Multiple conformations of a single SNAREpin between two nanodisc membranes reveal diverse pre-fusion states

Abstract

SNAREpins must be formed between two membranes to allow vesicle fusion, a required process for neurotransmitter release. Although its post-fusion structure has been well characterized pre-fusion conformations have been elusive. We used single molecule FRET and EPR to investigate the SNAREpin assembled between two nanodisc membranes. The SNAREpin shows at least three distinct dynamic states, which might represent pre-fusion intermediates. While the N-terminal half above the conserved ionic layer maintains a robust helical bundle structure the membrane-proximal C-terminal half shows either high FRET representing a helical bundle (45%), low FRET reflecting a frayed conformation (39%), or mid FRET revealing an yet unidentified structure (16%). It is generally thought that SNAREpins are trapped at a partially zipped conformation in the pre-fusion state, and complete SNARE assembly happens concomitantly with membrane fusion. However, our results show that the complete SNARE complex can be formed without membrane fusion, which suggests that the complete SNAREpin formation could precede membrane fusion, providing an ideal access to the fusion regulators such as complexins and synaptotagmin 1.

Synaptotagmin-1 Is an Antagonist for Munc18-1 in SNARE Zippering

“…2C in Ref. 41), but we did not observe an appreciable mid-FRET population for the NN. In contrast, the SNAREpin with the CC gave two major low and high FRET distributions with some population at mid-FRET (Fig.…”

“…Protein Expression and Purification-Protein expression and purification were described previously (41,43). Briefly, all recombinant proteins were expressed in Escherichia coli BL21 (DE3).…”

“…The v-SNARE-reconstituted nanodiscs (v-discs) were then flowed into the flow cell to allow docking and trans-SNAREpin formation (Fig. 1B) (41).…”

“…In this work, we investigated, using single molecule FRET (smFRET), the conformational changes of a trans-SNARE com-plex (or SNAREpin) assembled between two nanodisc membranes (41) induced by Munc18-1. We also studied the effect of Munc18-1 on SNARE-dependent proteoliposome lipid mixing and on the Ca 2ϩ -triggered fusion pore opening in well defined in vitro settings (39,42) to dissect the Munc18-1 function in the presence of Syt1.…”

“…Fluorophore Labeling of the Single Cysteine Mutants-The fluorophore labeling of the single cysteine mutants of syntaxin-1a and VAMP2 was described previously (41,44). Briefly, the mutants were purified as described above for syntaxin-1a and VAMP2 except that the cleavage buffer contained 2.5 mM DTT.…”