@article {Radko-Juettner2024-wm, title = {Targeting DCAF5 suppresses SMARCB1-mutant cancer by stabilizing SWI/SNF}, journal = {Nature}, year = {2024}, abstract = {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{\textendash}3. We report that the little-studied gene DDB1{\textendash}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.}, author = {Radko-Juettner, Sandi and Yue, Hong and Myers, Jacquelyn A and Carter, Raymond D and Robertson, Alexis N and Mittal, Priya and Zhu, Zhexin and Hansen, Baranda S and Donovan, Katherine A and Hunkeler, Moritz and Rosikiewicz, Wojciech and Wu, Zhiping and McReynolds, Meghan G and Roy Burman, Shourya S and Schmoker, Anna M and Mageed, Nada and Brown, Scott A and Mobley, Robert J and Partridge, Janet F and Stewart, Elizabeth A and Pruett-Miller, Shondra M and Behnam Nabet and Peng, Junmin and Gray, Nathanael S and Fischer, Eric S and Roberts, Charles W M} } @article {Johnson2024-al, title = {Structure and assembly of a bacterial gasdermin pore}, journal = {Nature}, year = {2024}, abstract = {In response to pathogen infection, gasdermin (GSDM) proteins form membrane pores that induce a host cell death process called pyroptosis1{\textendash}3. Studies of human and mouse GSDM pores have revealed the functions and architectures of assemblies comprising 24 to 33 protomers4{\textendash}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 {\textquoteleft}slinky{\textquoteright}-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.}, author = {Johnson, Alex G and Mayer, Megan L and Schaefer, Stefan L and McNamara-Bordewick, Nora K and Hummer, Gerhard and Kranzusch, Philip J} } @article { doi:10.1126/science.adk4422, title = {Continuous evolution of compact protein degradation tags regulated by selective molecular glues}, journal = {Science}, volume = {383}, number = {6688}, year = {2024}, pages = {eadk4422}, abstract = {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{\textendash}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{\textendash}electron microscopy structures of SD40 in complex with ligand-bound cereblon revealed mechanistic insights into the molecular basis of SD40{\textquoteright}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. {\textemdash}Di Jiang Rapid evolution of molecular glue interfaces yields a compact small molecule{\textendash}triggered degron with high target protein specificity.}, doi = {10.1126/science.adk4422}, url = {https://www.science.org/doi/abs/10.1126/science.adk4422}, author = {Jaron A. M. Mercer and Stephan J. DeCarlo and Roy Burman, Shourya S. and Vedagopuram Sreekanth and Andrew T. Nelson and Hunkeler, Moritz and Peter J. Chen and Katherine A. Donovan and Praveen Kokkonda and Praveen K. Tiwari and Veronika M. Shoba and Arghya Deb and Amit Choudhary and Fischer, Eric S. and Liu, David R.} } @article { doi:10.1126/sciadv.adk3074, title = {Modulating tumoral exosomes and fibroblast phenotype using nanoliposomes augments cancer immunotherapy}, journal = {Science Advances}, volume = {10}, number = {9}, year = {2024}, pages = {eadk3074}, abstract = {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{\textendash}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){\textendash}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{\textendash}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.}, doi = {10.1126/sciadv.adk3074}, url = {https://www.science.org/doi/abs/10.1126/sciadv.adk3074}, author = {May S. Freag and Mostafa T. Mohammed and Arpita Kulkarni and Hagar E. Emam and Krishna P. Maremanda and Ahmed O. Elzoghby} } @article {Benning2024-yu, title = {Helical reconstruction of VP39 reveals principles for baculovirus nucleocapsid assembly}, journal = {Nature Communications}, volume = {15}, number = {1}, year = {2024}, pages = {250}, abstract = {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.}, author = {Benning, Friederike M C and Jenni, Simon and Garcia, Coby Y and Nguyen, Tran H and Zhang, Xuewu and Chao, Luke H} } @article {Coelho2024-pi, title = {The eRF1 degrader SRI-41315 acts as a molecular glue at the ribosomal decoding center}, journal = {Nature Chemical Biology}, year = {2024}, abstract = {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.