CAND1 orchestrates CRLs through rock and roll.

Proper regulation of protein degradation is essential for cell physiology. In the current issue of Cell, Baek et al. elucidated how a large class of ubiquitin ligase, known as CRL, is assembled and disassembled through a key regulator, CAND1.

Structural insights into Ubr1-mediated N-degron polyubiquitination.

The N-degron pathway targets proteins that bear a destabilizing residue at the N terminus for proteasome-dependent degradation1. In yeast, Ubr1-a single-subunit E3 ligase-is responsible for the Arg/N-degron pathway2. How Ubr1 mediates the initiation of ubiquitination and the elongation of the ubiquitin chain in a linkage-specific manner through a single E2 ubiquitin-conjugating enzyme (Ubc2) remains unknown. Here we developed chemical strategies to mimic the reaction intermediates of the first and second ubiquitin transfer steps, and determined the cryo-electron microscopy structures of Ubr1 in complex with Ubc2, ubiquitin and two N-degron peptides, representing the initiation and elongation steps of ubiquitination. Key structural elements, including a Ubc2-binding region and an acceptor ubiquitin-binding loop on Ubr1, were identified and characterized. These structures provide mechanistic insights into the initiation and elongation of ubiquitination catalysed by Ubr1.

Fast and automated protein-DNA/RNA macromolecular complex modeling from cryo-EM maps.

Cryo-electron microscopy (cryo-EM) allows a macromolecular structure such as protein-DNA/RNA complexes to be reconstructed in a three-dimensional coulomb potential map. The structural information of these macromolecular complexes forms the foundation for understanding the molecular mechanism including many human diseases. However, the model building of large macromolecular complexes is often difficult and time-consuming. We recently developed DeepTracer-2.0, an artificial-intelligence-based pipeline that can build amino acid and nucleic acid backbones from a single cryo-EM map, and even predict the best-fitting residues according to the density of side chains. The experiments showed improved accuracy and efficiency when benchmarking the performance on independent experimental maps of protein-DNA/RNA complexes and demonstrated the promising future of macromolecular modeling from cryo-EM maps. Our method and pipeline could benefit researchers worldwide who work in molecular biomedicine and drug discovery, and substantially increase the throughput of the cryo-EM model building. The pipeline has been integrated into the web portal https://deeptracer.uw.edu/.

An engineered hypercompact CRISPR-Cas12f system with boosted gene-editing activity.

Compact CRISPR-Cas systems offer versatile treatment options for genetic disorders, but their application is often limited by modest gene-editing activity. Here we present enAsCas12f, an engineered RNA-guided DNA endonuclease up to 11.3-fold more potent than its parent protein, AsCas12f, and one-third of the size of SpCas9. enAsCas12f shows higher DNA cleavage activity than wild-type AsCas12f in vitro and functions broadly in human cells, delivering up to 69.8% insertions and deletions at user-specified genomic loci. Minimal off-target editing is observed with enAsCas12f, suggesting that boosted on-target activity does not impair genome-wide specificity. We determine the cryo-electron microscopy (cryo-EM) structure of the AsCas12f-sgRNA-DNA complex at a resolution of 2.9 Å, which reveals dimerization-mediated substrate recognition and cleavage. Structure-guided single guide RNA (sgRNA) engineering leads to sgRNA-v2, which is 33% shorter than the full-length sgRNA, but with on par activity. Together, the engineered hypercompact AsCas12f system enables robust and faithful gene editing in mammalian cells.

A novel membrane complex is required for docking and regulated exocytosis of lysosome-related organelles in Tetrahymena thermophila.

