Synaptotagmin 1 and SNAREs Form a Complex That Is Structurally Heterogeneous

doi:10.1016/j.jmb.2010.11.015

Journal of Molecular Biology

Volume 405, Issue 3, 21 January 2011, Pages 696-706

https://doi.org/10.1016/j.jmb.2010.11.015 Get rights and content

Abstract

Synaptotagmin 1 (syt1) functions as a Ca²⁺-sensor for neuronal exocytosis. Here, site-directed spin labeling was used to examine the complex formed between a soluble fragment of syt1, which contains its two C2 domains, and the neuronal core soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex. Changes in electron paramagnetic resonance lineshape and accessibility for spin-labeled syt1 mutants indicate that in solution, the assembled core SNARE complex contacts syt1 in several regions. For the C2B domain, contact occurs in the polybasic face and sites opposite the Ca²⁺-binding loops. For the C2A domain, contact is seen with the SNARE complex in a region near loop 2. Double electron-electron resonance was used to estimate distances between the two C2 domains of syt1. These distances have broad distributions in solution, which do not significantly change when syt1 is fully associated with the core SNARE complex. The broad distance distributions indicate that syt1 is structurally heterogeneous when bound to the SNAREs and does not assume a well-defined structure. Simulated annealing using electron paramagnetic resonance-derived distance restraints produces a family of syt1 structures where the Ca²⁺-binding regions of each domain face in roughly opposite directions. The results suggest that when associated with the SNAREs, syt1 is configured to bind opposing bilayers, but that the syt1/SNARE complex samples multiple conformational states.

Graphical Abstract

Research Highlights

► Synaptotagmin 1 is structurally heterogeneous when bound to SNAREs. ► When bound to SNAREs, C2A and C2B are aligned to bind opposing bilayers. ► SNAREs interact with synaptotagmin 1 at multiple sites on both C2A and C2B.

Introduction

In the central nervous system, the release of neurotransmitter occurs through a highly regulated Ca²⁺-dependent membrane fusion event that releases the contents of the synaptic vesicle into the synaptic cleft. Although a large number of proteins act to facilitate this fusion process, soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins are thought to make up the core of the fusion machinery. SNAREs include plasma membrane-associated syntaxin, vesicle-associated synaptobrevin, and SNAP-25,¹ and they form a four-helix bundle that acts to drive the vesicle and plasma membranes together.2, 3 However, SNAREs are not directly responsible for sensing Ca²⁺, and in neuronal exocytosis, there is compelling evidence that synaptotagmin 1 (syt1) functions as a Ca²⁺ sensor and is responsible for the fast synchronous release of neurotransmitter.4, 5, 6

Synaptotagmin 1 is one of several proteins, including complexins, Munc18, and CAPS, which regulate fusion and/or assembly of the SNARE complex. At the present time, the mechanisms and points at which these proteins function are not precisely known. Synaptotagmin 1 binds and penetrates membranes in a Ca²⁺-dependent manner,7, 8 and it has been observed to bind the assembled SNARE complex5, 9 as well as the individual SNARE proteins syntaxin and SNAP-25.10, 11, 12 A current popular view in the field is that fusion is controlled by SNARE zippering,¹ which is generally believed to proceed from the N-terminal to the C-terminal membrane anchors of syntaxin and synaptobrevin, ultimately forming a close junction between opposing membranes. However, it is not known whether syt1 acts to regulate and facilitate SNARE assembly or whether it acts on the membrane interface to assist the SNAREs in overcoming energetic barriers to membrane fusion.

Several distinctly different mechanisms for syt1 function have been proposed. For example, syt1 might regulate fusion by interacting with the SNAREs or intermediates in the SNARE assembly process, and it has been proposed to interact with individual SNAREs and promote SNARE assembly.¹³ In some measurements, complexin is observed to be inhibitory to the fusion process, and syt1 has been proposed to act by displacing complexin from the SNARE complex upon the arrival of a Ca²⁺ signal, thereby promoting zippering of the SNARE complex.¹⁴ It has been reported that syt1 forms a ternary complex with SNAREs and membranes in the presence of Ca²⁺,¹⁵ and it has been proposed that syt1 might act in such a complex by stabilizing the SNAREs and by facilitating their zippering on the membrane surface.¹⁶ Synaptotagmin 1 has also been observed to bridge bilayers17, 18, 19 and to promote the aggregation of vesicles in a Ca²⁺-dependent manner; a process that might facilitate the formation of a close junction between vesicle and plasma membranes. Synaptotagmin also perturbs bilayers, and it has been proposed to act by altering bilayer curvature and assisting in the formation of the hemi-fusion intermediate.²⁰

