The primed SNARE-complexin-synaptotagmin complex for neuronal exocytosis

Abstract

Synaptotagmin, complexin, and neuronal SNARE (soluble N-ethylmaleimide sensitive factor attachment protein receptor) proteins mediate evoked synchronous neurotransmitter release, but the molecular mechanisms mediating the cooperation between these molecules remain unclear. Here we determine crystal structures of the primed pre-fusion SNARE-complexin-synaptotagmin-1 complex. These structures reveal an unexpected tripartite interface between synaptotagmin-1 and both the SNARE complex and complexin. Simultaneously, a second synaptotagmin-1 molecule interacts with the other side of the SNARE complex via the previously identified primary interface. Mutations that disrupt either interface in solution also severely impair evoked synchronous release in neurons, suggesting that both interfaces are essential for the primed pre-fusion state. Ca²⁺ binding to the synaptotagmin-1 molecules unlocks the complex, allows full zippering of the SNARE complex, and triggers membrane fusion. The tripartite SNARE-complexin-synaptotagmin-1 complex at a synaptic vesicle docking site has to be unlocked for triggered fusion to start, explaining the cooperation between complexin and synaptotagmin-1 in synchronizing evoked release on the sub-millisecond timescale.

Extended Data Figure 1. Crystal packing and B-factors

a and b, Views of the crystal lattice of the Syt1-SNARE-Cpx-Syt1 C2AB (a) and Syt1-SNARE-Cpx-Syt1 C2B (b) crystal structures. The red rectangles highlight the C-terminal ends of the SNARE complexes. They are unstructured and form no crystal contacts in either crystal form. c and d, B-factor coloured cartoon representations of the Syt1-SNARE-Cpx-Syt1 C2AB (c) and Syt1-SNARE-Cpx-Syt1 C2B (d) crystal structures (the second syt1 molecule is related to the first by crystallographic symmetry). Both the primary and tripartite interfaces have low B-factors. e and f, Views of the molecular packing arrangements around the SNARE (coloured) and Syt1 C2B (orange) components that form that primary interface in the SNARE-Syt1 crystal structure (e, PDB code 5CCH) and the Syt1-SNARE-Cpx-Syt1 C2AB crystal structure (f). The molecular packing arrangements around the SNARE and Syt1 C2B components that form that primary interface are vastly different these crystal structures, illustrating that the formation of the primary interface is entirely independent from crystal packing environments. We also note that the protein constructs and crystallization conditions were very different for these structures.

Extended Data Figure 2. Additional details of the Syt1-SNARE-Cpx-Syt1 tripartite interface and primary sequence alignment

a, Close-up view of the contact interface between a pseudo-helix in Syt1 C2B (including the 3₁₀ helix T3 situated between β5 and β6, and the loop between α-helices β7 and HB), syntaxin-1A and SNAP-25_N. Syt1 Lys354 and SNAP-25 Glu24 as well as Syt1 Glu350 and SNAP-25 Arg17 interact via salt bridges. Syt1 Gln353 and syntaxin-1A His199, Syt1 Glu350 and SNAP-25 Gln20 form separate hydrogen bonds. b, Close-up view of the contact site between the HB α-helix of the Syt1 C2B domain and the Cpx central α-helix. Interacting residues are shown as sticks and are labeled, while water molecules are shown as red balls. Dashed lines indicate hydrogen bonds or salt bridges. c, Sequence alignment of rat synaptotagmin isoforms, rat Doc2A/B, and rabphilin3A, for residues around the SNARE-Cpx-Syt1 tripartite interface. The arrows show the residues involved in specific sidechain interactions in the tripartite interface (see also discussion the text). The red stars show the mutated residues used in this study. The alignment was performed using Clustal Omega (http://www.ebi.ac.uk/Tools/msa/clustalo/). The figure was prepared with Boxshade3.21 (http://www.ch.embnet.org/software/BOX_form.html). d, Cartoon representation of the known crystal structures of C2A and C2B domains of synaptotagmin isoforms, Doc2 isoforms, rabphilin3A (Rph), and Munc13-1. The black arrows indicate the presence of the HA or T3 helices in these structures.