}, author = {Coelho, Jo{\~a}o P L and Yip, Matthew CJ and Oltion, Keely and Taunton, Jack and Shao, Sichen} } @article {Pahil2024-vp, title = {A new antibiotic traps lipopolysaccharide in its intermembrane transporter}, journal = {Nature}, year = {2024}, abstract = {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{\textendash}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.}, author = {Pahil, Karanbir S and Gilman, Morgan S A and Baidin, Vadim and Clairfeuille, Thomas and Mattei, Patrizio and Bieniossek, Christoph and Dey, Fabian and Muri, Dieter and Baettig, Remo and Lobritz, Michael and Bradley, Kenneth and Kruse, Andrew C and Kahne, Daniel} } @article {doi:10.1021/acs.biochem.3c00425, title = {Reconstitution of the Alzheimer{\textquoteright}s Disease Tau Core Structure from Recombinant Tau297{\textendash}391 Yields Variable Quaternary Structures as Seen by Negative Stain and Cryo-EM}, journal = {Biochemistry}, year = {2023}, note = {PMID: 38154792}, pages = {null}, doi = {10.1021/acs.biochem.3c00425}, url = {https://doi.org/10.1021/acs.biochem.3c00425}, author = {Glynn, Calina and Chun, Joshua E. and Donahue, Cameron C. and Nadler, Monica J. S. and Fan, Zhanyun and Hyman, Bradley T.} } @article { doi:10.1073/pnas.2316964120, title = {Protective human antibodies against a conserved epitope in pre- and postfusion influenza hemagglutinin}, journal = {Proceedings of the National Academy of Sciences}, volume = {121}, number = {1}, year = {2023}, pages = {e2316964120}, abstract = {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{\textendash}dependent protection against lethal IAV and IBV infections. Strategies to elicit similar Abs routinely might contribute to more effective influenza vaccines.}, doi = {10.1073/pnas.2316964120}, url = {https://www.pnas.org/doi/abs/10.1073/pnas.2316964120}, author = {Joel Finney and Moseman, Annie Park and Susan Kong and Watanabe, Akiko and Song, Shengli and Walsh, Richard M. and Kuraoka, Masayuki and Ryutaro Kotaki and E. Ashley Moseman and McCarthy, Kevin R. and Dongmei Liao and Xiaoe Liang and Xiaoyan Nie and Olivia Lavidor and Richard Abbott and Harrison, Stephen C. and Kelsoe, Garnett} } @article {Markert2023, title = {Structure of the complete Saccharomyces cerevisiae Rpd3S-nucleosome complex}, journal = {Nature Communications}, volume = {14}, number = {1}, year = {2023}, month = {Dec}, pages = {8128}, abstract = {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{\textendash}H2B acidic patch. Direct regulation via the Sin3 subunit coordinates binding of the acetylated histone substrate to achieve substrate specificity.}, issn = {2041-1723}, doi = {10.1038/s41467-023-43968-8}, url = {https://doi.org/10.1038/s41467-023-43968-8}, author = {Markert, Jonathan W. and Seychelle M. Vos and Lucas Farnung} } @article {Zhang2023, title = {FOXP3 recognizes microsatellites and bridges DNA through multimerization}, journal = {Nature}, year = {2023}, month = {Nov}, abstract = {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{\textendash}5. However, the molecular mechanisms of FOXP3 remain unclear. Here we here show that FOXP3 uses the forkhead domain{\textendash}-a DNA-binding domain that is commonly thought to function as a monomer or dimer{\textendash}-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 {\textquoteleft}side rails{\textquoteright} bridged by five pairs of FOXP3 molecules, with each pair forming a {\textquoteleft}rung{\textquoteright}. 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.}, issn = {1476-4687}, doi = {10.1038/s41586-023-06793-z}, url = {https://doi.org/10.1038/s41586-023-06793-z}, author = {Zhang, Wenxiang and Leng, Fangwei and Wang, Xi and Ramirez, Ricardo N. and Park, Jinseok and Benoist, Christophe and Sun Hur} } @article {Antine2023, title = {Structural basis of Gabija anti-phage defence and viral immune evasion}, journal = {Nature}, year = {2023}, month = {Nov}, abstract = {Bacteria encode hundreds of diverse defense systems that protect from viral infection and inhibit phage propagation1{\textendash}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.