Structural Biology Research Of Doc2b/Munc13-1 Complex Roles In Synaptic Exocytosis

Neurons communication depends on neurotransmitters released by synaptic vesicles.During the neurotransmitters releasing,the neurotransmitters loaded by synaptic vesicles undergo transporting,docking process and finally fusion with plasma membrane.Then the neurotransmitters are secreted into the synaptic cleft and acted on postsynaptic membrane receptors.Membrane fusion is a precise regulation process mediated by SNARE complex assembly and triggered by Ca²⁺.The assembly of SNARE complex starts with the Syntaxin-1 closed by Munc18-1.Munc13-1 acts as a key factor to catalyze the opening of the closed conformation of Syntaxin-1 to promote the formation of the SNARE complex.However,whether there is any proteins couold regulate the activity of Munc13-1.Doc2,as a candidate factor,is an important presynaptic small molecule protein.Its N-terminal MID domain can specifically interact with Munc13-1.In the Doc2 knock out neurons,spontaneous neurotransmitter release is reduced by 50%-75%,and synchronous release is not affected.In chromaffin cells,overexpression of Doc2 enhances vesicle fusion.While,infusion of excessive Doc2 MID peptides in the mouse dorsal root ganglion inhibits neurotransmitter release.The above results indicate that the interaction between Doc2 and Munc13-1 is involved in the process of synaptic vesicles Exocytosis,but the mechanism of this interaction and how it affects synaptic function are still unknown.In this article,we explored the interaction mechanism between Doc2 and Munc13-1.First,combining with GST pull down experiment,we verified the interaction between subtype Doc2b in Doc2 family and Munc13-1 MUN domain,and proved the minimal Doc2b binding region is the BC subdomain of MUN.Meanwhile,we observed that Munc13-1 could induce Doc2b to transfer to plasma membrane upon the stimulation of phorbol ester in HEK293T cells.Secondly,we found that the Doc2b MID domain is a necessary but not the strongest Doc2b-binding region by size exclusion chromatography and isothermal titration calorimetry(ITC)experiments.This conclusion provides guidance for the crystallization of the Doc2b/Munc13-1 complex.Finally,we obtained the Doc2b/Munc13-1 complex crystal after large-scale crystallization screening.Above all,this work preliminary clarified the interaction mechanism between Doc2b and Munc13-1.In the meanwhile,this work lay a good foundation for acquiring the crystal structure of the Doc2b/Munc13-1 complex and fully elucidating the molecular mechanism of the interaction between Doc2b and Munc13-1.