Extended Data Figure 3. The Syt1 C2B mutants and the SNARE^Q complex are well folded

a, Top panels: CD spectra of WT and mutant Syt1 C2B domains in the absence of Ca²⁺. Bottom panels: CD thermal melting curves, monitored at 216 nm in the absence of Ca²⁺ (black) and in the presence of 5 mM Ca²⁺ (red). The specified melting temperatures were estimated by the mid-point of the melting curves (Methods). b, CD spectra of WT SNARE complex (left) and SNARE^Q (right). c, CD thermal melting curves of WT SNARE complex (black circle) and SNARE^Q complex (red triangle), monitored at 220 nm.

Extended Data Figure 4. ITC binding data and analyses

a–k, Differential power traces and heats of injection traces of the specified samples in the syringe and the cell of the ITC instrument. The experimental conditions are described in the Methods. For panels j-l, three independent experimental repeats were performed, and all ITC data curves are shown; shown are means ± s.e.m. for three independent repeat experiments.. For all other ITC experiments, the error bars were obtained from a fit of the data points of the particular ITC-experiments. The schemas in the insets summarize the mutations used in the particular experiments. The ITC experiments produce well-determined n vales for the following experiments: C2B^KA titrated into the SNARE complex (n=0.94±0.02), Cpx^48-73 titrated into the SNARE complex (n=0.97±0.02) or the SNARE^Q complex (n=0.98±0.01). For the other ITC experiments, it was difficult to achieve high enough concentrations of the injected sample to obtain optimal conditions for reliable determination of n. N.D., not detectable. Experimental conditions are described in the Methods.

Extended Data Figure 5. Superposition of observed interactions between the Syt1-C2B domain and the SNARE complex in solution and in crystal structures

a, Close-up view of a small contact between the polybasic region of the Syt1 C2B domain and the SNARE complex in the Syt1-SNARE-Cpx-Syt1 crystal structures. Interacting residues are shown as sticks and are labeled. Dashed lines indicate hydrogen bonds or salt bridges. b, Shown are the primary interface (PDB code 5CCG (ref. ), and structures in this work), the small contact shown in panel (a), and the deposited 5 conformers derived from solution NMR experiments that involve the polybasic region of Syt1 C2B (sticks-and-balls indicate residues of the polybasic region of Syt1 C2B). These NMR studies revealed that there are dynamic binding modes between the polybasic region of Syt1 C2B and the SNARE complex in solution. Although other interfaces between Syt1 and SNARE complex have been observed (e.g., secondary and tertiary interfaces in the crystal structure PDB code 5CCG), the ITC data (Extended Data Fig. 4) suggest that these are the only interactions and interfaces that occur in solution (see text).

Extended Data Figure 6. Additional analysis of electrophysiology experiments in neuronal cultures

a, Quantification of IPSC charge transfer (from the same neurons as in Fig. 4a). b, Recordings of miniature IPSCs (mIPSCs) from cultured cortical neurons with Syt1 conditional KO and Syt7 constitutive KO infected with lentiviruses expressing ΔCre/Cre recombinase and WT Syt1 or Syt1 mutants. Left to right: sample traces, cumulative probability plot of inter-event intervals, and quantification of event frequency, quantification of the amplitude and 10%–90% rise time. c, Quantification of IPSC charge transfer (from the same neurons as in Fig. 4b). d and e, mIPSCs (d) and mEPSCs (e) from cultured cortical neurons infected with lentiviruses expressing Syt1 mutants. Left to right: sample traces, cumulative probability plot of inter-event intervals, and quantification of event frequency, quantification of the amplitude and 10%–90% rise time. f, Quantification of IPSC charge transfer (from the same neurons as in Fig. 4d). g and h, Recordings of mIPSCs and mEPSCs from cultured cortical neurons infected with lentiviruses expressing Syt1^WT or Syt1^DA, without or with lentiviruses expressing Cpx1/2 shRNAs (Cpx1/2 DKD). Left to right: sample traces, cumulative probability plot of inter-event intervals, and quantification of event frequency, quantification of the amplitude and 10%–90% rise time. Shown are means ± s.e.m; the number of neurons/independent cultures are indicated. Statistical significance was assessed by Student’s t test (*P < 0.05; **P < 0.01; ***P < 0.001; NS, no significant difference) with respect to either the Cre (red) or the Cre+Syt1 group (black) in panels a and b, either the control (black) or the Syt1^DA group (red) in panels c–e, between Syt1^WT and Syt1^DA with or without Cpx1/2 DKD in panels f–h.

