Browsing by Subject "SNARE Proteins"
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Item Analysis of Synaptotagmin-SNARE Complex Interactions by One-Dimensional NMR Spectroscopy(2013-06-05) Zhou, Amy 1986-; Rice, Luke M.; Rizo-Rey, José; Brautigam, Chad A.; Yu, HongtaoThe mechanism of calcium-triggered neurotransmitter release is mediated by numerous proteins at the neuronal synapse. The SNARE proteins form a complex that mediates fusion between the synaptic vesicle and plasma membrane. The protein synaptotagmin-1 is the major sensor for the calcium concentration. Synaptotagmin-1 and the SNARE complex are therefore believed to interact in order to couple the calcium concentration to membrane fusion. The detailed mechanism of the interaction is still unclear, because the technical difficulties in probing the system have rendered it intractable with respect to traditional biochemical and biophysical methods. I will present an analytical method based on one-dimensional NMR spectroscopy that overcomes these limitations. The experiments are based on the current working model that synaptotagmin-1 facilitates the SNAREs’ role in membrane fusion in a calcium-dependent manner. My data suggest that the synaptotagmin-1/SNARE complex interaction is calcium-dependent and mediated primarily by the synaptotagmin C2B domain. Further, the polybasic region of C2B constitutes the primary binding site, while the two arginine residues at the bottom of the domain mediate additional interactions that lead to aggregation and precipitation. These results help clarify the complex mechanism of synaptotagmin-1/SNARE coupling, as well as to illustrate the usefulness of 1D NMR to study such protein-protein interactions. Alternative methods to probe such interactions are explored. The advantage of a related competition assay lies in its sampling of the primary binding site with little interference of the other binding mode(s). However, experimental artifacts hindered the application of the assay to my system. A diffusion-based method is another route for studying protein-protein interactions, provided there is sufficient dynamic range to allow for meaningful interpretations. The SNARE-, calcium-, and lipid-binding profiles of an extended synaptotagmin-like protein (E-Syt) were also characterized. E-Syts are of interest because they can shed light on the evolution of proteins in the synaptotagmin family. In addition, they can reveal general governing principles of tandem C2-domain proteins which often function in signal transduction and membrane trafficking.Item Competition Between Synaptotagmin 1 and Complexin for SNARE Complex Binding, Controls Fast Synaptic Vesicle Exocytosis(2007-05-23) Tang, Jiong; Südhof, Thomas C.Calcium binding to synaptotagmin 1 triggers fast exocytosis of synaptic vesicles that were primed for release by SNARE complex assembly. Besides synaptotagmin 1, fast Ca2+- triggered exocytosis requires complexins. Synaptotagmin 1 and complexins both bind to assembled SNARE complexes, but it is unclear how their functions are coupled. To clarify previous debates on calcium dependent and independent binding between synaptotagmin 1 and SNARE proteins, I systematically examined the interactions between synaptotagmin 1 and purified SNARE monomer, heterodimer and core complex separately. This would avoid the problem of doing binding assays in an undefined protein mixture. We found the calcium dependency of synaptotagmin 1 and SNARE interactions relied on the accurate binding conditions that include protein concentration and ionic strength. In addition, at physiological conditions, calcium dependent binding is favored. Based on this system, I discovered the competition between complexin and synaptotagmin 1 for SNARE complex binding. Although in hydrophilic environment, complexin shows much higher affinity for SNARE complex than synaptotagmin 1, synaptotagmin 1 can more efficiently replace complexin from membrane embedded SNARE complex in a strictly calcium dependent manner. Expression of synaptic vesicle targeted complexin (by fusion to synaptobrevin 2) in cultured cortical neurons severely blocks fast synchronous release, but not asynchronous release, which is very similar to that of synaptotagmin 1 knockout mice. Based on electrophysiological data and biochemical confirmation of competition, we suggest that the phenotype could result from the replacement of synaptotagmin 1 from SNARE complex by local high concentration of fused complexin. We propose our model as: complexin