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Single-molecule FRET–derived model of the synaptotagmin 1–SNARE fusion complex

Nature Structural & Molecular Biology volume 17, pages 318–324 (2010)Cite this article

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Abstract

Synchronous neurotransmission is triggered when Ca2+ binds to synaptotagmin 1 (Syt1), a synaptic-vesicle protein that interacts with SNAREs and membranes. We used single-molecule fluorescence resonance energy transfer (FRET) between synaptotagmin's two C2 domains to determine that their conformation consists of multiple states with occasional transitions, consistent with domains in random relative motion. SNARE binding results in narrower intrasynaptotagmin FRET distributions and less frequent transitions between states. We obtained an experimentally determined model of the elusive Syt1–SNARE complex using a multibody docking approach with 34 FRET-derived distances as restraints. The Ca2+-binding loops point away from the SNARE complex, so they may interact with the same membrane. The loop arrangement is similar to that of the crystal structure of SNARE-induced Ca2+-bound Syt3, suggesting a common mechanism by which the interaction between synaptotagmins and SNAREs aids in Ca2+-triggered fusion.

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Figure 1: smFRET reveals Syt1 conformations.
Figure 2: smFRET shows that Syt1 conformations are dynamic.
Figure 3: Syt1 binds to SNARE complexes reconstituted in 100% phosphatidylcholine (PC) bilayers more strongly at decreased ionic strength of the buffer.
Figure 4: smFRET-derived model of the Syt1–SNARE complex.

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Acknowledgements

We thank M. Bowen, G. Schröder, T. Südhof and J. Rizo for comments, the US National Institutes of Health for support to A.T.B. (RO1-MH63105) and the Burroughs Wellcome Fund for a CASI award to K.R.W. A.T.B. presented this work at the first Paul B. Sigler lecture at Yale University on 23 March 2009.

Author information

Author notes

  1. Steven Chu

    Present address: Present address: United States Department of Energy, Washington, DC, USA.,

Authors and Affiliations

  1. Department of Physics, North Carolina State University, Raleigh, North Carolina, USA

    Ucheor B Choi & Keith R Weninger

  2. The Howard Hughes Medical Institute, Stanford University, Stanford, California, USA

    Pavel Strop, Marija Vrljic & Axel T Brunger

  3. Department of Molecular and Cellular Physiology, Stanford University, Stanford, California, USA

    Pavel Strop, Marija Vrljic & Axel T Brunger

  4. Department of Neurology and Neurological Sciences, Stanford University, Stanford, California, USA

    Pavel Strop, Marija Vrljic & Axel T Brunger

  5. Department of Structural Biology, Stanford University, Stanford, California, USA

    Pavel Strop, Marija Vrljic & Axel T Brunger

  6. Department of Photon Science, Stanford University, Stanford, California, USA

    Pavel Strop, Marija Vrljic & Axel T Brunger

  7. Department of Physics, University of California, Berkeley, California, USA

    Steven Chu

  8. Department of Molecular and Cell Biology, University of California, Berkeley, California, USA

    Steven Chu

  9. Lawrence Berkeley National Laboratory, Berkeley, California, USA

    Steven Chu

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Contributions

U.B.C. prepared all samples and performed all experimental measurements; P.S., M.V. and A.T.B. performed all simulations; all authors contributed to formulation of the experimental design, interpretation of results and preparation of the manuscript.

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Correspondence to Axel T Brunger or Keith R Weninger.

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Choi, U., Strop, P., Vrljic, M. et al. Single-molecule FRET–derived model of the synaptotagmin 1–SNARE fusion complex. Nat Struct Mol Biol 17, 318–324 (2010). https://doi.org/10.1038/nsmb.1763

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