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Stimulator of interferon genes (STING) functions downstream of cyclic GMP-AMP synthase in DNA sensing or as a direct receptor for bacterial cyclic dinucleotides and small molecules to activate immunity during infection, cancer and immunotherapy1–10. Precise regulation of STING is essential to ensure balanced immune responses and prevent detrimental autoinflammation11–16. After activation, STING, a transmembrane protein, traffics from the endoplasmic reticulum to the Golgi, where its phosphorylation by the protein kinase TBK1 enables signal transduction17–20. The mechanism that ends STING signalling at the Golgi remains unknown. Here we show that adaptor protein complex 1 (AP-1) controls the termination of STING-dependent immune activation. We find that AP-1 sorts phosphorylated STING into clathrin-coated transport vesicles for delivery to the endolysosomal system, where STING is degraded21. We identify a highly conserved dileucine motif in the cytosolic C-terminal tail (CTT) of STING that, together with TBK1-dependent CTT phosphorylation, dictates the AP-1 engagement of STING. A cryo-electron microscopy structure of AP-1 in complex with phosphorylated STING explains the enhanced recognition of TBK1-activated STING. We show that suppression of AP-1 exacerbates STING-induced immune responses. Our results reveal a structural mechanism of negative regulation of STING and establish that the initiation of signalling is inextricably associated with its termination to enable transient activation of immunity.

Lentiviruses express non-enzymatic accessory proteins whose function is to subvert cellular machinery in the infected host. The HIV-1 accessory protein Nef hijacks clathrin adaptors to degrade or mislocalize host proteins involved in antiviral defenses. Here, we investigate the interaction between Nef and clathrin-mediated endocytosis (CME), a major pathway for membrane protein internalization in mammalian cells, using quantitative live-cell microscopy in genome-edited Jurkat cells. Nef is recruited to CME sites on the plasma membrane, and this recruitment correlates with an increase in the recruitment and lifetime of CME coat protein AP-2 and late-arriving CME protein dynamin2. Furthermore, we find that CME sites that recruit Nef are more likely to recruit dynamin2, suggesting that Nef recruitment to CME sites promotes CME site maturation to ensure high efficiency in host protein downregulation.

The assembly of the autophagy initiation machinery nucleates autophagosome biogenesis, including in the PINK1- and Parkin-dependent mitophagy pathway implicated in Parkinson’s disease. The structural interaction between the sole transmembrane autophagy protein, autophagy-related protein 9A (ATG9A), and components of the Unc-51–like autophagy activating kinase (ULK1) complex is one of the major missing links needed to complete a structural map of autophagy initiation. We determined the 2.4-Å x-ray crystallographic structure of the ternary structure of ATG9A carboxyl-terminal tail bound to the ATG13:ATG101 Hop1/Rev7/Mad2 (HORMA) dimer, which is part of the ULK1 complex. We term the interacting portion of the extreme carboxyl-terminal part of the ATG9A tail the “HORMA dimer–interacting region” (HDIR). This structure shows that the HDIR binds to the HORMA domain of ATG101 by β sheet complementation such that the ATG9A tail resides in a deep cleft at the ATG13:ATG101 interface. Disruption of this complex in cells impairs damage-induced PINK1/Parkin mitophagy mediated by the cargo receptor NDP52.

The transcription factor TFEB is a master regulator of lysosomal biogenesis and autophagy1. The phosphorylation of TFEB by the mechanistic target of rapamycin complex 1 (mTORC1)2–5 is unique in its mTORC1 substrate recruitment mechanism, which is strictly dependent on the amino acid-mediated activation of the RagC GTPase activating protein FLCN6,7. TFEB lacks the TOR signalling motif responsible for the recruitment of other mTORC1 substrates. We used cryogenic-electron microscopy to determine the structure of TFEB as presented to mTORC1 for phosphorylation, which we refer to as the ‘megacomplex’. Two full Rag–Ragulator complexes present each molecule of TFEB to the mTOR active site. One Rag–Ragulator complex is bound to Raptor in the canonical mode seen previously in the absence of TFEB. A second Rag–Ragulator complex (non-canonical) docks onto the first through a RagC GDP-dependent contact with the second Ragulator complex. The non-canonical Rag dimer binds the first helix of TFEB with a RagCGDP-dependent aspartate clamp in the cleft between the Rag G domains. In cellulo mutation of the clamp drives TFEB constitutively into the nucleus while having no effect on mTORC1 localization. The remainder of the 108-amino acid TFEB docking domain winds around Raptor and then back to RagA. The double use of RagC GDP contacts in both Rag dimers explains the strong dependence of TFEB phosphorylation on FLCN and the RagC GDP state.