In the ciliate Tetrahymena thermophila, lysosome-related organelles called mucocysts accumulate at the cell periphery where they secrete their contents in response to extracellular events, a phenomenon called regulated exocytosis. The molecular bases underlying regulated exocytosis have been extensively described in animals but it is not clear whether similar mechanisms exist in ciliates or their sister lineage, the Apicomplexan parasites, which together belong to the ecologically and medically important superphylum Alveolata. Beginning with a T. thermophila mutant in mucocyst exocytosis, we used a forward genetic approach to uncover MDL1 (Mucocyst Discharge with a LamG domain), a novel gene that is essential for regulated exocytosis of mucocysts. Mdl1p is a 40 kDa membrane glycoprotein that localizes to mucocysts, and specifically to a tip domain that contacts the plasma membrane when the mucocyst is docked. This sub-localization of Mdl1p, which occurs prior to docking, underscores a functional asymmetry in mucocysts that is strikingly similar to that of highly polarized secretory organelles in other Alveolates. A mis-sense mutation in the LamG domain results in mucocysts that dock but only undergo inefficient exocytosis. In contrast, complete knockout of MDL1 largely prevents mucocyst docking itself. Mdl1p is physically associated with 9 other proteins, all of them novel and largely restricted to Alveolates, and sedimentation analysis supports the idea that they form a large complex. Analysis of three other members of this putative complex, called MDD (for Mucocyst Docking and Discharge), shows that they also localize to mucocysts. Negative staining of purified MDD complexes revealed distinct particles with a central channel. Our results uncover a novel macromolecular complex whose subunits are conserved within alveolates but not in other lineages, that is essential for regulated exocytosis in T. thermophila.

A LcDOF5.6-LcRbohD regulatory module controls the reactive oxygen species-mediated fruitlet abscission in litchi.

Reactive oxygen species (ROS) have been emerging as a key regulator in plant organ abscission. However, the mechanism underlying the regulation of ROS homeostasis in the abscission zone (AZ) is not completely established. Here, we report that a DOF (DNA binding with one finger) transcription factor LcDOF5.6 can suppress the litchi fruitlet abscission through repressing the ROS accumulation in fruitlet AZ (FAZ). The expression of LcRbohD, a homolog of the Arabidopsis RBOHs that are critical for ROS production, was significantly increased during the litchi fruitlet abscission, in parallel with an increased accumulation of ROS in FAZ. In contrast, silencing of LcRbohD reduced the ROS accumulation in FAZ and decreased the fruitlet abscission in litchi. Using in vitro and in vivo assays, we revealed that LcDOF5.6 was shown to inhibit the expression of LcRbohD via direct binding to its promoter. Consistently, silencing of LcDOF5.6 increased the expression of LcRbohD, concurrently with higher ROS accumulation in FAZ and increased fruitlet abscission. Furthermore, the expression of key genes (LcIDL1, LcHSL2, LcACO2, LcACS1, and LcEIL3) in INFLORESCENCE DEFICIENT IN ABSCISSION signaling and ethylene pathways were altered in LcRbohD-silenced and LcDOF5.6-silenced FAZ cells. Taken together, our results demonstrate an important role of the LcDOF5.6-LcRbohD module during litchi fruitlet abscission. Our findings provide new insights into the molecular regulatory network of organ abscission.

MicroRNA482/2118 is lineage-specifically involved in gibberellin signalling via the regulation of GID1 expression by targeting noncoding PHAS genes and subsequently instigated phasiRNAs.

MicroRNA482/2118 (miR482/2118) is a 22-nt miRNA superfamily, with conserved functions in disease resistance and plant development. It usually instigates the production of phased small interfering RNAs (phasiRNAs) from its targets to expand or reinforce its silencing effect. Using a new high-quality reference genome sequence and comprehensive small RNA profiling, we characterized a newly evolved regulatory pathway of miR482/2118 in litchi. In this pathway, miR482/2118 cleaved a novel noncoding trans-acting gene (LcTASL1) and triggered phasiRNAs to regulate the expression of gibberellin (GA) receptor gene GIBBERELLIN INSENSITIVE DWARF1 (GID1) in trans; another trans-acting gene LcTASL2, targeted by LcTASL1-derived phasiRNAs, produced phasiRNAs as well to target LcGID1 to reinforce the silencing effect of LcTASL1. We found this miR482/2118-TASL-GID1 pathway was likely involved in fruit development, especially the seed development in litchi. In vivo construction of the miR482a-TASL-GID1 pathway in Arabidopsis could lead to defects in flower and silique development, analogous to the phenotype of gid1 mutants. Finally, we found that a GA-responsive transcription factor, LcGAMYB33, could regulate LcMIR482/2118 as a feedback mechanism of the sRNA-silencing pathway. Our results deciphered a lineage-specifically evolved regulatory module of miR482/2118, demonstrating the high dynamics of miR482/2118 function in plants.

