Neurons communicate with each other at synaptic contacts by releasing neurotransmitters from synaptic vesicles (SVs). This process is tightly controlled by activity-dependent changes in the presynaptic Ca²⁺ concentration and can occur in less than a millisecond after the neuronal spike (Südhof, 2013; Kaeser and Regehr, 2014). SV fusion is catalyzed by presynaptic SNARE proteins. The SNAREs on the opposing membranes (VAMP2 on the synaptic vesicle membrane; Syntaxin and SNAP25 on the presynaptic plasma membrane) assemble into a four-helix bundle that catalyzes fusion by forcing the two membranes together (Söllner et al., 1993; Weber et al., 1998). Related SNARE proteins are universally involved in intracellular transport pathways and by themselves can constitutively catalyze fusion (Weber et al., 1998; McNew et al., 2000). As such, Ca²⁺-evoked neurotransmitter release occurs from the readily releasable pool (RRP) of vesicles docked/primed at the presynaptic active zone (Südhof, 2013; Kaeser and Regehr, 2014). The current view is that at a single RRP vesicle, the SNARE complexes are firmly held (‘clamped’) in a partially assembled state (SNAREpins) close to the point of triggering fusion. Upon Ca²⁺ influx, multiple SNAREpins are synchronously activated to drive ultrafast SV fusion and neurotransmitter release (Südhof and Rothman, 2009; Südhof, 2013; Rizo and Xu, 2015; Rothman et al., 2017; Brunger et al., 2019).

It is well-established that the late stages of SV fusion are tightly regulated by two synaptic proteins – the presynaptic Ca²⁺ release sensor Synaptotagmin-1 (Syt1) and Complexin (CPX) (Südhof, 2013; Südhof and Rothman, 2009; Rizo and Xu, 2015; Brunger et al., 2019). CPX is an evolutionarily conserved cytosolic protein that bind and regulate synaptic SNARE complex assembly (McMahon et al., 1995; Huntwork and Littleton, 2007; Martin et al., 2011; Trimbuch and Rosenmund, 2016; Mohrmann et al., 2015). Biochemical and biophysical analyses show that CPX promotes the initial stages of SNARE assembly but then blocks complete assembly (Li et al., 2011; Kümmel et al., 2011; Lai et al., 2014; Krishnakumar et al., 2015). Thus, it can both facilitate and subsequently inhibit SV fusion. CPX contain distinct domains that mediate the dual clamp/activator function (Xue et al., 2007; Giraudo et al., 2008; Trimbuch and Rosenmund, 2016; Mohrmann et al., 2015). The largely unstructured N-terminal domain (residues 1–26 of mammalian CPX1) activates Ca²⁺-regulated vesicular release (Xue et al., 2010; Lai et al., 2016) while the α-helical accessory domain (CPX_acc, residues 26–48) serves as the primary clamping domain (Xue et al., 2007; Giraudo et al., 2008; Maximov et al., 2009; Yang et al., 2010; Kümmel et al., 2011; Cho et al., 2014). A central helical sequence within CPX (CPX_cen, residues 48–70) binds the groove between pre-assembled Syntaxin and VAMP2 and is essential for both function (Chen et al., 2002; Xue et al., 2007; Giraudo et al., 2008; Maximov et al., 2009). The remainder c-terminal portion (residues 71–134) has been shown to preferentially associate with curved lipid membrane via an amphipathic helical region and promotes the clamping function (Kaeser-Woo et al., 2012; Wragg et al., 2013; Gong et al., 2016).

The relative strength of CPX facilitatory vs inhibitory activities differs across species (Yang et al., 2013; Trimbuch and Rosenmund, 2016; Mohrmann et al., 2015; Xue et al., 2009). As a result of this intricate balance, genetic perturbations of CPX can produce apparently contradictory effects in different systems. For example, knockout (KO) of CPX in neuromuscular synapses of C. elegans and Drosophila results in increased spontaneous release, decreased evoked release with overall reduction in the RPP size (Huntwork and Littleton, 2007; Cho et al., 2014; Martin et al., 2011; Hobson et al., 2011; Wragg et al., 2013). In model mammalian synapses, CPX KO abates both spontaneous and evoked release with no significant change in the RRP size (Reim et al., 2001; Xue et al., 2008; López-Murcia et al., 2019) but acute CPX knockdown (KD) reduces synaptic strength, but also increases spontaneous release with a concomitant reduction in the number of primed vesicles (Maximov et al., 2009; Yang et al., 2010; Kaeser-Woo et al., 2012; Yang et al., 2013). Some of the apparent discrepancies might be related to the perturbation method used (Yang et al., 2013); nonetheless, the physiological role of mammalian CPX in regulating SV fusion and the underlying mechanisms remains in the center of debate (Mohrmann et al., 2015; Trimbuch and Rosenmund, 2016).