At the present time, there is limited structural information to support the specific models that have been proposed. Crystal structures for the assembled core SNARE complex,² a SNARE–complexin complex,²¹ as well as the fully zippered SNARE complex containing the transmembrane segments are available.²² There are also crystal structures and solution NMR structures for the individual C2 domains of syntaptotagmin 1,23, 24 as well as a crystal structure for a soluble fragment of syt1 containing the two C2 domains (syt1C2AB).²⁵ Information on syt1 associated with SNAREs or membranes and SNAREs is more limited and has been challenging to obtain. Site-directed spin labeling (SDSL) and electron paramagnetic resonance (EPR) spectroscopy have provided information on how the C2 domains of synaptotagmin 1 are positioned on the membrane interface and penetrate bilayers,8, 26, 27 and these data are qualitatively consistent with the results of fluorescence spectroscopy.⁷ Continuous-wave (CW) EPR²⁸ as well as long-range distance constraints from pulse EPR show that the two domains in syt1C2AB are flexibly linked in solution and remain flexibly linked when bound to bilayers.¹⁹ EPR-derived restraints for membrane-associated C2AB demonstrate that it bridges bilayers, and the structures that best satisfy the distance constraints orient the two C2 domains so that their Ca²⁺-binding loops associate with opposing bilayers.¹⁹ Recent single-molecule fluorescence resonance energy transfer (smFRET) data are generally consistent with these EPR data and indicate that the two domains are flexibly linked.²⁹

A number of approaches have been used to investigate the syt1–SNARE interaction. NMR chemical shift changes in ¹⁵N heteronuclear single quantum coherence spectra of syt1C2AB indicate that, in solution, syt1 interacts with the assembled SNARE complex primarily through the polybasic face of the C2B domain of syt1.¹⁵ This interaction is also seen by CW EPR in solution where immobilization of spin labels in the polybasic face of C2B is observed due to tertiary contact of the SNAREs with this face of C2B.²⁸ More recent measurements using smFRET suggest that an important interaction between the SNAREs and syt1 takes place on C2B at a site opposite the Ca²⁺-binding loops (Fig. 1), and that the two domains are positioned to interact with the same membrane surface.²⁹ This work supports a hypothesis where syt1 acts upon bilayers while simultaneously interacting with SNAREs. However, these measurements were carried out on a supported bilayer surface that contained only phosphatidylcholine and lacked negatively charged lipids, such as phosphatidylserine. Thus, membrane attachment of the C2 domains syt1 should not take place under the conditions of these experiments.

In the present work, we present the results of both CW and pulse EPR measurements of the interaction between syt1 and the soluble core SNARE complex. The data indicate that in solution, the two C2 domains remain flexibly linked when bound to the SNAREs, consistent with the previous smFRET result. However, simulated annealing using the EPR-derived distance constraints indicates that the most populated structure for syt1C2AB when associated with the SNAREs is one where the two C2 domains face in opposite directions. A scan of sites around the surface of syt1C2AB indicates that the polybasic face of C2B is an important site of contact with the SNAREs, although several other points of contact between syt1C2AB and the SNAREs are detected. It was not possible to dock syt1 to the SNAREs and satisfy the observed points of contact, a result which indicates that the syt1/SNARE complex samples multiple conformational states. The implications of these findings for syt1-mediated membrane fusion are discussed.

Section snippets

EPR spectra provide evidence for multiple sites of contact between syt1C2AB and the soluble core–SNARE complex

EPR spectra from the spin-labeled side chain, R1, are sensitive to the local environment at the labeled site. Lineshapes are influenced by the steric interactions made by R1, by the backbone dynamics of the site to which R1 is attached, and by polarity.30, 31, 32 Because the EPR spectrum is sensitive tertiary contact of the nitroxide side chain, protein–protein interactions have been successfully probed by SDSL and EPR spectroscopy.33, 34 Here, the spin-labeled side chain R1 was incorporated

Discussion

In spite of a significant level of effort, the mechanism by which syt1 mediates Ca²⁺-triggered synchronous neurotransmitter release is not well understood. In part, this difficulty is due to the fact that syt1 makes interactions with both bilayers and SNAREs and the fact that conventional methods have failed to provide a structure for the syt1–SNARE complex. In the work presented here, we examined the interactions between syt1 and the core SNARE complex using SDSL and determined an average