}, issn = {1476-4687}, doi = {10.1038/s41586-023-06855-2}, url = {https://doi.org/10.1038/s41586-023-06855-2}, author = {Sadie P. Antine and Alex G. Johnson and Mooney, Sarah E. and Leavitt, Azita and Megan L. Mayer and Erez Yirmiya and Gil Amitai and Sorek, Rotem and Kranzusch, Philip J.} } @article {Devant2023-sx, title = {Structural insights into cytokine cleavage by inflammatory caspase-4}, journal = {Nature}, volume = {624}, number = {7991}, year = {2023}, pages = {451{\textendash}459}, abstract = {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{\textendash}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.}, author = {Devant, Pascal and Dong, Ying and Mintseris, Julian and Ma, Weiyi and Gygi, Steven P and Wu, Hao and Kagan, Jonathan C} } @article {Cho2023-xp, title = {Structure of the human ATAD2 AAA+ histone chaperone reveals mechanism of regulation and inter-subunit communication}, journal = {Communications Biology}, volume = {6}, number = {1}, year = {2023}, pages = {993}, abstract = {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.}, author = {Cho, Carol and Ganser, Christian and Uchihashi, Takayuki and Kato, Koichi and Song, Ji-Joon} } @article {Walsh2023, title = {Structure of the preholoproteasome reveals late steps in proteasome core particle biogenesis}, journal = {Nature Structural \& Molecular Biology}, year = {2023}, month = {Aug}, abstract = {Assembly of the proteasome{\textquoteright}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.}, issn = {1545-9985}, doi = {10.1038/s41594-023-01081-w}, url = {https://doi.org/10.1038/s41594-023-01081-w}, author = {Walsh, Richard M. and Rawson, Shaun and Schnell, Helena M. and Velez, Benjamin and Rajakumar, Tamayanthi and Hanna, John} } @article {ref1, title = {TMEM63 proteins function as monomeric high-threshold mechanosensitive ion channels}, journal = {Neuron}, year = {2023}, month = {2023/08/04}, publisher = {Elsevier}, abstract = {OSCA/TMEM63s form mechanically activated (MA) ion channels in plants and animals, respectively. OSCAs and related TMEM16s and transmembrane channel-like (TMC) proteins form homodimers with two pores. Here, we uncover an unanticipated monomeric configuration of TMEM63 proteins. Structures of TMEM63A and TMEM63B (referred to as TMEM63s) revealed a single highly restricted pore. Functional analyses demonstrated that TMEM63s are bona fide mechanosensitive ion channels, characterized by small conductance and high thresholds. TMEM63s possess evolutionary variations in the intracellular linker IL2, which mediates dimerization in OSCAs. Replacement of OSCA1.2 IL2 with TMEM63A IL2 or mutations to key variable residues resulted in monomeric OSCA1.2 and MA currents with significantly higher thresholds. Structural analyses revealed substantial conformational differences in the mechano-sensing domain IL2 and gating helix TM6 between TMEM63s and OSCA1.2. Our studies reveal that mechanosensitivity in OSCA/TMEM63 channels is affected by oligomerization and suggest gating mechanisms that may be shared by OSCA/TMEM63, TMEM16, and TMC channels.}, issn = {0896-6273}, doi = {10.1016/j.neuron.2023.07.006}, url = {https://doi.org/10.1016/j.neuron.2023.07.006}, author = {Wang Zheng and Rawson, Shaun and Shen, Zhangfei and Tamilselvan, Elakkiya and Smith, Harper E. and Halford, Julia and Shen, Chen and Murthy, Swetha E. and Ulbrich, Maximilian H. and Sotomayor, Marcos and Tian-Min Fu and Jeffrey R. Holt} } @article {Park2023, title = {Cryo-EM structure of a RAS/RAF recruitment complex}, journal = {Nature Communications}, volume = {14}, number = {1}, year = {2023}, month = {Jul}, pages = {4580}, abstract = {RAF-family kinases are activated by recruitment to the plasma membrane by GTP-bound RAS, whereupon they initiate signaling through the MAP kinase cascade. Prior structural studies of KRAS with RAF have focused on the isolated RAS-binding and cysteine-rich domains of RAF (RBD and CRD, respectively), which interact directly with RAS. Here we describe cryo-EM structures of a KRAS bound to intact BRAF in an autoinhibited state with MEK1 and a 14-3-3 dimer. Analysis of this KRAS/BRAF/MEK1/14-3-3 complex reveals KRAS bound to the RAS-binding domain of BRAF, captured in two orientations. Core autoinhibitory interactions in the complex are unperturbed by binding of KRAS and in vitro activation studies confirm that KRAS binding is insufficient to activate BRAF, absent membrane recruitment. These structures illustrate the separability of binding and activation of BRAF by RAS and suggest stabilization of this pre-activation intermediate as an alternative therapeutic strategy to blocking binding of KRAS.