The interpretation of the physiological experiments can be limited by presence of the different CPX isoforms and possible compensatory homeostatic mechanisms. As such, the experiments in live synapses need to be complemented with a reductionist approach where the variables are limited, and the components can be rigorously controlled or altered. It is our hypothesis that the most direct mechanistic insight can be obtained from fully controlled cell-free systems. We have described a biochemically defined fusion setup based on a pore-spanning lipid bilayer setup that is well-suited for this purpose (Ramakrishnan et al., 2018; Ramakrishnan et al., 2019; Ramakrishnan et al., 2020).

Using this in vitro setup, which allows for precision study of the single-vesicle fusion kinetics, we recently demonstrated that mammalian CPX (mCPX), along with Syt1 and SNAREs, are essential and sufficient to achieve Ca²⁺-regulated fusion under physiologically relevant conditions (Ramakrishnan et al., 2020). Our data revealed that mCPX and Syt1 act co-operatively to clamp the SNARE assembly process and produce a pool of docked vesicles. The study also revealed that there are at least two types of clamped SNAREpins under a docked vesicle – a small subset that are reversibly clamped by binding to Syt1 (which we termed ‘central’) and a larger population that are thought to be free of Syt1 and require mCPX for clamping (termed ‘peripheral’). We further established that Syt1s’ ability to oligomerize and bind SNAREpins via the ‘primary’ binding site on SNAP25 is key to its ability to clamp central SNAREpins and that the activation of these Syt1-associated SNAREpins is sufficient to elicit rapid, Ca²⁺-synchronized vesicle fusion (Ramakrishnan et al., 2020).

Building on this work, here we use a systematic in vitro reconstitution strategy to obtain new and direct insights into the molecular basis of mCPX clamping function and its role in establishing Ca²⁺-regulated release. We report that mCPX inhibitory function requires a delay in overall fusion kinetics and involves well-defined interaction of the accessory-central helical fragments with the SNAREpins. The accessory-central helical domains also stimulate Ca²⁺-triggered vesicle fusion from the clamped state. Overall, we find that under physiologically-relevant conditions, mCPX is essential to generate/maintain a pool of docked vesicles and to promote Ca²⁺-triggered rapid ( < 10ms) and synchronous fusion of the docked vesicles.

To dissect the mCPX clamping functionality, we used physiologically relevant reconstitution conditions similar to our previous work (Ramakrishnan et al., 2020). Typically, we used small unilamellar vesicles (SUV) with ~70 copies (outward facing) of VAMP2 (vSUV) without or with ~25 copies Syt1 (Syt1-vSUV) (Figure 1—figure supplement 1). We employed pre-formed t-SNAREs (1:1 complex of Syntaxin1 and SNAP-25) in the planar bilayers (containing 15% PS and 3% PIP2) to both simplify the experimental approach and to bypass the requirement of SNARE-assembling chaperones, Munc18 and Munc13 (Baker and Hughson, 2016). Mammalian CPX1 (wild type or variants) was included in solution, typically at 2 μM unless noted otherwise (Figure 1—figure supplement 2). We used fluorescently labeled lipid (2% ATTO647N-PE) to track docking, clamping and spontaneous fusion of individual vesicles and a content dye (sulforhodamine B) to study Ca²⁺-triggered fusion of docked vesicles from the clamped state.

To focus on the ‘clamping’ of constitutive fusion events, we monitored large ensembles of vesicles to determine the percent remaining unfused as a function of time elapsed after docking and quantified as ‘survival percentages’ (Ramakrishnan et al., 2019; Ramakrishnan et al., 2020; Ramakrishnan et al., 2018). Docked immobile vesicles that remained un-fused during the initial 10-min observation period were defined as ‘clamped’ and the ‘docking-to-fusion’ delay enabled us to quantify the strength of the fusion clamp (Ramakrishnan et al., 2019; Ramakrishnan et al., 2020; Ramakrishnan et al., 2018). Since we track the fate of single vesicles, this analysis allowed us to examine the ‘clamping’ mechanism, independent of any alteration in the preceding docking sub-step.

Our earlier results showed that Syt1 alone can meaningfully delay but not stably clamp SNARE-mediated fusion. Similarly, mCPX, on its own, is ineffective in clamping SNARE-driven vesicle fusion. In fact, both Syt1 and mCPX are needed to produce a stably ‘clamped’ state which can then be reversed by Ca²⁺ (Ramakrishnan et al., 2020). It is possible that Syt1 and mCPX1 either act jointly to generate a new intermediate state in the SNARE assembly pathway or operate sequentially, with the kinetic delay introduced by Syt1 enabling mCPX to arrest SNARE assembly. To distinguish between these possibilities, we developed a mimic for the Syt1 clamp – a lipid-conjugated ssDNA that is capable of regulating SNARE-driven fusion in situ. Without directly interacting with the SNAREs, the specific base-pair hybridization of the complementary ssDNA reconstituted into the SUVs and the planar bilayer introduces a steric barrier which is expected to, and indeed does delay fusion (Figure 1, Figure 1—figure supplement 3). Moreover, this docking-to-fusion delay could be varied by adjusting the number of ssDNA molecules (Figure 1—figure supplement 3).

