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PMID: 26280336 Published · ppublish English Journal Article Research Support, N.I.H., Extramural Research Support, Non-U.S. Gov't Research Support, U.S. Gov't, Non-P.H.S.

Architecture of the synaptotagmin-SNARE machinery for neuronal exocytosis.

Nature ·Vol. 525 ·No. 7567 ·2015-09-03 ·Pages 62-7

Zhou Q, Lai Y, Bacaj T, Zhao M, Lyubimov AY, Uervirojnangkoorn M, Zeldin OB, Brewster AS, Sauter NK, Cohen AE, Soltis SM, Alonso-Mori R, Chollet M, Lemke HT, Pfuetzner RA, Choi UB, Weis WI, Diao J, Südhof TC, Brunger AT

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 Ca(2+)- and Mg(2+)-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, Ca(2+)-independent and conserved interface. Tests of this interface by mutagenesis suggest that it is essential for Ca(2+)-triggered neurotransmitter release in mouse hippocampal neuronal synapses and for Ca(2+)-triggered vesicle fusion in a reconstituted system. We propose that this interface forms before Ca(2+) triggering, moves en bloc as Ca(2+) 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.

MeSH Terms
Animals Binding Sites/genetics Calcium/chemistry,metabolism Cell Membrane/metabolism Crystallography, X-Ray Electrons Exocytosis Hippocampus/cytology Lasers Magnesium/chemistry,metabolism Membrane Fusion Mice Models, Biological Models, Molecular Mutation/genetics Neurons/chemistry,cytology,metabolism SNARE Proteins/chemistry,genetics,metabolism Synaptic Transmission Synaptic Vesicles/chemistry,metabolism Synaptotagmins/chemistry,genetics,metabolism
Chemicals
SNARE Proteins Synaptotagmins Magnesium Calcium
Authors & Affiliations
20 authors, click to expand affiliations / ORCID
Zhou Qiangjun
Department of Molecular and Cellular Physiology, Howard Hughes Medical Institute, Stanford University, Stanford, California 94305, USA. | Departments of Neurology and Neurological Sciences, Photon Science, and Structural Biology, Stanford University, Stanford, California 94305, USA.
Lai Ying
Department of Molecular and Cellular Physiology, Howard Hughes Medical Institute, Stanford University, Stanford, California 94305, USA. | Departments of Neurology and Neurological Sciences, Photon Science, and Structural Biology, Stanford University, Stanford, California 94305, USA.
Bacaj Taulant
Department of Molecular and Cellular Physiology, Howard Hughes Medical Institute, Stanford University, Stanford, California 94305, USA.
Zhao Minglei
Department of Molecular and Cellular Physiology, Howard Hughes Medical Institute, Stanford University, Stanford, California 94305, USA. | Departments of Neurology and Neurological Sciences, Photon Science, and Structural Biology, Stanford University, Stanford, California 94305, USA.
Lyubimov Artem Y
Department of Molecular and Cellular Physiology, Howard Hughes Medical Institute, Stanford University, Stanford, California 94305, USA. | Departments of Neurology and Neurological Sciences, Photon Science, and Structural Biology, Stanford University, Stanford, California 94305, USA.
Uervirojnangkoorn Monarin
Department of Molecular and Cellular Physiology, Howard Hughes Medical Institute, Stanford University, Stanford, California 94305, USA. | Departments of Neurology and Neurological Sciences, Photon Science, and Structural Biology, Stanford University, Stanford, California 94305, USA.
Zeldin Oliver B
Department of Molecular and Cellular Physiology, Howard Hughes Medical Institute, Stanford University, Stanford, California 94305, USA. | Departments of Neurology and Neurological Sciences, Photon Science, and Structural Biology, Stanford University, Stanford, California 94305, USA.
Brewster Aaron S
Physical Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley, California 94720, USA.
Sauter Nicholas K
Physical Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley, California 94720, USA.
Cohen Aina E
SLAC National Accelerator Laboratory, Stanford, California 94305, USA.
Soltis S Michael
SLAC National Accelerator Laboratory, Stanford, California 94305, USA.
Alonso-Mori Roberto
SLAC National Accelerator Laboratory, Stanford, California 94305, USA.
Chollet Matthieu
SLAC National Accelerator Laboratory, Stanford, California 94305, USA.
Lemke Henrik T
SLAC National Accelerator Laboratory, Stanford, California 94305, USA.