Small GTPases act as molecular switches and control numerous cellular processes by their binding and hydrolysis of guanosine triphosphate (GTP). The activity of small GTPases is coordinated by guanine nucleotide exchange factors (GEFs) and GTPase activating proteins (GAPs). Recent structural and functional studies have characterized a subset of GAPs whose catalytic units consist of longin domains. Longin domain containing GAPs regulate small GTPases that facilitate nutrient signalling, autophagy, vesicular trafficking and lysosome homeostasis. All known examples in this GAP family function as part of larger multiprotein complexes. The three characterized mammalian protein complexes in this class are FLCN:FNIP, GATOR1 and C9orf72:SMCR8. Each complex carries out a unique cellular function by regulating distinct small GTPases. In this article, we explore the roles of longin domain GAPs in nutrient sensing, membrane dynamic, vesicular trafficking and disease. Through a structural lens, we examine the mechanism of each longin domain GAP and highlight potential therapeutic applications.

The AP adaptor complexes are best known for forming the inner layer of clathrin coats on spherical vesicles. AP complexes also have many clathrin-independent roles in tubulovesicular membrane traffic, whose structural and mechanistic basis has been a mystery. HIV-1 Nef hijacks the AP-1 complex to sequester MHC-I internally, evading immune detection. We found that AP-1:Arf1:Nef:MHC-I forms a coat on tubulated membranes in the absence of clathrin, and determined its structure by cryo-ET. The coat assembles both laterally and axially via an Arf1 dimer interface not seen before. Nef recruits MHC-I, but is not essential for the underlying AP-1:Arf1 lattice. Consistent with a role for AP-1:Arf1 coated tubules as intermediates in clathrin coated vesicle formation, AP-1 positive tubules are enriched in cells upon clathrin knockdown, with or without Nef. Nef localizes preferentially to AP-1 tubules in cells, explaining how Nef can sequester MHC-I. The coat contact residues are conserved across Arf isoforms and across the Arf-dependent AP adaptors AP-1, 3, and 4. These findings reveal that AP complexes can self-assemble with Arf1 into tubular coats in the absence of clathrin or other scaffolding factors. The AP-1:Arf1 coat defines the structural basis of a broader class of tubulovesicular membrane coats, as an intermediate in clathrin vesicle formation from internal membranes, and as a MHC-I sequestration mechanism in HIV-1 infection.

The mechanistic target of rapamycin complex 1 (mTORC1) regulates cell growth and catabolism in response to nutrients through phosphorylation of key substrates. The tumor suppressor folliculin (FLCN) is a RagC/D guanosine triphosphatase (GTPase)–activating protein (GAP) that regulates mTORC1 phosphorylation of MiT-TFE transcription factors, controlling lysosome biogenesis and autophagy. We determined the cryo–electron microscopy structure of the active FLCN complex (AFC) containing FLCN, FNIP2, the N-terminal tail of SLC38A9, the RagA GDP :RagC GDP.BeFx- GTPase dimer, and the Ragulator scaffold. Relative to the inactive lysosomal FLCN complex structure, FLCN reorients by 90°, breaks contact with RagA, and makes previously unseen contacts with RagC that position its Arg 164 finger for catalysis. Disruption of the AFC-specific interfaces of FLCN and FNIP2 with RagC eliminated GAP activity and led to nuclear retention of TFE3, with no effect on mTORC1 substrates S6K or 4E-BP1. The structure provides a basis for regulation of an mTORC1 substrate-specific pathway and a roadmap to discover MiT-TFE family selective mTORC1 antagonists.

In the late stages of the HIV-1 life cycle, membrane localization and self-assembly of the Gag polyproteins induce membrane deformation and budding. However, release of the immature virion requires direct interaction between Gag lattice and upstream ESCRT machinery at the budding site, followed by assembly of the downstream ESCRT-III factors, culminating in membrane scission. In this work, using “bottom-up” coarse-grained (CG) molecular dynamics (MD) simulations we investigated the interactions between Gag and different upstream ESCRT components to delineate the molecular organization of proteins at the membrane neck of the HIV-1 budding site. We developed CG models of upstream ESCRT proteins and HIV-1 structural protein Gag based on experimental structural data and extensive all-atom MD simulations. We find that ESCRT-I proteins bound to the immature Gag lattice can recruit multiple copies of ESCRT-II coating the membrane neck. ESCRT-I can effectively oligomerize to higher-order complexes both in absence of ESCRT-II and when multiple copies of ESCRT-II are localized at the bud neck. The ESCRT-I/II supercomplexes observed in our simulations exhibit predominantly extended conformations. Importantly, the ESCRT-I/II supercomplex modulates the membrane mechanical properties at the budding site by decreasing the overall Gaussian curvature of membrane neck. Our findings serve to elucidate a network of interactions between the upstream ESCRT machinery, immature Gag lattice, and membrane bud neck that regulate the protein assemblies and enable bud neck constriction.

