Many large molecular machines are too elaborate to assemble spontaneously and are built through ordered pathways orchestrated by dedicated chaperones. During assembly of the core particle (CP) of the proteasome, where protein degradation occurs, its six active sites are simultaneously activated via cleavage of N-terminal propeptides. Such activation is autocatalytic and coupled to fusion of two half-CP intermediates, which protects cells by preventing activation until enclosure of the active sites within the CP interior. Here we uncover key mechanistic aspects of autocatalytic activation, which proceeds through alignment of the $\beta$5 and $\beta$2 catalytic triad residues, respectively, with these triads being misaligned before fusion. This mechanism contrasts with most other zymogens, in which catalytic centers are preformed. Our data also clarify the mechanism by which individual subunits can be added in a precise, temporally ordered manner. This work informs two decades-old mysteries in the proteasome field, with broader implications for protease biology and multisubunit complex assembly.

Gasdermin D (GSDMD) is the common effector for cytokine secretion and pyroptosis downstream of inflammasome activation and was previously shown to form large transmembrane pores upon cleavage by inflammatory caspases to generate the GSDMD N-terminal domain (GSDMD-NT)1-10. Here we report that GSDMD Cys191 is S-palmitoylated and palmitoylation is required for pore formation. S-palmitoylation, which does not affect GSDMD cleavage, is augmented by mitochondria-generated reactive oxygen species (ROS). Surprisingly, cleavage-deficient D275A GSDMD is also palmitoylated after inflammasome stimulation or treatment with ROS activators, and causes pyroptosis, although less efficiently than palmitoylated GSDMD-NT. Palmitoylated, but not unpalmitoylated, full-length GSDMD induces liposome leakage, and forms a pore similar in structure to GSDMD-NT pores shown by cryogenic electron microscopy. zDHHC5 and zDHHC9 are the major palmitoyltransferases that mediate GSDMD palmitoylation, and their expression is upregulated by inflammasome activation and ROS. The other human gasdermins are also palmitoylated in their N-termini. These data challenge the concept that cleavage is the only trigger for GSDMD activation. They suggest that reversible palmitoylation is a checkpoint for pore formation by both GSDMD-NT and intact GSDMD that serves as a general switch for the activation of this pore-forming family.

Rotaviruses infect cells by delivering into the cytosol a transcriptionally active inner capsid particle (a "double-layer particle": DLP). Delivery is the function of a third, outer layer, which drives uptake from the cell surface into small vesicles from which the DLPs escape. In published work, we followed stages of rhesus rotavirus (RRV) entry by live-cell imaging and correlated them with structures from cryogenic electron microscopy and tomography (cryo-EM and cryo-ET). The virus appears to wrap itself in membrane, leading to complete engulfment and loss of Ca2+ from the vesicle produced by the wrapping. One of the outer-layer proteins, VP7, is a Ca2+-stabilized trimer; loss of Ca2+ releases both VP7 and the other outer-layer protein, VP4, from the particle. VP4, activated by cleavage into VP8* and VP5*, is a trimer that undergoes a large-scale conformational rearrangement, reminiscent of the transition that viral fusion proteins undergo to penetrate a membrane. The rearrangement of VP5* thrusts a 250-residue, C-terminal segment of each of the three subunits outward, while allowing the protein to remain attached to the virus particle and to the cell being infected. We proposed that this segment inserts into the membrane of the target cell, enabling Ca2+ to cross. In the work reported here, we show the validity of key aspects of this proposed sequence. By cryo-EM studies of liposome-attached virions ("triple-layer particles": TLPs) and single-particle fluorescence imaging of liposome-attached TLPs, we confirm insertion of the VP4 C-terminal segment into the membrane and ensuing generation of a Ca2+ "leak". The results allow us to formulate a molecular description of early events in entry. We also discuss our observations in the context of other work on double-strand RNA virus entry.

