The NMR Analysis On The Regulatory Mechanism Of SNAP25 Loop In SNARE Complex Assembly

These SNARE complex include the plasma-membrane-associated proteins syntaxin and SNAP25,and the vesicular protein syb2.In neuronal exocytosis,SNARE proteins assemble into four alpha helices SNARE complexes,which provide the force for vesicle fusion.As a peripheral membrane protein,SNAP25 is different from other SNAREs.It has two alpha helical SNARE motifs and a flexible loop region between them.Due to its flexibility,the SNAP25 loop can respond the environment change timely and regulate the SNARE complex assembly.But the mechanism remains poorly understood because it was purposely deleted in almost all structural studies.Nuclear magnetic resonance(NMR)spectroscopy as a very useful tool provides high-resolution,atomic-level description of the structural changes,which helps to understand the conformational change of SNAP25 loop region in the assembly of SNARE complex.The SNARE complex assembles on the surface of plasma membrane.It is reported that syntaxin and syb2 would interact with membrane and convert to a prefcusion-state.SNAP25 binds membrane through the loop region,while how membrane influences the SNAP25 conformation is still not clear.We analyzed the structure of SNAP25 loop in aqueous buffer and irn membrane environment by NMR spectroscopy and CD spectropolarimetery.We found that in aqueous buffer SNAP25 loop region is flexible.However,when exposed to membrane environment,SNAP25 loop converts to a membrane-binding conformation which is more rigid and enhancs the interaction between SNAP25 and syntaxin.Thus,we suggested a model that the disorder-to-order conformational change of SNAP25 induced by membrane-binding could endow SNAP25 with binding sites for syntaxin,which promotes the assembly of SNARE complex.Phosphorylation is the most abundant post-translational modification,physiological experiment reported that SNAP25 T138 could be phosphorylated by the protein kinase A(PKA),which inhibits the SNARE complex assembly,but the specific mechanism is not clear yet.We utilized the liquid NMR to detect the phosphorylation of T138 in SNAP25 and its loop region catalyzed by PKA in vitro.We found that phosphorylation of T138 induces the conformation change of SNAP25 loop,which prevents the interaction between SNAP25 loop region and syntaxin.Furthermore,the SNARE complex assembly assay was used to detect weather the PKA-induced phosphorylation would influence the SNARE complex assembly.The results showed that PKA-induced phosphorylation inhibits the SNARE complex assembly.Based on the above results,we can draw a conclusion that PKA-induced T138 phosphorylation leads to the conformation change of SNAP25,which inhibits the interaction between SNAP25 and syntaxin.furthermore prevents the SNARE complex assembly.Zn2+ is the second most abundant trace metal in humans,regulating an enormous number of biological functions,while excess Zn2+induces damage of cells.Free Zn2+accumulate in cellular vesicles for storage and transfer into cell trough endocytosis.The physiological research shows that excess Zn2+has a negative feedback regulation for endocytosis,while the mechanism is not clear yet.Using the NMR spectra,we found that the Zn''-binding of SNAP25 induces a disorder-to-order transition in the C-terminal of SNAP25 loop region,which expands the interaction interface of SNAP25 and syntaxin further leads to a more stable mismatched t-SNARE complex.This mismatched t-SNARE complex inhibits the N-terminal of SNARE motifs interaction with syb2,which prevents the SNARE complex assembly.Liquid-liquid phase separation(LLPS)is a phenomenon that proteins form separated phase,dispersed phase(solute rich droplets)and a continuous phase(solute lean).We found that SNAP25 can form the LLPS in vitro,and the diameter of protein droplets is around 30 um.Using the scanning electron microscope(SEM),we found that in the protein droplets SNAP25 presents as the 50 nm oligomers.Furthermore,we found that mutation of R135,R136 in the C-terminal of SNAP25 loop to the aspartic acids inhibits the aggregation and LLPS of SNAP25,which indicated that the charge influences the LLPS of SNAP25.For the next step,we will analyze how the LLPS of SNAP25 influence the biological functions.