Exploring the Two Coupled Conformational Changes That Activate the Munc18-1/Syntaxin-1 Complex.
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ABSTRACT:
SUBMITTER: Gong J
PROVIDER: S-EPMC8728020 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:The Sec1/munc18 protein family is essential for vesicle fusion in eukaryotic cells via binding to SNARE proteins. Protein kinase C modulates these interactions by phosphorylating munc18a thereby reducing its affinity to one of the central SNARE members, syntaxin-1a. The established hypothesis is that the reduced affinity of the phosphorylated munc18a to syntaxin-1a is a result of local electrostatic repulsion between the two proteins, which interferes with their compatibility. The current study challenges this paradigm and offers a novel mechanistic explanation by revealing a syntaxin-non-binding conformation of munc18a that is induced by the phosphomimetic mutations. In the present study, using molecular dynamics simulations, we explored the dynamics of the wild-type munc18a versus phosphomimetic mutant munc18a. We focused on the structural changes that occur in the cavity between domains 3a and 1, which serves as the main syntaxin-binding site. The results of the simulations suggest that the free wild-type munc18a exhibits a dynamic equilibrium between several conformations differing in the size of its cavity (the main syntaxin-binding site). The flexibility of the cavity's size might facilitate the binding or unbinding of syntaxin. In silico insertion of phosphomimetic mutations into the munc18a structure induces the formation of a conformation where the syntaxin-binding area is rigid and blocked as a result of interactions between residues located on both sides of the cavity. Therefore, we suggest that the reduced affinity of the phosphomimetic mutant/phosphorylated munc18a is a result of the closed-cavity conformation, which makes syntaxin binding energetically and sterically unfavorable. The current study demonstrates the potential of phosphorylation, an essential biological process, to serve as a driving force for dramatic conformational changes of proteins modulating their affinity to target proteins.
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Project description:In neuronal exocytosis, SNARE assembly into a stable four-helix bundle drives membrane fusion. Previous studies have revealed that the SM protein Munc18-1 plays a critical role for precise SNARE assembly with the help of Munc13-1, but the underlying mechanism remains unclear. Here, we used single-molecule FRET assays with a nanodisc membrane reconstitution system to investigate the conformational dynamics of SNARE/Munc18-1 complexes in multiple intermediate steps towards the SNARE complex. We found that single Munc18-1 proteins induce the closed conformation of syntaxin-1 not only in the free syntaxin-1 but also in the t-SNARE (syntaxin-1/SNAP-25) complex. These results implicate that Munc18-1 may act as a gatekeeper for both binary and ternary SNARE complex formation by locking the syntaxin-1 in a cleft of Munc18-1. Furthermore, the kinetic analysis of the opening/closing transition reveals that the closed syntaxin-1 in the syntaxin-1/SNAP-25/Munc18-1 complex is less stable than that in the closed syntaxin-1/Munc18-1 complex, which is manifested by the infrequent closing transition, indicating that the conformational equilibrium of the ternary complex is biased toward the open conformation of syntaxin-1 compared with the binary complex.
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Project description:Priming of synaptic vesicles involves Munc13-catalyzed transition of the Munc18-1/syntaxin-1 complex to the SNARE complex in the presence of SNAP-25 and synaptobrevin-2; Munc13 drives opening of syntaxin-1 via the MUN domain while Munc18-1 primes SNARE assembly via domain 3a. However, the underlying mechanism remains unclear. In this study, we have identified a number of residues in domain 3a of Munc18-1 that are crucial for Munc13 and Munc18-1 actions in SNARE complex assembly and synaptic vesicle priming. Our results showed that two residues (Q301/K308) at the side of domain 3a mediate the interaction between the Munc18-1/syntaxin-1 complex and the MUN domain. This interaction enables the MUN domain to drive the opening of syntaxin-1 linker region, thereby leading to the extension of domain 3a and promoting synaptobrevin-2 binding. In addition, we identified two residues (K332/K333) at the bottom of domain 3a that mediate the interaction between Munc18-1 and the SNARE motif of syntaxin-1. This interaction ensures Munc18-1 to persistently associate with syntaxin-1 during the conformational change of syntaxin-1 from closed to open, which reinforces the role of Munc18-1 in templating SNARE assembly. Taken together, our data suggest a mechanism by which Munc13 activates the Munc18-1/syntaxin-1 complex and enables Munc18-1 to prime SNARE assembly.