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Data and summary statistics for DNA-regulated fusion assay.

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We then assessed the effect of mCPX on ssDNA-regulated fusion of vSUV in the absence of Ca²⁺ (Figure 1). mCPX was able to near-completely arrest spontaneous fusion of vSUV to generate stably docked vesicles, provided that the rate of SNARE-mediated fusion was sterically delayed by ~20 copies of ssDNA (Figure 1, Figure 1—figure supplement 4). The majority of the vSUVs were immobile following docking to the t-SNARE-containing suspended bilayer (Figure 1, Figure 1—figure supplement 4), and they rarely fused over the initial observation period. In contrast, little or no inhibition was observed in control experiments with ~5 copies of ssDNA that did not introduce a detectable delay in the fusion process, as all docked vesicles proceeded to fuse spontaneously typically within 1–2 s (Figure 1, Figure 1—figure supplement 4). This suggests that it is the delay in fusion per se that is necessary for the mCPX inhibitory function, and importantly that the mCPX clamp is not dependent or influenced by the ssDNA molecules (Figure 1—figure supplement 4). Thus, our data indicates that Syt1 and mCPX likely act sequentially to produce a synergistic clamp, with the delay introduced by Syt1 meta-stable clamp enabling CPX to bind and block the full assembly of the SNARE complex.

Next, we investigated the role of the distinct domains of mCPX in establishing the fusion block using Syt1 containing vSUV (Syt1-vSUVs). On their own, a majority (~80%) Syt1-vSUVs that docked to the t-SNARE containing bilayer surface were mobile and fused on an average 5–6 s after docking, while a small fraction (~20%) were immobile and stably clamped (Figure 2A and B). Inclusion of 2 μM wild-type mCPX (mCPX^WT) enhanced the vesicle docking rate, with an ~ three-fold increase in the total number of stably docked vesicles and >95% of Syt1-vSUVs remaining immobile post-docking (Figure 2A and B). This is consistent with our earlier findings (Ramakrishnan et al., 2020). A truncation mutant (mCPX^26-134) lacking the unstructured N-terminal domain had very little or no effect on the vesicle docking rate or the fusion clamp, with vesicle behavior near identical to CPX^WT (Figure 2A and B). Deletion of the CPX_acc in addition to N-terminal domain (mCPX^48-134) increased the number of docked vesicles (~ two-fold) but abrogated the inhibitory function with majority of the docked vesicles proceeding to fuse spontaneously (Figure 2A and B). Targeted mutations in CPX_cen (R48A Y52A K69A Y70A; mCPX^4A) that disrupt its interaction with the SNAREpins completely abolished both the stimulatory effect on vesicle docking and the fusion clamp (Figure 2A and B). In fact, both the CPX_acc deletion (mCPX^48-134) and CPX_cen modifications (mCPX^4A) resulted in complete loss of mCPX inhibitory function and could not be rescued even at highest concentration (20 μM) tested (Figure 2C, Figure 2—figure supplement 1). Deletion of the c-terminal domain (mCPX^26-83) lowered the clamping efficiency (Figure 2A and B) with ~50% vesicles clamped under the standard experimental conditions (2 μM mCPX^26-83). However, the inhibitory function was rescued simply by raising the concentration and was completely restored at 20 μM mCPX^26-83 (Figure 2C, Figure 2—figure supplement 1).

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Data and summary statistics of docking and survival analysis for CPX mutants.

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Altogether, we conclude that the CPX_cen-SNAREpin interaction promotes vesicle docking, and this interaction along with CPX_acc are critical for mCPX mediated clamping under physiologically relevant experimental conditions. The c-terminal domain plays an auxiliary role and contributes to the mCPX inhibitory function likely by concentrating it on vesicle surfaces due to its curvature-binding region. Supporting this, a CPX mutant (CPX^L117W) that enhances the curved membrane association of the c-terminal domain (Seiler et al., 2009) increased the clamping efficiency as compared to CPX^WT (Figure 2—figure supplement 2).