The transcription factor TFEB is a master regulator of lysosomal biogenesis and autophagy. The phosphorylation of TFEB by the mechanistic target of rapamycin complex 1 (mTORC1) is unique in its mTORC1 substrate recruitment mechanism, which is strictly dependent on the amino-acid-mediated activation of the RagC GAP FLCN. TFEB lacks the TOR signaling (TOS) motif responsible for the recruitment of other mTORC1 substrates. We used cryo-electron microscopy (cryo-EM) to determine the structure of TFEB as presented to mTORC1 for phosphorylation. Two full Rag-Ragulator complexes present each molecule of TFEB to the mTOR active site. One Rag-Ragulator complex is bound to Raptor in the canonical mode seen previously in the absence of TFEB. A second Rag-Ragulator complex (non-canonical) docks onto the first via a RagC GDP-dependent contact with the second Ragulator complex. The non-canonical Rag dimer binds the first helix of TFEB in a RagCGDP-dependent aspartate clamp in the cleft between the Rag G domains. Mutation of the clamp drives TFEB constitutively into the nucleus whilst having no effect on mTORC1 localization. The remainder of the 108-amino acid TFEB docking domain winds around Raptor and then back to RagA. This structure presents the phosphorylatable Ser residues of TFEB to the mTORC1 active site in a suitable geometry for their phosphorylation. The double use of RagC GDP contacts in both Rag dimers explains the strong dependence of TFEB phosphorylation on FLCN and the RagC GDP state.

The endosomal sorting complex required for transport (ESCRT) machinery is centrally involved in the repair of damage to both the plasma and lysosome membranes. ESCRT recruitment to sites of damage occurs on a fast time scale, and Ca 2+ has been proposed to play a key signaling role in the process. Here, we show that the Ca 2+ -binding regulatory protein ALG-2 binds directly to negatively charged membranes in a Ca 2+ -dependent manner. Next, by monitoring the colocalization of ALIX with ALG-2 on negatively charged membranes, we show that ALG-2 recruits ALIX to the membrane. Furthermore, we show that ALIX recruitment to the membrane orchestrates the downstream assembly of late-acting CHMP4B, CHMP3, and CHMP2A subunits along with the AAA + ATPase VPS4B. Finally, we show that ALG-2 can also recruit the ESCRT-III machinery to the membrane via the canonical ESCRT-I/II pathway. Our reconstitution experiments delineate the minimal sets of components needed to assemble the entire membrane repair machinery and open an avenue for the mechanistic understanding of endolysosomal membrane repair.

The endosomal sorting complexes required for transport (ESCRT) system is an ancient and ubiquitous membrane scission machinery that catalyzes the budding and scission of membranes. ESCRT-mediated scission events, exemplified by those involved in the budding of HIV-1, are usually directed away from the cytosol (“reverse topology”), but they can also be directed toward the cytosol (“normal topology”). The ESCRT-III subunits CHMP1B and IST1 can coat and constrict positively curved membrane tubes, suggesting that these subunits could catalyze normal topology membrane severing. CHMP1B and IST1 bind and recruit the microtubule-severing AAA + ATPase spastin, a close relative of VPS4, suggesting that spastin could have a VPS4-like role in normal-topology membrane scission. Here, we reconstituted the process in vitro using membrane nanotubes pulled from giant unilamellar vesicles using an optical trap in order to determine whether CHMP1B and IST1 are capable of membrane severing on their own or in concert with VPS4 or spastin. CHMP1B and IST1 copolymerize on membrane nanotubes, forming stable scaffolds that constrict the tubes, but do not, on their own, lead to scission. However, CHMP1B–IST1 scaffolded tubes were severed when an additional extensional force was applied, consistent with a friction-driven scission mechanism. We found that spastin colocalized with CHMP1B-enriched sites but did not disassemble the CHMP1B–IST1 coat from the membrane. VPS4 resolubilized CHMP1B and IST1 without leading to scission. These observations show that the CHMP1B–IST1 ESCRT-III combination is capable of severing membranes by a friction-driven mechanism that is independent of VPS4 and spastin.