Mutagenesis, protein expression, and purification

DNA of rat syt1 (P21707) was obtained from Dr. Carl Creutz (Pharmacology Department, University of Virginia) in the pGEX-KG vector encoding amino acid residues 96−421 (syt1C2AB).⁴⁵ The single native cysteine residue at position 277 was mutated to alanine by typical PCR strategies. A QuickChange Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA) was used to produce single- and double-cysteine mutants of C2AB, and all cysteine substitutions were confirmed by DNA sequencing.

The expression

Acknowledgements

We would like to thank Christian Altenbach (UCLA) for providing the LabVIEW programs used to process and simulate EPR spectra. We also acknowledge the assistance of the laboratories of Lukas Tamm (University of Virginia) and Reinhard Jahn (Max-Plank-Institute Göttingen, Germany) for help with the expression, purification, and reconstitution of the soluble SNARE complex. This work was supported by a grant from the National Institutes of Health, NIGMS, GM 072694.

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Release of neuronal transmitters from nerve terminals is triggered by the molecular Ca²⁺ sensor synaptotagmin 1 (Syt1). Syt1 is a transmembrane protein attached to the synaptic vesicle (SV), and its cytosolic region comprises two domains, C2A and C2B, which are thought to penetrate into lipid bilayers upon Ca²⁺ binding. Before fusion, SVs become attached to the presynaptic membrane (PM) by the four-helical SNARE complex, which is thought to bind the C2B domain in vivo. To understand how the interactions of Syt1 with lipid bilayers and the SNARE complex trigger fusion, we performed molecular dynamics (MD) simulations at a microsecond scale. We investigated how the isolated C2 modules and the C2AB tandem of Syt1 interact with membranes mimicking either SV or PM. The simulations showed that the C2AB tandem can either bridge SV and PM or insert into PM with its Ca²⁺-bound tips and that the latter configuration is more favorable. Surprisingly, C2 domains did not cooperate in penetrating into PM but instead mutually hindered their insertion into the bilayer. To test whether the interaction of Syt1 with lipid bilayers could be affected by the C2B-SNARE attachment, we performed systematic conformational analysis of the C2AB-SNARE complex. Notably, we found that the C2B-SNARE interface precludes the coupling of C2 domains and promotes their insertion into PM. We performed the MD simulations of the prefusion protein complex positioned between the lipid bilayers mimicking PM and SV, and our results demonstrated in silico that the presence of the Ca²⁺ bound C2AB tandem promotes lipid merging. Altogether, our MD simulations elucidated the role of the Syt1-SNARE interactions in the fusion process and produced the dynamic all-atom model of the prefusion protein-lipid complex.
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Membrane bridging would promote proximity of vesicle and plasma membrane SNARE proteins for complex assembly for fusion. In addition, synaptotagmin-1 interacts directly with SNARE protein complexes potentially promoting C-terminal zippering of SNARE complexes [133–138]. Some studies indicate that the polybasic region in C2B mediates SNARE binding by synaptotagmin-1 [139,140] but it appears that SNARE binding and PI(4,5)P2 associations can occur simultaneously [80,114,119].
PI(4,5)P₂ participates directly in priming and possibly in fusion steps of Ca^2 +-triggered vesicle exocytosis. High concentration nanodomains of PI(4,5)P₂ reside on the plasma membrane of neuroendocrine cells. A subset of vesicles that co-localize with PI(4,5)P₂ domains appear to undergo preferential exocytosis in stimulated cells. PI(4,5)P₂ directly regulates vesicle exocytosis by recruiting and activating PI(4,5)P₂-binding proteins that regulate SNARE protein function including CAPS, Munc13-1/2, synaptotagmin-1, and other C2 domain-containing proteins. These PI(4,5)P₂ effector proteins are coincidence detectors that engage in multiple interactions at vesicle exocytic sites. The SNARE protein syntaxin-1 also binds to PI(4,5)P₂, which promotes clustering, but an activating role for PI(4,5)P₂ in syntaxin-1 function remains to be fully characterized. Similar principles underlie polarized constitutive vesicle fusion mediated in part by the PI(4,5)P₂-binding subunits of the exocyst complex (Sec3, Exo70). Overall, focal vesicle exocytosis occurs at sites landmarked by PI(4,5)P₂, which serves to recruit and/or activate multifunctional PI(4,5)P₂-binding proteins. This article is part of a Special Issue entitled Phosphoinositides.
Calcium binding promotes conformational flexibility of the neuronal Ca<sup>2+</sup> sensor synaptotagmin
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Thus, a crystallography study (23) revealed tightly interacting domains, whereas optical studies suggested that in the solution C2 domains do not interact and can rotate freely (26). Furthermore, electron paramagnetic resonance studies suggest that Syt1 remains structurally heterogeneous even upon binding to the SNARE complex (42), although fluorescence resonance energy transfer studies (24,43) argue that Syt1 adopts a fixed conformation upon binding to the SNARE complex, with the C2B domain of Syt1 playing a primary role in these interactions. Our analysis revealed that in the solution Ca2+-unbound form of Syt1 exists as an ensemble of conformers with tightly coupled domains.
