data collection at LCLS-XPP. R.A.P. and Y.L. expressed and purified proteins for the single vesicle–vesicle experiments. U.B.C. helped with the comparison between the crystal structure and the smFRET data. Q.Z., M.Z., Y.L., T.B., T.C.S. and A.T.B. wrote the manuscript.

Competing interests

The authors declare no competing financial interests.

Corresponding authors

Correspondence to Thomas C. Südhof or Axel T. Brunger.

Extended data

Supplementary information

Videos

  1. 1.

    Structure of the Syt1-SNARE complex

    The entire asymmetric unit of the long unit cell crystal form consists of two SNARE complexes and three Syt1 C2AB fragments. One of the two SNARE complexes interacts with two Syt1 C2AB fragments in the asymmetric unit, and a symmetry related C2AB fragment (designated as Complex I). The other SNARE complex just interacts with the C2B domain of one Syt1 C2AB fragment (designated as Complex II). The diffraction data for this crystal structure were collected at the X-ray free electron laser (XFEL) Linac Coherent Light Source (LCLS) at SLAC Accelerator Laboratory at Stanford University.

  2. 2.

    Interfaces between Syt1 C2 domains and SNARE complexes

    At the start of the video, Complex I (long unit cell crystal form) is shown along with its symmetry mate, followed by close-up views of three different interfaces between Syt1 C2 domains and the SNARE complex, as well as the interface between two C2 domains.

About this article

Publication history

Received

Accepted

Published

DOI

https://doi.org/10.1038/nature14975

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.