Entropic forces drive self-organization and membrane fusion by SNARE proteins

Abstract

SNARE proteins are the core of the cell's fusion machinery and mediate virtually all known intracellular membrane fusion reactions on which exocytosis and trafficking depend. Fusion is catalyzed when vesicle-associated v-SNAREs form trans-SNARE complexes ("SNAREpins") with target membrane-associated t-SNAREs, a zippering-like process releasing ∼65 kT per SNAREpin. Fusion requires several SNAREpins, but how they cooperate is unknown and reports of the number required vary widely. To capture the collective behavior on the long timescales of fusion, we developed a highly coarse-grained model that retains key biophysical SNARE properties such as the zippering energy landscape and the surface charge distribution. In simulations the ∼65-kT zippering energy was almost entirely dissipated, with fully assembled SNARE motifs but uncomplexed linker domains. The SNAREpins self-organized into a circular cluster at the fusion site, driven by entropic forces that originate in steric-electrostatic interactions among SNAREpins and membranes. Cooperative entropic forces expanded the cluster and pulled the membranes together at the center point with high force. We find that there is no critical number of SNAREs required for fusion, but instead the fusion rate increases rapidly with the number of SNAREpins due to increasing entropic forces. We hypothesize that this principle finds physiological use to boost fusion rates to meet the demanding timescales of neurotransmission, exploiting the large number of v-SNAREs available in synaptic vesicles. Once in an unfettered cluster, we estimate ≥15 SNAREpins are required for fusion within the ∼1-ms timescale of neurotransmitter release.SNARE | membrane fusion | exocytosis | entropic force | neurotransmitter release E xocytosis and trafficking in cells depend on membrane fusion reactions, almost all of which are mediated by SNARE proteins (1-3). Fusion is catalyzed by the assembly of vesicleassociated v-SNAREs and target membrane-associated t-SNAREs into coiled coils of α-helices to form trans-SNARE complexes ("SNAREpins"). Assembly initiates at the N-terminal ends of the SNARE motifs and propagates toward the C-terminal transmembrane domains (TMDs) in a zipper-like fashion, pulling the membranes into close proximity and triggering fusion by a mechanism that remains poorly understood.An unanswered question is how SNARE proteins deliver the energy required to fuse membranes, estimated to be ∼40-140 kT (4-6). A common view is that the ∼65 kT per SNAREpin released during zippering (7) provides the necessary energy, but how this is coupled to the membranes is unclear given that the uncomplexed v-SNARE is largely unstructured and presumably flexible (8). It has been proposed that the linker domains (LDs) connecting the SNARE motifs to the TMDs are stiff, so that some zippering energy could be stored as LD bending energy that could deform and fuse membranes (3, 9). Such an effect would presumably be abolished by even a single flexible region. However, EPR spectroscopy suggests linkers are partial...

SNARE‐mediated membrane fusion is a two‐stage process driven by entropic forces

“…Here we addressed this challenge by developing a molecular dynamics description with SNAREs systematically but radically coarse‐grained, to such a degree that we could simulate the collective behavior of up to 20 SNAREpins on timescales up to ~100 μs. The results are consistent with our earlier study of the long time equilibrated behavior of the SNAREpins using a Monte Carlo approach which can only describe equilibrium . There we estimated fusion times using a scaling argument for the timescales and by fitting to an in vitro measurement of fusion time .…”

“…), consistent with the large spread in reported SNARE requirements for fusion . Given that any number can catalyze fusion, different apparent requirements may emerge from different experimental methods depending on the timescale probed . Further, these conclusions suggest that, in the interests of efficiency, the number of SNAREpins used at synaptic terminals to achieve membrane fusion may equal the minimum number required to achieve the sub‐millisecond timescales necessary for proper neurotransmission.…”

“…How then do the SNAREs accomplish fusion? In a recent simulation study, we showed that the nanoscale confinement of the membranes imposed by full SNARE zippering is sufficient in itself to catalyze fusion, driven by entropic forces among SNAREpins and membranes [29]. Experiment and mathematical modeling also suggest a post-fusion role for such entropic forces, whereby SNAREs regulate the size of the fusion pore and its release kinetics [32].…”

The fusion pore, 60 years after the first cartoon

“…However, zippering of several nearby SNARE complexes likely leads to formation of a ring‐shaped arrangement followed by entropically driven radial expansion. This expansion will pull the membranes together with high force depending on the number of SNARE complexes . Given the increased distance between the SNARE complexes, this will likely produce a hemifused zone as observed in the experiments described above.…”

“…At least three copies of SNAP25 appear to be required for fast fusion in chromaffin cells . It has been suggested that the fusion rate may increase with increasing numbers of SNARE complexes and that activation of fusion within 1 ms may require a cluster of at least 15 SNARE complexes . These results suggest that fusion pores may be formed by a variable number of SNARE complexes leading to fusion pore properties that depend on this number.…”