Molecular mechanism of fusion pore formation driven by the neuronal SNARE complex

Abstract

Release of neurotransmitters from synaptic vesicles begins with a narrow fusion pore, the structure of which remains unresolved. To obtain a structural model of the fusion pore, we performed coarsegrained molecular dynamics simulations of fusion between a nanodisc and a planar bilayer bridged by four partially unzipped SNARE complexes. The simulations revealed that zipping of SNARE complexes pulls the polar C-terminal residues of the synaptobrevin 2 and syntaxin 1A transmembrane domains to form a hydrophilic core between the two distal leaflets, inducing fusion pore formation. The estimated conductances of these fusion pores are in good agreement with experimental values. Two SNARE protein mutants inhibiting fusion experimentally produced no fusion pore formation. In simulations in which the nanodisc was replaced by a 40-nm vesicle, an extended hemifusion diaphragm formed but a fusion pore did not, indicating that restricted SNARE mobility is required for rapid fusion pore formation. Accordingly, rapid fusion pore formation also occurred in the 40-nm vesicle system when SNARE mobility was restricted by external forces. Removal of the restriction is required for fusion pore expansion. membrane fusion | exocytosis | transmitter release | molecular dynamics | nanodisc T ransmitter release from secretory vesicles begins with the formation of a narrow fusion pore (1). The soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins form a minimal membrane fusion machinery (2). The v-SNARE synaptobrevin-2 (syb2/VAMP2) and the t-SNARE syntaxin-1 (stx1) are anchored to the vesicle and plasma membrane, respectively, by their helical transmembrane domains (TMDs) (3), while the t-SNARE SNAP-25 is anchored at the plasma membrane by four palmitoylated cysteines (4). The SNARE motifs of syb2, stx1, and SNAP-25 associate via zippering of conserved heptad repeats to form a tight four-helix bundled trans-SNARE complex (5). The zippering of SNAREs from the membrane-distal N termini toward the membrane-proximal C termini is believed to pull the two opposing membranes together to drive fusion (6).A number of studies in which either SNARE transmembrane domains were replaced by lipid anchors (7-9) or a TMD was partially deleted (10), or in which the C terminus of the syb2 TMD was extended by polar residues (11), indicated that fusion was inhibited or even arrested, pointing to an important function of the TMD in fusion pore formation. The precise nanomechanical mechanism of fusion and the structure of the nascent pore remain unclear. One model proposes a lipid-based fusion pore (12), while other studies have suggested a proteinaceous fusion pore with the TMDs of stx1 and syb2 lining the fusion pore, like an ion channel or gap junction pore (9). Recent experiments using varied-sized nanodiscs (NDs) (13) support the hypothesis that the fusion pore is a hybrid structure composed of both lipids and proteins (14); however, there is still no structural model of the fusion pore.Conventional in vitro reconstitution e...

Resolving kinetic intermediates during the regulated assembly and disassembly of fusion pores

“…These finding suggest that v-SNARE density (ND3 S , 0.022 syb2/nm 2 , ND3 L : 0.0042 syb2/nm 2 ) and not just copy number is a crucial parameter for pore formation. Indeed, a recent modeling study suggested that restricting the mobility of SNAREs, to increase their relative density, facilitates pore formation 25 . In sharp contrast, when syt1 was co-reconstituted into ND3 L , robust pore formation occurred upon addition of Ca 2+ (Fig.…”

Influenza hemagglutinin drives viral entry via two sequential intramembrane mechanisms

“…Within this slice, lipids from the original stalk structure were maintained, and the remainder of the small vesicle was replaced with the large vesicle. This protocol was designed in analogy to that previously performed for nascent fusion pores (20). This hybrid stalk structure was then equilibrated using position restraints on 1) the phosphate groups of all lipids involved in the stalk, 2) the hemagglutinin fusion peptides, and 3) the hemagglutinin transmembrane domains.…”

Computational Modeling of Realistic Cell Membranes

“…Additional details of the fusion pathway were resolved in simulations of SNARE mediated fusion between bilayers and nanodisks. 587 In the work of Pinot et al, 588 a combination of in vivo, in vitro, and in silico experiments were used to show the combined effect of lipids and proteins in shaping membranes during fission. In particular, the role of polyunsaturated lipids in membrane vesiculation by dynamin and endophilin was revealed.…”