Synaptotagmin 1 (Syt1) is a synaptic vesicle protein that serves as a calcium sensor of neuronal secretion. It is established that calcium binding to Syt1 triggers vesicle fusion and release of neuronal transmitters, however, the dynamics of this process is not fully understood. To investigate how Ca²⁺ binding affects Syt1 conformational dynamics, we performed prolonged molecular dynamics (MD) simulations of Ca²⁺-unbound and Ca²⁺-bound forms of Syt1. MD simulations were performed at a microsecond scale and combined with Monte Carlo sampling. We found that in the absence of Ca²⁺ Syt1 structure in the solution is represented by an ensemble of conformational states with tightly coupled domains. To investigate the effect of Ca²⁺ binding, we used two different strategies to generate a molecular model of a Ca²⁺-bound form of Syt1. First, we employed subsequent replacements of monovalent cations transiently captured within Syt1 Ca²⁺-binding pockets by Ca²⁺ ions. Second, we performed MD simulations of Syt1 at elevated Ca²⁺ levels. All the simulations produced Syt1 structures bound to four Ca²⁺ ions, two ions chelated at the binding pocket of each domain. MD simulations of the Ca²⁺-bound form of Syt1 revealed that Syt1 conformational flexibility drastically increased upon Ca²⁺ binding. In the presence of Ca²⁺, the separation between domains increased, and interdomain rotations became more frequent. These findings suggest that Ca²⁺ binding to Syt1 may induce major changes in the Syt1 conformational state, which in turn may initiate the fusion process.
The juxtamembrane linker of full-length synaptotagmin 1 controls oligomerization and calcium-dependent membrane binding
2014, Journal of Biological Chemistry
Synaptotagmin 1 (Syt1) is the calcium sensor for synchronous neurotransmitter release. The two C2 domains of Syt1, which may mediate fusion by bridging the vesicle and plasma membranes, are connected to the vesicle membrane by a 60-residue linker. Here, we use site-directed spin labeling and a novel total internal reflection fluorescence vesicle binding assay to characterize the juxtamembrane linker and to test the ability of reconstituted full-length Syt1 to interact with opposing membrane surfaces. EPR spectroscopy demonstrates that the majority of the linker interacts with the membrane interface, thereby limiting the extension of the C2A and C2B domains into the cytoplasm. Pulse dipolar EPR spectroscopy provides evidence that purified full-length Syt1 is oligomerized in the membrane, and mutagenesis indicates that a glycine zipper/GXXXG motif within the linker helps mediate oligomerization. The total internal reflection fluorescence-based vesicle binding assay demonstrates that full-length Syt1 that is reconstituted into supported lipid bilayers will capture vesicles containing negatively charged lipid in a Ca²⁺-dependent manner. Moreover, the rate of vesicle capture increases with Syt1 density, and mutations in the GXXXG motif that inhibit oligomerization of Syt1 reduce the rate of vesicle capture. This work demonstrates that modifications within the 60-residue linker modulate both the oligomerization of Syt1 and its ability to interact with opposing bilayers. In addition to controlling its activity, the oligomerization of Syt1 may play a role in organizing proteins within the active zone of membrane fusion.
Coupling exo- and endocytosis: An essential role for PIP <inf>2</inf> at the synapse
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These residues are thought to form a basic patch on C2B allowing pre-adsorption to PIP2 prior to the Ca2+ signal, thus producing an enhanced coupling of synaptotagmin to the release machinery [57,59,93,94]. However, this site has also been proposed to mediate interactions with the SNARE proteins syntaxin and SNAP-25 in a Ca2+-independent manner [95,96], and also to the full SNARE complex (although this interaction seems dependent on Ca2+) [97–99]. It should also be noted that the C2A domain has been reported to bind to SNAP-25 within the SNARE complex in a Ca2+-dependent manner, but with low affinity [100].
Chemical synapses are specialist points of contact between two neurons, where information transfer takes place. Communication occurs through the release of neurotransmitter substances from small synaptic vesicles in the presynaptic terminal, which fuse with the presynaptic plasma membrane in response to neuronal stimulation. However, as neurons in the central nervous system typically only possess ~ 200 vesicles, high levels of release would quickly lead to a depletion in the number of vesicles, as well as leading to an increase in the area of the presynaptic plasma membrane (and possible misalignment with postsynaptic structures). Hence, synaptic vesicle fusion is tightly coupled to a local recycling of synaptic vesicles. For a long time, however, the exact molecular mechanisms coupling fusion and subsequent recycling remained unclear. Recent work now indicates a unique role for the plasma membrane lipid phosphatidylinositol 4,5-bisphosphate (PIP₂), acting together with the vesicular protein synaptotagmin, in coupling these two processes. In this work, we review the evidence for such a mechanism and discuss both the possible advantages and disadvantages for vesicle recycling (and hence signal transduction) in the nervous system. This article is part of a Special Issue entitled Lipids and Vesicular Transport.
GPCR mediated regulation of synaptic transmission
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Further, NMR data demonstrated a primary interaction interface with SNARE may be through a polybasic region on the C2B domain (Dai et al., 2007). Most recently, electron paramagnetic resonance spectroscopy data reveal a structurally heterogeneous interaction maintaining the polybasic and C2B domain interactions, but also a site near loop 2 of the calcium binding site in C2A; an interaction not seen in the other two studies mentioned (Lai et al., 2011). These authors suggest that this new binding mode allows for simultaneous interaction with the SNARE complex as well as two membrane surfaces in apposition such as the cell membrane and the docked synaptic vesicle.
Synaptic transmission is a finely regulated mechanism of neuronal communication. The release of neurotransmitter at the synapse is not only the reflection of membrane depolarization events, but rather, is the summation of interactions between ion channels, G protein coupled receptors, second messengers, and the exocytotic machinery itself which exposes the components within a synaptic vesicle to the synaptic cleft. The focus of this review is to explore the role of G protein signaling as it relates to neurotransmission, as well as to discuss the recently determined inhibitory mechanism of Gβγ dimers acting directly on the exocytotic machinery proteins to inhibit neurotransmitter release.

