Conformational dynamics of SNARE recycling mediated by NSF

Abstract

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.