Pfuetzner Richard A
Department of Molecular and Cellular Physiology, Howard Hughes Medical Institute, Stanford University, Stanford, California 94305, USA. | Departments of Neurology and Neurological Sciences, Photon Science, and Structural Biology, Stanford University, Stanford, California 94305, USA.
Choi Ucheor B
Department of Molecular and Cellular Physiology, Howard Hughes Medical Institute, Stanford University, Stanford, California 94305, USA. | Departments of Neurology and Neurological Sciences, Photon Science, and Structural Biology, Stanford University, Stanford, California 94305, USA.
Weis William I
Departments of Structural Biology, Molecular and Cellular Physiology, and Photon Science, Stanford University, Stanford, California 94305, USA.
Diao Jiajie
Department of Molecular and Cellular Physiology, Howard Hughes Medical Institute, Stanford University, Stanford, California 94305, USA. | Departments of Neurology and Neurological Sciences, Photon Science, and Structural Biology, Stanford University, Stanford, California 94305, USA.
Südhof Thomas C
Department of Molecular and Cellular Physiology, Howard Hughes Medical Institute, Stanford University, Stanford, California 94305, USA.
Brunger Axel T
Department of Molecular and Cellular Physiology, Howard Hughes Medical Institute, Stanford University, Stanford, California 94305, USA. | Departments of Neurology and Neurological Sciences, Photon Science, and Structural Biology, Stanford University, Stanford, California 94305, USA.
References (84)
84 references, click to expand
  1. Structure of human synaptotagmin 1 C2AB in the absence of Ca2+ reveals a novel domain association.
    Biochemistry. 2007 Nov 13;46(45):13041-8 PMID: 17956130
  2. Mechanism of phospholipid binding by the C2A-domain of synaptotagmin I.
    Biochemistry. 1998 Sep 8;37(36):12395-403 PMID: 9730811
  3. Potential for biomolecular imaging with femtosecond X-ray pulses.
    Nature. 2000 Aug 17;406(6797):752-7 PMID: 10963603
  4. Synaptotagmin I: a major Ca2+ sensor for transmitter release at a central synapse.
    Cell. 1994 Nov 18;79(4):717-27 PMID: 7954835
  5. Three-dimensional structure of the synaptotagmin 1 C2B-domain: synaptotagmin 1 as a phospholipid binding machine.
    Neuron. 2001 Dec 20;32(6):1057-69 PMID: 11754837
  6. New Python-based methods for data processing.
    Acta Crystallogr D Biol Crystallogr. 2013 Jul;69(Pt 7):1274-82 PMID: 23793153
  7. Conformational dynamics of calcium-triggered activation of fusion by synaptotagmin.
    Biophys J. 2013 Dec 3;105(11):2507-16 PMID: 24314081
  8. Close membrane-membrane proximity induced by Ca(2+)-dependent multivalent binding of synaptotagmin-1 to phospholipids.
    Nat Struct Mol Biol. 2006 Mar;13(3):209-17 PMID: 16491093
  9. Syntaxin: a synaptic protein implicated in docking of synaptic vesicles at presynaptic active zones.
    Science. 1992 Jul 10;257(5067):255-9 PMID: 1321498
  10. Synaptotagmin-1 docks secretory vesicles to syntaxin-1/SNAP-25 acceptor complexes.
    Cell. 2009 Sep 4;138(5):935-46 PMID: 19716167
  11. Dynamic Ca2+-dependent stimulation of vesicle fusion by membrane-anchored synaptotagmin 1.
    Science. 2010 May 7;328(5979):760-3 PMID: 20448186
  12. The Janus-faced nature of the C(2)B domain is fundamental for synaptotagmin-1 function.
    Nat Struct Mol Biol. 2008 Nov;15(11):1160-8 PMID: 18953334
  13. Synaptotagmin I functions as a calcium regulator of release probability.
    Nature. 2001 Mar 1;410(6824):41-9 PMID: 11242035
  14. Phaser crystallographic software.
    J Appl Crystallogr. 2007 Aug 1;40(Pt 4):658-674 PMID: 19461840
  15. Version 1.2 of the Crystallography and NMR system.
    Nat Protoc. 2007;2(11):2728-33 PMID: 18007608
  16. MolProbity: all-atom structure validation for macromolecular crystallography.
    Acta Crystallogr D Biol Crystallogr. 2010 Jan;66(Pt 1):12-21 PMID: 20057044
  17. Crystal structure of a SNARE complex involved in synaptic exocytosis at 2.4 A resolution.
    Nature. 1998 Sep 24;395(6700):347-53 PMID: 9759724
  18. Post-translational modifications and lipid binding profile of insect cell-expressed full-length mammalian synaptotagmin 1.
