Neurotransmitter release via synaptic vesicle exocytosis is mediated by the dynamic assembly and disassembly of the neuronal SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) complex, which consists of syntaxin-1, SNAP-25, and synaptobrevin-2. Despite their importance, the molecular mechanism of SNARE complex recycling remains unclear. Individual SNARE proteins are intrinsically disordered and undergo a disorder-to-order transition, which assembles into a highly stable four-helix bundle, providing the energy required to drive membrane fusion between the synaptic vesicle and the plasma membrane. The AAA+ protein NSF later disassembles the SNARE complex to maintain a pool of the individually functional SNARE proteins to be utilized for recurring rounds of synaptic vesicle fusion. Using single-molecule fluorescence resonance energy transfer (smFRET), we examined the stepwise conformational dynamics of individual SNARE proteins during NSF-mediated disassembly and reassembly of the SNARE complex. Interestingly, the disorder-to-order transition of the SNARE proteins during SNARE complex assembly was reversible by NSF-mediated disassembly, where the SNARE chaperone Munc18 preserved the intrinsically disordered state of SNAP-25 and synaptobrevin-2 by locking syntaxin-1 in an inhibiting closed conformation. Moreover, we observed a transient "entangled" conformation of SNAP-25 during the reassembly process of the SNARE complex. Together, NSF acts as a protein quality control mechanism for efficient membrane fusion via proper assembly of the SNARE complex.

Neurotransmitter release of synaptic vesicles relies on the assembly of the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex, consisting of syntaxin and SNAP-25 on the plasma membrane and synaptobrevin on the synaptic vesicle. The formation of the SNARE complex progressively zippers towards the membranes, which drives membrane fusion between the plasma membrane and the synaptic vesicle. However, the underlying molecular mechanism of SNARE complex regulation is unclear. In this study, we investigated the syntaxin-3b isoform found in the retinal ribbon synapses using single-molecule fluorescence resonance energy transfer (smFRET) to monitor the conformational changes of syntaxin-3b that modulate the SNARE complex formation. We found that syntaxin-3b is predominantly in a self-inhibiting closed conformation, inefficiently forming the ternary SNARE complex. Conversely, a phosphomimetic mutation (T14E) at the N-terminal region of syntaxin-3b promoted the open conformation, similar to the constitutively open form of syntaxin LE mutant. When syntaxin-3b is bound to Munc18-1, SNARE complex formation is almost completely blocked. Surprisingly, the T14E mutation of syntaxin-3b partially abolishes Munc18-1 regulation, acting as a conformational switch to trigger SNARE complex assembly. Thus, we suggest a model where the conformational change of syntaxin-3b induced by phosphorylation initiates the release of neurotransmitters in the ribbon synapses.

SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins play key roles in cellular processes involved in vesicle trafficking and neurotransmitter release of synaptic vesicles. Neuronal SNAREs are composed of syntaxin-1A, SNAP-25A and synaptobrevin-2, forming a highly stable four-helix bundle to provide the energy necessary to drive membrane fusion between the synaptic vesicle and the plasma membrane. However, the mechanistic details underlying SNARE complex disassembly is largely unknown. NSF (N-ethylmaleimide-sensitive factor), a member of AAA+ ATPase family, together with αSNAP (alpha soluble NSF attachment protein), disassembles the post-fusion SNARE complex to maintain the pool of individual SNARE proteins for recurring rounds of vesicle fusion. Using smFRET (single-molecule fluorescence resonance energy transfer), we monitored the conformational dynamics of individual SNARE proteins during the NSF-mediated disassembly process. Interestingly, all three SNARE proteins underwent order-disorder transitions during disassembly and vice versa during re-assembly into SNARE complex. In addition, we discovered a unique intermediate conformational pathway of SNAP-25A during NSF-mediated disassembly process. This notion is in line with recent findings where the N-terminus of SNAP-25A engages through the D1 pore of NSF, likely involving conformational changes. Taken together, we suggest a novel conformational pathway essential for regulating SNARE complex disassembly as well as assembly during synaptic vesicle fusion.