}, issn = {2041-1723}, doi = {10.1038/s41467-023-40299-6}, url = {https://doi.org/10.1038/s41467-023-40299-6}, author = {Park, Eun Young and Rawson, Shaun and Schmoker, Anna and Kim, Byeong-Won and Oh, Sehee and Song, KangKang and Jeon, Hyesung and Eck, Michael J.} } @article {ref1, title = {Structural basis for membrane-proximal proteolysis of substrates by ADAM10}, journal = {Cell}, year = {2023}, month = {2023/08/01}, publisher = {Elsevier}, abstract = {The endopeptidase ADAM10 is a critical catalyst for the regulated proteolysis of key drivers of mammalian development, physiology, and non-amyloidogenic cleavage of APP as the primary $\alpha$-secretase. ADAM10 function requires the formation of a complex with a C8-tetraspanin protein, but how tetraspanin binding enables positioning of the enzyme active site for membrane-proximal cleavage remains unknown. We present here a cryo-EM structure of a vFab-ADAM10-Tspan15 complex, which shows that Tspan15 binding relieves ADAM10 autoinhibition and acts as a molecular measuring stick to position the enzyme active site about 20 \AA from the plasma membrane for membrane-proximal substrate cleavage. Cell-based assays of N-cadherin shedding establish that the positioning of the active site by the interface between the ADAM10 catalytic domain and the bound tetraspanin influences selection of the preferred cleavage site. Together, these studies reveal the molecular mechanism underlying ADAM10 proteolysis at membrane-proximal sites and offer a roadmap for its modulation in disease.}, issn = {0092-8674}, doi = {10.1016/j.cell.2023.06.026}, url = {https://doi.org/10.1016/j.cell.2023.06.026}, author = {Lipper, Colin H. and Egan, Emily D. and Gabriel, Khal-Hentz and Blacklow, Stephen C.} } @article {Zhang2023, title = {Structural and functional characteristics of the SARS-CoV-2 Omicron subvariant BA.2 spike protein}, journal = {Nature Structural \& Molecular Biology}, volume = {30}, number = {7}, year = {2023}, month = {Jul}, pages = {980-990}, abstract = {The Omicron subvariant BA.2 has become the dominant circulating strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in many countries. Here, we have characterized structural, functional and antigenic properties of the full-length BA.2 spike (S) protein and compared replication of the authentic virus in cell culture and an animal model with previously prevalent variants. BA.2{\th}inspace}S can fuse membranes slightly more efficiently than Omicron BA.1, but still less efficiently than other previous variants. Both BA.1 and BA.2 viruses replicated substantially faster in animal lungs than the early G614 (B.1) strain in the absence of pre-existing immunity, possibly explaining the increased transmissibility despite their functionally compromised spikes. As in BA.1, mutations in the BA.2{\th}inspace}S remodel its antigenic surfaces, leading to strong resistance to neutralizing antibodies. These results suggest that both immune evasion and replicative advantage may contribute to the heightened transmissibility of the Omicron subvariants.}, issn = {1545-9985}, doi = {10.1038/s41594-023-01023-6}, url = {https://doi.org/10.1038/s41594-023-01023-6}, author = {Zhang, Jun and Tang, Weichun and Gao, Hailong and Lavine, Christy L. and Shi, Wei and Peng, Hanqin and Zhu, Haisun and Anand, Krishna and Kosikova, Matina and Kwon, Hyung Joon and Tong, Pei and Gautam, Avneesh and Rits-Volloch, Sophia and Wang, Shaowei and Megan L. Mayer and Wesemann, Duane R. and Seaman, Michael S. and Lu, Jianming and Xiao, Tianshu and Xie, Hang and Chen, Bing} } @article {Shlosman2023, title = {Allosteric activation of cell wall synthesis during bacterial growth}, journal = {Nature Communications}, volume = {14}, number = {1}, year = {2023}, month = {Jun}, pages = {3439}, abstract = {The peptidoglycan (PG) cell wall protects bacteria against osmotic lysis and determines cell