    Biochemistry. 2011 Nov 22;50(46):9998-10012 PMID: 21928778
  19. Studying calcium-triggered vesicle fusion in a single vesicle-vesicle content and lipid-mixing system.
    Nat Protoc. 2013 Jan;8(1):1-16 PMID: 23222454
  20. Synaptotagmin increases the dynamic range of synapses by driving Ca²+-evoked release and by clamping a near-linear remaining Ca²+ sensor.
    Neuron. 2011 Feb 24;69(4):736-48 PMID: 21338883
  21. The principle of membrane fusion in the cell (Nobel lecture).
    Angew Chem Int Ed Engl. 2014 Nov 17;53(47):12676-94 PMID: 25087728
  22. The synaptotagmin 1 linker may function as an electrostatic zipper that opens for docking but closes for fusion pore opening.
    Biochem J. 2013 Nov 15;456(1):25-33 PMID: 24001110
  23. High resolution structure, stability, and synaptotagmin binding of a truncated neuronal SNARE complex.
    J Biol Chem. 2003 Mar 7;278(10):8630-6 PMID: 12496247
  24. Structure of the first C2 domain of synaptotagmin I: a novel Ca2+/phospholipid-binding fold.
    Cell. 1995 Mar 24;80(6):929-38 PMID: 7697723
  25. Synaptotagmin-1 and synaptotagmin-7 trigger synchronous and asynchronous phases of neurotransmitter release.
    Neuron. 2013 Nov 20;80(4):947-59 PMID: 24267651
  26. Synaptotagmin: a calcium sensor on the synaptic vesicle surface.
    Science. 1992 May 15;256(5059):1021-5 PMID: 1589771
  27. How synaptotagmin promotes membrane fusion.
    Science. 2007 May 25;316(5828):1205-8 PMID: 17478680
  28. Protein production by auto-induction in high density shaking cultures.
    Protein Expr Purif. 2005 May;41(1):207-34 PMID: 15915565
  29. Dynamic binding mode of a Synaptotagmin-1-SNARE complex in solution.
    Nat Struct Mol Biol. 2015 Jul;22(7):555-64 PMID: 26030874
  30. Complexin inhibits spontaneous release and synchronizes Ca2+-triggered synaptic vesicle fusion by distinct mechanisms.
    Elife. 2014;3:e03756 PMID: 25122624
  31. Data Exploration Toolkit for serial diffraction experiments.
    Acta Crystallogr D Biol Crystallogr. 2015 Feb;71(Pt 2):352-6 PMID: 25664746
  32. Mutations in the second C2 domain of synaptotagmin disrupt synaptic transmission at Drosophila neuromuscular junctions.
