Structural Biology Research Of The Interaction Between Doc2? And Munc13-1 In Synaptic Vesicle Exocytosis

Neurons contact with each other via a specific structure called synapses.Synaptic exocytosis is a quite elaborate process,which is regulated by multiple interactions,including protein-protein,protein-membrane and membrane–membrane interactions.Munc13-1 and Doc2?play essential roles in regulating neurotransmitter release.Munc13-1 promotes transition of syntaxin-1 from closed conformation to open conformation,accelerating assembly of the SNARE complex.Doc2?is considered to be a Ca²⁺sensor in asynchronous and spontaneous release.Disruption of the interaction between Doc2?and Munc13-1 inhibits release in PC12 and cholinergic synapse,indicating the physiological significance of this interaction.However,the mechanism underlies this interaction remains unclear.Understanding this specific mechanism is of great significance to better explore the function of Doc2?and Munc13-1 in neurotransmitter release.Cotranslocation of Doc2?and Munc13-1 in Neuro-2a cells suggested that Doc2?can interact with Munc13-1 in vivo.The Neuro-2a cells were subjected to 70 mM KCl applications which caused elevation of[Ca²⁺]_i and translocation of Doc2?to the plasma membrane.Through the interaction with Munc13-1,Doc2?recruited Munc13-1 to the plasma membrane.According to early studies,the minimum interacting regions of the Doc2?-Munc13-1 are the Mid domain?residues 13-37?of Doc2?and the MUN domain?residues 851–1461?of Munc13-1.The interaction between Doc2?and MUN domain has been proved by GST pull-down assay.MUN domain is a large domain,which is divided into four subdomains?A,B,C,D?.We designed a series of MUN domain truncations to further determine the interaction sites and narrowed down the interaction region of the MUN domain to MUN BC?residues 1011-1407?.In this issue,we seeked for the specific binding sites in MUN BC subdomain through Site-Directed Mutagenesis technique;on the other hand,we expected to obtain protein crystals of MUN⁹³³/Mid complex to elucidate the mechanism of the interaction.The crystal structure of MUN⁹³³ has been resolved.The MUN⁹³³ is well-characterized and can interact with Doc2?.To obtain the crystal of MUN⁹³³/Mid complex,two different methods were carried out:?1?acquiring crystal of MUN⁹³³ first,then adding Mid domain protein to the MUN⁹³³ crystals;?2?purifying and crystallizing MUN⁹³³/Mid complex directly.After mass screening,we finally obtained the MUN⁹³³/Mid crystals good enough for X-ray diffraction.This paper verified the interaction between Doc2?and Munc13-1 in vitro and in vivo and narrowed down the interaction region of the MUN domain to MUN BC?residues1011-1407?through GST pull-down assays.We tried to cocrystalize MUN⁹³³ and Doc2?Mid domain and finally obtained the crystal competent for X-ray diffraction,thus paving the way for better understanding physiological significance of the interaction between Doc2?and Munc13-1.