Studies Of The Interaction Between Sec1/Munc18 (Sm) Protein And SNARE Protein SNAP-25

Fusion of synaptic vesicles with the presynaptic membrane releases neurotransmitters into the synaptic cleft,allowing neurotransmitters to bind to postsynaptic membrane receptors to complete the transmission of chemical signals between neurons.The SNARE proteins syntaxin-1,synaptobrevin-2 and SNAP-25 mediate the fusion of synaptic vesicles with the plasma membrane by assembling into a four-helix bundle called the SNARE complex.This process is strictly regulated by a series of proteins,such as Sec1/Munc18?SM?protein Munc18 and UNC13/Munc13 protein Munc13-1.Munc18 and syntaxin-1cound form the complex to initiate SNARE complex assembly.SM protein Vps33?a subunit of HOPS complex?orientates Nyv1?synaptobrevin-2 homolog in yeast?and Vam3?syntaxin-1 homolog in yeast?to template vacuolar SNARE complex formation.Recent studies have demonstrated that Sec1/Munc18?SM?family protein Munc18 could also serve as a template to interact with syntaxin-1 and synaptobrevin-2,thus facilitating SNARE complex assembly as Vps33 does.However,how SNAP-25 will participate in Munc18-templated SNARE complex assembly remains unclear.We utilized structural biology approaches to explore the interaction properties between the Squid Munc18homolog s-Sec1 and SNAP-25.This work includes the following aspects:1.Native-PAGE and fluorescence anisotropy experiments showed that there is an interaction between s-Sec1 and SNAP-25,further,the second SNARE motif of SNAP-25?SN2?contributes to the interaction.2.Recombinant fusion proteins of s-Sec1 and SN2 were constructed and linkered through a 15-amino acid glycine/serine linker(GS¹⁵)or a 23-amino acid linker(linker²³).3.Recombinant fusion proteins SN2-GS¹⁵-Sec1 and SN2-linker²³-Sec1 were expressed and purified for crystallization experiments.4.Crystals of SN2-GS¹⁵-Sec1 were obtained and suitable for X-ray diffration.The date sets are integrated and solved at a resolution of 3.2?,but the final model only contains isolated s-Sec1,without proper electron densities of SN2.This work preliminarily explores the actions of Sec1/Munc18?SM?protein Munc18in regulating SNARE complex assembly by interacting with SNARE protein SNAP-25.Elucidating the interaction between s-Sec1 and SNAP-25 would shed new light on the regulation mechanism of Sec1/Munc18?SM?protein Munc18 in SNARE complex assembly and neurotransmitter release.