Molecular determinants of complexin clamping and activation function
- Yale Nanobiology Institute, United States
- Department of Cell Biology, Yale University School of Medicine, United States
- Department of Pathology, Yale University School of Medicine, United States
- Departments of Neurology, Yale University School of Medicine, United States
Figures
Figure 1 with 4 supplements
Syt1 and mCPX act sequentially to arrest SNARE-driven fusion.
(A) Schematic of the programmable DNA-based mimetic used to simulate the Syt1 clamp on the SNARE-driven fusion. Annealing of the complementary ssDNA reconstituted into the SUV and the bilayer in dsDNA sterically counters the polarized SNARE assembly process and introduces a docking-to-fusion delay reminiscent of Syt1 (B) Survival analysis (Kaplan-Meier plot) curve shows that a nominal dock-to-fusion delay introduced by 20 copies of ssDNA (purple) allows mCPX to arrest spontaneous fusion of vSUVs. In contrast, no clamping was observed with 5 copies of ssDNA (yellow) which created no appreciable delay in the fusion kinetics. This suggests a sequentially mode of action for Syt1 and mCPX, wherein the kinetic delay introduced by Syt1 enables mCPX to block SNARE-driven fusion. Data was obtained from a minimum of three independent experiments, with at least 100 vesicles analyzed for each condition. A representative survival curve is shown for clarity.
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Figure 1—source data 1
Data and summary statistics for DNA-regulated fusion assay.
- https://cdn.elifesciences.org/articles/71938/elife-71938-fig1-data1-v1.xlsx
Figure 1—figure supplement 1
Coomassie-stained SDS-PAGE analysis of the proteins used in this study.
(A) Gel image of the Syt1-VAMP2 containing SUVs and t-SNAREs complex (1:1 Syntaxin1A and SNAP25) reconstituted into the free-standing bilayer. In most experiments we used SUVs containing 73 ± 6 and 25 ± 4 copies of outward-facing VAMP2 and Syt1. We used SUV containing 13 ± 4 and 25 ± 4 copies of VAMP2 and Syt1 for experiments under low-copy VAMP2 conditions. (B) Gel image of mammalian CPX wild-type, mutants and orthologs used in the study.
Figure 1—figure supplement 2
End-point (10 min) survival analysis shows that the 5 min pre-incubation of mCPX (2 μM) with either the t-SNARE containing bilayer (orange bar) or Syt1-vSUVs (green bar) does not affect its overall clamping ability.
No change in the clamping function was observed with mCPX wild type (mCPXWT) or with mutants, mCPX26-83 (lacking the c-terminal membrane binding domain) and mCPXL117W (with enhanced membrane binding property). The average values and standard deviations from three independent experiments (with ~100 vesicles in total) are shown.
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Figure 1—figure supplement 2—source data 1
Data and summary statistics of CPX incubation analysis.
- https://cdn.elifesciences.org/articles/71938/elife-71938-fig1-figsupp2-data1-v1.xlsx
Figure 1—figure supplement 3
DNA-hybridization regulates SNARE-mediated membrane fusion.
(A) Complementary single-stranded DNA (ssDNA) sequences were conjugated to cholesterol with tri-ethylene glycol spacer were incubated with pre-formed VAMP2 or t-SNARE SUVs with mild agitation and subsequently purified using float-up with a discontinuous Nycodenz gradient (B) Representative Coomassie and Sybr-green stained gels showing the incorporation of defined number of ssDNA into v-SUVs. The number of ssDNA incorporated into the vSUVs were controlled using a defined input ssDNA: lipid ratio. (C) Bulk lipid mixing assay (Weber et al., 1998) with ssDNA incorporated v- and t-SUVs shows that SNARE-driven fusion is not appreciably affected up to relatively high ssDNA density ( > 50 copies of ssDNA per vesicle). Representative fusion curves measured by dequenching of NBD-fluorescence is shown for clarity.
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Figure 1—figure supplement 3—source data 1
Data for DNA regulation bulk fusion assay.
