Conformational Dynamics of SNARE Recycling Mediated by NSF.

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.