Biophysical and structural studies have demonstrated that binding of the CPX_cen to the SNAREpins positions the CPX_acc to effectively block complete SNARE assembly (Kümmel et al., 2011; Giraudo et al., 2008; Krishnakumar et al., 2015). While the precise mode of action is under debate, there is evidence that this involves specific interactions of CPX_acc with the c-terminal region of the SNAREpins (Kümmel et al., 2011; Malsam et al., 2020). Critical information about these inter-molecular interactions was provided by the X-ray structure of mCPX bound to a mimetic of a pre-fusion half-zippered SNAREpins (Kümmel et al., 2011). It revealed that the CPX_cen is anchored to one SNARE complex, while its CPX_acc extends away and binds to the t-SNARE in a second SNARE complex in a site normally occupied by the C-terminus of the VAMP2 helix (Kümmel et al., 2011; Krishnakumar et al., 2015). This trans-insertion model suggest a straightforward mechanism by which CPX_acc can block the complete assembly of the SNARE complex (Kümmel et al., 2011; Krishnakumar et al., 2015).

To ascertain if the hydrophobic CPX_acc-t-SNARE binding interfaces observed in the crystal structure are involved in clamping in our in vitro system, we tested known CPX mutants designed to either enhance (D27L E34F R37A, ‘super-clamp’ mutant mCPX^SC) or weaken (A30E A31E L41 A44E, ‘non-clamp’ mutant 1 mCPX^NC1) this interaction (Giraudo et al., 2009; Kümmel et al., 2011). Survival analysis of Syt1-vSUVs showed that the binding interface mutants indeed alter the inhibitory activity of CPX as predicted (Figure 3A, Figure 3—figure supplement 1). The mCPX^NC1 abrogated the fusion clamp and was inactive even at higher (20 μM) concentration (Figure 3A, Figure 3—figure supplement 1). In contrast, mCPX^SC increased the clamping efficiency and produced stably docked vesicles at lower concentrations (IC₅₀ ~0.5 μM) compared to the mCPX^WT (IC₅₀ ~1 μM) (Figure 3B, Figure 3—figure supplement 1). These findings strongly support the notion that the CPX_acc-t-SNARE interactions observed in the pre-fusion mCPX-SNAREpin crystal is relevant for the CPX clamping function and is physiologically relevant.

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Data and summary statistics of survival analysis for ceCPX, dmCPX and mCPX mutants.

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Another key feature of the pre-fusion crystal structure is that the mCPX helix (CPX_cen +CPX_acc) forms a rigid bridge between two SNARE complexes (Kümmel et al., 2011; Krishnakumar et al., 2015). To test whether the rigidity of mCPX is important for clamping, we used a mCPX mutant (mCPX^GP) having a helix-breaking linker (GPGP) inserted between CPX_cen and CPX_acc. We found that disrupting the continuous helix indeed reduced the clamping efficiency (Figure 3A, Figure 3—figure supplement 1) indicating that the continuity and rigidity of the CPX helix is mechanistically important for its inhibitory function. This is also consistent with other previous studies (Chen et al., 2002; Xue et al., 2007; Cho et al., 2014; Radoff et al., 2014).

Recently, site-specific photo-crosslinking studies in a reconstituted fusion assay revealed that CPX_acc (of closely related mammalian isoform CPXII) binds to the c-terminal portions of SNAP25 and VAMP2 and both interactions are important for the mCPX inhibitory function (Malsam et al., 2020). The binding interface for SNAP25 was nearly identical to CPX_acc-t-SNARE interface observed in the crystal structure while the opposite side of the CPX_acc was found to interact with VAMP2 (Malsam et al., 2020). Note that this portion of VAMP2 was missing in the pre-fusion SNAREpin mimetic used for in the crystal structural analysis (Kümmel et al., 2011). To understand if the aforementioned CPX_acc-VAMP2 interaction is also part of the clamping mechanism in our cell-free system, we used a mCPX mutant (K33E R37E A40K A44E; non-clamp mutant 2, mCPX^NC2) that reverses the charge on key binding residues and is thus expected to disrupt this interaction (Malsam et al., 2020). mCPX^NC2 also failed to clamp spontaneous fusion of Syt1-vSUVs in our in vitro assay (Figure 3A, Figure 3—figure supplement 1) and was phenotypically analogous to the t-SNARE non-binding mutant (mCPX^NC1). This indicated the CPX_acc interacts with both t- and v-SNAREs to block full-zippering. As expected, because their central helix is unaltered, the majority of CPX_acc mutants tested retained the ability to promote vesicle docking process albeit lower than mCPX^WT (Figure 3—figure supplement 2).