Increased DNA methylation of the splicing regulator SR45 suppresses seed abortion in litchi.

The gene regulatory networks that govern seed development are complex, yet very little is known about the genes and processes that are controlled by DNA methylation. Here, we performed single-base resolution DNA methylome analysis and found that CHH methylation increased significantly throughout seed development in litchi. Based on the association analysis of differentially methylated regions and weighted gene co-expression network analysis (WGCNA), 46 genes were identified as essential DNA methylation-regulated candidate genes involved in litchi seed development, including LcSR45, a homolog of the serine/arginine-rich (SR) splicing regulator SR45. LcSR45 is predominately expressed in the funicle, embryo, and seed integument, and displayed increased CHH methylation in the promoter during seed development. Notably, silencing of LcSR45 in a seed-aborted litchi cultivar significantly improved normal seed development, whereas the ectopic expression of LcSR45 in Arabidopsis caused seed abortion. Furthermore, LcSR45-dependent alternative splicing events were found to regulate genes involved in seed development. Together, our findings demonstrate that LcSR45 is hypermethylated, and plays a detrimental role in litchi seed development, indicating a global increase in DNA methylation at this stage.

Molecular assemblies of the catalytic domain of SOS with KRas and oncogenic mutants.

Ras is regulated by a specific guanine nucleotide exchange factor Son of Sevenless (SOS), which facilitates the exchange of inactive, GDP-bound Ras with GTP. The catalytic activity of SOS is also allosterically modulated by an active Ras (Ras-GTP). However, it remains poorly understood how oncogenic Ras mutants interact with SOS and modulate its activity. Here, native ion mobility-mass spectrometry is employed to monitor the assembly of the catalytic domain of SOS (SOScat) with KRas and three cancer-associated mutants (G12C, G13D, and Q61H), leading to the discovery of different molecular assemblies and distinct conformers of SOScat engaging KRas. We also find KRasG13D exhibits high affinity for SOScat and is a potent allosteric modulator of its activity. A structure of the KRasG13D•SOScat complex was determined using cryogenic electron microscopy providing insight into the enhanced affinity of the mutant protein. In addition, we find that KRasG13D-GTP can allosterically increase the nucleotide exchange rate of KRas at the active site more than twofold compared to KRas-GTP. Furthermore, small-molecule Ras•SOS disruptors fail to dissociate KRasG13D•SOScat complexes, underscoring the need for more potent disruptors. Taken together, a better understanding of the interaction between oncogenic Ras mutants and SOS will provide avenues for improved therapeutic interventions.

The transcriptional control of LcIDL1-LcHSL2 complex by LcARF5 integrates auxin and ethylene signaling for litchi fruitlet abscission.

At the physiological level, the interplay between auxin and ethylene has long been recognized as crucial for the regulation of organ abscission in plants. However, the underlying molecular mechanisms remain unknown. Here, we identified transcription factors involved in indoleacetic acid (IAA) and ethylene (ET) signaling that directly regulate the expression of INFLORESCENCE DEFICIENT IN ABSCISSION (IDA) and its receptor HAESA (HAE), which are key components initiating abscission. Specifically, litchi IDA-like 1 (LcIDL1) interacts with the receptor HAESA-like 2 (LcHSL2). Through in vitro and in vivo experiments, we determined that the auxin response factor LcARF5 directly binds and activates both LcIDL1 and LcHSL2. Furthermore, we found that the ETHYLENE INSENSITIVE 3-like transcription factor LcEIL3 directly binds and activates LcIDL1. The expression of IDA and HSL2 homologs was enhanced in LcARF5 and LcEIL3 transgenic Arabidopsis plants, but reduced in ein3 eil1 mutants. Consistently, the expressions of LcIDL1 and LcHSL2 were significantly decreased in LcARF5- and LcEIL3-silenced fruitlet abscission zones (FAZ), which correlated with a lower rate of fruitlet abscission. Depletion of auxin led to an increase in 1-aminocyclopropane-1-carboxylic acid (the precursor of ethylene) levels in the litchi FAZ, followed by abscission activation. Throughout this process, LcARF5 and LcEIL3 were induced in the FAZ. Collectively, our findings suggest that the molecular interactions between litchi AUXIN RESPONSE FACTOR 5 (LcARF5)-LcIDL1/LcHSL2 and LcEIL3-LcIDL1 signaling modules play a role in regulating fruitlet abscission in litchi and provide a long-sought mechanistic explanation for how the interplay between auxin and ethylene is translated into the molecular events that initiate abscission.

