Dynamic binding mode of a Synaptotagmin-1–SNARE complex in solution

Brewer, Kyle D.; Bacaj, Taulant; Cavalli, Andrea; Camilloni, Carlo; Swarbrick, James; Liu, Jin; Zhou, Amy; Zhou, Peng; Barlow, Νicholas; Xu, Junjie; Seven, Alpay B.; Prinslow, Eric A; Voleti, Rashmi; Häußinger, Daniel; Bonvin, Alexandre M. J. J.; Tomchick, Diana R.; Vendruscolo, Michele; Graham, Bimbil; Südhof, Thomas C.; Rizo, Josep

doi:10.1038/nsmb.3035

Cited by 138 publications

(262 citation statements)

References 70 publications

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“…2a–d, Supplementary Video 2). We also tested potential interactions that involve the polybasic region of Syt1 C2B by mutating two Lys residues (K326A/K327A, referred to as KA mutant) in order to disrupt any dynamic binding modes involving the highly charged polybasic region 20,22 . All mutants of the Syt1 C2B domain are properly folded (Fig.…”

Section: Specific Mutations Disrupt the Tripartite Interfacementioning

confidence: 99%

The primed SNARE–complexin–synaptotagmin complex for neuronal exocytosis

Zhou

Wang

et al. 2017

Nature

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Summary Synaptotagmin, complexin and neuronal SNARE proteins mediate evoked synchronous neurotransmitter release, but the molecular mechanisms mediating the cooperation between these molecules remain unclear. Here, we determined crystal structures of the primed pre-fusion SNARE-complexin-synaptotagmin-1 complex. These structures reveal an unexpected tripartite interface between synaptotagmin-1 and both the SNARE complex and complexin. Simultaneously, a second synaptotagmin-1 molecule interacted with the other side of the SNARE complex via the previously identified primary interface. Mutations that disrupt either interface in solution also severely impaired evoked synchronous release in neurons, suggesting that both interfaces are essential for the primed pre-fusion state. Ca2+ binding to the synaptotagmin-1 molecules unlocks the complex, allows full zippering of the SNARE complex, and triggers membrane fusion. The tripartite SNARE-complexin-synaptotagmin-1 complex at a synaptic vesicle docking site has to be unlocked for triggered fusion to commence, explaining the cooperation between complexin and synaptotagmin-1 in synchronizing evoked release on the sub-millisecond timescale.

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Section: Specific Mutations Disrupt the Tripartite Interfacementioning

confidence: 99%

The primed SNARE–complexin–synaptotagmin complex for neuronal exocytosis

Zhou

Wang

et al. 2017

Nature

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270

501

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“…Neuronal SNARE proteins form a ternary complex consisting of synaptobrevin/vesicle-associated membrane protein (VAMP2), syntaxin, and synaptosomal-associated protein . The main isoform synaptotagmin-1 is involved in synchronous release, and forms a conserved Ca 2+ -independent interface with the ternary SNARE complex (9), along with Ca 2+ -dependent interactions with the plasma membrane, and potentially other interfaces with the SNARE complex (10). Complexin is a small cytosolic α-helical protein abundant in the presynaptic terminal (11) that interacts with the SNARE complex (12) and the membrane (13).…”

Section: +mentioning

confidence: 99%

N-terminal domain of complexin independently activates calcium-triggered fusion

Lai

Choi

Zhang

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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Complexin activates Ca 2+-triggered neurotransmitter release and regulates spontaneous release in the presynaptic terminal by cooperating with the neuronal soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) and the Ca 2+ -sensor synaptotagmin. The N-terminal domain of complexin is important for activation, but its molecular mechanism is still poorly understood. Here, we observed that a split pair of N-terminal and central domain fragments of complexin is sufficient to activate Ca 2+ -triggered release using a reconstituted single-vesicle fusion assay, suggesting that the N-terminal domain acts as an independent module within the synaptic fusion machinery. The N-terminal domain can also interact independently with membranes, which is enhanced by a cooperative interaction with the neuronal SNARE complex. We show by mutagenesis that membrane binding of the N-terminal domain is essential for activation of Ca 2+ -triggered fusion. Consistent with the membrane-binding property, the N-terminal domain can be substituted by the influenza virus hemagglutinin fusion peptide, and this chimera also activates Ca -triggered release (5-8). Neuronal SNARE proteins form a ternary complex consisting of synaptobrevin/vesicle-associated membrane protein (VAMP2), syntaxin, and synaptosomal-associated protein 25 (SNAP-25). The main isoform synaptotagmin-1 is involved in synchronous release, and forms a conserved Ca 2+ -independent interface with the ternary SNARE complex (9), along with Ca 2+

