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The primed SNARE–complexin–synaptotagmin complex for neuronal exocytosis

Nature volume 548, pages 420425 (24 August 2017) | Download Citation

Abstract

Synaptotagmin, complexin, and neuronal SNARE (soluble N-ethylmaleimide sensitive factor attachment protein receptor) proteins mediate evoked synchronous neurotransmitter release, but the molecular mechanisms mediating the cooperation between these molecules remain unclear. Here we determine crystal structures of the primed pre-fusion SNARE–complexin–synaptotagmin-1 complex. These structures reveal an unexpected tripartite interface between synaptotagmin-1 and both the SNARE complex and complexin. Simultaneously, a second synaptotagmin-1 molecule interacts with the other side of the SNARE complex via the previously identified primary interface. Mutations that disrupt either interface in solution also severely impair evoked synchronous release in neurons, suggesting that both interfaces are essential for the primed pre-fusion state. Ca2+ binding to the synaptotagmin-1 molecules unlocks the complex, allows full zippering of the SNARE complex, and triggers membrane fusion. The tripartite SNARE–complexin–synaptotagmin-1 complex at a synaptic vesicle docking site has to be unlocked for triggered fusion to start, explaining the cooperation between complexin and synaptotagmin-1 in synchronizing evoked release on the sub-millisecond timescale.

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Acknowledgements

We thank P. Gipson, J. Leitz, A. Lyubimov, and W. I. Weis for discussions, S. Muennich and S. Pokutta for assistance with ITC, and the National Institutes of Health (NIH) for support (R37 MH63105 to A.T.B.; P50 MH086403 to T.C.S.). Crystal diffraction screening and data collection were performed at synchrotron facilities provided by the Advanced Photon Source at Argonne National Laboratory, the Stanford Synchrotron Radiation Lightsource, California, and the Advanced Light Source, Berkeley, California, funded by Department of Energy (DOE) under contracts DE-AC02-06CH11357 (Advanced Photon Source), DE-AC02-76SF00515 (Stanford Synchrotron Radiation Lightsource), and DE-AC02-05CH11231 (ALS). We thank the staff at these beamlines for help with diffraction data collection. The Northeastern Collaborative Access Team (NECAT) beamlines are funded by the National Institute of General Medical Sciences (NIGMS) from the NIH (P41 GM103403). The Pilatus 6M detector at the 24-ID-C beam line is funded by an NIH-ORIP HEI grant (S10 RR029205). The Stanford Synchrotron Radiation Lightsource Structural Molecular Biology Program including beam line BL12-2 is supported by the DOE Office of Biological and Environmental Research, and by the NIH NIGMS (P41 GM103393). The contents of this publication are solely the responsibility of the authors and do not necessarily represent the official views of NIGMS or NIH.

Author information

Author notes

    • Minglei Zhao

    Present address: Department of Biochemistry & Molecular Biology, The University of Chicago, Chicago, Illinois 60637, USA.

    • Qiangjun Zhou
    •  & Peng Zhou

    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
    • , Peng Zhou
    • , Austin L. Wang
    • , Dick Wu
    • , Minglei Zhao
    • , Thomas C. Südhof
    •  & Axel T. Brunger
  2. Department of Neurology and Neurological Sciences, Department of Structural Biology, Department of Photon Science, Stanford University, Stanford, California 94305, USA

    • Qiangjun Zhou
    • , Austin L. Wang
    • , Minglei Zhao
    •  & Axel T. Brunger

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Contributions

Q.Z., P.Z., T.C.S., and A.T.B. designed experiments. Q.Z. performed biochemical and structural studies. P.Z. and Q.Z. performed electrophysiological studies. A.L.W. assisted with protein purification. D.W. generated cKO mice. M.Z. helped with crystallographic data collection. Q.Z, P.Z., T.C.S., and A.T.B. wrote the manuscript.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Axel T. Brunger.

Reviewer Information Nature thanks R. Heidelberger, C. Montecucco and the other anonymous reviewer(s) for their contribution to the peer review of this work.

Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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    Reporting Summary

Videos

  1. 1.

    Structure of the Syt1-SNARE-Cpx-Syt1 complex

    During the first 35 sec, a rotating view of the cartoon representation of the Syt1-SNARE-Cpx-Syt1 complex is shown. Subsequently, the interacting residues of the two interfaces are shown, and then, at 58 sec, the residues of the Ca2+ binding sites (from 58 to 1 min 12 sec) and polybasic regions (from 1 min 13 sec to 1 min 28 sec) of two Syt1 C2B domains are also shown in stick-and-ball representation. At 1 min 29 sec, the molecular surfaces of the molecules are shown. For clarity, we omitted the Syt1 C2A domains after 35 sec.

  2. 2.

    Locations of the mutations

    Rotating view of the cartoon representation of the Syt1-SNARE-Cpx-Syut1 complex with mutated residues shown in stick-and-ball representation. For clarity, we omitted the Syt1 C2A domains.

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https://doi.org/10.1038/nature23484

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