All-atom molecular dynamics simulations of Synaptotagmin-SNARE-complexin complexes bridging a vesicle and a flat lipid bilayer

  1. Josep Rizo
  2. Levent Sari
  3. Yife Qi
  4. Wonpil Im
  5. Milo M Lin

  • Curated by eLife

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    Evaluation Summary:

    This study investigates the release machinery of synaptic vesicles prior to SNARE-mediated fusion using atomistically detailed molecular dynamics simulations. While the approach provides an unparalleled perspective on a complex process that has eluded direct experimental access, the physiological relevance of the conclusions is not clear yet because of the short duration and necessary molecular approximations and assumptions underlying the simulations. The work will be of interest to all who study vesicle fusions.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #2 agreed to share their name with the authors.)

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Abstract

Synaptic vesicles are primed into a state that is ready for fast neurotransmitter release upon Ca 2+ -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 C 2 B 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 Ca 2+ influx.

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  1. Version published to 10.7554/elife.76356 on eLife
  2. eLife

    Author Response

    Reviewer #2 (Public Review):

    Rizo et al. present all-atom (AA) molecular dynamics simulations of molecular components of the neurotransmitter (NT) release machinery. Evoked NT release is triggered by machinery that senses calcium and responds by fusing the vesicular and plasma membranes to release NTs via a fusion pore. Synaptotagmin is the calcium sensor and the SNARE proteins are the core of the fusion machinery. Complexin is another molecular component, among others.

    Simulations were performed with 4 trans-SNARE complexes bridging 2 membranes with realistic lipid compositions, either 2 planar, or 1 planar and 1 vesicular. Other simulations incorporate also the C2A and C2B domains of Synaptotagmin-1 (Syt), and the accessory and central helices of Complexin-1 (Cpx). The authors' aim is to study the vesicle-release machinery system in its "primed" state, in which fusion is blocked ("clamped") before the influx of calcium which triggers fusion of the membranes and release. The planar membrane is 26 nm x 26 nm (sometimes a little larger) and the vesicle diameter 26 nm. The duration of each of the simulations of 2-5 million atoms was typically about 0.5 µsec.

    Some of the major conclusions the authors declare are as follows. (i) The juxtamembrane domains (linker domains, LDs) are unstructured in the trans-SNARE complexes. (ii) SNAREs on their own pull the membranes together and squash them into an extended contact zone (ECZ) (observed in simulations with SNAREs only) as seen in experiments (Hernandez et al, 2012). (iii) Their AA simulations are argued to support a model previously proposed by this group (voleti et al., 2020) of the primed state that clamps the fusion machinery, in which C2B binds the SNARE complex via the primary interface from the crystal structure (Zhou et al., 2015), with the C2B polybasic face binding the planar membrane, while a Cpx fragment binds the opposite side of the SNARE complex, based on an earlier crystal structure (Chen et al, 2002). In simulations, the structure was robust on the timescales probed. An orientation with the Cpx accessory helix impinging on the vesicle emerged, suggestive of a role in clamping fusion. The simulations implicate several residues as critical, consistent with earlier mutation studies. Two runs produced similar results.

    This is a very nice study which offers important information and insights about possible structures in the primed NT release machinery. To my knowledge, this is the most extensive AA model of a plausible NT machinery to date. The conclusion that the LDs are unstructured is interesting, contradicting prior MARTINI studies assuming helices were continuous from the SNARE complex into the LDs, and equally interesting is the finding of an ECZ with SNAREs pushed aside, in accord with previous coarse-grained studies. The outcome of the simulations of the voleti et al. C2B-SNARE-Cpx model is informative, yielding the preferred orientation and supporting the primary interface and Cpx-SNARE interactions implied by crystal structures.

    My main concerns are about the validity of the conclusions presented, given the AA results. AA simulations are extremely valuable, but have limited ability to probe the big questions about how the multi-component NT machinery cooperatively unclamps, fuses and releases on msec and greater timescales. I do believe a marriage of very short timescale methods (AA, MARTINI etc) and ultra coarse-grained methods is needed to understand these fascinating systems. This manuscript makes no reference to methods that probe these long timescales, and may sometimes overstate what can be concluded from their AA results. For example, their findings for the voleti C2B-SNARE-Cpx model do not, as far as I can see, obviously suggest that this structure clamps fusion. Similarly, simulations with Cpx removed and Ca2+ bound to the C2 domains were clearly worthwhile but inconclusive, as SNAREs were not released after ~ 400 ns of simulation. In both cases, uncertainties originate in the running time limitations of AA methods.

    We very much appreciate the summary of the paper and agree with these criticisms. We had already highlighted the limitations of our simulations and have further emphasized these limitations by pointing out the absence of key components in the revised manuscript (see response to point 2a of Essential Revisions). We also agree that coarse-grained simulations can offer important insights and allow simulations at much longer time scales, which makes them complementary to all-atom simulations. We realize that, in our attempt to emphasize the advantages of all-atom simulations, we did not do justice to the work on SNARE-mediate membrane fusion performed with continuum and coarsegrained simulations, and failed to mention important contributions in this field. We now mention several of these contributions and discuss the complementary role that distinct types of simulations of this system can play in the future (see our answer to point 1b of Essential Revisions above).

