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Architecture of the synaptotagmin–SNARE machinery for neuronal exocytosis

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 Ca2+- and Mg2+-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, Ca2+-independent and conserved interface. Tests of this interface by mutagenesis suggest that it is essential for Ca2+-triggered neurotransmitter release in mouse hippocampal neuronal synapses and for Ca2+-triggered vesicle fusion in a reconstituted system. We propose that this interface forms before Ca2+ triggering, moves en bloc as Ca2+ 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.

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Figure 1: Crystal structure of the Syt1–SNARE complex.
Figure 2: Primary interface between the Syt1 C2B domain and the neuronal SNARE complex.
Figure 3: Mutations of the primary interface affect binding and Ca2+-triggered single vesicle–vesicle fusion.
Figure 4: Mutations of the primary interface impair Syt1 function in Ca2+-triggered release.
Figure 5: Model of the role of the primary Syt1–SNARE interface.

Accession codes

Primary accessions

Protein Data Bank

Data deposits

The coordinates of the atomic models and corresponding structure factors of the Syt1–SNARE complexes have been deposited in the Protein Data Bank under the accession codes 5CCG, 5CCH, 5CCI and 5CCJ.

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Acknowledgements

We thank M. S. Padolina for help with protein purification, the Northeastern Collaborative Access Team (supported by NIH P41 GM103403) at Advanced Photon Source for X-ray data collection, and the SSRL/LCLS scientists E. L. Baxter, P. Ehrensberger, T. I. Eriksson, Y. Feng, M. Hollenbeck, E. G. Kovaleva, S. E. McPhillips, S. Nelson, J. Song, Y. Tsai, V. Vinetsky and D. Zhu for their invaluable assistance with data collection at the LCLS XPP facility. Use of the Stanford Synchrotron Radiation Lightsource (SSRL) and Linac Coherent Light Source (LCLS), SLAC National Accelerator Laboratory, is supported by the US Department of Energy, Office of Science, Office of Basic Energy Sciences under contract no. DE-AC02-76SF00515. The SSRL Structural Molecular Biology Program is supported by the DOE Office of Biological and Environmental Research, and by the National Institutes of Health, National Institute of General Medical Sciences (including P41GM103393). Portions of this research were carried out at the Linac Coherent Light Source (LCLS) at the SLAC National Accelerator Laboratory. LCLS is an Office of Science User Facility operated for the US Department of Energy Office of Science by Stanford University. This research was supported in part by the National Institutes of Health (R37MH63105 to A.T.B.; MH086403 to T.C.S.; GM095887 and GM102520 to N.K.S and A.S.B); and by a HHMI Collaborative Innovation Award (HCIA) to A.T.B. and W.I.W.

Author information

Authors and Affiliations

Authors

Contributions

Q.Z. designed, expressed, purified and crystallized the Syt1–SNARE complexes and the Syt1 C2B quintuple mutant, performed CD experiments, and designed the mutants to disrupt the primary interface. Q.Z. and M.Z. collected all diffraction data, determined and refined the crystal structures. Y.L. and J.D. performed the reconstituted single vesicle–vesicle experiments. T.B. performed the co-immunoprecipitation and electrophysiological experiments of neuronal cultures. A.Y.L., M.U., O.B.Z., N.K.S., A.S.B., W.I.W. and A.T.B. analysed and processed the LCLS diffraction data. A.E.C. and S.M.S. designed the goniometer-based setup at LCLS-XPP and helped with data collection. R.A.-M., M.C. and H.T.L. helped with data collection at LCLS-XPP. R.A.P. and Y.L. expressed and purified proteins for the single vesicle–vesicle experiments. U.B.C. helped with the comparison between the crystal structure and the smFRET data. Q.Z., M.Z., Y.L., T.B., T.C.S. and A.T.B. wrote the manuscript.

Corresponding authors

Correspondence to Thomas C. Südhof or Axel T. Brunger.

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Competing interests

The authors declare no competing financial interests.