CPX_cen is broadly conserved with ~75% amino acid sequence identity across diverse species, whereas CPX_acc is highly divergent with ~25% sequence identity (Figure 3—figure supplement 3). Nonetheless, cross-species rescue experiments have been largely successful, and in fact, CPX_acc could be exchanged without impairing function in mammalian, fly and nematode synapses (Xue et al., 2009; Cho et al., 2014; Radoff et al., 2014). This raises the question whether the distinct CPX_acc-SNARE interactions that are vital for mCPX inhibitory functionality in our in vitro assays are physiologically relevant. To address this, we examined the clamping ability of the C. elegans (ceCPX) and Drosophila (dmCPX) orthologs of mCPX in our in vitro reconstituted assay. Under standard experimental conditions (2 μM CPX), both ceCPX and dmCPX were able to promote vesicle docking (Figure 3—figure supplement 2) but were considerably less efficient (~15% and ~ 30%, respectively) in preventing spontaneous fusion of Syt1-vSUV (Figure 3C) as compared near-complete ( > 95%) fusion clamp observed with mCPX (Figure 2B and C). Interestingly, simply increasing the concentrations improved the clamping efficacy of both dmCPX and ceCPX, with ~60–70% of docked vesicles stably-clamped at 20 μM concentration (Figure 3C) and remained Ca²⁺-sensitive (Figure 3—figure supplement 4).

This suggests that specific molecular interactions of CPX_acc with SNAREs likely increase the potency of the mCPX inhibitory function and that this effect may be occluded at high concentrations of CPX. To verify this, we examined the effect of the mCPX mutant wherein the endogenous CPX_acc domain (residues 26–48) is replaced with an artificial alpha helix based on a Glu-Ala-Ala-Lys (EAAK) motif repeated seven times (Radoff et al., 2014). Noteworthy, this construct (mCPX^EAAK) was able to fully-restore CPX inhibitory functionality in C. elegans neuromuscular synapses (Radoff et al., 2014). In our in vitro assay, CPX^EAAK enhanced initial docking (Figure 3—figure supplement 2) but failed to clamp spontaneous fusion (~10% efficiency) under standard experimental conditions (2 μM CPX) and was moderately effective (~50% efficiency) at higher (20 μM CPX) concentration (Figure 3C, Figure 3—figure supplement 4). We note that the accessory helix of mCPX^EAAK is more hydrophobic in nature and interestingly resembles couple of the gain-of-function ‘super-clamp’ mutations with residue Asp-27 and Glu-34 replaced with Ala (Figure 3—figure supplement 3). This could potentially explain mCPX^EAAK ability to partially clamp vesicle fusion at high (20 μM) CPX concentration. Overall, our data supports the notion the specific CPX_acc-SNARE interaction is functionally relevant and likely enhances CPX inhibitory function.

Finally, we evaluated the probability and rate of Ca²⁺-triggered fusion from the clamped state in the presence and absence of mCPX. We used Syt1-vSUV loaded with Sulforhodamine B (fluorescent content marker) to track full-fusion events and lipid-conjugated Ca²⁺ indicator (Calcium green C24) attached to the planar bilayer to estimate the time of arrival of Ca²⁺ at/near the docked vesicles (Figure 4A). Consistent with our previous study, the influx of free Ca²⁺ (100 μM) triggered simultaneous fusion of >90% of the Syt1/mCPX-clamped vesicles (Figure 4B). These vesicles fused rapidly and synchronously, with a characteristic time-constant (τ) of ~11 msec following the arrival of Ca²⁺ locally (Figure 4C). Considering that the majority of Ca²⁺-triggered fusion occurs within a single frame (13 ms), we suspect that the true Ca²⁺-driven fusion rate is likely <10 ms.

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Data and summary statistics of effect of mCPX mutants on calcium activation of fusion.

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In absence of mCPX, we observed a relatively small number of docked vesicles prior to Ca²⁺ influx and this precluded any meaningful quantitative analysis. Hence, to obtain stably docked vesicles without mCPX, we used low VAMP2 conditions that is, SUVs containing ~13 copies of VAMP2 and ~25 copies of Syt1 (Figure 1—figure supplement 1). We have previously demonstrated that under these conditions, Syt1 alone is sufficient to produce stably-clamped vesicles (Ramakrishnan et al., 2019) and that is what we observe, with >95% of docked vesicles immobile post-docking. Addition of Ca²⁺ (100 μM) triggered rapid and synchronous fusion of ~70% of these Syt1-clamped vesicles (with τ ~ 11 msec) as compared to >90% fusion of Syt1/mCPX-clamped vesicles (Figure 4B and C). Besides mCPX, the number of SNAREpins available on a given vesicle is also different between the two conditions (~13 VAMP2 in Syt1-alone vs. ~70 VAMP2 in Syt1/CPX). Hence, to verify that the observed effect is directly attributable to mCPX, we tested and confirmed that the inclusion of mCPX under low VAMP2 conditions increased the Ca²⁺-triggered fusion probability (~90%) from the clamped state (Figure 4B). This indicated that besides clamping vesicle fusion, mCPX also promotes Ca²⁺-triggered vesicle fusion from the clamped state.