    Reviewer #3 (Public Review):

    Rizo and colleagues revisit several mechanistic questions centered on the roles of SNARE proteins, synaptotagmin 1 and complexin in catalyzing membrane fusion. This effort is purely simulation based with several impressive all-atom simulations of two closely apposed lipid bilayers harboring 4 mostly assembled SNARE complexes with and without Cpx1 and Syt1. The simulations explore only about half a microsecond of elapsed time and fail to capture the act of membrane fusion itself, perhaps due to this short time window imposed by computational limitations. The authors discuss various behaviors of the SNARE proteins and accessory proteins, comparing and contrasting their conformations with those derived from past crystallographic and NMR studies.

    Strengths: There are several attractive features of this study. All-atom simulations of SNARE-mediated fusion will necessarily involve many millions of atoms and thus few if any studies of this ambitious scope have been published. Most past computational work in this arena has either been at the coarse-grained level (which has limitations as pointed out by the authors) or has focused purely on a single SNARE complex rather than trying to capture a more realistic fusion/pre-fusion state. And the questions posed in this study are extremely difficult if not impossible to answer via conventional structural, in vitro biochemical and in vivo functional experimental approaches.

    Weaknesses: As is the case with all simulations, many realistic aspects of SNAREmediated fusion and the various proteins involved were omitted from the simulations for practical reasons. And several of these omissions may have large impacts on the results and conclusions. These omissions include pieces of the SNARE proteins, Cpx1, and Syt1 that are known to impact synaptic transmission but were not included to minimize the number of atoms simulated. Divalent cation interactions with anionic phospholipids were omitted even though these interactions likely have a large influence on the energy barrier for membrane fusion. Also, each simulation was performed only once, so the reader has no sense of how representative or accurate the presented results are. And importantly, the simulations never captured a bone fide fusion event, which seems like a critical aspect of modeling the prefusion state. Given that even the fastest known synapses require 50100 microseconds to convert a calcium influx into vesicle fusion, it is perhaps not surprising that no fusion events were observed in a 200-700 nanosecond simulation window across the handful of simulations performed in this study. Regardless of these omissions, the authors generated a large amount of simulated data and attempted to reconcile interesting observations with known protein structures and past functional data.

    We agree that our simulations have multiple caveats and in the revised version we now mention the absence of key components (see response to point 2a of Essential Revisions). However, as explained above, the simulations do reveal several interesting observations, and we place particular emphasis on those that correlate with experimental data. We note that the observation of an extended vesicle-flat bilayer interface correlates with EM data and that in this case we performed two simulations, one of 520 ns at 310 K and another of 454 ns simulation at 325 K. For the primed synaptotagmin-1-SNARE-complexin-1 complex, we performed two simulations with four complexes each, for a total of eight complexes, and the key features that we highlight were observed in all of these eight complexes.

  3. eLife

    Evaluation Summary:

    This study investigates the release machinery of synaptic vesicles prior to SNARE-mediated fusion using atomistically detailed molecular dynamics simulations. While the approach provides an unparalleled perspective on a complex process that has eluded direct experimental access, the physiological relevance of the conclusions is not clear yet because of the short duration and necessary molecular approximations and assumptions underlying the simulations. The work will be of interest to all who study vesicle fusions.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #2 agreed to share their name with the authors.)

  4. eLife

    Reviewer #1 (Public Review):

    Rizo et al. use all-atom molecular dynamics simulation to visualize the pre-fusion primed state during synaptic vesicle fusion. To this end, they incorporate some of the recent structural and functional data of SNARE and associated proteins, namely Synptotagmin-1 and Complexin to build their initial condition for the simulations. The data shed some new light on the potential organization of the fusion intermediate, but whether the observed protein organization is part of a true functional pathway or the product of the initial conditions chosen and the applied computational constraint is unclear, especially since none of the conditions tested result in membrane fusion.

  5. eLife

    Reviewer #2 (Public Review):

    Rizo et al. present all-atom (AA) molecular dynamics simulations of molecular components of the neurotransmitter (NT) release machinery. Evoked NT release is triggered by machinery that senses calcium and responds by fusing the vesicular and plasma membranes to release NTs via a fusion pore. Synaptotagmin is the calcium sensor and the SNARE proteins are the core of the fusion machinery. Complexin is another molecular component, among others.

    Simulations were performed with 4 trans-SNARE complexes bridging 2 membranes with realistic lipid compositions, either 2 planar, or 1 planar and 1 vesicular. Other simulations incorporate also the C2A and C2B domains of Synaptotagmin-1 (Syt), and the accessory and central helices of Complexin-1 (Cpx). The authors' aim is to study the vesicle-release machinery system in its "primed" state, in which fusion is blocked ("clamped") before the influx of calcium which triggers fusion of the membranes and release. The planar membrane is 26 nm x 26 nm (sometimes a little larger) and the vesicle diameter 26 nm. The duration of each of the simulations of 2-5 million atoms was typically about 0.5 µsec.

