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Evolutionarily conserved SNARE (soluble N -ethylmaleimide sensitive factor attachment protein receptors) proteins form a complex that drives membrane fusion in eukaryotes. The ATPase NSF ( N -ethylmaleimide sensitive factor), together with SNAPs (soluble NSF attachment protein), disassembles the SNARE complex into its protein components, making individual SNAREs available for subsequent rounds of fusion. Here we report structures of ATP- and ADP-bound NSF, and the NSF/SNAP/SNARE (20S) supercomplex determined by single-particle electron cryomicroscopy at near-atomic to sub-nanometre resolution without imposing symmetry. Large, potentially force-generating, conformational differences exist between ATP- and ADP-bound NSF. The 20S supercomplex exhibits broken symmetry, transitioning from six-fold symmetry of the NSF ATPase domains to pseudo four-fold symmetry of the SNARE complex. SNAPs interact with the SNARE complex with an opposite structural twist, suggesting an unwinding mechanism. The interfaces between NSF, SNAPs, and SNAREs exhibit characteristic electrostatic patterns, suggesting how one NSF/SNAP species can act on many different SNARE complexes. Using single-particle electron cryomicroscopy, several structures are reported which illuminate the mechanisms of action of the ATPase NSF that disassembles the SNARE complex into individual protein components. SNARE protein recycling mechanism In a variety of cellular processes — including neurotransmitter release, hormone release and vesicle trafficking — the evolutionarily conserved SNARE proteins form a complex that drives fusion between membranes of two cellular compartments. Once fusion occurs, these complexes are disassembled by the ATPase enzyme NSF and the SNAP adaptor proteins to recycle individual SNAREs for another round of membrane fusion. This study reports the use of single-particle electron cryomicroscopy to determine sub-nanometre to near-atomic resolution structures of NSF and the 20S complex. This paper reports the structures of full-length NSF in ATP- and ADP-bound states, and those of the roughly 660-kilodalton NSF/SNAP/SNARE (20S) super-complex involving two different SNARE complexes. The authors' data provide unprecedented details of the inner-workings of these essential molecular machines.

The recycling of SNARE proteins following complex formation and membrane fusion is an essential process in eukaryotic trafficking. A highly conserved AAA+ protein, NSF (N-ethylmaleimide sensitive factor) and an adaptor protein, SNAP (soluble NSF attachment protein), disassemble the SNARE complex. We report electron-cryomicroscopy structures of the complex of NSF, αSNAP, and the full-length soluble neuronal SNARE complex (composed of syntaxin-1A, synaptobrevin-2, SNAP-25A) in the presence of ATP under non-hydrolyzing conditions at ~3.9 Å resolution. These structures reveal electrostatic interactions by which two αSNAP molecules interface with a specific surface of the SNARE complex. This interaction positions the SNAREs such that the 15 N-terminal residues of SNAP-25A are loaded into the D1 ring pore of NSF via a spiral pattern of interactions between a conserved tyrosine NSF residue and SNAP-25A backbone atoms. This loading process likely precedes ATP hydrolysis. Subsequent ATP hydrolysis then drives complete disassembly.

Synaptic vesicles containing neurotransmitters fuse with the plasma membrane upon the arrival of an action potential at the active zone. Multiple proteins organize trans-SNARE complex assembly and priming, leading to fusion. One target membrane SNARE, syntaxin, forms nanodomains at the active zone, and another, SNAP-25, enters non-fusogenic complexes with it. Here, we reveal mechanistic details of AAA+ protein NSF (N-ethylmaleimide sensitive factor) and SNAP (soluble NSF attachment protein) action before fusion. We show that syntaxin clusters are conserved, that NSF colocalizes with them, and characterize SNARE populations that may exist within or near them using cryo-EM. Supercomplexes of NSF, α-SNAP, and either a syntaxin tetramer or one of two binary complexes of syntaxin—SNAP-25 reveal atomic details of SNARE processing and show how sequential ATP hydrolysis drives disassembly. These results suggest a functional role for syntaxin clusters as reservoirs and a corresponding role for NSF in syntaxin liberation and SNARE protein quality control preceding fusion. NSF hydrolyzes ATP to disassemble SNARE complexes. Here, the authors find NSF colocalizes with syntaxin nanodomains, reveal disassembly of syntaxin oligomers and other pre-fusion cis-SNARE complexes by NSF, and show how sequential hydrolysis drives disassembly.