shape, making this structure a key antibiotic target. Peptidoglycan is a polymer of glycan chains connected by peptide crosslinks, and its synthesis requires precise spatiotemporal coordination between glycan polymerization and crosslinking. However, the molecular mechanism by which these reactions are initiated and coupled is unclear. Here we use single-molecule FRET and cryo-EM to show that an essential PG synthase (RodA-PBP2) responsible for bacterial elongation undergoes dynamic exchange between closed and open states. Structural opening couples the activation of polymerization and crosslinking and is essential in vivo. Given the high conservation of this family of synthases, the opening motion that we uncovered likely represents a conserved regulatory mechanism that controls the activation of PG synthesis during other cellular processes, including cell division.}, issn = {2041-1723}, doi = {10.1038/s41467-023-39037-9}, url = {https://doi.org/10.1038/s41467-023-39037-9}, author = {Shlosman, Irina and Fivenson, Elayne M. and Gilman, Morgan S.A. and Sisley, Tyler A. and Walker, Suzanne and Bernhardt, Thomas G. and Kruse, Andrew C. and Loparo, Joseph J.} } @article {Shi2023, title = {Cryo-EM structure of SARS-CoV-2 postfusion spike in membrane}, journal = {Nature}, volume = {619}, number = {7969}, year = {2023}, month = {Jul}, pages = {403-409}, abstract = {The entry of SARS-CoV-2 into host cells depends on the refolding of the virus-encoded spike protein from a prefusion conformation, which is metastable after cleavage, to a lower-energy stable postfusion conformation1,2. This transition overcomes kinetic barriers for fusion of viral and target cell membranes3,4. Here we report a cryogenic electron microscopy (cryo-EM) structure of the intact postfusion spike in a lipid bilayer that represents the single-membrane product of the fusion reaction. The structure provides structural definition of the functionally critical membrane-interacting segments, including the fusion peptide and transmembrane anchor. The internal fusion peptide forms a hairpin-like wedge that spans almost the entire lipid bilayer and the transmembrane segment wraps around the fusion peptide at the last stage of membrane fusion. These results advance our understanding of the spike protein in a membrane environment and may guide development of intervention strategies.}, issn = {1476-4687}, doi = {10.1038/s41586-023-06273-4}, url = {https://doi.org/10.1038/s41586-023-06273-4}, author = {Shi, Wei and Cai, Yongfei and Zhu, Haisun and Peng, Hanqin and Voyer, Jewel and Rits-Volloch, Sophia and Cao, Hong and Megan L. Mayer and Song, KangKang and Xu, Chen and Lu, Jianming and Zhang, Jun and Chen, Bing} } @article {Walton2023, title = {Axonemal structures reveal mechanoregulatory and disease mechanisms}, journal = {Nature}, volume = {618}, number = {7965}, year = {2023}, month = {Jun}, pages = {625-633}, abstract = {Motile cilia and flagella beat rhythmically on the surface of cells to power the flow of fluid and to enable spermatozoa and unicellular eukaryotes to swim. In humans, defective ciliary motility can lead to male infertility and a congenital disorder called primary ciliary dyskinesia (PCD), in which impaired clearance of mucus by the cilia causes chronic respiratory infections1. Ciliary movement is generated by the axoneme, a molecular machine consisting of microtubules, ATP-powered dynein motors and regulatory complexes2. The size and complexity of the axoneme has so far prevented the development of an atomic model, hindering efforts to understand how it functions. Here we capitalize on recent developments in artificial intelligence-enabled structure prediction and cryo-electron microscopy (cryo-EM) to determine the structure of the 96-nm modular repeats of axonemes from the flagella of the alga Chlamydomonas reinhardtii and human respiratory cilia. Our atomic models provide insights into the conservation and specialization of axonemes, the interconnectivity between dyneins and their regulators, and the mechanisms that maintain axonemal periodicity. Correlated conformational changes in mechanoregulatory complexes with their associated axonemal dynein motors provide a mechanism for the long-hypothesized mechanotransduction pathway to regulate ciliary motility. Structures of respiratory-cilia doublet microtubules from four individuals with PCD reveal how the loss of individual docking factors can selectively eradicate periodically repeating structures.