binding promotes the assembly of SNARE complex and further stabilizes it. As a result, vesicles are activated into a "superprimed" metastable state, and are clamped at the same time waiting for triggering signals. Synaptotagmin 1 replaces complexin and releases this clamp through SNARE complex binding upon calcium entry. The simultaneous binding of synaptotagmin 1 with SNARE complex and phospholipids finally triggers membrane fusion and vesicle release.Item Dissecting Roles for the Macromolecular Machinery Involved in Neurotransmitter Release(2019-01-15) Prinslow, Eric Andrew; Rosenbaum, Daniel M.; Rizo-Rey, José; Rice, Luke M.; Yu, HongtaoNeurotransmitter release is a tightly regulated process that involves synaptic vesicle docking at presynaptic active zones, priming of the vesicles to a release-ready state, and calcium evoked fusion of the vesicle and plasma membranes. The probability of release is modulated by plastic changes that depend on synaptic activity; these changes shape the properties of neural networks and underlie multiple forms of information processing in the brain. Elucidating the mechanisms of neurotransmitter release and its regulation is thus critical for understanding brain function and establishing fundamental principles of neuronal communication. I have investigated the mechanism by which neurotransmitters send messages between neurons as a specific model system to study the general mechanism of intracellular membrane fusion. In one project, I investigated whether trans-SNARE complexes can be disassembled by NSF-αSNAP. I showed that trans-SNARE complex formation in the presence of NSF-αSNAP requires both Munc18-1 and Munc13-1, and is facilitated by synaptotagmin-1. I proposed a model whereby Munc18-1 and Munc13-1 are critical for mediating vesicle priming as well as precluding de-priming by preventing trans-SNARE complex disassembly. Complexin-1 also impaired de-priming, while synaptotagmin-1 may have assisted in priming and hindered de-priming. Additionally, I used various biophysical approaches including ITC and NMR to shed light into how Complexin has dual roles in fusion. One of my projects investigated the inhibitory role of Complexin and solved a controversy over conflicting ITC data. Another project focused on the Complexin N-terminal and C-terminal domains to try and develop a complete model of how Complexin functions that incorporates all of its known interactions and activating/inhibiting properties. I observed cooperative interactions between Complexin, the SNARE complex, and lipids by forming SNARE complexes anchored on nanodiscs and liposomes. Such cooperative binding of Complexin to membranes and SNAREs may be critical for releasing the inhibition caused by the accessory helix, although the molecular mechanism of action has yet to be determined. Overall, these experiments highlight the importance of interactions between numerous accessory proteins and the trans-SNARE for proper regulation of SNARE complex formation, and therefore fusion.Item Mechanism of Synaptotagmin Action in Neurotransmitter Release(2005-12-19) Arac-Ozkan, Demet; Rizo-Rey, JoséNeurotransmitter release occurs by fusion of the synaptic vesicle membrane with the plasma membrane. Formation of a highly stable complex, known as the SNARE (soluble NSF-attachment protein receptors) complex, brings the two membranes close in space. SNARE complex formation is required but probably not sufficient for fusion to occur. An increase in the local Ca2+ concentration at the synaptic terminal rapidly triggers neurotransmitter release. The mechanism of Ca2+ action is still unknown. Synaptotagmin 1, a brain-specific vesicular transmembrane protein, is the Ca2+ sensor in neurons. It has two cytoplasmic C2 domains (C2A and C2B) that bind Ca2+. Both C2 domains interact with negatively charged phospholipids in a Ca2+ dependent manner. The interaction of synaptotagmin 1 with the SNARE complex is also reported. We investigated whether the interaction of synaptotagmin 1 with membranes or with the SNARE complex is critical for membrane fusion. A new method to detect protein-protein interactions by 1D NMR spectroscopy was developed. Either the 13C signal of the SNARE complex or synaptotagmin 1 was monitored to perform competition experiments between SNAREs and lipid vesicles for binding to synaptotagmin 1. In the presence of both lipids and the SNARE complex, synaptotagmin 1 binds to lipids but cannot bind to the SNARE complex. This result suggests that Ca2+-dependent