- https://cdn.elifesciences.org/articles/71938/elife-71938-fig1-figsupp3-data1-v1.xlsx
Figure 1—figure supplement 4
Delay in fusion kinetics required for CPX clamping.
(A, B) Under physiologically-relevant conditions (SUVs reconstituted with ~70 copies of VAMP2), delay in fusion kinetics introduced by 20 copies of ssDNA was required to produce a stable clamp. Titration experiment showed that clamping ability of mCPX similar to those observed with Syt1 (Figure 2—figure supplement 2). In contrast, no clamping was observed even at the highest concentration (20 mM) of mCPX tested with 5 copies of ssDNA. (C) Under these conditions, clamping function of mCPX required the CPXacc and the rigid CPXacc-CPXcen helical portion as both deletion of CPXacc (CPX48-134) and a helix breaking mutation (CPXGP) introduced between CPXcen and CPXacc abrogated the fusion clamp. (D) Under low VAMP2 conditions (SUVs containing ~12 copies of VAMP2), 20 copies of ssDNA alone was able to sufficiently arrest fusion and this fusion clamp was further stabilized by inclusion of 2 μM mCPX. Averages and data from three independent experiments (with ~100 vesicles in total) are shown.
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Figure 1—figure supplement 4—source data 1
Data and summary statistics of optimizing the DNA-regulated fusion assay.
- https://cdn.elifesciences.org/articles/71938/elife-71938-fig1-figsupp4-data1-v1.xlsx
Figure 2 with 2 supplements
Molecular determinants of Complexin clamping function.
The effect of mCPX mutants on docking and clamping of spontaneous fusion was assessed using a single-vesicle analysis with a pore-spanning bilayer setup. (A) Inclusion of mCPX increases the number of docked Syt1-vSUVs and this stimulatory effect is greatly reduced when the interaction of the CPXcen to the SNAREpins is disrupted targeted mutations (mCPX4A). In contrast, deletion of the N-terminal domain (CPX26-134) or accessory helix (CPX48-134) or the c-terminal portion (CPX26-83) exhibit limited effect of the vesicle docking. In all cases, a mutant form of VAMP2 (VAMP24X) which eliminated fusion was used to unambiguously estimate the number of docked vesicles after the 10 min interaction phase. (B) The time between docking and fusion was measured for each docked vesicle and the results for the whole population are presented as a survival curve (Kaplan-Meier plots). Syt1-vSUVs (black curve) are diffusively mobile upon docking and fuse spontaneous with a half-time of ~5 s. Addition of soluble mCPX (red curve) fully arrest fusion to produce stably docked SUVs that attach and remain in place during the entire period of observation. CPX mutants with impaired SNARE interaction (mCPX4A, green curve) or lacking the accessory helical domain (mCPX48-134, yellow curve) fail to clamp fusion whilst the removal of c-terminal portion (mCPX26-83, purple curve) produces a partial clamping phenotype. The N-terminal domain is not involved in establishing the fusion clamp (C) End-point analysis at 10 s post-docking shows that the both the accessory helix deletion (mCPX48-134) and CPXcen modifications (mCPX4A) result in complete loss of inhibitory function and cannot be rescued even at 20 μM concentration. In contrast, the clamping function of the c-terminal deletion mutant (mCPX26-83) is fully restored at high CPX concentration. The average values and standard deviations from three independent experiments (with ~300 vesicles in total) are shown. **p < 0.01; *** p < 0.001 using the Student’s t-test.
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Figure 2—source data 1
Data and summary statistics of docking and survival analysis for CPX mutants.
- https://cdn.elifesciences.org/articles/71938/elife-71938-fig2-data1-v1.xlsx
Figure 2—figure supplement 1
Survival analysis (Kaplan-Meier plots) of Syt1-vSUVs shows that the loss of clamping phenotypes observed with CPX mutant with impaired SNARE interaction (mCPX4A, green curve) or lacking the accessory helical domain (mCPX48-134, blue curve) is not rescued at high (20 μM) CPX concentrations.