K29-linked ubiquitin signaling regulates proteotoxic stress response and cell cycle.

Protein ubiquitination shows remarkable topological and functional diversity through the polymerization of ubiquitin via different linkages. Deciphering the cellular ubiquitin code is of central importance to understand the physiology of the cell. However, our understanding of its function is rather limited due to the lack of specific binders as tools to detect K29-linked polyubiquitin. In this study, we screened and characterized a synthetic antigen-binding fragment, termed sAB-K29, that can specifically recognize K29-linked polyubiquitin using chemically synthesized K29-linked diubiquitin. We further determined the crystal structure of this fragment bound to the K29-linked diubiquitin, which revealed the molecular basis of specificity. Using sAB-K29 as a tool, we uncovered that K29-linked ubiquitination is involved in different kinds of cellular proteotoxic stress response as well as cell cycle regulation. In particular, we showed that K29-linked ubiquitination is enriched in the midbody and downregulation of the K29-linked ubiquitination signal arrests cells in G1/S phase.

Selective regulation of human TRAAK channels by biologically active phospholipids.

TRAAK is an ion channel from the two-pore domain potassium (K2P) channel family with roles in maintaining the resting membrane potential and fast action potential conduction. Regulated by a wide range of physical and chemical stimuli, the affinity and selectivity of K2P4.1 toward lipids remains poorly understood. Here we show the two isoforms of K2P4.1 have distinct binding preferences for lipids dependent on acyl chain length and position on the glycerol backbone. The channel can also discriminate the fatty acid linkage at the SN1 position. Of the 33 lipids interrogated using native mass spectrometry, phosphatidic acid had the lowest equilibrium dissociation constants for both isoforms of K2P4.1. Liposome potassium flux assays with K2P4.1 reconstituted in defined lipid environments show that those containing phosphatidic acid activate the channel in a dose-dependent fashion. Our results begin to define the molecular requirements for the specific binding of lipids to K2P4.1.

Inhibiting HIF-1 signaling alleviates HTRA1-induced RPE senescence in retinal degeneration.

BACKGROUND: Age-related macular degeneration (AMD), characterized by the degeneration of retinal pigment epithelium (RPE) and photoreceptors, is the leading cause of irreversible vision impairment among the elderly. RPE senescence is an important contributor to AMD and has become a potential target for AMD therapy. HTRA1 is one of the most significant susceptibility genes in AMD, however, the correlation between HTRA1 and RPE senescence hasn't been investigated in the pathogenesis of AMD. METHODS: Western blotting and immunohistochemistry were used to detect HTRA1 expression in WT and transgenic mice overexpressing human HTRA1 (hHTRA1-Tg mice). RT-qPCR was used to detect the SASP in hHTRA1-Tg mice and ARPE-19 cells infected with HTRA1. TEM, SA-ß-gal was used to detect the mitochondria and senescence in RPE. Retinal degeneration of mice was investigated by fundus photography, FFA, SD-OCT and ERG. The RNA-Seq dataset of ARPE-19 cells treated with adv-HTRA1 versus adv-NC were analyzed. Mitochondrial respiration and glycolytic capacity in ARPE-19 cells were measured using OCR and ECAR. Hypoxia of ARPE-19 cells was detected using EF5 Hypoxia Detection Kit. KC7F2 was used to reduce the HIF1α expression both in vitro and in vivo. RESULTS: In our study, we found that RPE senescence was facilitated in hHTRA1-Tg mice. And hHTRA1-Tg mice became more susceptible to NaIO3 in the development of oxidative stress-induced retinal degeneration. Similarly, overexpression of HTRA1 in ARPE-19 cells accelerated cellular senescence. Our RNA-seq revealed an overlap between HTRA1-induced differentially expressed genes associated with aging and those involved in mitochondrial function and hypoxia response in ARPE-19 cells. HTRA1 overexpression in ARPE-19 cells impaired mitochondrial function and augmented glycolytic capacity. Importantly, upregulation of HTRA1 remarkably activated HIF-1 signaling, shown as promoting HIF1α expression which mainly located in the nucleus. HIF1α translation inhibitor KC7F2 significantly prevented HTRA1-induced cellular senescence in ARPE-19 cells, as well as improved the visual function in hHTRA1-Tg mice treated with NaIO3. CONCLUSIONS: Our study showed elevated HTRA1 contributes to the pathogenesis of AMD by promoting cellular senescence in RPE through damaging mitochondrial function and activating HIF-1 signaling. It also pointed out that inhibition of HIF-1 signaling might serve as a potential therapeutic strategy for AMD. Video Abstract.

