Author response: All-atom molecular dynamics simulations of Synaptotagmin-SNARE-complexin complexes bridging a vesicle and a flat lipid bilayer
Year: 2022
Type: peer-review
Abstract: Article Figures and data Abstract Editor's evaluation Introduction Results Discussion Methods Data availability References Decision letter Author response Article and author information Metrics Abstract Synaptic vesicles are primed into a state that is ready for fast neurotransmitter release upon Ca2+-binding to Synaptotagmin-1. This state likely includes trans-SNARE complexes between the vesicle and plasma membranes that are bound to Synaptotagmin-1 and complexins. However, the nature of this state and the steps leading to membrane fusion are unclear, in part because of the difficulty of studying this dynamic process experimentally. To shed light into these questions, we performed all-atom molecular dynamics simulations of systems containing trans-SNARE complexes between two flat bilayers or a vesicle and a flat bilayer with or without fragments of Synaptotagmin-1 and/or complexin-1. Our results need to be interpreted with caution because of the limited simulation times and the absence of key components, but suggest mechanistic features that may control release and help visualize potential states of the primed Synaptotagmin-1-SNARE-complexin-1 complex. The simulations suggest that SNAREs alone induce formation of extended membrane-membrane contact interfaces that may fuse slowly, and that the primed state contains macromolecular assemblies of trans-SNARE complexes bound to the Synaptotagmin-1 C2B domain and complexin-1 in a spring-loaded configuration that prevents premature membrane merger and formation of extended interfaces, but keeps the system ready for fast fusion upon Ca2+ influx. Editor's evaluation Using all-atom molecular dynamics simulations to visualize the pre-fusion primed state during synaptic vesicle fusion is very original and this approach will certainly be used by others in the future. This work provides new insights into the protein organization prior to vesicle fusion that will help better understand the mechanisms of vesicle priming and evoked-release. https://doi.org/10.7554/eLife.76356.sa0 Decision letter Reviews on Sciety eLife's review process Introduction The release of neurotransmitters by Ca2+-triggered synaptic vesicle exocytosis is key for communication between neurons. The high speed of this process arises in part because synaptic vesicles are first tethered at the plasma membrane and undergo priming processes that leave them ready for fast fusion when an action potential induces Ca2+ influx (Südhof, 2013). Research for over three decades has led to extensive characterization of the neurotransmitter release machinery (Brunger et al., 2018; Rizo, 2022), allowing reconstitution of basic features of synaptic vesicle fusion with the central components of this machinery (Lai et al., 2017; Liu et al., 2016; Ma et al., 2013) and uncovering key aspects of the underlying mechanisms. The SNARE proteins syntaxin-1, SNAP-25 and synaptobrevin form a tight four-helix bundle called the SNARE complex that assembles (zippers) from the N- to the C-terminus to bring the vesicle and plasma membranes together, and is key for membrane fusion (Hanson et al., 1997b; Poirier et al., 1998; Söllner et al., 1993; Sutton et al., 1998). This complex is disassembled by NSF and SNAPs (no relation to SNAP-25) to recycle the SNAREs for another round of fusion (Mayer et al., 1996; Söllner et al., 1993). Munc18-1 and Munc13-1 play central roles in synaptic vesicle priming by organizing assembly of the SNARE complex via an NSF-SNAP-resistant pathway (Ma et al., 2013; Prinslow et al., 2019) whereby Munc18-1 first binds to a self-inhibited ‘closed’ conformation of syntaxin-1 (Dulubova et al., 1999; Misura et al., 2000). Munc13-1 later opens syntaxin-1 (Ma et al., 2011; Yang et al., 2015) while bridging the vesicle and plasma membranes (Quade et al., 2019; Xu et al., 2017), and Munc18-1 forms a template for SNARE complex assembly (Baker et al., 2015; Jiao et al., 2018; Parisotto et al., 2014; Sitarska et al., 2017). Synaptotagmin-1 (Syt1) acts as the Ca2+ sensor that triggers fast release (Fernández-Chacón et al., 2001) in a tight interplay with complexins whereby both Syt1 and complexins play inhibitory and active roles (Giraudo et al., 2006; Reim et al., 2001; Schaub et al., 2006; Tang et al., 2006). The Ca2+ sensor function is performed by the two C2 domains of Syt1 (C2A and