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Molecular mechanism of the synaptotagmin–SNARE interaction in Ca2+-triggered vesicle fusion

Nature Structural & Molecular Biology volume 17, pages 325–331 (2010)Cite this article

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Abstract

In neurons, SNAREs, synaptotagmin and other factors catalyze Ca2+-triggered fusion of vesicles with the plasma membrane. The molecular mechanism of this process, especially the interaction between synaptotagmin and SNAREs, remains an enigma. Here we characterized this interaction by single-molecule fluorescence microscopy and crystallography. The two rigid Ca2+-binding domains of synaptotagmin 3 (Syt3) undergo large relative motions in solution. Interaction with SNARE complex amplifies a particular state of the two domains that is further enhanced by Ca2+. This state is represented by the first SNARE-induced Ca2+-bound crystal structure of a synaptotagmin fragment containing both domains. The arrangement of the Ca2+-binding loops of this structure of Syt3 matches that of SNARE-bound Syt1, suggesting a conserved feature of synaptotagmins. The loops resemble the membrane-interacting loops of certain viral fusion proteins in the postfusion state, suggesting unexpected similarities between both fusion systems.

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Figure 1: Crystal structure of SNARE-induced Ca2+-bound Syt3.
Figure 2: Comparison of the structural arrangement of the Syt3 Ca2+-binding loops with Semliki Forest virus E1 fusion loops and rhesus rotavirus VP5 membrane-interacting loops.
Figure 3: Uncomplexed Syt3 conformational dynamics in solution.
Figure 4: Syt3 C2AB shows a variety of FRET states and dynamic behaviors.
Figure 5: Syt3 C2AB conformational dynamics in the presence of SNARE complex.
Figure 6: Model of a general Ca2+-SNARE-synaptotagmin fusion–triggering mechanism.

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References

  1. Jahn, R. & Scheller, R.H. SNAREs–engines for membrane fusion. Nat. Rev. Mol. Cell Biol. 7, 631–643 (2006).

    Article  CAS  Google Scholar 

  2. Harrison, S.C. Viral membrane fusion. Nat. Struct. Mol. Biol. 15, 690–698 (2008).

    Article  CAS  Google Scholar 

  3. Sudhof, T.C. The synaptic vesicle cycle. Annu. Rev. Neurosci. 27, 509–547 (2004).

    Article  Google Scholar 

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

    Article  CAS  Google Scholar 

  5. 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 

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

    Article  CAS  Google Scholar 

  7. Sugita, S., Shin, O.H., Han, W., Lao, Y. & Sudhof, T.C. Synaptotagmins form a hierarchy of exocytotic Ca2+ sensors with distinct Ca2+ affinities. EMBO J. 21, 270–280 (2002).

    Article  CAS  Google Scholar 

  8. Herrick, D.Z., Sterbling, S., Rasch, K.A., Hinderliter, A. & Cafiso, D.S. Position of synaptotagmin I at the membrane interface: cooperative interactions of tandem C2 domains. Biochemistry 45, 9668–9674 (2006).

    Article  CAS  Google Scholar 

  9. Hui, E., Bai, J. & Chapman, E.R. Ca2+-triggered simultaneous membrane penetration of the tandem C2-domains of synaptotagmin I. Biophys. J. 91, 1767–1777 (2006).

    Article  CAS  Google Scholar 

  10. Martens, S., Kozlov, M.M. & McMahon, H.T. How synaptotagmin promotes membrane fusion. Science 316, 1205–1208 (2007).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  12. Stein, A., Radhakrishnan, A., Riedel, D., Fasshauer, D. & Jahn, R. Synaptotagmin activates membrane fusion through a Ca2+-dependent trans interaction with phospholipids. Nat. Struct. Mol. Biol. 14, 904–911 (2007).

    Article  CAS  Google Scholar 

  13. Bhalla, A., Chicka, M.C. & Chapman, E.R. Analysis of the synaptotagmin family during reconstituted membrane fusion. Uncovering a class of inhibitory isoforms. J. Biol. Chem. 283, 21799–21807 (2008).

    Article  CAS  Google Scholar 

  14. Mittelsteadt, T. et al. Differential mRNA expression patterns of the synaptotagmin gene family in the rodent brain. J. Comp. Neurol. 512, 514–528 (2009).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  18. Choi, U. et al. Single molecule FRET derived model of the synaptotagmin 1–SNARE fusion complex. Nat. Struct. Mol. Biol. advance online publication, doi:10.1038/nsmb.1763 (21 February 2010).

  19. Fuson, K.L., Montes, M., Robert, J.J. & Sutton, R.B. Structure of human synaptotagmin 1 C2AB in the absence of Ca2+ reveals a novel domain association. Biochemistry 46, 13041–13048 (2007).

    Article  CAS  Google Scholar 

  20. Sutton, R.B., Ernst, J.A. & Brunger, A.T. Crystal structure of the cytosolic C2A–C2B domains of synaptotagmin III. Implications for Ca+2-independent snare complex interaction. J. Cell Biol. 147, 589–598 (1999).