membrane binding is the primary activity of synaptotagmin 1. We investigated the mechanism of Ca2+-dependent phospholipid binding to synaptotagmin 1 C2 domains. A combination of crosslinking and FRET experiments showed that synaptotagmin 1 does not oligomerize upon Ca2+-dependent binding to phospholipid vesicles. Intriguingly, it binds to two membranes simultaneously and brings them into close proximity as visualized by cryo-EM experiments. We showed that the isolated C2B domain is sufficient to induce close membrane proximity. Mutational analysis suggested that the abundance of basic residues around the C2B surface, which generates a highly positive electrostatic potential together with the bound Ca2+ ions, is essential for this activity. We suggest that the ability of the C2B domain to bring membranes into close proximity can explain why the C2B domain has a more critical function in vivo than the C2A domain.nnItem Munc18-1 Is a Master Orchestrator of Synaptic SNARE Assembly(2022-05) Stepien, Karolina Paulina; Yu, Hongtao; Rosen, Michael K.; Jiang, Youxing; Rizo-Rey, JoséNeurotransmitters are released by tightly controlled synaptic vesicle exocytosis at the active zone of presynaptic nerve terminals. This process requires primed fusion-competent vesicles and coordinated calcium-secretion coupling, and all these steps are governed by a sophisticated protein apparatus. SNAREs and Munc18-1 are the core components of this apparatus: SNAREs form SNARE complexes that bridge the vesicle and plasma membranes and Munc18-1 coordinates SNARE assembly. Productive formation of SNARE complexes is topologically complicated and requires overcoming multiple energy barriers. This work elucidates distinctive molecular mechanisms of how Munc18-1 orchestrates SNARE assembly. Reproducing the steps that lead to neuronal exocytosis with purified proteins and synthetic lipid vesicles has become an irreplaceable tool to decipher mechanistic details underlying synaptic membrane fusion. In one project, I created a new, much slower system by reconstituting a limited number of SNAREs. This new tool not only allowed to study the activity of the calcium sensor, synaptotagmin-1, showing its stimulatory function, but also provided a way to control the number of SNARE complexes. The latter is important as emerging evidence suggests that the number of primed SNARE complexes in neurons is small. My study shows that liposome fusion with few SNAREs requires Munc18-1, synaptotagmin-1, and Munc13-1. While the requirement of Munc13-1 for synaptic fusion can be partially bypassed by SNARE or Munc18-1 mutations, the absence of Munc18-1 has not been rescued by any means so far. In my next project, I elucidated the fundamental mechanism that completely prevents synaptic vesicle fusion unless Munc18-1 is present. Using fluorescence and nuclear resonance spectroscopy, I showed that another protein, αSNAP, completely blocks SNARE-dependent liposome fusion through diverse interactions with SNAREs and that such inhibition can only be overcome by Munc18-1 upon binding to closed syntaxin-1, which in turns initiates SNARE assembly. My third study described another mechanism underlying the essential role of Munc18-1 in SNARE assembly. I solved cryo-electron microscope structures of Munc18-1 bound simultaneously to its two cognate SNAREs, showing structural details of how Munc18-1 organizes formation of productive SNARE complexes. Overall, I described multiple mechanisms of how Munc18-1 functions at virtually every step of SNARE assembly.Item Regulation of SNARE-Mediated Synaptic Vesicle Release by Synaptotagmins and Complexins(2010-11-02) Kaeser-Woo, Yea Jin; Südhof, Thomas C.In the brain, neurons communicate with each other by synaptic transmission. This process includes release of neurotransmitter from vesicles in the presynaptic neuron into the synaptic cleft, and sensing of these neurotransmitters by the postsynaptic neuron with specific receptors. Long-lasting changes in the strength of synaptic contacts between neurons in the human brain, a process that is referred to as long-term synaptic plasticity, are the cellular correlates that underlie learning and memory. Synaptic transmission is initiated when an action potential arrives at the presynaptic terminal, and induces Ca2+ influx through voltage-gated Ca2+ channels. The SNARE (Soluble N-ethylmaleimide-sensitive-factor Attachment Protein Receptors) complex is the core component of the fusion machinery in the presynaptic terminal, as it forms a physical