C2B), which form most of its cytoplasmic region and bind multiple Ca2+ ions via loops at the tip of β-sandwich structures (Fernandez et al., 2001; Sutton et al., 1995; Ubach et al., 1998; Figure 1—figure supplement 1A). These loops also mediate Ca2+-dependent binding of both C2 domains to phospholipids, which is critical for release (Fernández-Chacón et al., 2001; Rhee et al., 2005). The C2B domain also binds to PIP2 through a polybasic region on the side of the β-sandwich (Bai et al., 2003), which induces binding to the plasma membrane. Moreover, the C2B domain can bind to the SNARE complex through three different surfaces (Brewer et al., 2015; Zhou et al., 2015; Zhou et al., 2017), although only binding through a so-called primary interface (Zhou et al., 2015) is firmly established as physiologically relevant (Guan et al., 2017; Voleti et al., 2020). Such binding is disrupted upon Ca2+-dependent binding of Syt1 to PIP2-containing membranes (Voleti et al., 2020). Complexin-1 binds tightly to the SNARE complex through a central α-helix that is preceded by an accessory helix (Chen et al., 2002) and may play a stimulatory role in release by promoting formation of a primed state with enhanced release probability (Chen et al., 2002), by protecting trans-SNARE complexes from disassembly by NSF and αSNAP (Prinslow et al., 2019) and/or by synchronizing Ca2+-triggered fusion mediated by the SNAREs and Syt1 (Diao et al., 2012). The complexin-1 accessory helix inhibits release (Xue et al., 2007), possibly because it causes steric clashes with the vesicle, hindering C-terminal assembly of the SNARE complex (Radoff et al., 2014; Trimbuch et al., 2014). These and other findings suggested that complexin-1 and Syt1 bind simultaneously to the SNARE complex in the primed state of synaptic vesicles, stabilizing this state and preventing premature fusion; in this model, Ca2+ influx relieves the inhibition by inducing dissociation of Syt1 from the SNAREs and enabling cooperation between Syt1 and the SNAREs in promoting fusion (Voleti et al., 2020). Despite this wealth of knowledge, fundamental questions remain about the mechanism of neurotransmitter release, particularly regarding how the SNAREs and Syt1 trigger fast, Ca2+-dependent membrane fusion. Major hurdles to address this question are the dynamic nature of this process and the fact that the protein complexes that trigger fusion are assembled between two membranes. Although important clues on the nature of the primed macromolecular assembly have been obtained with structural studies of soluble proteins or complexes anchored on one membrane (Chen et al., 2002; Grushin et al., 2019; Voleti et al., 2020; Zhou et al., 2015), this assembly is most likely affected by its location between two membranes. This feature strongly hinders the possibility of crystallization, while application of NMR spectroscopy for structure elucidation is hampered by the large size of any reconstituted two-membrane system (Voleti et al., 2021). Conversely, the small size of the SNAREs, Syt1 and complexin-1 hinders visualization by cryo-EM. Moreover, it is extremely challenging to capture transient states formed during the pathway to Ca2+-triggered membrane fusion experimentally. Molecular dynamics (MD) simulations offer a powerful tool to analyze dynamic biomolecular processes and model cellular membranes (Marrink et al., 2019). Simulations using continuum and/or coarse-grained representations have provided important insights into SNARE-mediated membrane fusion (Fortoul et al., 2015; Kasson et al., 2006; Manca et al., 2019; McDargh et al., 2018; Mostafavi et al., 2017; Risselada et al., 2011; Sharma and Lindau, 2018). Continuum models can access the longest timescales, but require experimental data or atomistic simulations to parameterize the material properties, and often need to constrain geometries or material properties due to lack of context-dependent parameters (Fortoul et al., 2015). Coarse-grained molecular simulation approaches are freed from some of these constraints but at the expense of reduced simulation speed, and are limited in their ability to capture certain entropic effects and protein conformational changes (see below). To date, coarse-grained models of SNARE-mediated fusion have accessed the low microsecond timescale (Kasson et al., 2006; Risselada et al., 2011; Sharma and Lindau, 2018). All-atom simulations are better suited to reproduce the finely-balanced network of interactions between proteins, Ca2+, and lipids that are