mTORC1 regulates cell growth and catabolism in response to fluctuations in nutrients through phosphorylation of key substrates. The tumor suppressor FLCN is a RagC GTPase activating protein (GAP) that regulates mTORC1 phosphorylation of TFEB, controlling lysosome biogenesis and autophagy. Here, we determined the cryo-EM structure of the active FLCN complex (AFC) containing FLCN, FNIP2, the N-terminal tail of SLC38A9, the RagAGDP:RagCGDP.BeFx- GTPase dimer, and the Ragulator scaffold. Relative to the inactive lysosomal FLCN complex (LFC) structure, FLCN reorients by 90°, breaks its contacts with RagA, and makes new contacts with RagC that position its Arg164 finger for catalysis. Disruption of the AFC-specific interfaces of FLCN and FNIP2 with RagC eliminated GAP activity in vitro and led to nuclear retention of TFE3, with no effect on mTORC1 phosphorylation of S6K or 4E-BP1. The structure thus provides a roadmap to discover TFEB-selective mTORC1 antagonists.One-Sentence SummaryThe cryo-EM structure of the active FLCN RagC GAP complex provides a structural basis for TFEB/TFE3 substrate-selective targeting of mTORC1.

How the highly curved phagophore membrane is stabilized during autophagy initiation is a major open question in autophagosome biogenesis. Here, we use in vitro reconstitution on membrane nanotubes and molecular dynamics simulations to investigate how core autophagy proteins in the LC3 (Microtubule-associated proteins 1A/1B light chain 3) lipidation cascade interact with curved membranes, providing insight into their possible roles in regulating membrane shape during autophagosome biogenesis. ATG12(Autophagy-related 12)–ATG5-ATG16L1 was up to 100-fold enriched on highly curved nanotubes relative to flat membranes. At high surface density, ATG12–ATG5-ATG16L1 binding increased the curvature of the nanotubes. While WIPI2 (WD repeat domain phosphoinositide-interacting protein 2) binding directs membrane recruitment, the amphipathic helix α2 of ATG16L1 is responsible for curvature sensitivity. Molecular dynamics simulations revealed that helix α2 of ATG16L1 inserts shallowly into the membrane, explaining its curvature-sensitive binding to the membrane. These observations show how the binding of the ATG12–ATG5-ATG16L1 complex to the early phagophore rim could stabilize membrane curvature and facilitate autophagosome growth.

Significance This study describes the discovery of a class of drug that can help the immune system eliminate hard-to-kill cells infected with HIV. HIV establishes a persistent infection for which there is no cure, necessitating the development of new approaches to enhance the clearance of HIV-infected cells. HIV encodes Nef, which down-regulates MHC-I expression in infected cells to impair immune-mediated clearance by cytotoxic T lymphocytes. We identified the plecomacrolide family of natural products as potent inhibitors of Nef, and concanamycin A restored MHC-I and enhanced the clearance of HIV-infected primary cells by cytotoxic T lymphocytes. Concanamycin A counteracted Nef from diverse clades of HIV targeting multiple allotypes of MHC-I, indicating the potential for broad therapeutic utility., Nef is an HIV-encoded accessory protein that enhances pathogenicity by down-regulating major histocompatibility class I (MHC-I) expression to evade killing by cytotoxic T lymphocytes (CTLs). A potent Nef inhibitor that restores MHC-I is needed to promote immune-mediated clearance of HIV-infected cells. We discovered that the plecomacrolide family of natural products restored MHC-I to the surface of Nef-expressing primary cells with variable potency. Concanamycin A (CMA) counteracted Nef at subnanomolar concentrations that did not interfere with lysosomal acidification or degradation and were nontoxic in primary cell cultures. CMA specifically reversed Nef-mediated down-regulation of MHC-I, but not CD4, and cells treated with CMA showed reduced formation of the Nef:MHC-I:AP-1 complex required for MHC-I down-regulation. CMA restored expression of diverse allotypes of MHC-I in Nef-expressing cells and inhibited Nef alleles from divergent clades of HIV and simian immunodeficiency virus, including from primary patient isolates. Lastly, we found that restoration of MHC-I in HIV-infected cells was accompanied by enhanced CTL-mediated clearance of infected cells comparable to genetic deletion of Nef. Thus, we propose CMA as a lead compound for therapeutic inhibition of Nef to enhance immune-mediated clearance of HIV-infected cells.

The Rag GTPases (Rags) recruit mTORC1 to the lysosomal membrane in response to nutrients, where it is then activated in response to energy and growth factor availability. The lysosomal folliculin (FLCN) complex (LFC) consists of the inactive Rag dimer, the pentameric scaffold Ragulator, and the FLCN:FNIP2 (FLCN-interacting protein 2) GTPase activating protein (GAP) complex, and prevents Rag dimer activation during amino acid starvation. How the LFC is disassembled upon amino acid refeeding is an outstanding question. Here we show that the cytoplasmic tail of the human lysosomal solute carrier family 38 member 9 (SLC38A9) destabilizes the LFC and thereby triggers GAP activity of FLCN:FNIP2 toward RagC. We present the cryo-EM structures of Rags in complex with their lysosomal anchor complex Ragulator and the cytoplasmic tail of SLC38A9 in the pre- and post-GTP hydrolysis state of RagC, which explain how SLC38A9 destabilizes the LFC and so promotes Rag dimer activation.