To identify the molecular aspects underlying mCPX stimulatory function, we examined the effect of mCPX mutants on Ca²⁺-triggered release under low VAMP2 conditions (Figure 4D). Deletion of the N-terminal alone (mCPX^26-134) or the N- and C-terminal domains (mCPX^26-83) had very little or no effect on the mCPX stimulatory function (Figure 4D). However, deletion of the CPX_acc in addition to N-terminal domain (mCPX^48-134) or disrupting the CPX_cen-SNARE interaction (mCPX^4A) abrogated the mCPX activation function (Figure 4D) suggesting that the CPX_cen and CPX_acc domains are crucial for mCPX’s stimulatory function. In contrast to their clamping function, disrupting the rigidity and continuity of the CPX_cen-CPX_acc helix with the GPGP insert (mCPX^GP) had no effect on the activation function (Figure 4D). Furthermore, the mCPX mutant with a randomized CPX_acc (CPX^EAAK) and the C. elegans ortholog (ceCPX), both of which lack the clamping functionality under the experimental conditions (Figure 3), retained the ability to promote Ca²⁺-triggered fusion of the docked vesicles (Figure 4D). Taken together, our data suggest that specific interactions of CPX_cen with SNAREpins are required for the mCPX stimulatory function and the CPX_acc can act independently of CPX_cen via a mechanism different from that involved in clamping vesicle fusion.

Our data indicates the mCPX is critical to produce the ‘clamped’ state and also contribute towards synchronizing fusion to Ca²⁺ influx. In addition, we find that the stimulatory and clamping functionality of mCPX are mechanistically separable. There is a long-standing debate over the role of CPX in establishing a fusion clamp and perhaps the best evidence in support has come from biochemical analyses (Giraudo et al., 2006; Giraudo et al., 2008; Kümmel et al., 2011; Lai et al., 2014) and physiological studies in invertebrate synapses (Huntwork and Littleton, 2007; Cho et al., 2014; Martin et al., 2011; Hobson et al., 2011). In the case of mammalian synapses, a role for CPX in blocking spontaneous release events remains controversial because KD/KO manipulations yield seemingly contradictory results and show neuron-specific differences (Xue et al., 2008; López-Murcia et al., 2019; Maximov et al., 2009; Yang et al., 2013). Here, using a fully defined albeit simplified cell-free system we provide compelling evidence that mCPX is an integral part of the overall clamping mechanism and delineate the molecular mechanism of mCPX inhibitory function. The distinct effects of different CPX truncation and targeted mutations match with data obtained from other reductionist or even physiological systems (Giraudo et al., 2006; Giraudo et al., 2008; Kümmel et al., 2011; Cho et al., 2014; Lai et al., 2014; Gong et al., 2016) forcefully arguing for the physiological relevance of results obtained from our in vitro reconstituted assay.

Our experiments indicate that mCPX inhibitory function entails distinct and specific interactions of the CPX_cen and CPX_acc domains with assembling SNAREpins, and that the c-terminal domain augments clamping function by increasing the local concentration and/or by proper orientation of CPX via interactions with the vesicle membrane (Figure 2). Our results indicate that CPX_cen binds in the groove between assembling Syntaxin and VAMP2 helices at the early stages of vesicle docking to stabilize the partially-zippered SNAREpins, consequently promote vesicle docking. This in turn positions CPX_acc to block further zippering of SNARE complex both by directly capturing the VAMP2 c-terminus and by simultaneously occupying its binding pocket on the t-SNARE. In line with earlier reports (Chen et al., 2002; Xue et al., 2007; Radoff et al., 2014; Cho et al., 2014; Kümmel et al., 2011), we find that a continuous, rigid CPX helix is essential for a stable fusion clamp. However, the precise configuration of this clamped state under the docked vesicles has been unclear. This is in large part due to the observed variability in the positioning of the CPX_acc (Choi et al., 2016; Malsam et al., 2020; Zhou et al., 2017; Kümmel et al., 2011). CPX_acc has been proposed to interact with c-terminal portion of the t-SNARE and VAMP2, both in a cis configuration that is CPX_cen and CPX_acc bound to the same SNAREpin (Choi et al., 2016; Malsam et al., 2020) or in a trans configuration that is CPX_cen and CPX_acc interacting with neighboring SNAREpins (Choi et al., 2016; Kümmel et al., 2011; Krishnakumar et al., 2015; Cho et al., 2014). We favor the trans insertion clamping model as this arrangement would enable CPX to regulate the distinct central and peripheral SNAREpin populations (Figure 5, see below).