Whereas oncogenes can potentially be inhibited with small molecules, the loss of tumour suppressors is more common and is problematic because the tumour-suppressor proteins are no longer present to be targeted. Notable examples include SMARCB1-mutant cancers, which are highly lethal malignancies driven by the inactivation of a subunit of SWI/SNF (also known as BAF) chromatin-remodelling complexes. Here, to generate mechanistic insights into the consequences of SMARCB1 mutation and to identify vulnerabilities, we contributed 14 SMARCB1-mutant cell lines to a near genome-wide CRISPR screen as part of the Cancer Dependency Map Project1–3. We report that the little-studied gene DDB1–CUL4-associated factor 5 (DCAF5) is required for the survival of SMARCB1-mutant cancers. We show that DCAF5 has a quality-control function for SWI/SNF complexes and promotes the degradation of incompletely assembled SWI/SNF complexes in the absence of SMARCB1. After depletion of DCAF5, SMARCB1-deficient SWI/SNF complexes reaccumulate, bind to target loci and restore SWI/SNF-mediated gene expression to levels that are sufficient to reverse the cancer state, including in vivo. Consequently, cancer results not from the loss of SMARCB1 function per se, but rather from DCAF5-mediated degradation of SWI/SNF complexes. These data indicate that therapeutic targeting of ubiquitin-mediated quality-control factors may effectively reverse the malignant state of some cancers driven by disruption of tumour suppressor complexes.

In response to pathogen infection, gasdermin (GSDM) proteins form membrane pores that induce a host cell death process called pyroptosis1–3. Studies of human and mouse GSDM pores have revealed the functions and architectures of assemblies comprising 24 to 33 protomers4–9, but the mechanism and evolutionary origin of membrane targeting and GSDM pore formation remain unknown. Here we determine a structure of a bacterial GSDM (bGSDM) pore and define a conserved mechanism of pore assembly. Engineering a panel of bGSDMs for site-specific proteolytic activation, we demonstrate that diverse bGSDMs form distinct pore sizes that range from smaller mammalian-like assemblies to exceptionally large pores containing more than 50 protomers. We determine a cryo-electron microscopy structure of a Vitiosangium bGSDM in an active `slinky'-like oligomeric conformation and analyse bGSDM pores in a native lipid environment to create an atomic-level model of a full 52-mer bGSDM pore. Combining our structural analysis with molecular dynamics simulations and cellular assays, our results support a stepwise model of GSDM pore assembly and suggest that a covalently bound palmitoyl can leave a hydrophobic sheath and insert into the membrane before formation of the membrane-spanning $\beta$-strand regions. These results reveal the diversity of GSDM pores found in nature and explain the function of an ancient post-translational modification in enabling programmed host cell death.

Conditional protein degradation tags (degrons) are usually >100 amino acids long or are triggered by small molecules with substantial off-target effects, thwarting their use as specific modulators of endogenous protein levels. We developed a phage-assisted continuous evolution platform for molecular glue complexes (MG-PACE) and evolved a 36–amino acid zinc finger (ZF) degron (SD40) that binds the ubiquitin ligase substrate receptor cereblon in complex with PT-179, an orthogonal thalidomide derivative. Endogenous proteins tagged in-frame with SD40 using prime editing are degraded by otherwise inert PT-179. Cryo–electron microscopy structures of SD40 in complex with ligand-bound cereblon revealed mechanistic insights into the molecular basis of SD40’s activity and specificity. Our efforts establish a system for continuous evolution of molecular glue complexes and provide ZF tags that overcome shortcomings associated with existing degrons. Degron tags enable rapid and tunable control of target protein levels using small molecules. The ability to develop tags with desirable properties could expand their use in research and biotechnology. Mercer et al. report a continuous evolution platform for generating high-affinity molecular glue complexes. Using this approach, the authors evolved a compact zinc-finger degron that engages the protein cereblon in the presence of thalidomide derivatives that avoid endogenous proteins, unlike the immunomodulatory drugs commonly used to trigger protein degradation. This work provides a compact orthogonal degron tag and a powerful system with which to engineer molecular glue interactions using diverse small molecules. —Di Jiang Rapid evolution of molecular glue interfaces yields a compact small molecule–triggered degron with high target protein specificity.