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Project description:During the priming step that leaves synaptic vesicles ready for neurotransmitter release, the SNARE syntaxin-1 transitions from a closed conformation that binds Munc18-1 tightly to an open conformation within the highly stable SNARE complex. Control of this conformational transition is important for brain function, but the underlying mechanism is unknown. NMR and fluorescence experiments now show that the Munc13-1 MUN domain, which plays a central role in vesicle priming, markedly accelerates the transition from the syntaxin-1-Munc18-1 complex to the SNARE complex. This activity depends on weak interactions of the MUN domain with the syntaxin-1 SNARE motif, and probably with Munc18-1. Together with available physiological data, these results provide a defined molecular basis for synaptic vesicle priming, and they illustrate how weak protein-protein interactions can play crucial biological roles by promoting transitions between high-affinity macromolecular assemblies.
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Project description:Priming of synaptic vesicle involves Munc13-catalyzed transition of the Munc18-1/syntaxin-1 complex to the SNARE complex in the presence of SNAP-25 and synaptobrevin-2; Munc13 drives opening of syntaxin-1 via the MUN domain while Munc18-1 primes SNARE assembly via domain 3a. However, the underlying mechanism remains unclear. In this study, we have identified a number of residues in domain 3a of Munc18-1 that are crucial for Munc13 and Munc18-1 actions in SNARE complex assembly and synaptic vesicle priming. Our results showed that two residues (Q301/K308) at the side of domain 3a mediate the interaction between the Munc18-1/syntaxin-1 complex and the MUN domain. This interaction enables the MUN domain to drive the opening of syntaxin-1 linker region, thereby leading to the extension of domain 3a and promoting synaptobrevin-2 binding. In addition, we identified two residues (K332/K333) at the bottom of domain 3a that mediate the interaction between Munc18-1 and the SNARE motif of syntaxin-1. This interaction ensures Munc18-1 to persistently associate with syntaxin-1 during the conformational change of syntaxin-1 from closed to open, which reinforces the role of Munc18-1 in templating SNARE assembly. Taken together, our data suggest a mechanism by which Munc13 activates the Munc18-1/Syx1 complex and enables Munc18-1 to prime SNARE assembly.
| S-SCDT-EMBOJ-2019-103631 | biostudies-other
Project description:Synaptotagmin-like protein 4-a (Slp4-a)/granuphilin-a is specifically localized on dense-core vesicles in certain neuroendocrine cells and negatively controls dense-core vesicle exocytosis through specific interaction with Rab27A. However, the precise molecular mechanism of its inhibitory effect on exocytosis has never been elucidated and is still a matter of controversy. Here we show by deletion and chimeric analyses that the linker domain of Slp4-a interacts with the Munc18-1.syntaxin-1a complex by directly binding to Munc18-1 and that this interaction promotes docking of dense-core vesicles to the plasma membrane in PC12 cells. Despite increasing the number of plasma membrane docked vesicles, expression of Slp4-a strongly inhibited high-KCl-induced dense-core vesicle exocytosis. The inhibitory effect by Slp4-a is absolutely dependent on the linker domain of Slp4-a, because substitution of the linker domain of Slp4-a by that of Slp5 (the closest isoform of Slp4-a that cannot bind the Munc18-1.syntaxin-1a complex) completely abrogated the inhibitory effect. Our findings reveal a novel docking machinery for dense-core vesicle exocytosis: Slp4-a simultaneously interacts with Rab27A and Munc18-1 on the dense-core vesicle and with syntaxin-1a in the plasma membrane.