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Noteworthy, we observe that the specific interactions of the CPX_acc with the synaptic SNARE proteins increase the potency of the clamp, and in accordance mCPX is ~2–3 fold more efficient in establishing the fusion clamp as compared to dmCPX or ceCPX under the same experimental conditions (Figure 3). However, the divergence in clamping ability among the mammalian, fly, and nematode CPXs is diminished at higher concentrations of CPX. This might explain the puzzling observation that in physiological analyses, when CPX is over-expressed, cross-species rescue experiments are largely successful yet CPX_acc-SNARE disrupting mutants’ exhibit limited effect on the CPX clamping ability (Yang et al., 2010; Cho et al., 2014; Radoff et al., 2014). Considering that the CPX_acc is highly divergent across different species, it is conceivable that CPX_acc has distinctively evolved to optimally bind and clamp the species-specific SNARE partners. Additional biochemical/structural studies are needed to address this question.

Overall, our data strongly argues that mCPX has an intrinsic capacity to inhibit SNARE-dependent fusion and under minimal conditions is required (along with Syt1) to generate and maintain a pool of release-ready vesicles. Indeed, functionality of mCPX observed in our in vitro system perfectly matches with physiological studies in model invertebrate systems (Martin et al., 2011; Hobson et al., 2011; Huntwork and Littleton, 2007; Cho et al., 2014). However, recent physiological studies in mammalian synapses reported that acute CPX loss reduces SV fusion probability but does not unclamp spontaneous fusion. Hence, they conclude that CPX is dispensable for ‘fusion clamping’ in mammalian neurons (López-Murcia et al., 2019). It is worth noting that under these conditions CPX removal abates both spontaneous and evoked neurotransmitter release without changing the number of docked vesicles (López-Murcia et al., 2019). This suggests that acute CPX loss likely affects the late-stage vesicle priming process, and it is possible this ‘loss-of-fusion’ phenotype occludes CPX role in regulating spontaneous fusion events. Indeed, rescue experiments in CPX1/2/3 triple-knockout mouse background show that the CPX_acc mutants enhances the spontaneous fusion events without altering evoked release, revealing that mCPX has a strong suppressive clamping function (Malsam et al., 2020). It is feasible mCPX also plays a more specialized role in mammalian synapses and is primarily involved in stabilizing newly primed synaptic vesicles and prevents their premature fusion (Dhara et al., 2014; Chang et al., 2015). In doing so, mCPX may function as a fusion clamp in an activity-dependent manner and is critical to blocking spontaneous/tonic and asynchronous vesicular release (Dhara et al., 2014; Chang et al., 2015; Yang et al., 2010) and indirectly promoting synchronous SV exocytosis.

mCPX on its own is ineffective in clamping SNARE-driven vesicle fusion, as the c-terminal portion of VAMP2 assembles into the SNARE complex far faster than free CPX can bind to prevent its zippering (Gao et al., 2012). As such, a delay in SNARE zippering is required for the CPX to bind and thereby block fusion. The fact that sufficient delay can be artificially provided by ~20 copies of DNA duplexes (Figure 1) suggest that under physiological conditions, Syt1 (and perhaps other proteins on the SV) might hinder the SNARE assembly by a simple steric mechanism, enabling mCPX to function as a fusion clamp. This is supported by the observation that the Syt1 clamp or the formation of the central SNAREpins are not strictly required for mCPX clamping function (Ramakrishnan et al., 2020).

Ca²⁺-activation studies (Figure 4) show that mCPX also contributes to Ca²⁺-triggered vesicle fusion from the clamped state. Reinforcing our earlier reports (Ramakrishnan et al., 2019; Ramakrishnan et al., 2020), we find that Syt1 and a small number of SNAREs are largely sufficient to get Ca²⁺-evoked fusion with ~70% of vesicles fusing in response to 100 μM Ca²⁺. Inclusion of mCPX increases the fusion probability with >90% Ca²⁺-triggered fusion from the clamped state (Figure 4). We do not observe any change in the fusion kinetics (τ~11ms) without or with mCPX (Figure 4), at least with our current time resolution of ~13 ms and persistent high Ca²⁺ levels as opposed to Ca²⁺-transients in the synapse.

Deletion/mutational analyses reveal that the α-helical CPX_cen and CPX_acc are the minimal domain required for the activation function (Figure 4). Specifically, the well-defined CPX_cen-SNARE interactions (Chen et al., 2002) was found to be critical for the stimulatory function and this effect is observed even low VAMP2 conditions that is with vesicles containing Syt1-clamped central SNAREpins only (Figure 4). This is in line with our previous finding that CPX_cen interaction with the SNAREs, independent of the clamping functionality, is important for Ca²⁺-evoked release in Drosophila neuromuscular junctions (Cho et al., 2014). Our data shows that CPX_acc also contributes to the activation function, but the underlying mechanism is unclear. CPX_acc could act indirectly by promoting CPX_cen binding (Radoff et al., 2014) or directly by interacting with the SNARE complex albeit in a manner different from the clamping interactions.