    Article  CAS  Google Scholar 

  21. Gibbons, D.L. et al. Conformational change and protein-protein interactions of the fusion protein of Semliki Forest virus. Nature 427, 320–325 (2004).

    Article  CAS  Google Scholar 

  22. Dormitzer, P.R., Nason, E.B., Prasad, B.V. & Harrison, S.C. Structural rearrangements in the membrane penetration protein of a non-enveloped virus. Nature 430, 1053–1058 (2004).

    Article  CAS  Google Scholar 

  23. Jarousse, N., Wilson, J.D., Arac, D., Rizo, J. & Kelly, R.B. Endocytosis of synaptotagmin 1 is mediated by a novel, tryptophan-containing motif. Traffic 4, 468–478 (2003).

    Article  CAS  Google Scholar 

  24. Kielian, M., Klimjack, M.R., Ghosh, S. & Duffus, W.A. Mechanisms of mutations inhibiting fusion and infection by Semliki Forest virus. J. Cell Biol. 134, 863–872 (1996).

    Article  CAS  Google Scholar 

  25. Dowling, W., Denisova, E., LaMonica, R. & Mackow, E.R. Selective membrane permeabilization by the rotavirus VP5* protein is abrogated by mutations in an internal hydrophobic domain. J. Virol. 74, 6368–6376 (2000).

    Article  CAS  Google Scholar 

  26. Joo, C., Balci, H., Ishitsuka, Y., Buranachai, C. & Ha, T. Advances in single-molecule fluorescence methods for molecular biology. Annu. Rev. Biochem. 77, 51–76 (2008).

    Article  CAS  Google Scholar 

  27. Huang, H. & Cafiso, D.S. Conformation and membrane position of the region linking the two C2 domains in synaptotagmin 1 by site-directed spin labeling. Biochemistry 47, 12380–12388 (2008).

    Article  CAS  Google Scholar 

  28. Weninger, K., Bowen, M.E., Choi, U.B., Chu, S. & Brunger, A.T. Accessory proteins stabilize the acceptor complex for synaptobrevin, the 1:1 syntaxin/SNAP-25 complex. Structure 16, 308–320 (2008).

    Article  CAS  Google Scholar 

  29. 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 

  30. 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 

  31. Sorensen, J.B. et al. Sequential N- to C-terminal SNARE complex assembly drives priming and fusion of secretory vesicles. EMBO J. 25, 955–966 (2006).

    Article  CAS  Google Scholar 

  32. Chan, Y.H., van Lengerich, B. & Boxer, S.G. Effects of linker sequences on vesicle fusion mediated by lipid-anchored DNA oligonucleotides. Proc. Natl. Acad. Sci. USA 106, 979–984 (2009).

    Article  CAS  Google Scholar 

  33. Jun, Y. & Wickner, W. Assays of vacuole fusion resolve the stages of docking, lipid mixing, and content mixing. Proc. Natl. Acad. Sci. USA 104, 13010–13015 (2007).

    Article  CAS  Google Scholar 

  34. Floyd, D.L., Ragains, J.R., Skehel, J.J., Harrison, S.C. & van Oijen, A.M. Single-particle kinetics of influenza virus membrane fusion. Proc. Natl. Acad. Sci. USA 105, 15382–15387 (2008).

    Article  CAS  Google Scholar 

  35. Katsov, K., Muller, M. & Schick, M. Field theoretic study of bilayer membrane fusion: II. Mechanism of a stalk-hole complex. Biophys. J. 90, 915–926 (2006).

    Article  CAS  Google Scholar 

  36. Zimmerberg, J. & Gawrisch, K. The physical chemistry of biological membranes. Nat. Chem. Biol. 2, 564–567 (2006).

    Article  CAS  Google Scholar 

  37. Giraudo, C.G. et al. SNAREs can promote complete fusion and hemifusion as alternative outcomes. J. Cell Biol. 170, 249–260 (2005).

    Article  CAS  Google Scholar 

  38. Kasson, P.M. et al. Ensemble molecular dynamics yields submillisecond kinetics and intermediates of membrane fusion. Proc. Natl. Acad. Sci. USA 103, 11916–11921 (2006).

    Article  CAS  Google Scholar 

  39. Katsov, K., Muller, M. & Schick, M. Field theoretic study of bilayer membrane fusion. I. Hemifusion mechanism. Biophys. J. 87, 3277–3290 (2004).

    Article  CAS  Google Scholar 

  40. Muller, M., Katsov, K. & Schick, M. A new mechanism of model membrane fusion determined from Monte Carlo simulation. Biophys. J. 85, 1611–1623 (2003).

    Article  CAS  Google Scholar 

  41. Chernomordik, L.V., Leikina, E., Frolov, V., Bronk, P. & Zimmerberg, J. An early stage of membrane fusion mediated by the low pH conformation of influenza hemagglutinin depends upon membrane lipids. J. Cell Biol. 136, 81–93 (1997).