The primed SNARE-complexin-synaptotagmin complex for neuronal exocytosis.

Synaptotagmin, complexin, and neuronal SNARE (soluble N-ethylmaleimide sensitive factor attachment protein receptor) proteins mediate evoked synchronous neurotransmitter release, but the molecular mechanisms mediating the cooperation between these molecules remain unclear. Here we determine crystal structures of the primed pre-fusion SNARE-complexin-synaptotagmin-1 complex. These structures reveal an unexpected tripartite interface between synaptotagmin-1 and both the SNARE complex and complexin. Simultaneously, a second synaptotagmin-1 molecule interacts with the other side of the SNARE complex via the previously identified primary interface. Mutations that disrupt either interface in solution also severely impair evoked synchronous release in neurons, suggesting that both interfaces are essential for the primed pre-fusion state. Ca2+ binding to the synaptotagmin-1 molecules unlocks the complex, allows full zippering of the SNARE complex, and triggers membrane fusion. The tripartite SNARE-complexin-synaptotagmin-1 complex at a synaptic vesicle docking site has to be unlocked for triggered fusion to start, explaining the cooperation between complexin and synaptotagmin-1 in synchronizing evoked release on the sub-millisecond timescale.

LcERF2 modulates cell wall metabolism by directly targeting a UDP-glucose-4-epimerase gene to regulate pedicel development and fruit abscission of litchi.

Elucidating the biochemical and molecular basis of premature abscission in fruit crops should help develop strategies to enhance fruit set and yield. Here, we report that LcERF2 contributes to differential abscission rates and responses to ethylene in Litchi chinensis (litchi). Reduced LcERF2 expression in litchi was observed to reduce fruit abscission, concurrent with enhanced pedicel growth and increased levels of hexoses, particularly galactose, as well as pectin abundance in the cell wall. Ecoptic expression of LcERF2 in Arabidopsis thaliana caused enhanced petal abscission, together with retarded plant growth and reduced pedicel galactose and pectin contents. Transcriptome analysis indicated that LcERF2 modulates the expression of genes involved in cell wall modification. Yeast one-hybrid, dual-luciferase reporter and electrophoretic mobility shift assays all demonstrated that a UDP-glucose-4-epimerase gene (LcUGE) was the direct downstream target of LcERF2. This result was further supported by a significant reduction in the expression of the A. thaliana homolog AtUGE2-4 in response to LcERF2 overexpression. Significantly reduced pedicel diameter and enhanced litchi fruit abscission were observed in response to LcUGE silencing. We conclude that LcERF2 mediates fruit abscission by orchestrating cell wall metabolism, and thus pedicel growth, in part by repressing the expression of LcUGE.

TCF7L2 promotes ER stress signaling in diabetic retinopathy.