Mutation of C9orf72 is the most prevalent defect associated with amyotrophic lateral sclerosis and frontotemporal degeneration1. Together with hexanucleotide-repeat expansion2,3, haploinsufficiency of C9orf72 contributes to neuronal dysfunction4-6. Here we determine the structure of the C9orf72-SMCR8-WDR41 complex by cryo-electron microscopy. C9orf72 and SMCR8 both contain longin and DENN (differentially expressed in normal and neoplastic cells) domains7, and WDR41 is a β-propeller protein that binds to SMCR8 such that the whole structure resembles an eye slip hook. Contacts between WDR41 and the DENN domain of SMCR8 drive the lysosomal localization of the complex in conditions of amino acid starvation. The structure suggested that C9orf72-SMCR8 is a GTPase-activating protein (GAP), and we found that C9orf72-SMCR8-WDR41 acts as a GAP for the ARF family of small GTPases. These data shed light on the function of C9orf72 in normal physiology, and in amyotrophic lateral sclerosis and frontotemporal degeneration.

Significance Autophagy (cellular self-eating) is essential for the health and survival of eukaryotic cells. Therapeutic autophagy induction is a major goal in the field. Rubicon inhibits autophagy and is a potential target for autophagy inducers. Rubicon is localized to its site of action in the cell by binding to the small GTPase Rab7. Here, we report a high-resolution structure of a large part of Rubicon, known as the Rubicon Homology (RH) domain. We show how the RH domain binds to Rab7 and show that the Rab7-binding residues of Rubicon are essential for Rubicon localization and autophagy inhibition. This provides a roadmap to block Rubicon localization and activity in order to upregulate autophagy., Rubicon is a potent negative regulator of autophagy and a potential target for autophagy-inducing therapeutics. Rubicon-mediated inhibition of autophagy requires the interaction of the C-terminal Rubicon homology (RH) domain of Rubicon with Rab7–GTP. Here we report the 2.8-Å crystal structure of the Rubicon RH domain in complex with Rab7–GTP. Our structure reveals a fold for the RH domain built around four zinc clusters. The switch regions of Rab7 insert into pockets on the surface of the RH domain in a mode that is distinct from those of other Rab–effector complexes. Rubicon residues at the dimer interface are required for Rubicon and Rab7 to colocalize in living cells. Mutation of Rubicon RH residues in the Rab7-binding site restores efficient autophagic flux in the presence of overexpressed Rubicon, validating the Rubicon RH domain as a promising therapeutic target.

The endosomal sorting complexes required for transport (ESCRT) system is an ancient and ubiquitous membrane scission machinery that catalyzes the budding and scission of membranes. ESCRT-mediated scission events, exemplified by those involved in the budding of HIV-1, are usually directed away from the cytosol (‘reverse-topology’), but they can also be directed towards the cytosol (‘normal-topology’). Of the ESCRT complexes 0-III, ESCRT-III is most directly implicated in membrane severing. Various subunits of ESCRT-III recruit the AAA+ ATPase VPS4, which is essential for ESCRT disassembly and reverse topology membrane scission. The ESCRT-III subunits CHMP1B and IST1 can coat and constrict positively curved membrane tubes, suggesting that these subunits could catalyze normal topology membrane severing, perhaps in conjunction with a AAA+ ATPase. CHMP1B and IST1 bind and recruit the microtubule-severing AAA+ ATPase spastin, a close relative of VPS4, suggesting that spastin could have a VPS4-like role in normal topology membrane scission. In order to determine whether CHMP1B and IST1 are capable of membrane severing on their own or in concert with VPS4 or spastin, we sought to reconstitute the process in vitro using membrane nanotubes pulled from giant unilamellar vesicles (GUVs) using an optical trap. CHMP1B and IST1 copolymerize on membrane nanotubes, forming stable scaffolds that constrict the tubes, but do not, on their own, lead to scission. However, CHMP1B-IST1-scaffolded tubes were severed when an additional extensional force was applied, consistent with a friction-driven scission mechanism. Spastin colocalized with CHMP1B enriched sites but did not disassemble the CHMP1B-IST1 coat from the membrane. VPS4 resolubilized CHMP1B and IST1 but did not lead to scission. These data show that the CHMP1B and IST1 tubular coat contributes to membrane scission. Constriction alone is insufficient for scission. However, the dynamical extension of the coated tube does lead to scission. Finally, we find that in the normal topology setting analyzed here, scission is independent of VPS4 and spastin. These observations show that the CHMP1B-IST1 ESCRT-III combination is capable of severing membranes by a friction-driven mechanism.