bridge between the vesicular membrane and the presynaptic target membrane that delivers the force to fuse the two membranes. Additional presynaptic proteins are required to activate or suppress neurotransmitter release which allows the presynaptic neuron to tightly control and regulate the process of neurotransmitter release. Among these proteins are synaptotagmins and complexins, two protein families that directly interact with the SNARE complex, and that are interdependent to each other in regulating SNARE-mediated synaptic vesicle release: complexin clamps neurotransmitter release until synaptotagmin is recruited by Ca2+ influx, and then it activates SNARE-mediated fusion process together with synaptotagmin. Here I describe the prospective in vivo function of synaptotagmin 12, a novel isoform of synaptotagmin, which lacks the typical Ca2+ binding residues of synaptotagmin, but instead contains a unique sequence motif which can be phosphorylated by cAMP-dependent protein kinase A. By using gene targeting method, I directly examined whether phosphorylation of synaptotagmin 12 is involved in presynaptic forms of long-term plasticity. In parallel, I developed a structure-function approach to functionally dissect how individual domains of complexin contribute to its dichotomic functions of clamping and activating neurotransmitter release. With this approach, we focused on how the accessory alpha-helix of complexin participates in SNARE-mediated synaptic fusion.Item Structural and Functional Studies of C2-Domain Proteins Involved In Neurotransmitter Release(2007-05-22) Dai, Han; Rizo-Rey, JoséNeurotransmitter release is mediated by synaptic vesicle exocytosis at presynaptic nerve terminals. This process is extremely fast and strictly regulated by the intracellular Ca2+ concentration. To achieve this exquisite regulation, many proteins are involved. The central fusion machinery includes the SNARE proteins synaptobrevin, syntaxin and SNAP-25, as well as Munc18. Besides these universal components, many other neuronal specific proteins are also involved in regulating Ca2+-triggered release. Interestingly, most of these regulatory proteins contain C2 domains, versatile protein modules with Ca2+-dependent and Ca2+-independent activities. However, the mechanism of regulation by these C2-domain proteins remains unclear. My research has focused on understanding the structural and functional properties of two types of C2-domain proteins, synaptotagmins and RIMs. With NMR spectroscopy and biochemical assays, we demonstrated that synaptotagmin 4 is a Ca2+ sensor in Drosophila but not in rat, in contrast to the prediction based on sequence alignments. X-ray crystallography revealed that changes in the orientations of critical Ca2+-ligands render the rat synaptotagmin 4 C2B domain unable to form full Ca2+-binding sites. We also analyzed the structural and biochemical properties of the RIM2 C2A domain. NMR spectroscopy and FRET experiments demonstrated no interaction between the RIM2 C2A domain and Ca2+, phospholipid, synaptotagmin 1, and SNAP-25. However, the crystal structure of RIM C2A domain exhibits a strikingly dipolar distribution of the surface electrostatic potential. Several lines of evidence from the crystal structure suggested a potential target binding site around the bottom 310-helix. With fluorescence microscopy and microfluidic channel technology, we demonstrated that synaptotagmin 1 binds simultaneously to phospholipids and the SNARE complex reconstituted in membranes in the presence of Ca2+, forming a quaternary SSCAP complex, and that the membrane penetration of synaptotagmin 1 into phospholipids is independent of the reconstituted SNARE complex. We also showed that synaptotagmin 1 displaces complexin from the reconstituted SNARE complex in the presence of Ca2+, and that the synaptotagmin 1 C2B domain is primarily responsible for SNARE binding. Moreover, NMR spectroscopy and site-directed mutagenesis studies yielded structural information of the potential binding interface, allowing us to use computational modeling and docking to generate a preliminary model of the SSCAP complex.Item Structural and Functional Studies of Munc18 and SNARE Proteins(2011-08-10) Xu, Yi; Rizo-Rey, JoséRelease of neurotransmitters is a tightly regulated process and a key event in interneuron communication. Release involves a series of steps, including vesicles docking to the active zone of the plasma membrane, priming to a readily releasable state, and Ca2+-triggered membrane