Conformational Dynamics of SNARE Proteins during NSF‐Mediated Disassembly

Abstract

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.