Complexin cross-links prefusion SNAREs into a zigzag array

Abstract

Complexin prevents SNAREs from releasing neurotransmitters until an action potential arrives at the synapse. To understand the mechanism for this inhibition, we determined the structure of complexin bound to a mimetic of a prefusion SNAREpin lacking the portion of the v-SNARE that zippers last to trigger fusion. The 'central helix' of complexin is anchored to one SNARE complex, while its 'accessory helix' extends away at ~45° and bridges to a second complex, occupying the vacant v-SNARE binding site to inhibit fusion. We expected the accessory helix to compete with the v-SNARE for t-SNARE binding but found instead that the interaction occurs intermolecularly. Thus, complexin organizes the SNAREs into a zigzag topology that, when interposed between the vesicle and plasma membranes, is incompatible with fusion.

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Figure 1: Structure of the prefusion CPX–SNARE complex.
Figure 2: Interacting surfaces of CPXacc and the t-SNAREs.
Figure 3: Characterization of the interaction of CPXacc with SNARE complexes by isothermal titration calorimetry.
Figure 4: FRET experiments probing CPX orientation in pre- and postfusion CPX–SNARE complexes.
Figure 5: Effects of CPX and VAMP2 mutations on clamping in cell-cell fusion assays.
Figure 6: Molecular models for CPX clamping.

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References

  1. 1

    Fatt, P. & Katz, B. Spontaneous subthreshold activity at motor nerve endings. J. Physiol. (Lond.) 117, 109–128 (1952).

  2. 2

    Palade, G.E. & Palay, S.L. Electron microscope observations of interneuronal and neuromuscular synapses. Anat. Rec. 118, 335–336 (1954).

  3. 3

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

  4. 4

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

  5. 5

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

  6. 6

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

  7. 7

    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).

  8. 8

    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).

  9. 9

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

  10. 10

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

  11. 11

    Geppert, M. et al. Synaptotagmin I: a major Ca 2 + sensor for transmitter release at a central synapse. Cell 79, 717–727 (1994).

  12. 12

    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 Ca 2 + -dependent soluble N-ethylmaleimide-sensitive factor attachment protein receptor complex binding in synaptic exocytosis. J. Neurosci. 26, 12556–12565 (2006).

  13. 13

    Domanska, M.K., Kiessling, V., Stein, A., Fasshauer, D. & Tamm, L.K. Single vesicle millisecond fusion kinetics reveals number of SNARE complexes optimal for fast SNARE-mediated membrane fusion. J. Biol. Chem. 284, 32158–32166 (2009).

  14. 14

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

  15. 15

    Liu, T., Tucker, W.C., Bhalla, A., Chapman, E.R. & Weisshaar, J.C. SNARE-driven, 25-millisecond vesicle fusion in vitro. Biophys. J. 89, 2458–2472 (2005).

  16. 16

    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).

  17. 17

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

  18. 18

    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).

  19. 19

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

  20. 20

    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).

  21. 21

    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).

  22. 22

    Cho, R.W., Song, Y. & Littleton, J.T. Comparative analysis of Drosophila and mammalian complexins as fusion clamps and facilitators of neurotransmitter release. Mol. Cell Neurosci. 45, 389–397 (2010).

  23. 23

    Huntwork, S. & Littleton, J.T. A complexin fusion clamp regulates spontaneous neurotransmitter release and synaptic growth. Nat. Neurosci. 10, 1235–1237 (2007).

  24. 24

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

  25. 25

    Hobson, R.J., Liu, Q., Watanabe, S. & Jorgensen, E.M. Complexin Maintains Vesicles in the Primed State in C. elegans. Curr. Biol. 21, 106–113 (2011).

  26. 26

    Martin, J.A., Hu, Z., Fenz, K.M., Fernandez, J. & Dittman, J.S. Complexin has opposite effects on two modes of synaptic vesicle fusion. Curr. Biol. 21, 97–105 (2011).

  27. 27

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

  28. 28

    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).

  29. 29

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

  30. 30

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

  31. 31

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

  32. 32

    Reim, K. et al. Complexins regulate a late step in Ca 2 + -dependent neurotransmitter release. Cell 104, 71–81 (2001).

  33. 33

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

  34. 34

    Lu, B., Song, S. & Shin, Y.K. Accessory alpha-helix of complexin I can displace VAMP2 locally in the complexin-SNARE quaternary complex. J. Mol. Biol. 396, 602–609 (2010).

  35. 35

    Melia, T.J. et al. Regulation of membrane fusion by the membrane-proximal coil of the t-SNARE during zippering of SNAREpins. J. Cell Biol. 158, 929–940 (2002).