}, issn = {1476-4687}, doi = {10.1038/s41586-023-06140-2}, url = {https://doi.org/10.1038/s41586-023-06140-2}, author = {Walton, Travis and Gui, Miao and Velkova, Simona and Fassad, Mahmoud R. and Hirst, Robert A. and Haarman, Eric and O{\textquoteright}Callaghan, Christopher and Bottier, Mathieu and Thomas Burgoyne and Mitchison, Hannah M. and Brown, Alan} } @article {Jones2023, title = {Tethered agonist activated ADGRF1 structure and signalling analysis reveal basis for G protein coupling}, journal = {Nature Communications}, volume = {14}, number = {1}, year = {2023}, month = {Apr}, pages = {2490}, abstract = {Adhesion G Protein Coupled Receptors (aGPCRs) have evolved an activation mechanism to translate extracellular force into liberation of a tethered agonist (TA) to effect cell signalling. We report here that ADGRF1 can signal through all major G protein classes and identify the structural basis for a previously reported G$\alpha$q preference by cryo-EM. Our structure shows that G$\alpha$q preference in ADGRF1 may derive from tighter packing at the conserved F569 of the TA, altering contacts between TM helix I and VII, with a concurrent rearrangement of TM helix VII and helix VIII at the site of G$\alpha$ recruitment. Mutational studies of the interface and of contact residues within the 7TM domain identify residues critical for signalling, and suggest that G$\alpha$s signalling is more sensitive to mutation of TA or binding site residues than G$\alpha$q. Our work advances the detailed molecular understanding of aGPCR TA activation, identifying features that potentially explain preferential signal modulation.}, issn = {2041-1723}, doi = {10.1038/s41467-023-38083-7}, url = {https://doi.org/10.1038/s41467-023-38083-7}, author = {Jones, Daniel T. D. and Dates, Andrew N. and Rawson, Shaun D. and Burruss, Maggie M. and Lipper, Colin H. and Blacklow, Stephen C.} } @article {Erlandson2023-mc, title = {The relaxin receptor RXFP1 signals through a mechanism of autoinhibition}, journal = {Nature Chemical Biology}, year = {2023}, abstract = {The relaxin family peptide receptor 1 (RXFP1) is the receptor for relaxin-2, an important regulator of reproductive and cardiovascular physiology. RXFP1 is a multi-domain G protein-coupled receptor (GPCR) with an ectodomain consisting of a low-density lipoprotein receptor class A (LDLa) module and leucine-rich repeats. The mechanism of RXFP1 signal transduction is clearly distinct from that of other GPCRs, but remains very poorly understood. In the present study, we determine the cryo-electron microscopy structure of active-state human RXFP1, bound to a single-chain version of the endogenous agonist relaxin-2 and the heterotrimeric Gs protein. Evolutionary coupling analysis and structure-guided functional experiments reveal that RXFP1 signals through a mechanism of autoinhibition. Our results explain how an unusual GPCR family functions, providing a path to rational drug development targeting the relaxin receptors.}, author = {Erlandson, Sarah C and Rawson, Shaun and Osei-Owusu, James and Brock, Kelly P and Liu, Xinyue and Paulo, Joao A and Mintseris, Julian and Gygi, Steven P and Marks, Debora S and Cong, Xiaojing and Kruse, Andrew C} } @article {PLUMMERMEDEIROS2023168038, title = {Activity and Structural Dynamics of Human ABCA1 in a Lipid Membrane}, journal = {Journal of Molecular Biology}, volume = {435}, number = {8}, year = {2023}, pages = {168038}, abstract = {The human ATP-binding cassette (ABC) transporter ABCA1 plays a critical role in lipid homeostasis as it extracts sterols and phospholipids from the plasma membrane for excretion to the extracellular apolipoprotein A-I and subsequent formation of high-density lipoprotein (HDL) particles. Deleterious mutations of ABCA1 lead to sterol accumulation and are associated with atherosclerosis, poor cardiovascular outcomes, cancer, and Alzheimer{\textquoteright}s disease. The mechanism by which ABCA1 