Autophagy is a cellular process that degrades cytoplasmic cargo by engulfing it in a double-membrane vesicle, known as the autophagosome, and delivering it to the lysosome. The ATG12–5–16L1 complex is responsible for conjugating members of the ubiquitin-like ATG8 protein family to phosphatidylethanolamine in the growing autophagosomal membrane, known as the phagophore. ATG12–5–16L1 is recruited to the phagophore by a subset of the phosphatidylinositol 3-phosphate-binding seven-bladedß -propeller WIPI proteins. We determined the crystal structure of WIPI2d in complex with the WIPI2 interacting region (W2IR) of ATG16L1 comprising residues 207–230 at 1.85 Å resolution. The structure shows that the ATG16L1 W2IR adopts an alpha helical conformation and binds in an electropositive and hydrophobic groove between WIPI2 ß-propeller blades 2 and 3. Mutation of residues at the interface reduces or blocks the recruitment of ATG12–5–16 L1 and the conjugation of the ATG8 protein LC3B to synthetic membranes. Interface mutants show a decrease in starvation-induced autophagy. Comparisons across the four human WIPIs suggest that WIPI1 and 2 belong to a W2IR-binding subclass responsible for localizing ATG12–5–16 L1 and driving ATG8 lipidation, whilst WIPI3 and 4 belong to a second W34IR-binding subclass responsible for localizing ATG2, and so directing lipid supply to the nascent phagophore. The structure provides a framework for understanding the regulatory node connecting two central events in autophagy initiation, the action of the autophagic PI 3-kinase complex on the one hand and ATG8 lipidation on the other.

Autophagy is a conserved eukaryotic pathway critical for cellular adaptation to changes in nutrition levels and stress. The class III phosphatidylinositol (PI)3-kinase complexes I and II (PI3KC3-C1 and -C2) are essential for autophagosome initiation and maturation, respectively, from highly curved vesicles. We used a cell-free reaction that reproduces a key autophagy initiation step, LC3 lipidation, as a biochemical readout to probe the role of autophagy-related gene (ATG)14, a PI3KC3-C1-specific subunit implicated in targeting the complex to autophagy initiation sites. We reconstituted LC3 lipidation with recombinant PI3KC3-C1, -C2, or various mutant derivatives added to extracts derived from a CRISPR/Cas9-generated ATG14-knockout cell line. Both complexes C1 and C2 require the C-terminal helix of VPS34 for activity on highly curved membranes. However, only complex C1 supports LC3 lipidation through the curvature-targeting amphipathic lipid packing sensor (ALPS) motif of ATG14. Furthermore, the ALPS motif and VPS34 catalytic activity are required for downstream recruitment of WD-repeat domain phosphoinositide-interacting protein (WIPI)2, a protein that binds phosphatidylinositol 3-phosphate and its product phosphatidylinositol 3, 5-bisphosphate, and a WIPI-binding protein, ATG2A, but do not affect membrane association of ATG3 and ATG16L1, enzymes contributing directly to LC3 lipidation. These data reveal the nuanced role of the ATG14 ALPS in membrane curvature sensing, suggesting that the ALPS has additional roles in supporting LC3 lipidation.

Autophagy is a cellular process that degrades cytoplasmic cargo by engulfing it in a double membrane vesicle, known as the autophagosome, and delivering it to the lysosome. The ATG12–5-16L1 complex is responsible for conjugating members of the ubiquitin-like ATG8 protein family to phosphatidylethanolamine in the growing autophagosomal membrane, known as the phagophore. ATG12–5-16L1 is recruited to the phagophore by a subset of the phosphatidylinositol 3-phosphate-binding seven bladed β-propeller WIPI proteins. We determined the crystal structure of WIPI2d in complex with the WIPI2 interacting region (W2IR) of ATG16L1 comprising residues 207-230 at 1.85 Å resolution. The structure shows that the ATG16L1 W2IR adopts an alpha helical conformation and binds in an electropositive and hydrophobic groove between WIPI2 β-propeller blades 2 and 3. Mutation of residues at the interface reduces or blocks the recruitment of ATG12–5-16L1 and the conjugation of the ATG8 protein LC3B to synthetic membranes. Interface mutants show a decrease in starvation-induced autophagy. Comparisons across the four human WIPIs suggest that WIPI1 and 2 belong to a W2IR-binding subclass responsible for localizing ATG12–5-16L1 and driving ATG8 lipidation, whilst WIPI3 and 4 belong to a second W34IR-binding subclass responsible for localizing ATG2, and so directing lipid supply to the nascent phagophore. The structure provides a framework for understanding the regulatory node connecting two central events in autophagy initiation, the action of the autophagic PI 3-kinase complex on the one hand, and ATG8 lipidation on the other.

