Prefusion Structures of Lipid-Bound SNARE Proteins Suggest Folding Pathways of Trans-SNARE Complex

Abstract

The assembly of three neuronal soluble N-ethylmaleimide-sensitive factor attachment protein (SNAP) receptor (SNARE) proteins synaptobrevin 2, syntaxin-1A, and SNAP-25 is the key step that leads to exocytotic fusion of synaptic vesicles. In the fully assembled SNARE complex, these three proteins form a coiled-coil four-helix bundle structure by interaction of their respective SNARE motifs. Although biochemical and mutational analyses strongly suggest that the heptad-repeat SNARE motifs zipper into the final structure in the N- to C-terminal direction, little is known about the prefusion state of individual membrane-bound SNAREs and whether they change conformation from the unzippered prefusion to the zippered postfusion state in a continuous or step-wise fashion in membrane environments. We have solved the solution NMR structures of micelle-bound synaptobrevin and syntaxin-1A in their prefusion conformations. In addition to their respective transmembrane helices, the SNARE motifs of both proteins have considerable degrees of helical content. For synaptobrevin, only the N-terminal half (residues 36-54) of the SNARE motif forms a transient helix, and the fraction of helical content and interfacial association decreases as the protein is moved from micelle to bicelle to bilayer environments, suggesting that membrane curvature affects the folding of synaptobrevin. For syntaxin, the SNARE motif consists of two well-ordered, membrane-bound helices separated by the “0-layer” residue. These unexpected structural orders of the N- and C-terminal halves of the prefusion SNARE motifs suggest the formation of partially zippered SNARE complex intermediates. Interferometric fluorescence measurements in lipid bilayers confirm that the open SNARE motif helices interact with lipid bilayers and that the assembly of SNARE complexes involves the segmented movements of N- and C-terminal halves of SNARE motifs in relation to the membrane surface.