Extended data figures and tables

Extended Data Figure 1 Purification and crystallization of the Syt1–SNARE complex.

a, Diagram of the Duet co-expression vectors (Novagen) that express the fragments of the neuronal SNARE complex and the C2AB-linker-SNAP-25_C chimaera used for purification and crystallization of the Syt1–SNARE37aa-linker complex. The rat syntaxin-1A and His-tagged rat synaptobrevin-2 fragments were cloned into the vector pACYCDuet-1; the C2AB-linker-SNAP-25_C chimaera and the SNAP-25_N fragment were cloned into the vector pETDuet-1 with amino acid ranges labelled. Dashed lines represent the 37-amino-acid linker (see Methods). b, The purified Syt1–SNARE37aa-linker complex eluted as a single peak during size-exclusion chromatography (profile on the left). Left gel: Coomassie-blue-stained SDS–PAGE gel of the purified Syt1–SNARE37aa-linker complex (unboiled and boiled). Right gel: Coomassie-blue-stained SDS–PAGE gel of dissolved crystals of the Syt1–SNARE complex that were grown over a period of 2 months starting from purified Syt1–SNARE37aa-linker (unboiled and boiled). Although Syt1 was initially covalently linked to SNAP-25_C, the linker was cleaved during crystallization. The comparison between boiled and unboiled lanes is a hallmark showing that neuronal SNARE complex is fully formed. c, Boiled Coomassie-blue-stained SDS–PAGE gel of the purified Syt1–SNARE37aa-linker complex in solution at ambient temperature at the specified time after purification. Cleavage is apparent on day one and progresses slowly over several days. d, Schema showing the commonly used vapour-diffusion technique: the drop contains a lower concentration of the precipitant than the reservoir. The crystallization of the quintuple mutant of Syt1 C2B is used as an example. e, Schema showing a reverse vapour-diffusion method that was used for crystallization of the Ca2+-bound Syt1–SNARE complex: the drop contains a higher concentration of the precipitant than the reservoir.

Extended Data Figure 2 Diffraction images, electron density maps and crystal packing of the Syt1–SNARE complex in the long unit cell crystal form.

a, Only one out of 85 screened crystals in the long unit cell crystal form diffracted to 4.1 Å resolution at the APS NE-CAT microfocus synchrotron beamline (a total of 105 crystals were screened with 20 that indexed in the short unit cell crystal form). b, A total of 61 out of 72 crystals in the long unit cell crystal form diffracted to at least 3.5 Å resolution at the LCLS XFEL (a total of 148 crystals were diffracted, out of those 113 crystals produced 578 images that could be processed; 35 crystals did not diffract or showed multiple lattices). These exposures were taken along the crystal c axis. The left upper pictures in a and b show images of loop-mounted crystals after X-ray exposure. c, mFoDFc annealed omit map (Methods) of the Ca2+-bound Syt1–SNARE complex in the long unit cell crystal form using diffraction data collected at the LCLS XFEL; omitted residues within region I of the primary interface (residues 335–340 in Syt1 and 159–166 in SNAP-25) are coloured cyan. The contour level is 2.3σ. df, Representative 2mFoDFc electron density maps of the Ca2+-bound Syt1–SNARE complex in the long unit cell crystal form using diffraction data collected at the LCLS XFEL. The contour level is 1.5σ. g, Views of the crystal lattice perpendicular to the bc (left) and to the ac (right) planes of the Ca2+-bound Syt1–SNARE complex in the long unit cell crystal form. The particular layer shown on the right corresponds to the red arrowhead in the left panel (only a slice corresponding to the layer is shown, creating the appearance of two disconnected groups of molecules—these groups are actually connected via interactions with the neighbouring layers). The red dashed oval indicates the ‘missing’ Syt1 C2AB fragment compared to the short unit cell crystal form (Extended Data Fig. 3d).

Extended Data Figure 3 Asymmetric unit, electron density maps and crystal packing of the Syt1–SNARE complex in the short unit cell crystal form.

a, Asymmetric unit of the Ca2+-bound Syt1–SNARE complex in the short unit cell crystal form at 3.6 Å resolution using diffraction data collected at the APS NE-CAT microfocus synchrotron beamline (Extended Data Table 1). The colour code is the same as in Fig. 1c. Two Syt1 C2AB fragments (distinguished by the designators I and I′) bind to the same SNARE complex in the asymmetric unit (see schema). b, mFoDFc annealed omit map (Methods) of the Ca2+-bound Syt1–SNARE complex in the short unit cell crystal form collected at the APS NE-CAT microfocus synchrotron beamline; omitted residues within region I of the primary interface (residues 335–340 in Syt1 and 159–166 in SNAP-25) are coloured cyan. The contour level is 2.3σ . Left side, without B -factor sharpening; right side, with B -factor sharpening. c , Representative 2 mFo DFc electron density map of the Ca2+-bound Syt1–SNARE complex for the short unit cell crystal form using diffraction data collected at the APS NE-CAT microfocus synchrotron beamline. The contour level is 1.5 σ . Left side, without B -factor sharpening; right side, with B -factor sharpening. b, c , The sharpening B -factor (−55 Å2) was set to make the lowest atomic B -factor of the short unit cell crystal form comparable to that of the long unit cell crystal form. Even with B -factor sharpening, the electron density map of the long unit cell crystal form collected at the LCLS XFEL is superior to that of the short unit cell crystal form collected at the APS NE-CAT microfocus synchrotron beamline. d , Views of the crystal lattice perpendicular to the bc (left) and to the ac (right) planes of the Ca2+-bound Syt1–SNARE complex in the short unit cell crystal form. The particular layer shown on the right corresponds to the red arrowhead in the left panel. The unit cell is outlined by a black box.