In contrast, increasing the CPX concentration fully-restores the inhibitory function of the c-terminal deletion mutant (mCPX26-83, purple curve). The average values and standard deviations from three independent experiments (with ~300 vesicles in total) are shown.
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Figure 2—figure supplement 1—source data 1
Data and statistics of survival analysis of CPX mutants at high concentration.
- https://cdn.elifesciences.org/articles/71938/elife-71938-fig2-figsupp1-data1-v1.xlsx
Figure 2—figure supplement 2
Dose-dependency analysis using Syt1-vSUVs shows that CPX mutant with a hydrophobic mutation (mCPXL117W, red curve) designed to improve its membrane association is more efficient in clamping fusion as compared to the CPXWT (black curve).
This implies that the c-terminal domain contributes to clamping function by increasing the local CPX concentration via membrane interaction. The average values and standard deviations from three to four independent experiments (with ~250 vesicles in total) are shown.
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Figure 2—figure supplement 2—source data 1
Data for titration analysis for CPX L117W mutant.
- https://cdn.elifesciences.org/articles/71938/elife-71938-fig2-figsupp2-data1-v1.xlsx
Figure 3 with 4 supplements
Specific interaction of mCPX accessory helix with SNAREs enhances its clamping function.
(A) End-point survival analysis (measured at 10 s post docking) using Syt1-vSUVs demonstrates that disrupting the binding of the CPXacc to either the t-SNAREs (CPXNC1) or the VAMP2 (CPXNC2) abrogates the clamping function, and that a helix breaking mutation (CPXGP) introduced between CPXcen and CPXacc also abrogates the fusion clamp. (B) In contrast, mutations designed to enhance the binding of CPXacc to t-SNAREs (CPXSC) increase the potency of the CPX clamp. This indicates efficient clamping by CPX requires a continuous rigid helix along with specific interaction of the CPXacc with the assembling SNARE complex. (C) Supporting this notion, survival analysis (Kaplan-Meier plots) shows that both Drosophila and C. elegans CPXs, which have very low sequence identity with the mCPX accessory domain, and a CPX mutant with a randomized accessory helical sequence (CPXEAAK) have poor clamping efficiency under standard (2 μM) experimental conditions and only partial clamping at higher (20 μM) concentration. The average values and standard deviations from three to four independent experiments (with ~250 vesicles in total) are shown. *** indicates p < 0.001 using the Student’s t-test.
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Figure 3—source data 1
Data and summary statistics of survival analysis for ceCPX, dmCPX and mCPX mutants.
- https://cdn.elifesciences.org/articles/71938/elife-71938-fig3-data1-v1.xlsx
Figure 3—figure supplement 1
Survival analysis (Kaplan-Meier plots) using Syt1-vSUVs and 2 μM CPX shows targeted mutations that disrupt the interaction of CPXacc with the t-SNARE (mCPXNC1, blue curve) or VAMP2 (mCPXNC2, green curve) abrogate the clamping function.
Similarly, GPGP mutations introduced between the CPXcen and CPXacc (mCPXGP, red curve) that breaks the continuity of the helical domain disrupts the clamping function. Overall, this argues that a rigid α-helical structure along with distinct interaction of the CPXacc with the assembling SNAREpins are required for its clamping function. Supporting this, CPXacc that strengths the t-SNARE binding stabilizes the fusion clamp and as such, enhances the clamping efficiency (Figure 2B). The average values and standard deviations from three independent experiments (with ~300 vesicles in total) are shown.
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Figure 3—figure supplement 1—source data 1
Data and summary statistics of survival analysis of mCPX mutants.
- https://cdn.elifesciences.org/articles/71938/elife-71938-fig3-figsupp1-data1-v1.xlsx
Figure 3—figure supplement 2
Docking analysis with Syt1-vSUVs show that CPXacc does not contribute significantly to stimulatory effect on vesicle docking.