Extended Data Figure 7. Syt1^DA increases mIPSC frequency in a Ca²⁺-dependent manner

Recordings of mIPSCs from cultured cortical neurons infected with lentiviruses expressing Syt1^WT or Syt1^DA, without or with 10 μM BAPTA-AM preincubated for 30 min at 37 °C. Sample traces (left), cumulative probability plot of inter-event intervals (middle), and quantification of event frequency, amplitude and 10%–90% rise time (right) of mIPSCs. Shown as means ± s.e.m; the number of neurons/independent cultures are indicated. Statistical significance was assessed by Student’s t test (**P < 0.01; ***P < 0.001; NS, no significant difference) with respect to either the Syt1^WT (black) or the Syt1^DA group (red). The absence of a dominant negative effect of the Syt1^DA group on spontaneous release in the presence of BAPTA-AM is consistent with the notion that spontaneous release largely depends on a different Ca²⁺-sensor. We speculate that this mini-release Ca²⁺ sensor may compete with Syt1 in binding to the tripartite interface. Elimination of the primary interface or presence of the Syt1^DA mutant would affect the binding equilibrium, possibly explaining the increase of spontaneous release. We note that the effect of the Syt1^DA mutant on spontaneous release (as assessed by mIPSCs and mEPSCs) is opposite to the effect on evoked release (as assessed by IPSCs and EPSCs) (Fig. 4).

Extended Data Figure 8. Structure comparison of the Syt1-SNARE-Cpx-Syt1 crystal structures and other known crystal structures

a–c, Conserved and variable regions of the SNARE-Cpx subcomplex in the Syt1-SNARE-Cpx-Syt1 C2B crystal structure (colored), and crystal structures of the SNARE-Cpx subcomplex (PDB code 1KIL, black and PDB code 3RK3, white). The interaction between the central a-helix of Cpx and the SNARE complex is essentially identical in all crystal structures, while the angle at which the accessory helix protrudes away from the SNARE complex is variable. Such variability was also observed by single molecule fluorescence resonance transfer experiments. d, The superposition of the Syt1 C2B domain of the Syt1-SNARE-Cpx-Syt1 C2AB crystal structure with crystal structures of uncomplexed C2AB fragments (PDB codes are indicated in the figure) illustrates variability of the position of the C2A domain relative to the C2B domain of Syt1.

Extended Data Figure 9. A possible supramoleular arrangement of one Syt1 C2B molecule bridging two SNARE complexes

Orthogonal views (cartoon presentation, left; schema, right) of an arrangement where one Syt1 C2B domain bridges two SNARE complexes via the primary and tripartite interfaces, respectively. Directions (N-terminus to C-terminus) of the α-helices of the SNARE complex and Cpx are indicated by crosses (pointing into the page) and dots (pointing out of the page). The bridged complex would be sandwiched between two membranes as suggested in the schema in the lower right panel.

Figure 1. Crystal structures of the Syt1-SNARE-Cpx-Syt1 complex

a, Domain diagrams. The numbers delineate the boundaries of the fragments used for crystallization. TM: transmembrane region. b, Cartoon representation of the Syt1-SNARE-Cpx-Syt1 C2AB crystal structure. The Syt1 C2B domain (orange), Cpx (cyan), and the SNARE complex (blue, red, green) form the tripartite interface. A second Syt1 C2B domain (grey, related by crystallographic symmetry) forms the primary interface with the SNARE complex. For clarity, we omitted the C2A domain of the Syt1 molecule involved in the primary interface (Extended Data Fig. 1c). c, Rotated view of panel b, showing a section that includes 3₁₀ helix T3 and α-helix HA of the Syt1 C2B domain. d, Cartoon representation of the Syt1-SNARE-Cpx-Syt1 C2B crystal structure. The molecular surface of the complete SNARE complex structure (PDB code 1N7S) is superimposed. e, Schema of the Syt1-SNARE-Cpx-Syt1 C2AB crystal structure. f, Close-up view of the tripartite interface (same orientation as in panel b), showing the continuation of α-helix HA of the Syt1 C2B domain into the central α-helix of Cpx. Helical dipoles are indicated by δ⁺ and δ⁻.

