Re‐examining how Munc13‐1 facilitates opening of syntaxin‐1

Abstract

Munc13-1 is crucial for neurotransmitter release and, together with Munc18-1, orchestrates assembly of the neuronal SNARE complex formed by syntaxin-1, SNAP-25, and synaptobrevin. Assembly starts with syntaxin-1 folded into a selfinhibited closed conformation that binds to Munc18-1. Munc13-1 is believed to catalyze the opening of syntaxin-1 to facilitate SNARE complex formation. However, different types of Munc13-1-syntaxin-1 interactions have been reported to underlie this activity, and the critical nature of Munc13-1 for release may arise because of its key role in bridging the vesicle and plasma membranes. To shed light into the mechanism of action of Munc13-1, we have used NMR spectroscopy, SNARE complex assembly experiments, and liposome fusion assays. We show that point mutations in a linker region of syntaxin-1 that forms intrinsic part of the closed conformation strongly impair stimulation of SNARE complex assembly and liposome fusion mediated by Munc13-1 fragments, even though binding of this linker region to Munc13-1 is barely detectable. Conversely, the syntaxin-1 SNARE motif clearly binds to Munc13-1, but a mutation that disrupts this interaction does not affect SNARE complex assembly or liposome fusion. We also show that Munc13-1 cannot be replaced by an artificial tethering factor to mediate liposome fusion. Overall, these results emphasize how very weak interactions can play fundamental roles in promoting conformational transitions and strongly support a model whereby the critical nature of Munc13-1 for neurotransmitter release arises not only from its ability to bridge two membranes but also from an active role in opening syntaxin-1 via interactions with the linker. K E Y W O R D S conformational transition, membrane fusion, Munc13, Munc18, neurotransmitter release, SNAREs, syntaxin-1

Ca2+-dependent release of synaptotagmin-1 from the SNARE complex on phosphatidylinositol 4,5-bisphosphate-containing membranes

“…However, the Syt1 oligomers are compatible with the state proposed in Figure 10A ( Tagliatti et al, 2020 ) and hence can be readily incorporated into the model of Figure 10C . Note also that, while our NMR studies could not detect binding of the Syt1 C 2 B domain to CpxSC through the tripartite interface ( Figures 2E and 3 ), it is premature to completely rule out the relevance of the tripartite structure given the potential functional importance of very weak interactions ( Magdziarek et al, 2020 ). Nevertheless, it is also premature to accept this structure as physiologically relevant, and further evidence for such relevance needs to be obtained, ideally using mutations that exclusively replace residues in the interface rather than interior residues that are important for protein stability.…”

“…This task is hindered by the fact that relevant interactions may be necessarily weak because of the very nature of this dynamic system, which is expected to undergo quick, drastic rearrangements during the events that lead to fast membrane fusion upon Ca 2+ influx. Weak interactions can be dramatically enhanced by co-localization within the confines of a primed synaptic vesicle, but they must be distinguished from other, perhaps stronger but irrelevant interactions that are detectable in vitro (Magdziarek et al, 2020). Another complicating aspect is that basic sequences such as the polybasic region and R398,R399 can bind to SNAREs and to membranes, both of which are acidic, and binding to a wrong molecule can occur in the absence of the bona fide target.…”

The Interaction of Munc18-1 Helix 11 and 12 with the Central Region of the VAMP2 SNARE Motif Is Essential for SNARE Templating and Synaptic Transmission

“…Importantly, Munc13-1 assists template complex formation by inducing a conformational change within the syntaxin-1 linker region between the Habc and SNARE domains (Ma et al, 2011;Ma et al, 2013;Yang et al, 2015;Wang et al, 2017;Magdziarek et al, 2020). Furthermore, Munc13-1 stabilizes the template complex by interacting with the membrane proximal linker region of VAMP2 (Wang et al, 2019;Shu et al, 2020).…”

“…Distinct mutations in domain 3a that favor the open conformation of Munc18-1 yield gain of function phenotypes ( Parisotto et al, 2014 ; Sitarska et al, 2017 ; Jiao et al, 2018 ). Importantly, Munc13-1 assists template complex formation by inducing a conformational change within the syntaxin-1 linker region between the Habc and SNARE domains ( Ma et al, 2011 , 2013 ; Yang et al, 2015 ; Wang et al, 2017 ; Magdziarek et al, 2020 ). Furthermore, Munc13-1 stabilizes the template complex by interacting with the membrane proximal linker region of VAMP2 ( Wang et al, 2019 ; Shu et al, 2020 ).…”

Phenotypic Characteristics and Copy Number Variants in a Cohort of Colombian Patients with VACTERL Association

“…Silencing of fcho1 in zebrafish embryos causes alterations in morphogenesis [Umasankar et al, 2012]. The UNC13A/MUNC13A-1 gene is evolutionarily conserved and involved in vesicle maturation during exocytosis in the synaptic process [Brose and Rosenmund, 2002;Magdziarek et al, 2020]. Homozygous mutations in this gene have been associated with microcephaly and abnormal cortical electrical activity [Engel et al, 2016].…”