expected to lead to membrane fusion but, because of the large size of the systems involved (millions of atoms), the low microsecond time scale has only recently become accessible through the most powerful available high-performance computing resources. In this context, it is worth noting that the delay from Ca2+ influx into the presynaptic terminal to observation of postsynaptic currents in rat cerebellar synapses at 38 °C is 60 μs (Sabatini and Regehr, 1996), and that multiple events occur within this time frame, including Ca2+ binding to the sensor, release of inhibitory interactions that hinder premature fusion, Ca2+-evoked synaptic vesicle fusion, opening of the fusion pore, diffusion of neurotransmitters through the synaptic cleft, binding of the neurotransmitters to their postsynaptic receptors and opening of the channels that underlie the postsynaptic currents. These observations suggest that the fusion step may occur in just a few microseconds and hence that it may be possible to recapitulate the initiation of Ca2+-dependent synaptic vesicle fusions in all-atom MD simulations starting with a properly designed initial configuration. Here, we present all-atom MD simulations with explicit water molecules of systems containing four trans-SNARE complexes bridging two flat bilayers or a vesicle and a flat bilayer, without or with fragments of Syt1 and/or complexin-1. Because of the limited simulation times and the absence of key components, our results cannot lead to definitive conclusions but they help visualize potential trajectories and intermediates along the pathway to fusion and reveal intriguing features, leading to predictions or hypotheses that can be tested experimentally and with additional simulations. Our data indicate that trans-SNARE complexes strongly pull two membranes together, as expected, but have a tendency to induce extended membrane-membrane adhesion interfaces that have been observed experimentally but fuse slowly (Hernandez et al., 2012; Witkowska et al., 2021). Our results also suggest that, in the primed state of synaptic vesicles, Syt1 and complexin-1 form a spring-loaded macromolecular assembly with trans-SNARE complexes that hinders formation of such extended contact interfactes and premature bilayer merger, but is ready for fast membrane fusion upon Ca2+ influx. Results Four trans-SNARE complexes between two flat lipid bilayers The possibility of observing membrane fusion in the low microsecond time scale in all-atom MD simulations depends critically on the choice of the starting configuration, but the exact nature of the primed state of synaptic vesicles is unknown. Hence, we used the structural and functional information available on this system to generate potential starting configurations. The MD simulations presented here involved systems ranging from 1.7 to 5.9 million atoms. While multiple replicas of each simulation should ideally be carried out to verify the consistency of the results, performing replicated simulations would have limited the number of systems that we could study. Moreover, each simulation included four-trans SNARE complexes bridging two lipid bilayers and the variability in the behavior of the complexes in each simulation already provided insights into the consistency of the observed behaviors. Hence, we chose to use the available high performance computing time to investigate systems with different components and/or distinct geometry, designing each new starting configuration according to what we had learned from the previous simulations. The simulations generated a large amount of data and it is impossible to describe a thorough analysis within the constraints of a single paper. Here, we present the main observations from the analyses that we have performed, and key files from the simulations are available in Dryad for further analyses. In all the systems that we built, the composition of the bilayer containing anchored synaptobrevin approximated the lipid composition of synaptic vesicles (Takamori et al., 2006) and that of the bilayer with anchored syntaxin-1 was based on the lipid composition of the plasma membrane (Chan et al., 2012; Table 1). Both bilayers had asymmetric lipid distributions in the two leaflets to mimic those present in vivo (Kobayashi and Menon, 2018). The first system that we built was designed to examine whether SNARE complexes alone can bend two flat lipid bilayers and initiate bilayer fusion. The system contained four trans-SNARE complexes between two square lipid