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^†: A.L.L., H.H., and D.Z.H. contributed equally to this work.

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Journal of Molecular Biology

Synaptotagmin 1 and SNAREs Form a Complex That Is Structurally Heterogeneous

Abstract

Graphical Abstract

Research Highlights

Introduction

Section snippets

EPR spectra provide evidence for multiple sites of contact between syt1C2AB and the soluble core–SNARE complex

Discussion

Mutagenesis, protein expression, and purification

Acknowledgements

Cell

Trends Cell Biol.

Trends Biochem. Sci.

J. Biol. Chem.

Neuron

J. Mol. Biol.

J. Mol. Biol.

J. Mol. Biol.

Neuron

Neuron

Trends Biochem. Sci.

Curr. Opin. Struct. Biol.

J. Mol. Biol.

J. Magn. Reson., Ser. A

J. Biol. Chem.

J. Magn. Reson.

Prog. Nucl. Magn. Reson. Spectrosc.

J. Magn. Reson.

SNAREs—engines for membrane fusion

Nat. Rev. Mol. Cell Biol.

Crystal structure of a SNARE complex involved in synaptic exocytosis at 2.4 A resolution

Nature

Synaptotagmin: a calcium sensor on the synaptic vesicle surface

Science

PIP2 increases the speed of response of synaptotagmin and steers its membrane-penetration activity toward the plasma membrane

Nat. Struct. Mol. Biol.

Position of synaptotagmin I at the membrane interface: cooperative interactions of tandem C2 domains

Biochemistry

How does synaptotagmin trigger neurotransmitter release?

Annu. Rev. Biochem.

Role of synaptotagmin in Ca2+-triggered exocytosis

Biochem. J.

Role of synaptotagmin in Ca²⁺-triggered exocytosis