α-SNAP Enhances SNARE Zippering by Stabilizing the SNARE Four-Helix Bundle

Intracellular membrane fusion is mediated by dynamic assembly and disassembly of soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein (SNAP) receptors (SNAREs). α-SNAP guides NSF to disassemble SNARE complexes after membrane fusion. Recent experiments showed that α-SNAP also dramatically enhances SNARE assembly and membrane fusion. How α-SNAP is involved in these opposing activities is not known. Here, we examine the effect of α-SNAP on the stepwise assembly of the synaptic SNARE complex using optical tweezers. We found that α-SNAP destabilized the linker domain (LD) of the SNARE complex but stabilized its C-terminal domain (CTD) through a conformational selection mechanism. In contrast, α-SNAP minimally affected assembly of the SNARE N-terminal domain (NTD), indicating that α-SNAP barely bound the partially assembled trans-SNARE complex. Thus, α-SNAP recognizes the folded CTD for SNARE disassembly with NSF and subtly modulates membrane fusion by altering the stabilities of the SNARE CTD and LD. [Display omitted] •α-SNAP destabilizes the SNARE linker domain•α-SNAP stabilizes the C-terminal domain by conformational selection•α-SNAP has no effect on N-terminal domain assembly•α-SNAP does not disassemble the partially zippered trans-SNARE complex Using a single-molecule approach, Ma et al. find that α-SNAP plays distinct roles in stepwise SNARE assembly: it destabilizes the SNARE linker domain, stabilizes the C-terminal domain, but hardly affects the N-terminal domain. The findings shed light on membrane fusion as well as α-SNAP’s opposing functions in SNARE assembly and disassembly.