Function Mechanism Of Munc13-1 MUN Domain In Synaptic Vesicle Priming

Neurotransmitter release mediated by Ca2+-triggered synaptic vesicle exocytosis is a fundamental process for synaptic transmission.The core machinery for this process includes the neuronal SNAREs syntaxin-1,SNAP-25 and synaptobrevin.The three SNAREs assemble into a tight SNARE complex that consists of a four-helix bundle,which can bring the membranes into close proximity.In addition,the Sec1-Munc18 protein Munc 18-1,which involved in SNARE complex assembly through its interactions with neuronal SNAREs,is essential for vesicle exocytosis.Rescent studies showed that UNC-13-Munc13s perform a crucial function in synaptic vesicle priming,and regulate SNARE complex assembly by starting with the Munc18-1-syntaxin-1 complex through its MUN domain.However,the structure of the MUN domain has not been solved,and the relevance between the priming function of UNC-13-Munc13s in vivo and the activity of the MUN domain in opening syntaxin-1 in vitro remains unclear.The main findings of this study were summarized as follows:Crystal structure of the rat Munc13-1 MUN domain at 2.9 A resolution was obtained.The structure reveals an elongated arch-shaped architecture formed by ?-helical bundles,with a highly conserved hydrophobic pocket located at the junction of B and C subdomains.Key residues responsible for Munc13-1 MU-N domain's activity in opening syntaxin-1 were identified.Using fluorescence resonance energy transfer(FRET)and reconstitution experiments,we identified two highly conserved residues Asn1128,Phe1131(NF)located in the middle of the MUN domain,as key sites for stimulation on SNARE complex assembly and SNARE complex dependent proteoliposome fusion in vitro.Essential role of residues NF in synaptic vesicle priming was demonstrated.Electrophysiological data recorded at cholinergic neuromuscular junctions(NMJs)of Caenorhabditis elegans showed that the residues NF are important for both spontaneous and evoked neurotransmitter release,and mutation of residues NF abrogates synaptic vesicle priming.These compelling evidence support the notion that the activity of the MUN domain in stimulating the transition from the Munc 18-1-closed syntaxin-1 complex to the SNARE complex underlies the crucial function of UNC-13-Muncl3s in synaptic vesicle priming.