The data presented here, taken together with our earlier report (Ramakrishnan et al., 2020), suggests a parsimonious model of how Syt1 and CPX could regulate SNARE-mediated fusion (Figure 5, Figure 5—figure supplement 1, Figure 5—figure supplement 2). We posit that under every docked vesicle, there are two types of SNAREpins – the central SNAREpins which are bound to Syt1 and are responsible for Ca²⁺-triggered release and peripheral SNAREpins which are not bound to Syt1 and thus, not directly regulated by Ca²⁺. We further suggest that the central and peripheral SNAREpins are equal in number and are assembled as a pair via a common, bridging molecule of CPX (Figure 5A). At the early stages of SV docking, Syt1 oligomers bind and clamp sub-set of central SNAREpins via the ‘primary’ interface (Ramakrishnan et al., 2020). CPX bind the Syt1-associated central SNAREpins via the CPX_cen which positions the CPX_acc helix to bind the t-SNAREs an oppositely-oriented SNAREpin occupying the space where the C-terminal half of VAMP2 would ordinarily zipper to drive fusion. In this way, CPX_acc acts to clamp the peripheral SNAREpin. This ‘bridging model’ (Figure 5, Figure 5—figure supplement 1) is based on the known ‘trans-clamping’ interaction observed in the pre-fusion CPX/SNAREpin crystal structure (Kümmel et al., 2011) and is validated by biochemical and functional analyses both previously (Cho et al., 2014; Krishnakumar et al., 2015; Krishnakumar et al., 2011; Kümmel et al., 2011) and in the current work. In addition, CPX_acc might also directly interact with the peripheral VAMP2 c-terminus to prevent its assembly (not shown in Figure 5).

As evidenced in the recent crystal structure (Zhou et al., 2017), CPX_cen binding to the central SNAREpins likely creates a new binding interface for second Syt1 to bind the same SNAREpins. Thus, mCPX could regulate Ca²⁺ triggered vesicle fusion via the ‘tripartite’ interface (Figure 5, Figure 5—figure supplement 1, Figure 5—figure supplement 2). Supporting this proposition, we have previously shown the ‘tripartite’ interface is not necessary to produce stably docked vesicles but is required for efficient Ca²⁺-triggered fusion from the clamped state (Ramakrishnan et al., 2020). In fact, disrupting binding of Syt1 to the tripartite interface lowers the fusion probability (~25%) similar to that observed with the removal of mCPX (Ramakrishnan et al., 2020). Furthermore, as the tripartite binding motif is largely conserved among different Synaptotagmin isoforms, so it is possible that mCPX binding could enable synergistically regulation of vesicular release by different calcium sensors (Volynski and Krishnakumar, 2018; Zhou et al., 2017). In addition to creating the ‘tripartite’ interface, mCPX binding might also promote vesicle fusion by stabilizing the full zippering SNARE complex. Obviously, this model is highly speculative and further functional studies (with higher temporal resolution, physiological Ca²⁺ dynamics and different calcium sensors) as well as high-resolution structural data of vesicle-membrane junctions are needed to dissect the precise role of mCPX and its synergistic action with Syt1 in regulating Ca²⁺-triggered vesicular fusion from the clamped state.

All data generated or analyzed during this study are included in the manuscript and supporting files. Source data files for Figures 1, 2, 3, 4, and gel blots associated supplements are provided.

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Author details

1. Manindra Bera

   1. Yale Nanobiology Institute, New Haven, United States
   2. Department of Cell Biology, Yale University School of Medicine, New Haven, United States

   Contribution

   Conceptualization, Formal analysis, Investigation, Methodology, Writing – original draft, Writing – review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0001-9297-8126

2. Sathish Ramakrishnan

   1. Yale Nanobiology Institute, New Haven, United States
   2. Department of Pathology, Yale University School of Medicine, New Haven, United States

   Contribution

   Conceptualization, Formal analysis, Investigation

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-7844-2234

3. Jeff Coleman

   1. Yale Nanobiology Institute, New Haven, United States
   2. Department of Cell Biology, Yale University School of Medicine, New Haven, United States

   Contribution

   Methodology, Resources

   Competing interests

   No competing interests declared

4. Shyam S Krishnakumar

   1. Yale Nanobiology Institute, New Haven, United States
   2. Departments of Neurology, Yale University School of Medicine, New Haven, United States

   Contribution

   Conceptualization, Formal analysis, Investigation, Supervision, Writing – original draft, Writing – review and editing

   For correspondence

   shyam.krishnakumar@yale.edu

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0001-6148-3251

5. James E Rothman

   1. Yale Nanobiology Institute, New Haven, United States
   2. Department of Cell Biology, Yale University School of Medicine, New Haven, United States

   Contribution

   Conceptualization, Investigation, Supervision, Writing – review and editing

   For correspondence

   james.rothman@yale.edu

   Competing interests

   No competing interests declared

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