Extended Data Figure 4 Single-molecule FRET efficiency distributions of the Syt1–SNARE complex versus FRET efficiency values calculated from the Syt1–SNARE interfaces observed in the crystal structure.

Shown are histograms of intermolecular single molecule FRET (smFRET) efficiency values that were measured between pairs of covalently attached organic labels on the Syt1 C2AB fragment and the SNARE complex28 (also shown as large spheres superimposed on the interfaces observed in the crystal structure). Arrowheads indicate FRET efficiencies calculated from the crystal structure of the Ca2+-bound Syt1–SNARE complex in the long unit cell crystal form (complex I) for the primary, secondary and tertiary interfaces, using the methods and approximations described in ref. 28 to simulate the positions of dye centres in order to calculate the FRET-efficiency values. Only the dye pair combinations between the nearest C2 domain (including the C2A–C2B linker) and the SNARE complex were calculated for the three interfaces. Note that owing to the presence of transitions between different states the histograms reflect a combined effect of interaction interfaces. The label at position A61 would have disrupted the tertiary interfaces between the C2A domain and the SNARE complex, explaining the discrepancy for these labels (indicated by open triangles). In retrospect, the top smFRET-derived model28 and the primary interface observed in the crystal structure primarily differed in the orientation of the C2B domain. Moreover, the top smFRET-derived model predicted the approximate location of the primary interface on the neuronal SNARE (see Fig. 4c in ref. 28).

Extended Data Figure 5 Comparison of the two crystal forms and the Ca2+- and Mg2+-bound crystal structures of the Syt1–SNARE complex.

a, Superposition of the primary interfaces of the Ca2+-bound Syt1–SNARE complex structure in the long unit cell crystal form (gold and bright orange) and in the short unit cell crystal form (white). The primary interface is very similar in both crystal forms: the r.m.s.d. for the primary interface between both crystal forms is 0.38 Å (bright orange) and 0.42 Å (white) for complex I and complex II, respectively (including Cα atoms of the SNARE complex and the Syt1 C2B (I) domain forming the interface). b, Superposition of complex I in the long unit cell crystal form with the asymmetric unit of the short unit cell crystal form, but only showing the secondary interface (light-blue shaded disk) between Syt1 C2B (I′) and the SNARE complex (I). The bottom panels show close-up views of the secondary interface: left, interacting residues (sticks and balls); right, a 90° rotated view of the view shown in the left panel. The Syt1 C2B (I′) domain is rotated by 16° between the two crystal forms and, as a consequence, the interactions between residues R281, K288 and R398 of the Syt1 C2B (I′) domain and residues E224 and E228 of syntaxin-1A are slightly changed by this rotation. Notably, residues Syt1 R281, K288 and R398 are involved in both the primary (Fig. 2) and secondary interfaces. c, Superposition of complex I in the long unit cell crystal form with the asymmetric unit of the short unit cell crystal form, showing all interfaces. d, Superposition of the Ca2+-bound (white) and Mg2+-bound (black) crystal structures of the Syt1–SNARE complex, both in the short unit cell crystal form. The lower left panel shows a close-up view of the primary interface, indicating that it is very similar in both the Ca2+- and Mg2+-bound crystal structures. The Syt1 C2B domain that forms the secondary interface (light-blue shaded disk) is rotated by 19° between the Ca2+- and Mg2+-bound complexes. The lower-right panel is a rotated view of the complex, also showing the tertiary interface (light-green shaded disk), and the C2A–C2B interface that involves asymmetry-related Syt1 C2A domain (I′) (grey shaded disk). e, B-factor coloured cartoon representations of the asymmetric units of the Ca2+-bound long unit cell crystal form (top), the Ca2+-bound short unit cell crystal form (bottom left), and the Mg2+-bound short unit cell crystal form (bottom right) of the Syt1–SNARE complex. Note that the primary interfaces have relatively low B-factors, similar to the majority of the structure, while parts of the C2A and C2B domains involved in the secondary and tertiary interfaces have higher B-factors, possibly indicating increased flexibility.