drives lipid movement is poorly understood, and a unified platform to produce active ABCA1 protein for both functional and structural studies has been missing. In this work, we established a stable expression system for both a human cell-based sterol export assay and protein purification for in vitro biochemical and structural studies. ABCA1 produced in this system was active in sterol export and displayed enhanced ATPase activity after reconstitution into a lipid bilayer. Our single-particle cryo-EM study of ABCA1 in nanodiscs showed protein induced membrane curvature, revealed multiple distinct conformations, and generated a structure of nanodisc-embedded ABCA1 at 4.0-{\r A} resolution representing a previously unknown conformation. Comparison of different ABCA1 structures and molecular dynamics simulations demonstrates both concerted domain movements and conformational variations within each domain. Taken together, our platform for producing and characterizing ABCA1 in a lipid membrane enabled us to gain important mechanistic and structural insights and paves the way for investigating modulators that target the functions of ABCA1.}, keywords = {ABC transporter, Cholesterol, cryo-electron microscopy, membrane transport, membrane transporter reconstitution}, issn = {0022-2836}, doi = {https://doi.org/10.1016/j.jmb.2023.168038}, url = {https://www.sciencedirect.com/science/article/pii/S0022283623000943}, author = {Ashlee M. Plummer-Medeiros and Alan T. Culbertson and Claudio L. Morales-Perez and Maofu Liao} } @article {doi:10.1021/jacs.2c13512, title = {Structural Basis of Sirtuin 6-Catalyzed Nucleosome Deacetylation}, journal = {Journal of the American Chemical Society}, year = {2023}, note = {PMID: 36930461}, pages = {null}, doi = {10.1021/jacs.2c13512}, url = {https://doi.org/10.1021/jacs.2c13512}, author = {Wang, Zhipeng A. and Markert, Jonathan W. and Whedon, Samuel D. and Yapa Abeywardana, Maheeshi and Lee, Kwangwoon and Jiang, Hanjie and Suarez, Carolay and Lin, Hening and Lucas Farnung and Philip A. Cole} } @article {DUNCANLOWEY2023, title = {Cryo-EM structure of the RADAR supramolecular anti-phage defense complex}, journal = {Cell}, year = {2023}, abstract = {Summary RADAR is a two-protein bacterial defense system that was reported to defend against phage by {\textquotedblleft}editing{\textquotedblright} messenger RNA. Here, we determine cryo-EM structures of the RADAR defense complex, revealing RdrA as a heptameric, two-layered AAA+ ATPase and RdrB as a dodecameric, hollow complex with twelve surface-exposed deaminase active sites. RdrA and RdrB join to form a giant assembly up to 10 MDa, with RdrA docked as a funnel over the RdrB active site. Surprisingly, our structures reveal an RdrB active site that targets mononucleotides. We show that RdrB catalyzes ATP-to-ITP conversion in\ vitro and induces the massive accumulation of inosine mononucleotides during phage infection in\ vivo, limiting phage replication. Our results define ATP mononucleotide deamination as a determinant of RADAR immunity and reveal supramolecular assembly of a nucleotide-modifying machine as a mechanism of anti-phage defense.}, keywords = {Adenosine Deaminase, anti-phage immunity, phage}, issn = {0092-8674}, doi = {https://doi.org/10.1016/j.cell.2023.01.012}, url = {https://www.sciencedirect.com/science/article/pii/S0092867423000429}, author = {Brianna Duncan-Lowey and Tal, Nitzan and Alex G. Johnson and Rawson, Shaun and Megan L. Mayer and Shany Doron and Millman, Adi and Sarah Melamed and Taya Fedorenko and Assaf Kacen and Brandis, Alexander and Tevie Mehlman and Gil Amitai and Sorek, Rotem and Kranzusch, Philip J.} } @article { doi:10.1126/science.ade5750, title = {Structures of BIRC6-client complexes provide a mechanism of Smac-mediated release of caspases}, journal = {Science}, year = {2023}, pages = {eade5750}, abstract = {Tight regulation of apoptosis is essential for metazoan development and prevents diseases such as cancer and neurodegeneration. Caspase activation is central to apoptosis and inhibitor of apoptosis (IAP) proteins are the principal actors that restrain caspase activity and are therefore attractive therapeutic targets. IAPs, in turn, are regulated by mitochondria-derived pro-apoptotic factors such as Smac and HtrA2. Through a series of cryo-electron microscopy (cryo-EM) structures of full-length human baculoviral IAP repeat-containing protein 6 (BIRC6) bound to Smac, caspases, and HtrA2, we provide a molecular understanding for BIRC6-mediated caspase inhibition and its release by Smac. The architecture of BIRC6, together with near-irreversible binding of Smac, elucidates how the IAP inhibitor Smac can effectively control a processive ubiquitin ligase to respond to apoptotic stimuli.