bilayers. The number of SNARE complexes was based on symmetry considerations and the finding that fast vesicle fusion typically observed in synapses requires at least three SNARE complexes (Mohrmann et al., 2010). For simplicity, the SNARE complexes contained the four SNARE motifs, the transmembrane (TM) sequences of syntaxin-1 and synaptobrevin, and the juxtamembrane linkers between their respective SNARE motif and TM region, but did not include the syntaxin-1 N-terminal region or the long linker between the two SNAP-25 SNARE motifs. Table 1 Size in atoms, length of productions MD simulations, temperature, speed of the simulations on Frontera at TACC and lipid composition of the flat bilayers and the vesicle of the different systems. qscff1699436 atoms750 ns310 K24 ns/day with 16 nodesUpper bilayerCHL1POPCPOPSSAPESDPESDPStotalUpper leaflet54046807212001200%453906100Lower leaflet54014684120240961226%4411.96.99.819.67.8Lower bilayerCHL1POPCPOPSSAPI2DSAPESDPESDPStotalUpper leaflet54013412060841681201226%44119.84.96.913.79.8Lower leaflet54053800488401200%454400470sqscff1700475 atoms270 ns310 K 24 ns/day with 16 nodesqscv3222393 atoms520 ns310 K23 ns/day with 32 nodes454 ns325 KVesicleCHL1POPCSAGLSAPESAPI2DSDPESDPSSOPStotalOuter leaflet1258296053402822101992779%45.310.6019.2010.17.67.2Inner leaflet8146680183099111766%46.137.8010.405.60.10.1Flat bilayerCHL1POPCSAGLSAPESAPI2DSDPESDPSSOPStotalUpper leaflet540961284601561201201188%45.48.117.15.113.110.110.1Lower leaflet5405161236084001188%45.443.41307.100prsg5056443 atoms336 ns310 K13 ns/day with 32 nodesFlat bilayerCHL1POPCSAGLSAPESAPI2DSDPESDPSSOPStotalUpper leaflet83015118132932621821811849%44.98.217.1514.29.89.8Lower leaflet81077418720126001800%4543140700prs25870280 atoms310 ns310 K16 ns/day 48 nodesFlat bilayerCHL1POPCSAGLSAPESAPI2DSDPESDPSSOPStotalUpper leaflet1035184231611153222302302300%458175141010Lower leaflet100996422880154002237%45.143.113.906.900prsncpxca5870246 atoms439 ns310 K16 ns/day 48 nodes Vesicle: same as qscv system. Flat bilayer: same as prs2 system. A key aspect in the design of realistic potential states of trans-SNARE complexes is the conformation of the juxtamembrane linkers of syntaxin-1 and synaptobrevin. Popular models of SNARE-mediated membrane fusion depicted continuous helices spanning the SNARE motifs, juxtamembrane linkers and TM regions for both synaptobrevin and syntaxin-1, envisioning that these helices can bend to accommodate the geometry of trans-SNARE complexes (Hanson et al., 1997a; Sutton et al., 1998; Weber et al., 1998). These models were supported by coarse-grained MD simulations that used the MARTINI force field and modeled the SNAREs in continuous helical conformations (Risselada et al., 2011). However, the intrinsic helical restraints enforced by the force field might bias the results and/or obscure the potential role of conformational changes in the dynamical coupling of the SNAREs to membrane fusion. Moreover, the bending of the helices required to form trans-SNARE complexes leads to unrealistic conformations that are expected to be unfavorable energetically because of their distorted geometry and are not commonly observed in protein structures. Thus, the helical restraints might have played a key role in membrane fusion in these simulations. Although continuous helices were observed in the crystal structure of a cis-SNARE complex that represents the configuration occurring after membrane fusion (Stein et al., 2009), the natural expectation is that the helical structure must break somewhere to accommodate the geometry of a trans-SNARE complex, most likely at the juxtamembrane linker. This expectation has been supported experimentally (Kim et al., 2002) and with all-atom MD simulations (Bykhovskaia, 2021). Moreover, helix continuity in the linkers is not required for neurotransmitter release (Kesavan et al., 2007; Zhou et al., 2013). Thus, to generate trans-SNARE complexes for our simulations we started with the crystal structure of the cis-SNARE complex but we did not impose restraints on the conformation of the juxtamembrane linkers. Since the N-terminal half of the SNARE four-helix bundle is more stable than the C-terminal half (Chen et al., 2002; Gao et al., 2012) and is more distal from the membrane, we imposed position restraints for only the N-terminal half. In addition, we used position restraints to force the TM regions of synaptobrevin and syntaxin-1 to designed locations for insertion in their corresponding bilayers. A short (1 ns) restrained MD simulation