The majority of crystal structures are determined by the method of molecular replacement (MR). The range of application of MR is limited mainly by the need for an accurate search model. In most cases, pre-existing experimentally determined structures are used as search models. In favorable cases, ab initio predicted structures have yielded search models adequate for molecular replacement. The ORF8 protein of SARS-CoV-2 represents a challenging case for MR using an ab initio prediction because ORF8 has an all β-sheet fold and few orthologs. We previously determined experimentally the structure of ORF8 using the single anomalous dispersion (SAD) phasing method, having been unable to find an MR solution to the crystallographic phase problem. Following a report of an accurate prediction of the ORF8 structure, we assessed whether the predicted model would have succeeded as an MR search model. A phase problem solution was found, and the resulting structure was refined, yielding structural parameters equivalent to the original experimental solution.

Macroautophagic clearance of cytosolic materials entails the initiation, growth and closure of autophagosomes. Cargo triggers the assembly of a web of cargo receptors and core machinery. Autophagy-related protein 9 (ATG9) vesicles seed the growing autophagosomal membrane, which is supplied by de novo phospholipid synthesis, phospholipid transport via ATG2 proteins and lipid flipping by ATG9. Autophagosomes close via ESCRT complexes. Here, we review recent discoveries that illuminate the molecular mechanisms of autophagosome formation and discuss emerging questions in this rapidly developing field. In this Review, Hurley and colleagues cover the most recent discoveries and the emerging molecular understanding of the mechanisms of autophagosome formation.

Viral infection and pathogenesis is mediated by host protein—viral protein complexes that are important targets for therapeutic intervention as they are potentially less prone to development of drug resistance. We have identified human, recombinant antibodies (Fabs) from a phage display library that bind to three HIV-host complexes. We used these Fabs to 1) stabilize the complexes for structural studies; and 2) facilitate characterization of the function of these complexes. Specifically, we generated recombinant Fabs to Vif-CBF-β-ELOB-ELOC (VCBC); ESCRT-I complex and AP2-complex. For each complex we measured binding affinities with KD values of Fabs ranging from 12–419 nM and performed negative stain electron microscopy (nsEM) to obtain low-resolution structures of the HIV-Fab complexes. Select Fabs were converted to scFvs to allow them to fold intracellularly and perturb HIV-host protein complex assembly without affecting other pathways. To identify these recombinant Fabs, we developed a rapid screening pipeline that uses quantitative ELISAs and nsEM to establish whether the Fabs have overlapping or independent epitopes. This pipeline approach is generally applicable to other particularly challenging antigens that are refractory to immunization strategies for antibody generation including multi-protein complexes providing specific, reproducible, and renewable antibody reagents for research and clinical applications. The curated antibodies described here are available to the scientific community for further structural and functional studies on these critical HIV host-factor proteins.

The endosomal sorting complexes required for transport (ESCRTs) I, -II and –III, and their associated factors are a collection of ∼20 proteins in yeast and ∼30 in mammals, responsible for severing membrane necks in processes that range from multivesicular body formation, HIV release and cytokinesis, to plasma and lysosomal membrane repair. ESCRTs are best known for ‘reverse-topology’ membrane scission, where they act on the inner surface of membrane necks, often when membranes are budded away from the cytosol. These events are driven by membrane-associated assemblies of dozens to hundreds of ESCRT molecules. ESCRT-III proteins form filaments with a variety of geometries and ESCRT-I has now been shown to also form helical structures. The complex nature of the system and the unusual topology of its action has made progress challenging, and led to controversies with regard to its underlying mechanism. This Review will focus on recent advances obtained by structural in vitro reconstitution and in silico mechanistic studies, and places them in their biological context. The field is converging towards a consensus on the broad outlines of a mechanism that is driven by a progressive ATP-dependent treadmilling exchange of ESCRT subunits, as well as compositional change and geometric transitions in ESCRT filaments.