    Some of the major conclusions the authors declare are as follows. (i) The juxtamembrane domains (linker domains, LDs) are unstructured in the trans-SNARE complexes. (ii) SNAREs on their own pull the membranes together and squash them into an extended contact zone (ECZ) (observed in simulations with SNAREs only) as seen in experiments (Hernandez et al, 2012). (iii) Their AA simulations are argued to support a model previously proposed by this group (voleti et al., 2020) of the primed state that clamps the fusion machinery, in which C2B binds the SNARE complex via the primary interface from the crystal structure (Zhou et al., 2015), with the C2B polybasic face binding the planar membrane, while a Cpx fragment binds the opposite side of the SNARE complex, based on an earlier crystal structure (Chen et al, 2002). In simulations, the structure was robust on the timescales probed. An orientation with the Cpx accessory helix impinging on the vesicle emerged, suggestive of a role in clamping fusion. The simulations implicate several residues as critical, consistent with earlier mutation studies. Two runs produced similar results.

    This is a very nice study which offers important information and insights about possible structures in the primed NT release machinery. To my knowledge, this is the most extensive AA model of a plausible NT machinery to date. The conclusion that the LDs are unstructured is interesting, contradicting prior MARTINI studies assuming helices were continuous from the SNARE complex into the LDs, and equally interesting is the finding of an ECZ with SNAREs pushed aside, in accord with previous coarse-grained studies. The outcome of the simulations of the voleti et al. C2B-SNARE-Cpx model is informative, yielding the preferred orientation and supporting the primary interface and Cpx-SNARE interactions implied by crystal structures.

    My main concerns are about the validity of the conclusions presented, given the AA results. AA simulations are extremely valuable, but have limited ability to probe the big questions about how the multi-component NT machinery cooperatively unclamps, fuses and releases on msec and greater timescales. I do believe a marriage of very short timescale methods (AA, MARTINI etc) and ultra coarse-grained methods is needed to understand these fascinating systems. This manuscript makes no reference to methods that probe these long timescales, and may sometimes overstate what can be concluded from their AA results. For example, their findings for the voleti C2B-SNARE-Cpx model do not, as far as I can see, obviously suggest that this structure clamps fusion. Similarly, simulations with Cpx removed and Ca2+ bound to the C2 domains were clearly worthwhile but inconclusive, as SNAREs were not released after ~ 400 ns of simulation. In both cases, uncertainties originate in the running time limitations of AA methods.

  6. eLife

    Reviewer #3 (Public Review):

    Rizo and colleagues revisit several mechanistic questions centered on the roles of SNARE proteins, synaptotagmin 1 and complexin in catalyzing membrane fusion. This effort is purely simulation based with several impressive all-atom simulations of two closely apposed lipid bilayers harboring 4 mostly assembled SNARE complexes with and without Cpx1 and Syt1. The simulations explore only about half a microsecond of elapsed time and fail to capture the act of membrane fusion itself, perhaps due to this short time window imposed by computational limitations. The authors discuss various behaviors of the SNARE proteins and accessory proteins, comparing and contrasting their conformations with those derived from past crystallographic and NMR studies.

    Strengths: There are several attractive features of this study. All-atom simulations of SNARE-mediated fusion will necessarily involve many millions of atoms and thus few if any studies of this ambitious scope have been published. Most past computational work in this arena has either been at the coarse-grained level (which has limitations as pointed out by the authors) or has focused purely on a single SNARE complex rather than trying to capture a more realistic fusion/pre-fusion state. And the questions posed in this study are extremely difficult if not impossible to answer via conventional structural, in vitro biochemical and in vivo functional experimental approaches.

    Weaknesses: As is the case with all simulations, many realistic aspects of SNARE-mediated fusion and the various proteins involved were omitted from the simulations for practical reasons. And several of these omissions may have large impacts on the results and conclusions. These omissions include pieces of the SNARE proteins, Cpx1, and Syt1 that are known to impact synaptic transmission but were not included to minimize the number of atoms simulated. Divalent cation interactions with anionic phospholipids were omitted even though these interactions likely have a large influence on the energy barrier for membrane fusion. Also, each simulation was performed only once, so the reader has no sense of how representative or accurate the presented results are. And importantly, the simulations never captured a bone fide fusion event, which seems like a critical aspect of modeling the prefusion state. Given that even the fastest known synapses require 50-100 microseconds to convert a calcium influx into vesicle fusion, it is perhaps not surprising that no fusion events were observed in a 200-700 nanosecond simulation window across the handful of simulations performed in this study. Regardless of these omissions, the authors generated a large amount of simulated data and attempted to reconcile interesting observations with known protein structures and past functional data.

  7. Version published to 10.1101/2021.12.29.474428v1 on bioRxiv