Extended Data Figure 6 Sequence alignments of Syt1, SNAP-25 and syntaxin-1A from different homologues.

a, Sequence alignment of Syt1 homologues, showing the sequences around the primary interface of the Syt1–SNARE complex. Note that rat Syt5 refers to UniProt ID Q925C0, zebrafish Syt9 refers to GeneBank accession number AAI52175, rat Syt9 refers to UniProt ID P47861, and human Syt9 refers to UniProt ID O00445. b, Electrostatic potential surfaces of the known crystal structures of synaptotagmin-1, synaptotagmin-3, synaptotagmin-4 and synaptotagmin-7; the dashed rectangles indicate the regions that correspond to the primary interface regions I and II of the Syt1–SNARE complex. c, Sequence alignment of different SNAP-25 homologues, showing the sequences around the primary interface of the Syt1–SNARE complex. d, Sequence alignment of different syntaxin homologues, showing a sequence range around the primary interface of the Syt1–SNARE complex. In all panels, the interacting residues of the primary interface are indicated by solid circles and coloured boxes for region I (cyan) and region II (red/orange).

Extended Data Figure 7 Syt1 mutants and SNARE complexes with SNAP-25 mutants are well folded.

a, Top panels: CD spectra of wild-type and mutant Syt1 C2B domains in the absence of Ca2+. Bottom panels: thermal denaturation was monitored by molar ellipticity at a wavelength of 216 nm in the absence of Ca2+ (black) and in the presence of 5 mM Ca2+ (red). The specified melting temperatures were estimated as the mid-point of the melting curves (Methods). b, Superposition of the Syt1 C2B domains from the Ca2+-bound Syt1–SNARE complex in the short unit cell crystal form (gold), the crystal structure of the quintuple mutant (R281A/E295A/Y338W/R398A/R399A) of the Syt1 C2B domain (green), and the crystal structure of the isolated Syt1 C2B domain (white, PDB code 2YOA). c, d, Representative m2FoDFc electron density maps of the crystal structure of the quintuple mutant of the Syt1 C2B domain (Extended Data Table 1) contoured at 2.0σ. The labels refer to the mutated residues. e, Overlay of SEC profiles of full-length Syt1 mutant proteins used in the single vesicle–vesicle fusion assay (Fig. 3d–g). f, Coomassie-blue-stained SDS–PAGE with and without boiling of neuronal SNARE complexes formed by full-length SNAP-25 and its mutants, syntaxin-1A and synaptobrevin-2, using the proteins that were used in the single vesicle–vesicle fusion assay (Methods).

Extended Data Figure 8 Probability of fusion versus time upon 500 μM Ca2+ injection and spontaneous fusion for Syt1 and SNAP-25 mutants.

Shown are the data that were used to generate Fig. 3d–g. The number of independent experiments and analysed events are provided in Extended Data Table 2. ad, Cumulative histograms of probability of fusion versus time for Syt1 mutants upon 500 μM Ca2+ injection (a) and spontaneous fusion (b), and SNAP-25 mutants upon 500 μM Ca2+ injection (c) and spontaneous fusion (d). eg, Control experiments: e, Ca2+-triggered fusion; f, spontaneous fusion with 3 mM ATP, without SNAP-25 or Syt1; and g, mock injection without Ca2+.

Extended Data Table 1 Crystallographic data and refinement statistics
Extended Data Table 2 Data summary table for the single vesicle–vesicle fusion experiments with Syt1 and SNAP-25 mutants

Supplementary information

Structure of the Syt1-SNARE complex

The entire asymmetric unit of the long unit cell crystal form consists of two SNARE complexes and three Syt1 C2AB fragments. One of the two SNARE complexes interacts with two Syt1 C2AB fragments in the asymmetric unit, and a symmetry related C2AB fragment (designated as Complex I). The other SNARE complex just interacts with the C2B domain of one Syt1 C2AB fragment (designated as Complex II). The diffraction data for this crystal structure were collected at the X-ray free electron laser (XFEL) Linac Coherent Light Source (LCLS) at SLAC Accelerator Laboratory at Stanford University. (MOV 21214 kb)

Interfaces between Syt1 C2 domains and SNARE complexes

At the start of the video, Complex I (long unit cell crystal form) is shown along with its symmetry mate, followed by close-up views of three different interfaces between Syt1 C2 domains and the SNARE complex, as well as the interface between two C2 domains. (MOV 28122 kb)

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Zhou, Q., Lai, Y., Bacaj, T. et al. Architecture of the synaptotagmin–SNARE machinery for neuronal exocytosis. Nature 525, 62–67 (2015). https://doi.org/10.1038/nature14975

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