in water was sufficient for this purpose and led to unstructured conformations for the juxtamembrane linkers without substantially altering the four-helix bundle even at the C-terminal half, which was not restrained (Figure 1—figure supplement 1B). Four copies of the resulting trans-SNARE complex were generated by translations and rotations (Figure 1—figure supplement 1C), and were merged with the two bilayers separated by 5 nm to generate the initial configuration of this system (Figure 1A). We then carried out an unrestrained production simulation of this system for 750 ns at 310 K. As expected, the two membranes became almost circular to minimize tension and were gradually drawn together by the SNAREs, although the minimal distance between the bilayers reached a plateau (Figure 1—figure supplement 1D, E). The two bilayers were actually drawn to each other on one side first (at about 110 ns, Figure 1B) and later on the other side (Figure 1C), leading to close packing of the lipids against the SNARE four-helix bundles (Figure 1D). It is noteworthy that the four SNARE complexes were zippered at the C-terminus to the same extent as in the initial configuration and that extensive interactions were established between the juxtamembrane linkers and the membranes during the simulations. Such interactions were not unexpected, as both linkers contain abundant basic residues, the synaptobrevin linker in addition contains hydrophobic residues, and both linkers were shown to interact with the adjacent membrane (Brewer et al., 2011; Kim et al., 2002). Since much of the SNARE four-helix bundle is negatively charged, these findings suggest that any electrostatic repulsion existing between the SNARE four-helix bundle and the membranes can be readily overcome by the high stability of the SNARE four-helix bundle and perhaps some contribution from the linker-bilayer interactions. During the 750 ns of the simulation we observed of the syntaxin-1 membrane, but the was and was fusion. These findings suggest that four trans-SNARE complexes are to fuse two flat bilayers in the 1 μs time Figure 1 with 1 supplement all MD simulations of four trans-SNARE complexes bridging two flat bilayers. configuration of the system with SNARE complexes only and of the MD simulation after 110 and ns The SNARE complexes are by in The lipids are shown as The scale in which is a than the length of the SNARE four-helix of the same MD simulation at ns all as configuration of the system containing four Syt1 molecules in addition to the four trans-SNARE complexes and of the simulation at ns SNARE complexes are by in and the molecules are shown as with in and C2B in The lipids are shown as The for the lipids Ca2+ ions are shown as To whether Syt1 might with the SNAREs in bending two flat bilayers to initiate membrane fusion, we performed another simulation with an system we included a spanning the two C2 domains of Syt1 bound to Ca2+ ions (Fernandez et al., 2001; Ubach et al., 1998; Figure During a ns production MD simulation of this we observed that the molecules the action of the trans-SNARE complexes in the two bilayers (Figure 1—figure supplement particularly when the C2 domains bind to one bilayer through the Ca2+-binding loops and to the other bilayer via the side of the which is basic (Figure Although such bridging might help in fusion et al., 2006) in a different configuration, it that such potential action would require a much time scale in this system and we did not this Four trans-SNARE complexes bridging a vesicle and a flat bilayer on the results from the simulations with four trans-SNARE complexes between two flat we that, synaptic vesicle fusion in the low microsecond time this speed might require the geometry occurring at small synaptic vesicles nm fuse with the plasma membrane. To this we built a system with four trans-SNARE complexes (Figure supplement bridging a vesicle and a flat bilayer (Figure The initial of the vesicle was as a between the system realistic and the size of the system to the time required for MD simulations. The vesicle was in molecular contact with the flat bilayer that the system was for fusion. Since the lipid of the vesicle was close to but not in an initial production MD The were in an process the vesicle was stable (see During this the flat bilayer became circular and the vesicle became nm (Figure supplement but the stable in production Figure with 1 supplement all MD simulation of four trans-SNARE complexes bridging a vesicle and a flat of the initial