Proteins with Tetratricopeptide-repeat (TPR) domains are encoded by large gene families and distributed in all plant lineages. In this study, the Soluble NSF-Attachment Protein (SNAP) subfamily of TPR containing proteins is characterized. In soybean, five members constitute the SNAP gene family: GmSNAP18, GmSNAP11, GmSNAP14, GmSNAP02, and GmSNAP09 . Recently, GmSNAP18 has been reported to mediate resistance to soybean cyst nematode (SCN). Using a population of recombinant inbred lines from resistant and susceptible parents, the divergence of the SNAP gene family is analysed over time. Phylogenetic analysis of SNAP genes from 22 diverse plant species showed that SNAPs were distributed in six monophyletic clades corresponding to the major plant lineages. Conservation of the four TPR motifs in all species, including ancestral lineages, supports the hypothesis that SNAPs were duplicated and derived from a common ancestor and unique gene still present in chlorophytic algae. Syntenic analysis of regions harbouring GmSNAP genes in soybean reveals that this family expanded from segmental and tandem duplications following a tetraploidization event. qRT-PCR analysis of GmSNAPs indicates a co-regulation following SCN infection. Finally, genetic analysis demonstrates that GmSNAP11 contributes to an additive resistance to SCN. Thus, GmSNAP11 is identified as a novel minor gene conferring resistance to SCN.

N-ethylmaleimide-sensitive factor （NSF） and a soluble NSF attachment proteins （a-SNAPs） work together within a 20S particle to disassemble and recycle the SNAP receptor （SNARE） complex after intracellular membrane fusion. To understand the disassembly mechanism of the SNARE complex by NSF and a-SNAP, we performed single-parti- cle cryo-electron microscopy analysis of 20S particles and determined the structure of the a-SNAP-SNARE assembly portion at a resolution of 7.35 ~. The structure illustrates that four a-SNAPs wrap around the single left-handed SNARE helical bundle as a right-handed cylindrical assembly within a 20S particle. A conserved hydrophobic patch connecting helices 9 and 10 of each a-SNAP forms a chock protruding into the groove of the SNARE four-helix bun- dle. Biochemical studies proved that this structural element was critical for SNARE complex disassembly. Our study suggests how four a-SNAPs may coordinate with the NSF to tear the SNARE complex into individual proteins.

R-SNAREs (soluble N-ethylmaleimide–sensitive factor receptor), Q-SNAREs, and Sec1/Munc18 (SM)-family proteins are essential for membrane fusion in exocytic and endocytic trafficking. The yeast vacuolar tethering/SM complex HOPS (homotypic fusion and vacuole protein sorting) increases the fusion of membranes bearing R-SNARE to those with 3Q-SNAREs far more than it enhances their trans-SNARE pairings. We now report that the fusion of these proteoliposomes is also supported by GST-PX or GST-FYVE, recombinant dimeric proteins which tether by binding the phosphoinositides in both membranes. GST-PX is purely a tether, as it supports fusion without SNARE recognition. GST-PX tethering supports the assembly of new, active SNARE complexes rather than enhancing the function of the fusion-inactive SNARE complexes which had spontaneously formed in the absence of a tether. When SNAREs are more disassembled, as by Sec17, Sec18, and ATP (adenosine triphosphate), HOPS is required, and GST-PX does not suffice. We propose a working model where tethering orients SNARE domains for parallel, active assembly.