    Article  CAS  Google Scholar 

  42. Melia, T.J., You, D., Tareste, D.C. & Rothman, J.E. Lipidic antagonists to SNARE-mediated fusion. J. Biol. Chem. 281, 29597–29605 (2006).

    Article  CAS  Google Scholar 

  43. Cooke, I.R. & Deserno, M. Coupling between lipid shape and membrane curvature. Biophys. J. 91, 487–495 (2006).

    Article  CAS  Google Scholar 

  44. Tian, A. & Baumgart, T. Sorting of lipids and proteins in membrane curvature gradients. Biophys. J. 96, 2676–2688 (2009).

    Article  CAS  Google Scholar 

  45. Wang, H. et al. Bilayer edge and curvature effects on partitioning of lipids by tail length: atomistic simulations. Biophys. J. 95, 2647–2657 (2008).

    Article  CAS  Google Scholar 

  46. Kemble, G.W., Danieli, T. & White, J.M. Lipid-anchored influenza hemagglutinin promotes hemifusion, not complete fusion. Cell 76, 383–391 (1994).

    Article  CAS  Google Scholar 

  47. Gilbert, J.M. & Greenberg, H.B. Virus-like particle-induced fusion from without in tissue culture cells: role of outer-layer proteins VP4 and VP7. J. Virol. 71, 4555–4563 (1997).

    CAS  PubMed  PubMed Central  Google Scholar 

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

    Article  CAS  Google Scholar 

  49. Otwinowski, Z.M.W. Processing of X-ray diffraction data collected in oscillation mode. Methods Enzymol. 276, 307–326 (1997).

    Article  CAS  Google Scholar 

  50. Emsley, P. & Cowtan, K. Coot: model-building tools for molecular graphics. Acta Crystallogr. D Biol. Crystallogr. 60, 2126–2132 (2004).

    Article  Google Scholar 

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

    Article  CAS  Google Scholar 

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

    Article  Google Scholar 

  53. Wozniak, A.K., Schroder, G.F., Grubmuller, H., Seidel, C.A. & Oesterhelt, F. Single-molecule FRET measures bends and kinks in DNA. Proc. Natl. Acad. Sci. USA 105, 18337–18342 (2008).

    Article  CAS  Google Scholar 

  54. Engh, R. & Huber, R. Accurate bond and ange parameters for X-ray protein structure refinement. Acta Crystallogr. A 47, 392–400 (1991).

    Article  Google Scholar 

  55. Brunger, A.T. X-PLOR, Version 3.1. A System for X-ray Crystallography and NMR. (Yale Unversity Press, New Haven, Connecticut, USA, 1992).

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Acknowledgements

We thank T. Südhof and J. Rizo for discussions, Y. Zhang, B. Cui, S. Solomatin, T.H. Lee and A. Persitidinis for discussions of single-molecule experimental setup and data analysis, Y. Abdiche and K. Lindquist for technical assistance and discussions of biosensor experiments, and the US National Institutes of Health for support to A.T.B (RO1-MH63105). A.T.B. presented this work at the first Paul B. Sigler lecture at Yale University on 23 March 2009.

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Author notes

  1. Pavel Strop & Steven Chu

    Present address: Present addresses: Rinat/Pfizer, Protein Engineering Department, South San Francisco, California, USA (P.S.) and US Department of Energy, Washington, DC, USA (S.C.).,

  2. Marija Vrljic and Pavel Strop: These authors contributed equally to this work.

Authors and Affiliations

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

    Marija Vrljic, Pavel Strop & Axel T Brunger

  2. Department of Neurology and Neurological Science, Stanford University, Stanford, California, USA

    Marija Vrljic, Pavel Strop & Axel T Brunger

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

    Marija Vrljic, Pavel Strop & Axel T Brunger

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

    Marija Vrljic, Pavel Strop & Axel T Brunger

  5. Howard Hughes Medical Institute, Stanford, California, USA

    Marija Vrljic, Pavel Strop & Axel T Brunger

  6. Protein Chemistry Department, Genentech Inc., South San Francisco, California, USA

    James A Ernst

  7. Department of Cell Physiology and Molecular Biophysics, Texas Tech University, Health Sciences Center, School of Medicine, Lubbock, Texas, USA

    R Bryan Sutton

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

    Steven Chu

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

    Steven Chu

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

    Steven Chu

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Contributions

J.A.E. and R.B.S. purified, crystallized and collected diffraction data; P.S. processed, solved and refined the Syt3 structure; M.V. collected and analyzed single-molecule data; M.V. and P.S. designed and purified proteins used in single-molecule studies and prepared the manuscript; A.T.B., M.V., P.S. and S.C. performed experimental design, data analysis and manuscript preparation.

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Correspondence to Axel T Brunger.

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Vrljic, M., Strop, P., Ernst, J. et al. Molecular mechanism of the synaptotagmin–SNARE interaction in Ca2+-triggered vesicle fusion. Nat Struct Mol Biol 17, 325–331 (2010). https://doi.org/10.1038/nsmb.1764

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