Complexin suppresses spontaneous exocytosis by capturing the membrane-proximal regions of VAMP2 and SNAP25

Malsam, Jörg; Baerfuss, Simon; Trimbuch, Thorsten; Zarebidaki, Fereshteh; Sonnen, Andreas F.‐P.; Wild, Klemens; Scheutzow, Andrea; Sinning, Irmgard; Briggs, John; Rosenmund, Christian; Soellner, Thomas H

doi:10.1101/849885

Cited by 3 publications

(3 citation statements)

References 88 publications

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“…This model was supported by electrophysiological experiments in mouse neurons [84] and by the observation that replacement of the complexin accessory helix with an unrelated sequence with high helix propensity preserves the inhibitory function of complexin in Caenorhabditis elegans [82], as this result strongly suggests that the inhibitory activity does not arise from specific protein interactions. Conversely, some evidence suggested that weak interactions of the complexin accessory helix with SNAP‐25 and synaptobrevin inhibit release [86]. It is plausible that transient complexin‐SNARE interactions that do not necessarily need to be specific may hinder C‐terminal zippering of the four‐helix bundle, and that such interactions may occur regardless of the specific sequence of the accessory helix.…”

Section: Interactions Of Complexins With Snares and Membranesmentioning

confidence: 99%

On the difficulties of characterizing weak protein interactions that are critical for neurotransmitter release

et al. 2022

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The mechanism of neurotransmitter release has been extensively characterized, showing that vesicle fusion is mediated by the SNARE complex formed by syntaxin‐1, SNAP‐25 and synaptobrevin. This complex is disassembled by N‐ethylmaleimide sensitive factor (NSF) and SNAPs to recycle the SNAREs, whereas Munc18‐1 and Munc13s organize SNARE complex assembly in an NSF‐SNAP‐resistant manner. Synaptotagmin‐1 acts as the Ca2+ sensor that triggers exocytosis in a tight interplay with the SNAREs and complexins. Here, we review technical aspects associated with investigation of protein interactions underlying these steps, which is hindered because the release machinery is assembled between two membranes and is highly dynamic. Moreover, weak interactions, which are difficult to characterize, play key roles in neurotransmitter release, for instance by lowering energy barriers that need to be overcome in this highly regulated process. We illustrate the crucial role that structural biology has played in uncovering mechanisms underlying neurotransmitter release, but also discuss the importance of considering the limitations of the techniques used, including lessons learned from research in our lab and others. In particular, we emphasize: (a) the promiscuity of some protein sequences, including membrane‐binding regions that can mediate irrelevant interactions with proteins in the absence of their native targets; (b) the need to ensure that weak interactions observed in crystal structures are biologically relevant; and (c) the limitations of isothermal titration calorimetry to analyze weak interactions. Finally, we stress that even studies that required re‐interpretation often helped to move the field forward by improving our understanding of the system and providing testable hypotheses.

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Section: Interactions Of Complexins With Snares and Membranesmentioning

confidence: 99%

On the difficulties of characterizing weak protein interactions that are critical for neurotransmitter release

et al. 2022

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“…Complexin-II clamps spontaneous Ca 2+ -independent membrane fusion in the reconstituted assay (i.e. fusion before addition of Ca 2+ ), via laterally binding the membrane-proximal Cterminal ends of SNAP25 and VAMP2 (Malsam et al, 2020). Remarkably, V48F and D166Y showed impaired clamping by complexin, as apparent by increased fusion before Ca 2+ addition (Fig.…”

Section: V48f and D166y Mutants Show Increased Partner Snare Interact...mentioning

confidence: 86%

SNAP25 disease mutations change the energy landscape for synaptic exocytosis due to aberrant SNARE interactions