    J Comp Neurol. 2001 Jul 16;436(1):4-16 PMID: 11413542
  33. Autonomous function of synaptotagmin 1 in triggering synchronous release independent of asynchronous release.
    Neuron. 2005 Nov 23;48(4):547-54 PMID: 16301172
  34. UCSF Chimera--a visualization system for exploratory research and analysis.
    J Comput Chem. 2004 Oct;25(13):1605-12 PMID: 15264254
  35. Overview of the CCP4 suite and current developments.
    Acta Crystallogr D Biol Crystallogr. 2011 Apr;67(Pt 4):235-42 PMID: 21460441
  36. Crystallography & NMR system: A new software suite for macromolecular structure determination.
    Acta Crystallogr D Biol Crystallogr. 1998 Sep 1;54(Pt 5):905-21 PMID: 9757107
  37. A single C2 domain from synaptotagmin I is sufficient for high affinity Ca2+/phospholipid binding.
    J Biol Chem. 1993 Dec 15;268(35):26386-90 PMID: 8253763
  38. Synaptic proteins promote calcium-triggered fast transition from point contact to full fusion.
    Elife. 2012;1:e00109 PMID: 23240085
  39. Accurate macromolecular structures using minimal measurements from X-ray free-electron lasers.
    Nat Methods. 2014 May;11(5):545-8 PMID: 24633409
  40. Crystallographic model quality at a glance.
    Acta Crystallogr D Biol Crystallogr. 2009 Mar;65(Pt 3):297-300 PMID: 19237753
  41. Membrane-bound orientation and position of the synaptotagmin C2B domain determined by site-directed spin labeling.
    Biochemistry. 2005 Jan 11;44(1):18-28 PMID: 15628842
  42. Lipid-anchored SNAREs lacking transmembrane regions fully support membrane fusion during neurotransmitter release.
    Neuron. 2013 Oct 16;80(2):470-83 PMID: 24120845
  43. Super-resolution biomolecular crystallography with low-resolution data.
    Nature. 2010 Apr 22;464(7292):1218-22 PMID: 20376006
  44. Synaptotagmin I functions as a calcium sensor to synchronize neurotransmitter release.
    Neuron. 2002 Dec 5;36(5):897-908 PMID: 12467593
  45. Synaptotagmin interaction with SNAP-25 governs vesicle docking, priming, and fusion triggering.
    J Neurosci. 2013 Sep 4;33(36):14417-30 PMID: 24005294
  46. Processive ATP-driven substrate disassembly by the N-ethylmaleimide-sensitive factor (NSF) molecular machine.
    J Biol Chem. 2013 Aug 9;288(32):23436-45 PMID: 23775070
  47. Synaptotagmin has an essential function in synaptic vesicle positioning for synchronous release in addition to its role as a calcium sensor.
    Neuron. 2009 Aug 27;63(4):482-96 PMID: 19709630
  48. Three-dimensional structure of the complexin/SNARE complex.
    Neuron. 2002 Jan 31;33(3):397-409 PMID: 11832227
  49. Synaptotagmin-mediated bending of the target membrane is a critical step in Ca(2+)-regulated fusion.
    Cell. 2009 Aug 21;138(4):709-21 PMID: 19703397
  50. Linker length and composition influence the flexibility of Oct-1 DNA binding.
    EMBO J. 1997 Apr 15;16(8):2043-53 PMID: 9155030
  51. Electrostatic interaction of internal Mg2+ with membrane PIP2 Seen with KCNQ K+ channels.
    J Gen Physiol. 2007 Sep;130(3):241-56 PMID: 17724161
  52. A distinct tethering step is vital for vacuole membrane fusion.
    Elife. 2014;3:e03251 PMID: 25255215
  53. Energetics of stalk intermediates in membrane fusion are controlled by lipid composition.
    Proc Natl Acad Sci U S A. 2012 Jun 19;109(25):E1609-18 PMID: 22589300
  54. In vitro system capable of differentiating fast Ca2+-triggered content mixing from lipid exchange for mechanistic studies of neurotransmitter release.
    Proc Natl Acad Sci U S A. 2011 Jul 19;108(29):E304-13 PMID: 21705659
  55. Augmenting neurotransmitter release by enhancing the apparent Ca2+ affinity of synaptotagmin 1.
    Proc Natl Acad Sci U S A. 2005 Dec 20;102(51):18664-9 PMID: 16352718
  56. Synaptotagmin-1, -2, and -9: Ca(2+) sensors for fast release that specify distinct presynaptic properties in subsets of neurons.
    Neuron. 2007 May 24;54(4):567-81 PMID: 17521570
  57. Neurotransmitter release: the last millisecond in the life of a synaptic vesicle.
    Neuron. 2013 Oct 30;80(3):675-90 PMID: 24183019
  58. Ca(2+)-dependent and -independent activities of neural and non-neural synaptotagmins.
    Nature. 1995 Jun 15;375(6532):594-9 PMID: 7791877
  59. PHENIX: building new software for automated crystallographic structure determination.
    Acta Crystallogr D Biol Crystallogr. 2002 Nov;58(Pt 11):1948-54 PMID: 12393927
  60. Monitoring synaptic transmission in primary neuronal cultures using local extracellular stimulation.
    J Neurosci Methods. 2007 Mar 30;161(1):75-87 PMID: 17118459
  61. The structure of an Arf-ArfGAP complex reveals a Ca2+ regulatory mechanism.
    Cell. 2010 May 28;141(5):812-21 PMID: 20510928
  62. Distinct roles for two synaptotagmin isoforms in synchronous and asynchronous transmitter release at zebrafish neuromuscular junction.
    Proc Natl Acad Sci U S A. 2010 Aug 3;107(31):13906-11 PMID: 20643933
  63. Enabling X-ray free electron laser crystallography for challenging biological systems from a limited number of crystals.
    Elife. 2015;4. doi: 10.7554/eLife.05421 PMID: 25781634
  64. Cell biology of Ca2+-triggered exocytosis.
    Curr Opin Cell Biol. 2010 Aug;22(4):496-505 PMID: 20561775
  65. Synaptotagmin C2B domain regulates Ca2+-triggered fusion in vitro: critical residues revealed by scanning alanine mutagenesis.