With exception of the t-SNARE binding mutant (mCPXNC1), all other accessory helix mutants tested at the standard 2 μM concentration were able to significantly increase the number of vesicles docked. mCPX orthologs from Drosophila (dmCPX) and C. elegans (ceCPX) were also able to promote vesicle docking and to levels comparable to mCPXWT. Considering that CPXcen is unaltered in mCPX mutants tested and shows high degree of sequence conservation in the orthologs, this data further supports the notion the CPXcen-SNARE interaction is the key to CPX ability to promote vesicle docking. In all experiments, VAMP24X mutant was used to enable unambiguous counting of stably docked vesicles. The average values and standard deviations from three independent experiments (with ~200 vesicles in total for each condition) are shown. *p < 0.05, ** p < 0.01 *** p < 0.001 using the Student’s t-test.
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Figure 3—figure supplement 2—source data 1
Data and summary statistics of docking analysis for ceCPX, dmCPX and mCPX mutants.
- https://cdn.elifesciences.org/articles/71938/elife-71938-fig3-figsupp2-data1-v1.xlsx
Figure 3—figure supplement 3
Sequence alignment of alpha helical CPXacc-CPXcen portion (mCPX residues 26–70) shows that the CPXcen is largely conserved while CPXacc is highly divergent across different species.
All identical residues are denoted by black asterix while conservative replacements are denoted as blue asterix. The conserved residues within the accessory helix involved in binding to t-SNAREs and VAMP2 are colored in green and red respectively. The low degree of conservation in the CPXacc region may explain the divergent clamping efficiency observed in our in vitro assay. The mCPXEAAK mutant has lowest level of sequence identity with mCPXacc but couple of key residues (highlighted in yellow) are replaced by Alanine residue, creating an extended hydrophobic pocket which is predicted to bind t-SNARE more efficiently. This could in theory explain mCPXEAAK ability to partially rescue clamping at high concentrations.
Figure 3—figure supplement 4
Syt1-vSUVs stably clamped at high concentration (20 μM) of Drosophila CPX (dmCPX), C.
elegans CPX (ceCPX) and mCPXEAAK remain Ca2+-sensitive and majority is triggered to fuse rapidly and synchronously following the addition of 1 mM Ca2+. In these experiments, fusion was attested by sudden increase, followed by diffusion of ATTO647N-PE fluorescence introduced in the Syt1-vSUVs. Average and standard deviations from three independent experiments (minimum of 100 vesicles under each condition) are shown.
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Figure 3—figure supplement 4—source data 1
Data for calcium sensitivity of ceCPX, dmCPX and mCPX mutant.
- https://cdn.elifesciences.org/articles/71938/elife-71938-fig3-figsupp4-data1-v1.xlsx
Figure 4
Complexin increases the probability of Ca2+-triggered vesicular release.
(A) The effect of mCPX on Ca2+-triggered fusion was assessed using a content-release assay with Sulforhodamine-B loaded vesicles. Sulforhodamine-B is largely self-quenched when encapsulated inside an SUV. Fusion of the vesicle results in dilution of the probe, which is accompanied by increasing fluorescence. The Ca2+-sensor dye, Calcium Green, introduced in the suspended bilayer (via a lipophilic 24-carbon alkyl chain) was used to monitor the arrival of Ca2+ at/near the docked vesicles. A representative fluorescence trace before and after the addition of 100 μM Ca2+ shows that the rise in Sulforhodamine-B (red curve) fluorescence intensity occurs within a single frame (13 ms) of Ca2+ binding to local Calcium green (green curve) (B) End-point analysis at 1 min post Ca2+-addition shows that >90% of all Syt1/mCPX-clamped vesicles (~70 copies of VAMP2 and ~25 copies of Syt1) fuse following Ca2+ addition as compared to ~70% of Syt1-clamped vesicles (~13 copies of VAMP2 and ~25 copies of Syt1). Inclusion of mCPX enhances the fusion probability even under the low-VAMP2 condition suggesting that mCPX promote Ca2+-triggered fusion independent of its clamping function. (C) Kinetic analysis shows that the clamped vesicles with or without mCPX fuse rapidly following Ca2+-addition with near identical time constant of ~11 ms. This represents the temporal resolution limit of our recordings (13 ms frame rate) and the true Ca2+-triggered fusion rate may well be below 10 ms. (D) Deletion and mutational analysis under low-VAMP2 conditions (SUVs with ~13 copies of VAMP2 and ~25 copies of Syt1) show that the deletion of CPXacc (CPX48-134, blue bar) or disruption of CPXcen-SNARE interaction (CPX4A, green bar) abrogate the stimulatory function, but deletion of the N-terminal portion (CPX26-134, yellow bar) or the c-terminal domain (CPX26-83, purple bar) has no effect. The stimulatory function does not require rigid CPXacc-CPXcen helix (mCPXGP, orange bar) nor clamping specific CPXacc-SNARE interaction as non-clamping CPXEAAK mutant (cyan bar) and C. elegans ortholog (ceCPX, brown bar) retain stimulatory function. The average values and standard deviations from three independent experiments (with ~100 vesicles in total) are shown. ** p < 0.01, ***p < 0.001 using the Student’s t-test.