system. of one of the trans-SNARE complexes in the initial system. of the system after a ns MD simulation at 310 K and a ns simulation at K. of the four trans-SNARE complexes after the ns MD simulation at 310 K and the ns simulation at K. In the SNARE complexes are by in The lipids are shown as models of the system in its initial configuration after the steps after ns at 310 K and after ns at 310 K and ns at K In of and the of molecules are shown as to the locations of lipid the system we performed a production of ns at 310 K. Although we observed of were of the bilayers that might the initiation of fusion. We the to K and carried out a production of ns in an to fusion, but observed The configuration that the vesicle to some extent to one side with to the flat bilayer during the simulations (Figure The four-helix bundles of the four trans-SNARE complexes assembled to the hydrophobic to as et al., at the of the simulations (Figure as in the initial configuration of the system (Figure of the SNARE complexes became to the flat bilayer (Figure the other three had as in the starting configuration (Figure and The juxtamembrane linkers of synaptobrevin and syntaxin-1 established extensive interactions with the lipids in the simulations. ns of the simulation at 310 we observed that the of the vesicle was resulting in an extended contact interface with the flat bilayer the of Figure with those of the initial in Figure To these findings we the number of between of the vesicle and the flat bilayer as a function of We a contact to each distance 1 which is a used to and electrostatic interactions between atoms. The results that the number of to about ns and then (Figure supplement 1D). The extended contact interface the of the simulation at and during this simulation the flat bilayer became to to the of the vesicle (Figure These finding with results obtained in analyses of fusion mediated by the SNAREs, which extended contact interfaces between the that are to as tight intermediates (Hernandez et al., 2012). These intermediates to membrane fusion, but fusion in the time scale (Hernandez et al., 2012; Witkowska et al., 2021). Hence, it is that such extended interfaces occur in the pathway that leads to fast Ca2+-triggered synaptic vesicle fusion. also that the required to initiate membrane fusion is expected to with the of the interface between the two as require more lipid molecules to be of reconstitution including additional components of the release machinery suggested that these additional components formation of extended contact interfaces, interfaces with contact between the bilayers that are to as interfaces et al., 2017). Simulations of the primed Synaptotagmin-1-SNARE-complexin-1 complex our simulations not out the possibility that SNAREs alone might be to induce membrane fusion in the low microsecond time as it is that other geometries might be more in inducing fusion. However, the of our results with the of reconstitution that fast fusion requires additional of a primed state of synaptic vesicles that is ready for fast release is the key to fast Ca2+-triggered fusion in The exact nature of this state is unclear, but Syt1 and are most likely bound to trans-SNARE complexes in this as both proteins bind to the SNARE complex and are critical for fast, Ca2+-triggered neurotransmitter release (Fernández-Chacón et al., 2001; Reim et al., A model of this state (Voleti et al., was based on crystal structures of the SNARE complex bound to a complexin-1 (Chen et al., 2002) or to the Syt1 C2B domain through the primary interface (Zhou et al., 2015), as as a structure of Syt1 bound to lipid SNARE complex et al., 2019). In this model, complexin-1 and the 1 C2B domain bind to of the SNARE four-helix and the C2B domain binds to the plasma membrane through a polybasic region on the side of the C2B domain to the primary However, the of this macromolecular assembly with to the vesicle and plasma and the extent to which the SNARE complex is are To insights into the nature of the primed complex that is ready for fast Ca2+-triggered membrane fusion, we built a system with four trans-SNARE complexes bridging a vesicle and a flat bilayer, each bound to a complexin-1 and the Syt1 as observed by (Chen et al., 2002; Zhou et al., 2015; to as primed The complexin-1
Source: eLife
Authors: Josep Rizo, Levent Sari, Yife Qi, Wonpil Im, Milo M Lin
Language: English
Subfield: Cell Biology
Domain: Life Sciences
Open Access status: gold
Estimated APC paid: $2,500
OpenAlex ID: W4283657399