    J Biol Chem. 2008 Nov 14;283(46):31763-75 PMID: 18784080
  66. Inference of macromolecular assemblies from crystalline state.
    J Mol Biol. 2007 Sep 21;372(3):774-97 PMID: 17681537
  67. Goniometer-based femtosecond crystallography with X-ray free electron lasers.
    Proc Natl Acad Sci U S A. 2014 Dec 2;111(48):17122-7 PMID: 25362050
  68. Controlling synaptotagmin activity by electrostatic screening.
    Nat Struct Mol Biol. 2012 Oct;19(10):991-7 PMID: 22940675
  69. Natively inhibited Trypanosoma brucei cathepsin B structure determined by using an X-ray laser.
    Science. 2013 Jan 11;339(6116):227-30 PMID: 23196907
  70. Reconstituted synaptotagmin I mediates vesicle docking, priming, and fusion.
    J Cell Biol. 2011 Dec 26;195(7):1159-70 PMID: 22184197
  71. Coot: model-building tools for molecular graphics.
    Acta Crystallogr D Biol Crystallogr. 2004 Dec;60(Pt 12 Pt 1):2126-32 PMID: 15572765
  72. Single reconstituted neuronal SNARE complexes zipper in three distinct stages.
    Science. 2012 Sep 14;337(6100):1340-3 PMID: 22903523
  73. Membrane fusion.
    Nat Struct Mol Biol. 2008 Jul;15(7):658-64 PMID: 18618939
  74. Conserved prefusion protein assembly in regulated exocytosis.
    Mol Biol Cell. 2006 Jan;17(1):283-94 PMID: 16267273
  75. A complexin/synaptotagmin 1 switch controls fast synaptic vesicle exocytosis.
    Cell. 2006 Sep 22;126(6):1175-87 PMID: 16990140
  76. Ca(2+)-independent syntaxin binding to the C(2)B effector region of synaptotagmin.
    Mol Cell Neurosci. 2012 Jan;49(1):1-8 PMID: 22008253
  77. Calcium sensitive ring-like oligomers formed by synaptotagmin.
    Proc Natl Acad Sci U S A. 2014 Sep 23;111(38):13966-71 PMID: 25201968
  78. SNAREpins: minimal machinery for membrane fusion.
    Cell. 1998 Mar 20;92(6):759-72 PMID: 9529252
  79. XDS.
    Acta Crystallogr D Biol Crystallogr. 2010 Feb;66(Pt 2):125-32 PMID: 20124692
  80. 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. 2006 Nov 29;26(48):12556-65 PMID: 17135417
  81. Synaptotagmin-1 functions as a Ca2+ sensor for spontaneous release.
    Nat Neurosci. 2009 Jun;12(6):759-66 PMID: 19412166
  82. A quaternary SNARE-synaptotagmin-Ca2+-phospholipid complex in neurotransmitter release.
    J Mol Biol. 2007 Mar 30;367(3):848-63 PMID: 17320903
  83. Single-molecule FRET-derived model of the synaptotagmin 1-SNARE fusion complex.
    Nat Struct Mol Biol. 2010 Mar;17(3):318-24 PMID: 20173763
  84. Improved crystal orientation and physical properties from single-shot XFEL stills.
    Acta Crystallogr D Biol Crystallogr. 2014 Dec 1;70(Pt 12):3299-309 PMID: 25478847
Article Info
Journal
Nature
Abbr.
Nature
ISSN
1476-4687
DOI
10.1038/nature14975
Published
2015-09-03
Epub
2015-00-17
Pages
62-7
Language
English
Region
England
NLM ID
0410462
PMCID
PMC4607316
Subset
IM
Grants
NIGMS NIH HHS · R01 GM102520 · United States
NIMH NIH HHS · MH086403 · United States
NIGMS NIH HHS · R01 GM077071 · United States
NIGMS NIH HHS · GM095887 · United States
NIMH NIH HHS · R37 MH063105 · United States
NIMH NIH HHS · R37MH63105 · United States
NIMH NIH HHS · P50 MH086403 · United States
Howard Hughes Medical Institute · United States
NIGMS NIH HHS · P41GM103393 · United States
NIGMS NIH HHS · GM102520 · United States
NIGMS NIH HHS · R01 GM095887 · United States
NIGMS NIH HHS · P41 GM103403 · United States
Databases
PDB
5CCG, 5CCH, 5CCI, 5CCJ
External Links
PubMed source DOI: 10.1038/nature14975 PMC Full Text View journal details
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