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Figure 4—source data 1
Data and summary statistics of effect of mCPX mutants on calcium activation of fusion.
- https://cdn.elifesciences.org/articles/71938/elife-71938-fig4-data1-v1.xlsx
Figure 5 with 2 supplements
Synergistic regulation of SNARE-mediated fusion by CPX and Syt1.
(A) Model of pre-fusion CPX-Syt-SNARE complex containing the central and peripheral SNAREpins connected via CPX trans-clamping interaction. The central SNAREpins, which are responsible for the Ca2+-triggered fusion, are bound to and sterically clamped by two Syt molecules - one independently at the ‘primary’ interface and other in the conjunction with CPXcen (red) at the ‘tripartite’ interface. The CPXacc (yellow) emanating from the central SNAREs reaches out to bind and clamp the peripheral SNAREpin (dark gray). This molecular model was generated using the X-ray crystal structures 5W5C (Zhou et al., 2017) and 3RL0 (Kümmel et al., 2011) (see Figure 5—figure supplement 1). Noteworthy, the positioning of peripheral SNAREpins in this model is likely to be flexible considering the inherent variability in the localization of CPXacc (B) Organization of pre-fusion CPX-Syt-SNARE complex at the synaptic vesicle-plasma membrane interface. In addition to the ‘bridging interaction’, the primary C2B domain (gray) also self-assembles to an oligomeric structure which strengthens the Syt1 clamp on the central SNAREpins. The SNAREpins are multi-colored, CPX is cyan and tripartite C2B is pink. Only a single cross-linked SNAREpins is shown, but multiple SNARE complexes are likely involved in driving rapid SV fusion (see Figure 5—figure supplement 2). We have omitted the transmembrane domains of SNAREs/Syt and the Syt C2A domains for clarity.
Figure 5—figure supplement 1
X-ray structure 3RL0 (orange) representing the CPX trans-clamping interaction (Kummel et al.Kümmel et al., 2011) was superimposed onto the crystal structure 5W5C (gray) of the primed CPX-Syt1-SNARE complex (Zhou et al., 2017) to generate the ‘bridging model’ shown in Figure 5A.
Figure 5—figure supplement 2
Possible organization of pre-fusion CPX-Syt-SNARE complexes under a docked vesicle.
Considering the topological constraints and recent cryo-electron tomography structural data (Li et al., 2019; Radhakrishnan et al., 2021), we imagine about 6 pairs of ‘central +peripheral’ SNAREpins might be arranged in a symmetrical fashion under a docked SV. Ca2+- binding to Syt1 would trigger the dissociation of this ‘clamped’ structure, allowing multiple ‘central’ SNAREpins to act cooperatively to drive ultra-fast fusion.
Additional files
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Source data 1
Combined data and summary statistics for all figures and figure supplements.
- https://cdn.elifesciences.org/articles/71938/elife-71938-data1-v1.zip
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Molecular determinants of complexin clamping and activation function
eLife 11:e71938.
https://doi.org/10.7554/eLife.71938