Diabetic retinopathy (DR) is one of the most common blindness in working-age adults. Transcription factor 7 like 2 (TCF7L2) is a susceptibility gene of DR, however, its roles in the pathogenesis of DR are still largely unknown. In this study, we found that TCF7L2 was mainly located in the cell nucleus of retinal ganglion cell layer (GCL) and inner nuclear layer (INL), while it was not expressed in the cell nucleus of retinal outer nuclear layer (ONL). Expression of TCF7L2 was significantly elevated in the retinas of db/db diabetic mice and oxygen-induced retinopathy (OIR) mice. Also, in Ad-hTCF7L2 treated hiPSCs-derived retinal progenitor cells (RPCs), activating transcription factor 6 (ATF6)-related endoplasmic reticulum (ER) stress signaling was remarkably activated. Moreover, knockdown of TCF7L2 significantly inhibited ATF6-related ER stress signaling. Furthermore, the data of endothelial permeability assay showed that RPCs pretreated with Ad-hTCF7L2 lead to enhanced monolayer permeability of human umbilical vein endothelial cells (HUVECs), and knockdown of TCF7L2 or ATF6 in RPCs could alleviate the monolayer permeability of HUVECs. Thus, our results showed that TCF7L2 could trigger ATF6-related ER stress signaling and promote vein endothelial cell permeability, which will provide important insight into the role of TCF7L2 in the pathogenesis of DR and contribute to designing potential therapies.

LcEIL2/3 are involved in fruitlet abscission via activating genes related to ethylene biosynthesis and cell wall remodeling in litchi.

Fruit crops are subject to precocious fruit abscission, during which the phytohormone ethylene (ET) acts as a major positive regulator. However, the molecular basis of ET-induced fruit abscission remains poorly understood. Here, we show that two ETHYLENE INSENSITIVE 3-like (EIL) homologs in litchi, LcEIL2 and LcEIL3, play a role in ET-activated fruitlet abscission. LcEIL2/3 were significantly upregulated in the fruit abscission zone (AZ) during the ET-induced fruitlet abscission in litchi. The presence of LcEIL2/3 in wild-type Arabidopsis and ein3 eil1 mutants can accelerate the floral organ abscission. Moreover, the electrophoretic mobility shift assay and dual luciferase reporter analysis illustrated that LcEIL2/3 directly interacted with the gene promoters to activate the expression of cell wall remodeling genes LcCEL2/8 and LcPG1/2, and ET biosynthetic genes LcACS1/4/7 and LcACO2/3. Furthermore, we showed that LcPG1/2 were expressed in the floral abscission zone of Arabidopsis, and constitutive expression of LcPG2 in Arabidopsis promoted the floral organ abscission. In conclusion, we propose that LcEIL2/3 are involved in ET-induced fruitlet abscission via controlling expression of genes related to ET biosynthesis and cell wall remodeling in litchi.

Xyloglucan endotransglucosylase/hydrolase genes LcXTH4/7/19 are involved in fruitlet abscission and are activated by LcEIL2/3 in litchi.

Organ abscission in plants requires the hydrolysis of cell wall components, mainly including celluloses, pectins, and xyloglucans. However, how the genes that encode those hydrolytic enzymes are regulated and their function in abscission remains unclear. Previously we revealed that two cellulase genes LcCEL2/8 and two polygalacturonase genes LcPG1/2 were responsible for the degradation of celluloses and pectins, respectively, during fruitlet abscission in litchi. Here, we further identified three xyloglucan endotransglucosylase/hydrolase genes (LcXTH4, LcXTH7, LcXTH19) that are also involved in this process. Nineteen LcXTHs, named LcXTH1-19, were identified in the litchi genome. Transcriptome data and qRT-PCR confirmed that LcXTH4/7/19 were significantly induced at the abscission zone (AZ) during fruitlet abscission in litchi. The GUS reporter driven by each promoter of LcXTH4/7/19 was specifically expressed at the floral abscission zone of Arabidopsis, and importantly ectopic expression of LcXTH19 in Arabidopsis resulted in precocious floral organ abscission. Moreover, electrophoretic mobility shift assay (EMSA) and dual-luciferase reporter analysis showed that the expression of LcXTH4/7/19 could be directly activated by two ETHYLENE INSENSITIVE 3-like (EIL) transcription factors LcEIL2/3. Collectively, we propose that LcXTH4/7/19 are involved in fruitlet abscission, and LcEIL2/3-mediated transcriptional regulation of diverse cell wall hydrolytic genes is responsible for this process in litchi.

Mechanistic insights into the recycling machine of the SNARE complex.

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.