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“…The system is exquisitely fine-tuned to increase the probability of fusion between synaptic vesicles and the plasma membrane by orders of magnitude upon Ca 2+ binding to Syt1. Synaptotagmins simultaneously interact with anionic phospholipid membranes (5) and the neuronal SNARE complex (6,7). The SNARE complex also interacts with complexin (Cpx), a small soluble protein that both activates evoked release and regulates spontaneous release (8,9).…”

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confidence: 99%

Morphologies of synaptic protein membrane fusion interfaces

Gipson

Fukuda

Danev

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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Neurotransmitter release is orchestrated by synaptic proteins, such as SNAREs, synaptotagmin, and complexin, but the molecular mechanisms remain unclear. We visualized functionally active synaptic proteins reconstituted into proteoliposomes and their interactions in a native membrane environment by electron cryotomography with a Volta phase plate for improved resolvability. The images revealed individual synaptic proteins and synaptic protein complex densities at prefusion contact sites between membranes. We observed distinct morphologies of individual synaptic proteins and their complexes. The minimal system, consisting of neuronal SNAREs and synaptotagmin-1, produced point and long-contact prefusion states. Morphologies and populations of these states changed as the regulatory factors complexin and Munc13 were added. Complexin increased the membrane separation, along with a higher propensity of point contacts. Further inclusion of the priming factor Munc13 exclusively restricted prefusion states to point contacts, all of which efficiently fused upon Ca 2+ triggering. We conclude that synaptic proteins have evolved to limit possible contact site assemblies and morphologies to those that promote fast Ca 2+ -triggered release. T he process of vesicle trafficking is central for transporting materials both inside and outside of cells and is essential for maintaining cellular homeostasis (1, 2). Synaptic vesicle fusion releases neurotransmitter molecules into the synaptic cleft upon an action potential-a fast (<5 ms) and highly regulated process. The neuronal SNARE proteins synaptobrevin-2/VAMP2 and syntaxin-1A are anchored in the synaptic vesicle and plasma membranes, respectively, whereas SNAP-25 is peripherally associated with the plasma membrane. The N-to C-terminal zippering of the trans SNARE complex between synaptic and plasma membranes provides the force for membrane juxtaposition and fusion (3), but SNAREs alone are not Ca 2+ -sensitive. Synaptotagmins are membrane-tethered proteins containing two Ca 2+ -binding C2 domains, and a subset of the 16 isoforms of mammalian synaptotagmins act as Ca 2+ sensors for neurotransmitter release: e.g., synaptotagmin-1 (Syt1) is the Ca 2+ sensor for evoked synchronous neurotransmitter release (4). The system is exquisitely fine-tuned to increase the probability of fusion between synaptic vesicles and the plasma membrane by orders of magnitude upon Ca 2+ binding to Syt1. Synaptotagmins simultaneously interact with anionic phospholipid membranes (5) and the neuronal SNARE complex (6, 7). The SNARE complex also interacts with complexin (Cpx), a small soluble protein that both activates evoked release and regulates spontaneous release (8, 9). Moreover, Cpx forms a tripartite complex with the SNARE complex and Syt1 (10). This tripartite complex is part of the prefusion, "primed" state of the synaptic vesicle fusion machinery. Both Munc13 and Munc18 are important for priming, and, at the molecular level, they facilitate proper SNARE complex assembly (11, 12).Although high-resol...

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Dynamic binding mode of a Synaptotagmin-1–SNARE complex in solution

Cited by 138 publications

References 70 publications

The primed SNARE–complexin–synaptotagmin complex for neuronal exocytosis

The primed SNARE–complexin–synaptotagmin complex for neuronal exocytosis

N-terminal domain of complexin independently activates calcium-triggered fusion

Morphologies of synaptic protein membrane fusion interfaces

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