The soluble N-ethylmaleimide-sensitive factor attachment protein alpha (α-SNAP) is essential for vesicle trafficking, coordinating trans-SNARE zippering and cis-SNARE disassembly. α-SNAP also regulates autophagy, apoptosis, calcium signaling, and AMPK activity. The hyh missense mutation M105I produces a distinctive neurodevelopmental phenotype, yet its pathogenic mechanism remains unclear. Because many α-SNAP functions rely on lipid binding, we examined whether M105I alters this property. In silico modeling revealed structural rearrangements that conceal the N-terminal hydrophobic loop, and molecular dynamics simulations predicted reduced binding free energy and weakened protein–lipid interactions. These predictions were validated in vitro and in hyh mouse brains, showing diminished membrane association, particularly at the plasma membrane. Liposome flotation assays with plasma-membrane–derived lipids confirmed that M105I directly impairs lipid binding and that membrane composition influences this interaction. Thus, defective lipid engagement emerges as a central determinant of α-SNAP dysfunction and likely contributes to the pathogenesis of hyh phenotype. Combining molecular dynamics, in vitro assays, and subcellular analyses in mouse brains reveals that the α-SNAP M105I mutation conceals a hydrophobic loop and reduces its affinity for plasma membrane lipids, uncovering the basis of the hyh phenotype.

At physiological protein levels, the slow HOPS- and SNARE-dependent fusion which occurs upon complete SNARE zippering is stimulated by Sec17 and Sec18:ATP without requiring ATP hydrolysis. To stimulate, Sec17 needs its central residues which bind the 0-layer of the SNARE complex and its N-terminal apolar loop. Adding a transmembrane anchor to the N-terminus of Sec17 bypasses this requirement for apolarity of the Sec17 loop, suggesting that the loop functions for membrane binding rather than to trigger bilayer rearrangement. In contrast, when complete C-terminal SNARE zippering is prevented, fusion strictly requires Sec18 and Sec17, and the Sec17 apolar loop has functions beyond membrane anchoring. Thus Sec17 and Sec18 act twice in the fusion cycle, binding to trans-SNARE complexes to accelerate fusion, then hydrolyzing ATP to disassemble cis-SNARE complexes.

The homotypic fusion of yeast vacuoles, each with 3Q‐ and 1R‐SNARE, requires SNARE chaperones (Sec17p/Sec18p and HOPS) and regulatory lipids (sterol, diacylglycerol and phosphoinositides). Pairs of liposomes of phosphatidylcholine/phosphatidylserine, bearing three vacuolar Q‐SNAREs on one and the R‐SNARE on the other, undergo slow lipid mixing, but this is unaffected by HOPS and inhibited by Sec17p/Sec18p. To study these essential fusion components, we reconstituted proteoliposomes of a more physiological composition, bearing vacuolar lipids and all four vacuolar SNAREs. Their fusion requires Sec17p/Sec18p and HOPS, and each regulatory lipid is important for rapid fusion. Although SNAREs can cause both fusion and lysis, fusion of these proteoliposomes with Sec17p/Sec18p and HOPS is not accompanied by lysis. Sec17p/Sec18p, which disassemble SNARE complexes, and HOPS, which promotes and proofreads SNARE assembly, act synergistically to form fusion‐competent SNARE complexes, and this synergy requires phosphoinositides. This is the first chemically defined model of the physiological interactions of these conserved fusion catalysts.

The AAA+ATPase p97/VCP, helped by adaptor proteins, exerts its essential role in cellular events such as endoplasmic reticulum‐associated protein degradation or the reassembly of Golgi, ER and the nuclear envelope after mitosis. Here, we report the three‐dimensional cryo‐electron microscopy structures at ∼20 Å resolution in two nucleotide states of the endogenous hexameric p97 in complex with a recombinant p47 trimer, one of the major p97 adaptor proteins involved in membrane fusion. Depending on the nucleotide state, we observe the p47 trimer to be in two distinct arrangements on top of the p97 hexamer. By combining the EM data with NMR and other biophysical measurements, we propose a model of ATP‐dependent p97(N) domain motions that lead to a rearrangement of p47 domains, which could result in the disassembly of target protein complexes.