Cancer cells program fibroblasts into cancer associated fibroblasts (CAFs) in a two-step manner. First, cancer cells secrete exosomes to program quiescent fibroblasts into activated CAFs. Second, cancer cells maintain the CAF phenotype via activation of signal transduction pathways. We rationalized that inhibiting this two-step process can normalize CAFs into quiescent fibroblasts and augment the efficacy of immunotherapy. We show that cancer cell–targeted nanoliposomes that inhibit sequential steps of exosome biogenesis and release from lung cancer cells block the differentiation of lung fibroblasts into CAFs. In parallel, we demonstrate that CAF-targeted nanoliposomes that block two distinct nodes in fibroblast growth factor receptor (FGFR)–Wnt/β-catenin signaling pathway can reverse activate CAFs into quiescent fibroblasts. Co-administration of both nanoliposomes significantly improves the infiltration of cytotoxic T cells and enhances the antitumor efficacy of αPD-L1 in immunocompetent lung cancer–bearing mice. Simultaneously blocking the tumoral exosome-mediated activation of fibroblasts and FGFR-Wnt/β-catenin signaling constitutes a promising approach to augment immunotherapy. Blocking exosome-mediated activation of fibroblasts and FGFR/β-catenin axis using nanoliposomes augments αPD-L1 immunotherapy.

Baculoviruses are insect-infecting pathogens with wide applications as biological pesticides, in vitro protein production vehicles and gene therapy tools. Its cylindrical nucleocapsid, which encapsulates and protects the circular double-stranded viral DNA encoding proteins for viral replication and entry, is formed by the highly conserved major capsid protein VP39. The mechanism for VP39 assembly remains unknown. We use electron cryomicroscopy to determine a 3.2 \AA helical reconstruction of an infectious nucleocapsid of Autographa californica multiple nucleopolyhedrovirus, revealing how dimers of VP39 assemble into a 14-stranded helical tube. We show that VP39 comprises a distinct protein fold conserved across baculoviruses, which includes a Zinc finger domain and a stabilizing intra-dimer sling. Analysis of sample polymorphism shows that VP39 assembles in several closely-related helical geometries. This VP39 reconstruction reveals general principles for baculoviral nucleocapsid assembly.

Translation termination is an essential cellular process, which is also of therapeutic interest for diseases that manifest from premature stop codons. In eukaryotes, translation termination requires eRF1, which recognizes stop codons, catalyzes the release of nascent proteins from ribosomes and facilitates ribosome recycling. The small molecule SRI-41315 triggers eRF1 degradation and enhances translational readthrough of premature stop codons. However, the mechanism of action of SRI-41315 on eRF1 and translation is not known. Here we report cryo-EM structures showing that SRI-41315 acts as a metal-dependent molecular glue between the N domain of eRF1 responsible for stop codon recognition and the ribosomal subunit interface near the decoding center. Retention of eRF1 on ribosomes by SRI-41315 leads to ribosome collisions, eRF1 ubiquitylation and a higher frequency of translation termination at near-cognate stop codons. Our findings reveal a new mechanism of release factor inhibition and additional implications for pharmacologically targeting eRF1.

Gram-negative bacteria are extraordinarily difficult to kill because their cytoplasmic membrane is surrounded by an outer membrane that blocks the entry of most antibiotics. The impenetrable nature of the outer membrane is due to the presence of a large, amphipathic glycolipid called lipopolysaccharide (LPS) in its outer leaflet1. Assembly of the outer membrane requires transport of LPS across a protein bridge that spans from the cytoplasmic membrane to the cell surface. Maintaining outer membrane integrity is essential for bacterial cell viability, and its disruption can increase susceptibility to other antibiotics2–6. Thus, inhibitors of the seven lipopolysaccharide transport (Lpt) proteins that form this transenvelope transporter have long been sought. A new class of antibiotics that targets the LPS transport machine in Acinetobacter was recently identified. Here, using structural, biochemical and genetic approaches, we show that these antibiotics trap a substrate-bound conformation of the LPS transporter that stalls this machine. The inhibitors accomplish this by recognizing a composite binding site made up of both the Lpt transporter and its LPS substrate. Collectively, our findings identify an unusual mechanism of lipid transport inhibition, reveal a druggable conformation of the Lpt transporter and provide the foundation for extending this class of antibiotics to other Gram-negative pathogens.