fusion. These steps are tightly controlled by an intricate protein machinery. Essential components of this machinery are proteins from the Sec1/Munc18 (SM) and SNARE (soluble N-ethylmaleimide sensitive factor attachment protein receptors) families. SNAREs function by forming a four-helix bundle called SNARE complex; assembly of the SNARE complex brings two membranes together and is key for membrane fusion. The function of SM proteins is less clear. The neuronal SM proteins Munc18-1 was identified and linked to synaptic vesicle fusion due to its tight binding to syntaxin-1. The strict requirement of Munc18-1 for release is illustrated by the observation that, in mice, deletion of Munc18-1 abolished neurotransmitter release completely. Munc18-1 binds to the closed conformation of syntaxin-1 as well as to assembled SNARE complexes containing open syntaxin-1. Analysis of point mutations on Munc18-1 showed that binding of Munc18-1 to the Habc domain of open syntaxin-1 is critical for synaptic vesicle priming but not for the release step. The fact that Munc18-1 and complexin-1 could bind simultaneously to the SNARE complex suggested Munc18-1 remained bound to a macrocomplex that is poised for Ca2+ triggering of fusion. Analysis using diverse biophysical approaches revealed that Munc18-1 indeed binds to the C-terminus of the synaptobrevin SNARE motif and to the SNARE four-helix bundle. Both interactions have similar affinities and the N-terminal region of syntaxin-1 competes with the SNARE four-helix bundle and synaptobrevin for Munc18-1 binding, suggesting that the interaction between Munc18-1 and the SNARE four-helix bundle involves the same cavity of Munc18-1 that binds to syntaxin-1. To directly test Munc18-1’s role in fusion, I reconstituted v- and t-SNAREs into separate liposomes. Fusion between these proteoliposomes in the presence of Munc18-1 was monitored with a lipid mixing assay. Intriguingly, I found that enhanced lipid mixing caused by rat Munc18-1 alone was observed. Biochemistry studies showed that denaturation of rat Munc18-1 or squid Munc18-1 causes membrane lipid mixing in the absence of SNAREs proteins.Item Structural and Functional Studies of the Munc13 MUN Domain and the RIM C2B Domain(2007-12-17) Guan, Rong; Rizo-Rey, JoséNeurotransmitter release is essential for normal brain function and is achieved through exocytosis of synaptic vesicles. Many proteins are involved the regulation of neurotransmission. The central fusion machinery includes the SNARE proteins and Munc18- 1. Besides these universal components, many other neuronal specific proteins are also involved in regulating Ca2+-triggered neurotransmitter release, such as the key priming factors RIMs and Munc13s. Munc13s are essential for vesicle priming. RIMs form a protein scaffold in the presynaptic nerve terminal. My studies have focused on the structures and functions of the Munc13 MUN domain and the RIM C2B domain. I have studied the structure and function of the Munc13 MUN domain. On one hand, I have tried to determine the three dimensional structure of the Munc13 MUN domain by Xray crystallography. I have successfully obtained crystals of the Munc13-1 MUN domain, Munc13-3 MUN domain and a fragment containing the Munc13-1 C1, C2B and MUN domains. These crystals will be further optimized to enable structure determination. On the other hand, I have tried to identify the binding partners of the MUN domain using various methods. Cross-linking experiments revealed an interaction between the Munc13-1 MUN domain and endogenous Munc18-1. In addition, cofloatation assays revealed an interaction between MUN and reconstituted SNARE complex. Detailed analysis using cofloatation assays suggested both MUN and complexin can compete with Munc18-1 for SNARE complex binding in a membrane environment. Our studies also suggested that the membrane environment can modulate the strength of protein-protein interactions remarkably, which emphasize the importance to include membranes in the studies of protein-protein interactions involved in neurotransmission. I have also analyzed the structural and biochemical properties of the RIM1 C2B domain. NMR spectroscopy and FRET experiments demonstrated no interaction between the RIM1 C2B domain and Ca2+, phospholipids, or its putative binding partners, synaptotagmin 1 and liprins. X-ray crystallography revealed the existence of a RIM1 C2B domain homodimer, which was confirmed by analytical ultracentrifugation and NMR spectroscopy. Our results suggested a model that