Figure 2. Close-up views of the SNARE-Cpx-Syt1 tripartite interface

a–d, Interacting residues are shown in stick representation and labelled, water molecules are shown as red balls, hydrogen bonds and salt bridges are shown as dashed lines. Molecular surfaces are shown in panels a and d. a, interactions between the α-helix HA of the Syt1 C2B domain and syntaxin-1A. Leu387 in Syt1 C2B protrudes into the cavity of syntaxin-1A and Ser391 in Syt1 C2B forms a water-mediated hydrogen bond with Gln207 in syntaxin-1A. b, Close-up view of the interactions between the N-terminal end of the α-helix HA of the Syt1 C2B domain and the C-terminal end of the Cpx central α-helix. The main chain carboxyl of Asp68 in Cpx forms hydrogen bonds with the main chain of Glu386 in the Syt1 C2B domain and is involved in water-mediated hydrogen bonds with the side chain of the same residue. The main chain carboxyl of Lys69 in Cpx interacts with side chain Oγ of Thr383 in the Syt1 C2B domain, while the main chain of Gly384 in Syt1 C2B domain is involved in water-mediated hydrogen bonds with the side chain of Glu211 in syntaxin-1A. c and d, Close-up views of a region of hydrophobic interactions involved in the tripartite interface. Hydrophobic residues are shown in sphere representation. Dashed lines in panel c indicate the depth of the section shown in panel d. Inset, schema showing the approximate locations of the mutations.

Figure 3. Probing the interfaces between the SNARE complex and the Syt1 C2B domain by ITC

a and c, Schema showing the approximate locations of the mutations used for ITC experiments in panel b and d, separately. b, ITC binding traces and dissociation constants (inset) of the Syt1 C2B domain and C2B^KA mutant titrated into the SNARE or SNARE^Q complex. d, Sample ITC binding traces and dissociation constants (inset) of the Syt1 C2B domain and its mutants titrated into the SNARE^Q complex or the SNARE^Q-Cpx complex. Three independent experimental repeats were performed, and standard deviations (SD) of the dissociation constants are provided. N.D., not detectable. Extended Data Fig. 4 shows further ITC data and analyses.

Figure 4. Ca²⁺-triggering of release by Syt1 requires both the SNARE-Cpx-Syt1 and Syt1-SNARE interfaces

a, Recordings of IPSCs from cultured cortical neurons with Syt1 conditional KO and Syt7 constitutive KO, infected with lentiviruses expressing ΔCre/Cre recombinase and WT Syt1 or Syt1 mutants. b and c, IPSCs and EPSCs from wildtype cultured cortical neurons infected with lentiviruses expressing Syt1 mutants. d and e, Recordings of IPSCs and EPSCs from cultured cortical neurons infected with lentiviruses expressing Syt1^WT or Syt1^DA, without or with lentiviruses expressing Cpx1/2 shRNAs (Cpx1/2 DKD). Sample traces (left) and quantification of peak amplitudes (right) of evoked IPSCs (a, b, d) or of EPSCs (c, e) elicited by single action potentials. Quantifications of IPSC charge transfer from the same neurons are shown in Extended Data Fig. 6a, c, f. Shown are means ± s.e.m; the number of neurons/independent cultures are indicated. Statistical significance was assessed by Student’s t test (**P < 0.01; ***P < 0.001; NS, no significant difference) with respect to either the Cre (red) or the Cre+Syt1 group (black) in panel a, either the control (black) or the Syt1^DA group (red) in panels b and c, between Syt1^WT and Syt1^DA with or without Cpx1/2 DKD in panels d and e.

Figure 5. Unlocking and triggering the primed Syt1-SNARE-Cpx-Syt1 complex

a, Recordings of IPSCs evoked by a 30-s application of 0.5 M hypertonic sucrose to induce exocytosis of RRP from cultured cortical neurons with Syt1 conditional KO and Syt7 constitutive KO. Cultures were infected with lentiviruses expressing ΔCre/Cre recombinase and WT Syt1 or Syt1 mutants. Sample traces (left) and summary graph of the IPSC charge transfers from RRP (right). Shown are means ± s.e.m; the number of neurons/independent cultures are indicated. Statistical significance was assessed by Student’s t test (*P < 0.05; **P < 0.01; ***P < 0.001) with respect to either the Cre (marked in red) or the Cre+Syt1 group (black). b and c, Orthogonal views (cartoon representation, upper; schema, lower) of the Syt1-SNARE-Cpx-Syt1 crystal structure with the membrane interacting elements of the primary Syt1/SNARE subcomplex located in a plane perpendicular to the page. d, Model of a primed Syt1-SNARE-Cpx-Syt1 (“Locked”) complex situated between the synaptic vesicle and plasma membranes. e, Upon unlocking and Ca²⁺-triggering, fusion occurs. For clarity, we omitted the transmembrane domains of the Syt1 molecules. Two or more such complexes are likely involved (e.g., Extended Data Fig. 9).