Phylogenetically and antigenically distinct influenza A and B viruses (IAV and IBV) circulate in human populations, causing widespread morbidity. Antibodies (Abs) that bind epitopes conserved in both IAV and IBV hemagglutinins (HAs) could protect against disease by diverse virus subtypes. Only one reported HA Ab, isolated from a combinatorial display library, protects against both IAV and IBV. Thus, there has been so far no information on the likelihood of finding naturally occurring human Abs that bind HAs of diverse IAV subtypes and IBV lineages. We have now recovered from several unrelated human donors five clonal Abs that bind a conserved epitope preferentially exposed in the postfusion conformation of IAV and IVB HA2. These Abs lack neutralizing activity in vitro but in mice provide strong, IgG subtype–dependent protection against lethal IAV and IBV infections. Strategies to elicit similar Abs routinely might contribute to more effective influenza vaccines.

Acetylation of histones is a key post-translational modification that guides gene expression regulation. In yeast, the class I histone deacetylase containing Rpd3S complex plays a critical role in the suppression of spurious transcription by removing histone acetylation from actively transcribed genes. The S. cerevisiae Rpd3S complex has five subunits (Rpd3, Sin3, Rco1, Eaf3, and Ume1) but its subunit stoichiometry and how the complex engages nucleosomes to achieve substrate specificity remains elusive. Here we report the cryo-EM structure of the complete Rpd3S complex bound to a nucleosome. Sin3 and two copies of subunits Rco1 and Eaf3 encircle the deacetylase subunit Rpd3 and coordinate the positioning of Ume1. The Rpd3S complex binds both trimethylated H3 tails at position lysine 36 and makes multiple additional contacts with the nucleosomal DNA and the H2A–H2B acidic patch. Direct regulation via the Sin3 subunit coordinates binding of the acetylated histone substrate to achieve substrate specificity.

FOXP3 is a transcription factor that is essential for the development of regulatory T cells, a branch of T cells that suppress excessive inflammation and autoimmunity1–5. However, the molecular mechanisms of FOXP3 remain unclear. Here we here show that FOXP3 uses the forkhead domain–-a DNA-binding domain that is commonly thought to function as a monomer or dimer–-to form a higher-order multimer after binding to TnG repeat microsatellites. The cryo-electron microscopy structure of FOXP3 in a complex with T3G repeats reveals a ladder-like architecture, whereby two double-stranded DNA molecules form the two `side rails' bridged by five pairs of FOXP3 molecules, with each pair forming a `rung'. Each FOXP3 subunit occupies TGTTTGT within the repeats in a manner that is indistinguishable from that of FOXP3 bound to the forkhead consensus motif (TGTTTAC). Mutations in the intra-rung interface impair TnG repeat recognition, DNA bridging and the cellular functions of FOXP3, all without affecting binding to the forkhead consensus motif. FOXP3 can tolerate variable inter-rung spacings, explaining its broad specificity for TnG-repeat-like sequences in vivo and in vitro. Both FOXP3 orthologues and paralogues show similar TnG repeat recognition and DNA bridging. These findings therefore reveal a mode of DNA recognition that involves transcription factor homomultimerization and DNA bridging, and further implicates microsatellites in transcriptional regulation and diseases.