RIM1 C2B dimerization might facilitate the Munc13 C2A homodimer to Munc13 C2A/RIM zinc finger heterodimer switch during synaptic vesicle priming.Item Structural Studies of Complexin/SNARE Interactions(2008-09-18) Lee, Daeho; Rizo-Rey, JoséVesicular neurotransmitter release is mediated by exocytosis of synaptic vesicles at the presynaptic active zone of nerve terminals. The Ca2+-triggered release process is extremely fast, lasts less than half a millisecond, and is tightly regulated by Ca2+. Action potentials cause Ca2+ influx through voltage-gated Ca2+ channels, which in turn triggers synaptic vesicle fusion. The typical sub-millisecond latency between an action potential and neurotransmitter release and the precise timing of the release process are crucial for information processing in the nervous system. To achieve this exquisite regulation, many proteins are involved. The goal of our investigations was to delineate interactions between complexin and SNARE components that lead to the formation of a primed minimal fusion machinery. We have generated and used new constructs of short forms of synaptobrevin and complexin, as well as constructs of SNAP-25 and syntaxin that have previously been shown to be part of the minimal fusion machinery. With NMR spectroscopy, the use of the short synaptobrevin constructs has led to experimental results suggesting at least two key intermediates during the docking/priming process that are independent of complexin. We found evidence for a modular assembly of the full SNARE complex. In the absence of Syb2-CT, the N-terminal half of SNARE complex forms a four-helix bundle, while the C-terminal half, starting just after the polar layer, is disordered. In the presence of the Syb2-CT, however, both halves of the SNARE complex form a four-helix bundle. It is interesting to note that Syb2 residues 29-84 are sufficient for formation of the fully assembled SNARE complex. This evidence strongly suggests the existence of at least two intermediates during the docking priming reaction. Furthermore, with NMR spectroscopy, we have found new evidence that complexin can bind to the t-SNARE complex, in contrast to earlier evidence suggesting that complexin regulates the fully assembled SNARE complex. We demonstrated that Cpx-FL binds the t-SNARE complex SN1/SN3/Syx1a(188-259) in solution, as wa suggested for membrane-bound t-SNAREs. Note, however, that the t-SNARE complex does not contain the large complexin-binding interface provided by Syb2. Furthermore, we found that Cpx-FL also binds t-SNARE sub-complexes formed by SN1/SN3, and SN1/Syx1a(188-259), while very little binding was observed between Cpx-FL and Syx1a(188-259) alone. This finding is particularly interesting, because the cryst structure of the fully assembled SNARE complex does not suggest any binding between Cpx26-83 and either SN1 or SN3, whereas the only common component in all of the above experiments was SN1 domain.Item Structure and Function of Proteins Involved in Regulated Secretion: Synaptotagmins and Complexin(2010-01-12) Craig, Timothy Kellogg; Rizo-Rey, JoséThe release of neurotransmitter from neurons is a tightly regulated process. There are a number of proteins required for membrane fusion to occur, and then there are regulatory proteins that allow membrane fusion to proceed at incredible speed with the precise timing necessary for complex functions such as sight, motor control, and conscious thought. This study will explore the role of three such regulators through biophysical and structural methods. There are a number of proteins that are essential for membrane fusion. The SNARE proteins are the plasma membrane protein Syntaxin, the vesicle membrane protein Synaptobrevin, and the plasma membrane associated protein SNAP25. These proteins form a tight complex called the SNARE complex that is required for neurotransmitter release. This complex bridges the vesicle and plasma membranes, bringing them into close proximity. Formation of this complex is thus an important point of regulation for the neurotransmitter release process. This SNARE complex serves not only to bridge two membranes, but also to become an anchoring point for a number of regulators of neurotransmitter release such as Complexin, and Synaptotagmin as well as other required proteins such as Munc13 and Munc18. Complexin is a small soluble protein that binds to the SNARE complex with high affinity and regulates the formation of the SNARE complex. Synaptotagmin is the calcium sensor for fast release of