| S-EPMC1446092 | biostudies-literature
Project description:Munc18-1 forms a template to organize assembly of the neuronal SNARE complex that triggers neurotransmitter release, binding first to a closed conformation of syntaxin-1 where its amino-terminal region interacts with the SNARE motif, and later binding to synaptobrevin. However, the mechanism of SNARE complex assembly remains unclear. Here, we report two cryo-EM structures of Munc18-1 bound to cross-linked syntaxin-1 and synaptobrevin. The structures allow visualization of how syntaxin-1 opens and reveal how part of the syntaxin-1 amino-terminal region can help nucleate interactions between the amino termini of the syntaxin-1 and synaptobrevin SNARE motifs, while their carboxyl termini bind to distal sites of Munc18-1. These observations, together with mutagenesis, SNARE complex assembly experiments, and fusion assays with reconstituted proteoliposomes, support a model whereby these interactions are critical to initiate SNARE complex assembly and multiple energy barriers enable diverse mechanisms for exquisite regulation of neurotransmitter release.
| S-EPMC9216511 | biostudies-literature
Project description:In neuronal and hormonal release, regulated exocytosis requires an essential set of proteins: the soluble N-ethylmaleimide sensitive-factor attachment receptor proteins (SNAREs) syntaxin 1, SNAP-25, VAMP, and their regulator, Munc18. Recently, it was found that Munc18-1 can interact with syntaxin 1 through distinct mechanisms: an inhibitory mode enveloping syntaxin (mode 1), sequestering it from SNARE protein interactions, and direct binding to an evolutionarily conserved N-terminal peptide of syntaxin (mode 2/3). The latter interaction has been proposed to control "priming" of the fusion reaction, defined using electrophysiology, but it is unknown how this interaction is regulated, and any dynamic effect at the molecular or vesicular level in cells remains undiscovered. We now show that a phosphorylation site in syntaxin 1 (Ser(14)) regulates the N-terminal interaction with Munc18-1. Probing syntaxin 1 association with Munc18-1, in real-time and in living cells, we found that modification of Ser(14) modulated the dynamics of this interaction, specifically at the plasma membrane. Destabilization of this dynamic interaction enhanced vesicle immobilization at the plasma membrane with a resulting inhibition of exocytosis.
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Project description:Syntaxin and Munc18 are, in tandem, essential for exocytosis in all eukaryotes. Recently, it was shown that Munc18 inhibition of neuronal syntaxin 1 can be overcome by arachidonic acid, indicating that this common second messenger acts to disrupt the syntaxin-Munc18 interaction. Here, we show that arachidonic acid can stimulate syntaxin 1 alone, indicating that it is syntaxin 1 that undergoes a structural change in the syntaxin 1-Munc18 complex. Arachidonic acid is incapable of dissociating Munc18 from syntaxin 1 and, crucially, Munc18 remains associated with syntaxin 1 after arachidonic-acid-induced syntaxin 1 binding to synaptosomal-associated protein 25 kDa (SNAP25). We also show that the same principle operates in the case of the ubiquitous syntaxin 3 isoform, highlighting the conserved nature of the mechanism of arachidonic acid action. Neuronal soluble N-ethyl maleimide sensitive factor attachment protein receptors (SNAREs) can be isolated from brain membranes in a complex with endogenous Munc18, consistent with a proposed function of Munc18 in vesicle docking and fusion.
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Project description:Neuronal exocytosis is mediated by SNARE proteins, which assemble into a highly stable four-helical bundle in a process that is not well understood. Here, electron paramagnetic resonance spectroscopy was used to examine how the t-SNAREs syntaxin and SNAP25 assemble in the presence and absence of the regulatory protein Munc18-1. Syntaxin and SNAP25 form a 2:1 complex, which is structurally heterogeneous and persists in the presence of excess SNAP25. Munc18-1 dissociates this 2:1 complex, but a 1:1 complex is retained where syntaxin is in a closed state. In the absence of an N-terminal fragment of syntaxin, Munc18-1 also stabilizes a 1:1 complex of sytaxin/SNAP25; however, syntaxin now samples an open state. These data demonstrate that the open-closed syntaxin equilibrium is shifted toward the open state when syntaxin and Munc18-1 are associated with SNAP25, and the results indicate that a syntaxin/SNAP25:Munc18-1 complex is a likely starting point for SNARE assembly.
| S-EPMC4775345 | biostudies-literature