Kádková,

Murach,

Østergaard

et al. 2024

eLife

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SNAP25 is one of three neuronal SNAREs driving synaptic vesicle exocytosis. We studied three mutations in SNAP25 that cause epileptic encephalopathy: V48F, and D166Y in the Synaptotagmin-1 (Syt1) binding interface, and I67N, which destabilizes the SNARE-complex. All three mutations reduced Syt1-dependent vesicle docking to SNARE-carrying liposomes and Ca2+-stimulated membrane fusion in vitro and in neurons. The V48F and D166Y mutants (with potency D166Y > V48F) led to reduced Readily Releasable Pool (RRP) size, due to increased spontaneous (mEPSC) release and decreased priming rates. These mutations lowered the energy barrier for fusion and increased the release probability, which are gain-of-function features not found in Syt1 knockout (KO) neurons; normalized mEPSC release rates were higher (potency D166Y>V48F) than in the Syt1 KO. These mutations (potency D166Y > V48F) increased spontaneous association to partner SNAREs, resulting in unregulated membrane fusion. In contrast, the I67N mutant decreased mEPSC frequency and EPSC amplitudes due to an increase in the apparent height of the energy barrier for fusion, whereas the RRP size was unaffected. This could be partly compensated by positive charges lowering the energy barrier. Overall, pathogenic mutations in SNAP25 cause complex changes in the energy landscape for priming and fusion.

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“…The crystal structure of a complexin-1-SNARE complex showed that SNARE binding is mediated by a central helix of complexin-1 that is preceded by a so-called accessory helix [29]. The central helix is critical for all complexin functions while the accessory helix is responsible at least in part for the inhibitory activity [38,39], likely because it hinders C-terminal zippering of the SNARE complex due to steric clashes with the vesicle [34,40] and/or interactions with the SNAREs [41,42]. N-and C-terminal sequences of complexins also contribute to their dual functions through interactions with the lipids and perhaps with the SNAREs [41,[43][44][45][46][47].…”

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confidence: 99%

Analysis of tripartite Synaptotagmin‐1‐SNARE‐complexin‐1 complexes in solution

et al. 2022

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Characterizing interactions of Synaptotagmin‐1 with the SNARE complex is crucial to understand the mechanism of neurotransmitter release. X‐ray crystallography revealed how the Synaptotagmin‐1 C2B domain binds to the SNARE complex through a so‐called primary interface and to a complexin‐1‐SNARE complex through a so‐called tripartite interface. Mutagenesis and electrophysiology supported the functional relevance of both interfaces, and extensive additional data validated the primary interface. However, ITC evidence suggesting that binding via the tripartite interface occurs in solution was called into question by subsequent NMR data. Here, we describe joint efforts to address this apparent contradiction. Using the same ITC approach with the same C2B domain mutant used previously (C2BKA‐Q) but including ion exchange chromatography to purify it, which is crucial to remove polyacidic contaminants, we were unable to observe the substantial endothermic ITC signal that was previously attributed to binding of this mutant to the complexin‐1‐SNARE complex through the tripartite interface. We were also unable to detect substantial populations of the tripartite interface in NMR analyses of the ITC samples or in measurements of paramagnetic relaxation effects, despite the high sensitivity of this method to detect weak protein complexes. However, these experiments do not rule out the possibility of very low affinity (KD > 1 mm) binding through this interface. These results emphasize the need to develop methods to characterize the structure of synaptotagmin‐1‐SNARE complexes between two membranes and to perform further structure–function analyses to establish the physiological relevance of the tripartite interface.

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Complexin suppresses spontaneous exocytosis by capturing the membrane-proximal regions of VAMP2 and SNAP25

Cited by 3 publications

References 88 publications

On the difficulties of characterizing weak protein interactions that are critical for neurotransmitter release

On the difficulties of characterizing weak protein interactions that are critical for neurotransmitter release

SNAP25 disease mutations change the energy landscape for synaptic exocytosis due to aberrant SNARE interactions

Analysis of tripartite Synaptotagmin‐1‐SNARE‐complexin‐1 complexes in solution

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