neurotransmitter. Here I present data showing that the N-terminus of Complexin is involved in a critical interaction with the C-terminus of the SNARE complex that is responsible for the excitatory effect of complexin in neurotransmitter release. Synaptotagmins work with Complexins to trigger rapid membrane fusion in response to calcium influx. Synaptotagmin VII is an important protein for the release of glucagon from islets of langerhans. The C2B domain of this protein is nearly 50% identical to the C2B domain of SytI, but when the C2B domains of SytVII and SytI are switched, the protein does not function correctly. In this study the structure of SytVII was determined by x-ray crystallography to 1.44Å resolution in order to determine if the C2B domain of SytVII is structurally different from other C2B domains. Additionally I crystallized and solved the structure of the C2A domain of Synaptotagmin IX in an effort to compare it to the C2 domains of the other members of the synaptotagmin family. This analysis resulted in the surprising conclusion that a high degree of structural similarity does not necessarily relate to interoperability of the domains.Item Studies on the Structure and Interactions of Synaptotagmin-1 and SNARE Proteins in Neurotransmitter Release(2014-11-14) Brewer, Kyle Daniel; Rosen, Michael K.; Rizo-Rey, José; Yu, Hongtao; Jiang, YouxingThe SNARE complex and synaptotagmin-1 are essential for Ca²⁺-evoked neurotransmitter release, yet the mechanism of how these proteins work together in membrane fusion is unclear. Dozens of studies performed over two decades have described different types of synaptotagmin-1/SNARE interactions and reported the individual structures of the SNAREs, the SNARE complex, and the C₂ domains that form most of the cytoplasmic region of synaptotagmin-1. However, a high-resolution structure of a synaptotagmin-1/SNARE complex, which is crucial to understand the mechanism of release, has not been reported. In this work, we explore methods to examine the biophysical properties of synaptotagmin-1 and the SNAREs, primarily using NMR. We first examine the conformation of synaptobrevin on nanodisc bilayers and find that that the N-terminal portion of the SNARE domain has a high propensity to remain unfolded on membranes. We next look at the conformation of synaptotagmin-1 on nanodiscs and demonstrate that although both C₂ domains primarily bind to the same membrane, a small population of antiparallel conformers also exist. Finally and most importantly, we look at the structure of synaptotagmin-1/SNARE complex. After overcoming many obstacles and failed approaches, we were able to obtain intermolecular restraints for this 66 kDa machinery by introducing lanthanide tags for measurement of pseudocontact shifts (PCSs). Computational analyses incorporating these restraints show that a static structure cannot fully explain all the PCS measurements, but the data can be fit with a dynamic ensemble of structures whereby a polybasic region of the synaptotagmin-1 C₂B domain binds to a polyacidic region formed by the syntaxin-1 and SNAP-25 SNARE motifs. The orientation of the synaptotagmin-1 C₂B domain with respect to the SNARE complex within the ensemble is expected to allow quick, simultaneous interaction with lipids on both membranes upon Ca²⁺ binding to bring the membranes together. Distinct mutations in the C₂B domain polybasic region caused differential disruptions of SNARE complex-binding that correlate with the impairment of neurotransmitter release caused by these mutations. Overall, these results and the architecture of the synaptagmin-1/SNARE complex revealed by our NMR data support the hypothesis that synaptotagmin-1 cooperates with the SNAREs by bringing membranes together to trigger fast fusion upon Ca²⁺ influx.Item Unraveling the Functions of Synaptotagmin and Munc13 in Neurotransmitter Release(2015-01-30) Seven, Alpay Burak; Rosen, Michael K.; Rizo-Rey, José; Albanesi, Joseph P.; Jiang, Qiu-XingNeurotransmitter release is a central event in interneuronal communication. The release machinery includes three SNAREs (soluble N-ethylmaleimide sensitive factor adaptor protein receptor) and Munc18-1 as core components. Munc13, synaptotagmin and complexin underlie the exquisitely tight regulation of synaptic exocytosis. The SNAREs form the SNARE complex that brings synaptic vesicles and plasma membrane together. This complex is disassembled by NSF/α-SNAP (soluble N-ethylmaleimide sensitive fusion protein/NSF attachment protein). Munc18 