  36. 36

    Walter, A.M., Wiederhold, K., Bruns, D., Fasshauer, D. & Sorensen, J.B. Synaptobrevin N-terminally bound to syntaxin-SNAP-25 defines the primed vesicle state in regulated exocytosis. J. Cell Biol. 188, 401–413 (2010).

  37. 37

    Ellena, J.F. et al. Dynamic structure of lipid-bound synaptobrevin suggests a nucleation-propagation mechanism for trans-SNARE complex formation. Proc. Natl. Acad. Sci. USA 106, 20306–20311 (2009).

  38. 38

    Yang, X., Kaeser-Woo, Y.J., Pang, Z.P., Xu, W. & Sudhof, T.C. Complexin clamps asynchronous release by blocking a secondary Ca 2 + sensor via its accessory alpha helix. Neuron 68, 907–920 (2010).

  39. 39

    Pabst, S. et al. Selective interaction of complexin with the neuronal SNARE complex. Determination of the binding regions. J. Biol. Chem. 275, 19808–19818 (2000).

  40. 40

    Krishnakumar, S.S. et al. A conformational switch in complexin is required for synaptotagmin to trigger synaptic fusion. Nat. Struct. Mol. Biol. doi:10.1038/nsmb.2103 (2011).

  41. 41

    Kuzmin, P.I., Zimmerberg, J., Chizmadzhev, Y.A. & Cohen, F.S. A quantitative model for membrane fusion based on low-energy intermediates. Proc. Natl. Acad. Sci. USA 98, 7235–7240 (2001).

  42. 42

    Chernomordik, L.V., Zimmerberg, J. & Kozlov, M.M. Membranes of the world unite! J. Cell Biol. 175, 201–207 (2006).

  43. 43

    Stein, A., Weber, G., Wahl, M.C. & Jahn, R. Helical extension of the neuronal SNARE complex into the membrane. Nature 460, 525–528 (2009).

  44. 44

    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).

  45. 45

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

  46. 46

    Chicka, M.C., Hui, E., Liu, H. & Chapman, E.R. Synaptotagmin arrests the SNARE complex before triggering fast, efficient membrane fusion in response to Ca 2 + . Nat. Struct. Mol. Biol. 15, 827–835 (2008).

  47. 47

    Doublié, S. Preparation of selenomethionyl proteins for phase determination. Methods Enzymol. 276, 523–530 (1997).

  48. 48

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

  49. 49

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

  50. 50

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

  51. 51

    Murshudov, G.N., Vagin, A.A. & Dodson, E.J. Refinement of macromolecular structures by the maximum-likelihood method. Acta Crystallogr. D Biol. Crystallogr. 53, 240–255 (1997).

  52. 52

    Kleywegt, G.J. & Jones, T.A. Where freedom is given, liberties are taken. Structure 3, 535–540 (1995).

  53. 53

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

  54. 54

    Lakowicz, J.R. Principles of Fluorescence Spectroscopy (Springer, New York; Berlin, 2006).

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Acknowledgements

We wish to thank the staffs of beamline X29 at the National Synchrotron Light Source, Brookhaven National Laboratory and of the Northeastern Collaborative Access Team (NE-CAT) facility at the Advanced Photon Source, Argonne National Laboratory, for their help in data collection; L. Khandan (Yale University) and S. Baguley (Yale University) for technical assistance; and J. Coleman (Yale University) for advice. We are grateful to E. Karatekin (Yale University) and D.W. Rodgers (University of Kentucky) for discussions regarding this manuscript. This work was supported by grants from the US National Institutes of Health to K.M.R. (R01GM080616) and to J.E.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.

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D.K. coordinated the experiments in this paper, was responsible for structure analysis and designed constructs for the functional analyses. S.S.K. and D.T.R. conducted the FRET experiments; F.L. conducted the ITC analysis and C.G.G. carried out the cell-cell fusion experiments. F.P. contributed to the analysis of the FRET and ITC data. D.K., J.E.R. and K.M.R. analyzed data and wrote this manuscript.

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Correspondence to James E Rothman or Karin M Reinisch.

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The authors declare no competing financial interests.

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Supplementary Figures 1–4, Supplementary Tables 1–3 and Supplementary Methods (PDF 1031 kb)

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Kümmel, D., Krishnakumar, S., Radoff, D. et al. Complexin cross-links prefusion SNAREs into a zigzag array. Nat Struct Mol Biol 18, 927–933 (2011). https://doi.org/10.1038/nsmb.2101

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