Bacteria encode hundreds of diverse defense systems that protect from viral infection and inhibit phage propagation1–5. Gabija is one of the most prevalent anti-phage defense systems, occurring in >15% of all sequenced bacterial and archaeal genomes1,6,7, but the molecular basis of how Gabija defends cells from viral infection remains poorly understood. Here we use X-ray crystallography and cryo-EM to define how Gabija proteins assemble into an \textasciitilde500 kDa supramolecular complex that degrades phage DNA. Gabija protein A (GajA) is a DNA endonuclease that tetramerizes to form the core of the anti-phage defense complex. Two sets of Gabija protein B (GajB) dimers dock at opposite sides of the complex and create a 4:4 GajAB assembly that is essential for phage resistance in vivo. We show that a phage-encoded protein Gabija anti-defense 1 (Gad1) directly binds the Gabija GajAB complex and inactivates defense. A cryo-EM structure of the virally inhibited state reveals that Gad1 forms an octameric web that encases the GajAB complex and inhibits DNA recognition and cleavage. Our results reveal the structural basis of assembly of the Gabija anti-phage defense complex and define a unique mechanism of viral immune evasion.

Inflammatory caspases are key enzymes in mammalian innate immunity that control the processing and release of interleukin-1 (IL-1)-family cytokines1,2. Despite the biological importance, the structural basis for inflammatory caspase-mediated cytokine processing has remained unclear. To date, catalytic cleavage of IL-1-family members, including pro-IL-1$\beta$ and pro-IL-18, has been attributed primarily to caspase-1 activities within canonical inflammasomes3. Here we demonstrate that the lipopolysaccharide receptor caspase-4 from humans and other mammalian species (except rodents) can cleave pro-IL-18 with an efficiency similar to pro-IL-1$\beta$ and pro-IL-18 cleavage by the prototypical IL-1-converting enzyme caspase-1. This ability of caspase-4 to cleave pro-IL-18, combined with its previously defined ability to cleave and activate the lytic pore-forming protein gasdermin D (GSDMD)4,5, enables human cells to bypass the need for canonical inflammasomes and caspase-1 for IL-18 release. The structure of the caspase-4–pro-IL-18 complex determined using cryogenic electron microscopy reveals that pro-lL-18 interacts with caspase-4 through two distinct interfaces: a protease exosite and an interface at the caspase-4 active site involving residues in the pro-domain of pro-IL-18, including the tetrapeptide caspase-recognition sequence6. The mechanisms revealed for cytokine substrate capture and cleavage differ from those observed for the caspase substrate GSDMD7,8. These findings provide a structural framework for the discussion of caspase activities in health and disease.

ATAD2 is a non-canonical ATP-dependent histone chaperone and a major cancer target. Despite widespread efforts to design drugs targeting the ATAD2 bromodomain, little is known about the overall structural organization and regulation of ATAD2. Here, we present the 3.1 \AA cryo-EM structure of human ATAD2 in the ATP state, showing a shallow hexameric spiral that binds a peptide substrate at the central pore. The spiral conformation is locked by an N-terminal linker domain (LD) that wedges between the seam subunits, thus limiting ATP-dependent symmetry breaking of the AAA+ ring. In contrast, structures of the ATAD2-histone H3/H4 complex show the LD undocked from the seam, suggesting that H3/H4 binding unlocks the AAA+ spiral by allosterically releasing the LD. These findings, together with the discovery of an inter-subunit signaling mechanism, reveal a unique regulatory mechanism for ATAD2 and lay the foundation for developing new ATAD2 inhibitors.

Assembly of the proteasome's core particle (CP), a barrel-shaped chamber of four stacked rings, requires five chaperones and five subunit propeptides. Fusion of two half-CP precursors yields a complete structure but remains immature until active site maturation. Here, using Saccharomyces cerevisiae, we report a high-resolution cryogenic electron microscopy structure of preholoproteasome, a post-fusion assembly intermediate. Our data reveal how CP midline-spanning interactions induce local changes in structure, facilitating maturation. Unexpectedly, we find that cleavage may not be sufficient for propeptide release, as residual interactions with chaperones such as Ump1 hold them in place. We evaluated previous models proposing that dynamic conformational changes in chaperones drive CP fusion and autocatalytic activation by comparing preholoproteasome to pre-fusion intermediates. Instead, the data suggest a scaffolding role for the chaperones Ump1 and Pba1/Pba2. Our data clarify key aspects of CP assembly, suggest that undiscovered mechanisms exist to explain CP fusion/activation, and have relevance for diseases of defective CP biogenesis.