binds to syntaxin, which keeps syntaxin in its closed confirmation and prevents to form the SNARE complex. Interactions between Munc13, Munc18, and syntaxin perform a vital role in regulation of the SNARE complex formation. Tight regulation of the release machinery requires other factors such as Ca2+ sensor Synaptotagmin-1 and negatively charged lipids. Synaptotagmin interacts with the SNARE complex and the negatively charged lipids, and initiates the synchronous fast release by sensing the Ca2+ influx. It is crucial to investigate functions of individual proteins to understand the mechanism of membrane fusion and neurotransmitter release. Therefore, we investigated the mechanism of membrane bridging by synaptotagmin. Our cryo-electron microscopy (cryo-EM) images showed that a majority of synaptotagmin fragment containing both C¬2A and C2B domains (C2AB) molecules bridge membranes directly. Fluorescence spectroscopy demonstrates that the bottom of the C2B domain contacts the membrane in a substantial population of membrane-bound synaptotagmin fragments. NMR analysis of C2AB-nanodiscs shows that a fraction of C2AB molecules binds to membranes with antiparallel orientations of the C2 domains. Together with previous studies, these results show that direct bridging constitutes the prevalent mechanism of membrane bridging by synaptotagmin, suggesting that this mechanism underlies the function of synaptotagmin-1 in neurotransmitter release. We have also discovered that Munc13-1 can bridge membranes in a Ca2+-independent manner, which shed light to the docking activity of Munc13-1. We also showed that Munc13-1 can cause efficient lipid mixing and slow content mixing together with Munc18-1 in the absence of synaptotagmin-1. Addition of synaptotagmin facilitates the fusion pore formation in content mixing assays. Recently, we were able to reconstitute key components of the release machinery. We are further investigating our observations with cryo-electron microscopy to understand the mechanism of membrane fusion.Item Unraveling the Role of SNARE Interactions in Neurotransmitter Release(2005-05-04) Chen, Xiaocheng; Kavalali, Ege T.The release of neurotransmitters by Ca2+-triggered synaptic vesicle exocytosis is tightly controlled by an intricate protein machinery. Essential components of this machinery are the synaptic vesicle protein synaptobrevin and the plasma membrane proteins syntaxin 1 and SNAP-25, which are collectively known as SNAREs and form a tight complex (the core complex). The assembly of the core complex may mediate membrane fusion. Complexin is a highly conserved cytoplasmic protein that binds tightly to the SNARE complex. Analysis of the interaction between complexin and the SNARE complex showed that complexin binds to the groove between the synaptobrevin and syntaxin helices, and the binding stabilizes the syntaxin/synaptobrevin interface. These results led to a model whereby complexin stabilizes the fully assembled SNARE complex, which is critical for the fast Ca2+-triggered neurotransmitter release. The N-terminal domain of syntaxin 1 folds back and forms a 'closed' conformation, which interacts with munc18-1, an essential protein in the neurotransmitter release. It has been proposed that the binding of munc18-1 might change the closed conformation. To test this model, I solved the solution structure of the N-terminal domain within the closed conformation of syntaxin 1 and structure comparisons showed that the N-terminal domain adopts the same conformation whether it is isolated, bound to Munc18-1, or within the closed conformation. Analysis of the Ca2+-binding properties of the core complex revealed that it contains several low affinity Ca2+ binding sites and most of them are nonspecific for Ca2+. A SNAP-25 mutation that causes a change in the Ca2+-dependence of secretion in chromaffin cells has no effect on the SNARE/synaptotagmin 1 interactions, but has a conspicuous effect on core complex assembly. Thus, the SNAREs are unlikely to directly act as Ca2+ sensors, but SNARE complex assembly is tightly coupled to Ca2+ sensing in neurotransmitter release. To directly test SNARE function, I reconstituted v- and t-SNAREs into separate liposomes and carefully characterized the proteoliposomes containing v- and t-SNAREs. Fusion between the v- and t-SNARE proteoliposomes was then monitored with a lipid mixing assay. Interestingly, little fusion was observed. The results suggest that the SNAREs alone are not sufficient to mediate membrane fusion.