Significance The structure of the SARS-CoV-2 ORF8 protein reveals two novel intermolecular interfaces layered onto an ORF7 fold. One is mediated by a disulfide bond, the other is noncovalent, and both are novel with respect to SARS-CoV. The structural analysis here establishes a molecular framework for understanding the rapid evolution of ORF8, its contributions to COVID-19 pathogenesis, and the potential for its neutralization by antibodies., The molecular basis for the severity and rapid spread of the COVID-19 disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is largely unknown. ORF8 is a rapidly evolving accessory protein that has been proposed to interfere with immune responses. The crystal structure of SARS-CoV-2 ORF8 was determined at 2.04-Å resolution by X-ray crystallography. The structure reveals a ∼60-residue core similar to SARS-CoV-2 ORF7a, with the addition of two dimerization interfaces unique to SARS-CoV-2 ORF8. A covalent disulfide-linked dimer is formed through an N-terminal sequence specific to SARS-CoV-2, while a separate noncovalent interface is formed by another SARS-CoV-2−specific sequence, 73YIDI76. Together, the presence of these interfaces shows how SARS-CoV-2 ORF8 can form unique large-scale assemblies not possible for SARS-CoV, potentially mediating unique immune suppression and evasion activities.

The mechanistic target of rapamycin complex 1 (mTORC1) couples cell growth to nutrient, energy and growth factor availability (1–3). mTORC1 is activated at the lysosomal membrane when amino acids are replete via the Rag guanosine triphosphatases (GTPases) (4–6). Rags exist in two stable states, an inactive (RagA/BGDP:RagC/DGTP) and active (RagA/BGTP:RagC/DGDP) state, during low and high cellular amino acid levels (4, 5). The lysosomal folliculin (FLCN) complex (LFC) consists of the inactive Rag dimer, the pentameric scaffold Ragulator (7, 8), and the FLCN:FNIP (FLCN-interacting protein) GTPase activating protein (GAP) complex (9), and prevents activation of the Rag dimer during amino acid starvation (10, 11). How the LFC is released upon amino acid refeeding is a major outstanding question in amino-acid dependent Rag activation. Here we show that the cytoplasmic tail of the lysosomal solute carrier family 38 member 9 (SLC38A9), a known Rag activator (12–14), destabilizes the LFC. By breaking up the LFC, SLC38A9 triggers the GAP activity of FLCN:FNIP toward RagC. We present the cryo electron microscopy (cryo-EM) structures of Rags in complex with their lysosomal anchor complex Ragulator and the cytoplasmic tail of SLC38A9 in the pre and post GTP hydrolysis state of RagC, which explain how SLC38A9 destabilizes the LFC and so promotes Rag dimer activation.

The ESCRT complexes drive membrane scission in HIV-1 release, autophagosome closure, MVB biogenesis, cytokinesis, and other cell processes. ESCRT-I is the most upstream complex and bridges the system to HIV-1 Gag in virus release. The crystal structure of the headpiece of human ESCRT-I comprising TSG101–VPS28–VPS37B–MVB12A was determined, revealing an ESCRT-I helical assembly with a 12 molecule repeat. Electron microscopy confirmed that ESCRT-I subcomplexes form helical filaments in solution. Mutation of VPS28 helical interface residues blocks filament formation in vitro and autophagosome closure and HIV-1 release in human cells. Coarse grained simulations of ESCRT assembly at HIV-1 budding sites suggest that formation of a 12-membered ring of ESCRT-I molecules is a geometry-dependent checkpoint during late stages of Gag assembly and HIV-1 budding, and templates ESCRT-III assembly for membrane scission. These data show that ESCRT-I is not merely a bridging adaptor, but has an essential scaffolding and mechanical role in its own right., Reporting Summary Further information on experimental design is available in the Nature Research Reporting Summary linked to this article.

Mutation of C9ORF72 is the most prevalent defect in amyotrophic lateral sclerosis (ALS) and frontal temporal degeneration (FTD). Together with hexanucleotide repeat expansion, haploinsufficiency of C9ORF72 contributes to neuronal dysfunction. We determined the structure of the SMCR8-C9orf72-WDR41 complex by cryo-EM. C9orf72 and SMCR8 are both longin-DENN domain proteins, while WDR41 is a beta-propeller protein that binds to SMCR8 such that the whole structure resembles an eye slip hook. Contacts between WDR41 and SMCR8DENN drive lysosomal localization in amino acid starvation. The structure suggested that SMCR8-C9orf72 was a small GTPase activating protein (GAP). We found that SMCR8-C9orf72-WDR41 is a GAP for Arf family small GTPases, and refer to it as the Lysosomal SMCR8-C9orf72 Arf GAP (“L-SCARF”) complex. These data rationalize the function of C9orf72 both in normal physiology and in ALS/FTD.

Prinz and Hurley preview work from Li and colleagues, which uses structural analyses to reveal a new method of lipid transfer by VPS13., All lipid transport proteins in eukaryotes are thought to shuttle lipids between cellular membranes. In this issue, Li et al. (2020. J. Cell Biol. https://doi.org/10.1083/jcb.202001161) show that Vps13 has a channel-like domain that may allow lipids to flow between closely apposed membranes at contact sites.