}, doi = {10.1126/science.ade5750}, url = {https://www.science.org/doi/abs/10.1126/science.ade5750}, author = {Hunkeler, Moritz and Jin, Cyrus Y. and Fischer, Eric S.} } @article {Paidimuddala2023, title = {Mechanism of NAIP{\textendash}-NLRC4 inflammasome activation revealed by cryo-EM structure of unliganded NAIP5}, journal = {Nature Structural \& Molecular Biology}, volume = {30}, number = {2}, year = {2023}, month = {Feb}, pages = {159-166}, abstract = {The nucleotide-binding domain (NBD), leucine rich repeat (LRR) domain containing protein family (NLR family) apoptosis inhibitory proteins (NAIPs) are cytosolic receptors that play critical roles in the host defense against bacterial infection. NAIPs interact with conserved bacterial ligands and activate the NLR family caspase recruitment domain containing protein 4 (NLRC4) to initiate the NAIP{\textendash}-NLRC4 inflammasome pathway. Here we found the process of NAIP activation is completely different from NLRC4. Our cryo-EM structure of unliganded mouse NAIP5 adopts an unprecedented wide-open conformation, with the nucleating surface fully exposed and accessible to recruit inactive NLRC4. Upon ligand binding, the winged helix domain (WHD) of NAIP5 undergoes roughly 20{\textdegree} rotation to form a steric clash with the inactive NLRC4, which triggers the conformational change of NLRC4 from inactive to active state. We also show the rotation of WHD places the 17{\textendash}18 loop at a position that directly bind the active NLRC4 and stabilize the NAIP5{\textendash}NLRC4 complex. Overall, these data provide structural mechanisms of inactive NAIP5, the process of NAIP5 activation and NAIP-dependent NLRC4 activation.}, issn = {1545-9985}, doi = {10.1038/s41594-022-00889-2}, url = {https://doi.org/10.1038/s41594-022-00889-2}, author = {Paidimuddala, Bhaskar and Cao, Jianhao and Nash, Grady and Xie, Qing and Wu, Hao and Zhang, Liman} } @article {Li2023, title = {Structural basis of regulated m7G tRNA modification by METTL1{\textendash}WDR4}, journal = {Nature}, volume = {613}, number = {7943}, year = {2023}, month = {Jan}, pages = {391-397}, abstract = {Chemical modifications of RNA have key roles in many biological processes1{\textendash}3. N7-methylguanosine (m7G) is required for integrity and stability of a large subset of tRNAs4{\textendash}7. The methyltransferase 1{\textendash}WD repeat-containing protein 4 (METTL1{\textendash}WDR4) complex is the methyltransferase that modifies G46 in the variable loop of certain tRNAs, and its dysregulation drives tumorigenesis in numerous cancer types8{\textendash}14. Mutations in WDR4 cause human developmental phenotypes including microcephaly15{\textendash}17. How METTL1{\textendash}WDR4 modifies tRNA substrates and is regulated remains elusive18. Here we show,\  through structural, biochemical and cellular studies of human METTL1{\textendash}WDR4, that WDR4 serves as a scaffold for METTL1 and the tRNA T-arm. Upon tRNA binding, the $\alpha$C region of METTL1 transforms into a helix, which together with the $\alpha$6 helix secures both ends of the tRNA variable loop. Unexpectedly, we find that the predicted disordered N-terminal region of METTL1 is part of the catalytic pocket and essential for methyltransferase activity. Furthermore, we reveal that S27 phosphorylation in the METTL1 N-terminal region inhibits methyltransferase activity by locally disrupting the catalytic centre. Our results provide a molecular understanding of tRNA substrate recognition and phosphorylation-mediated regulation of METTL1{\textendash}WDR4, and reveal the presumed disordered N-terminal region of METTL1 as a nexus of methyltransferase activity.}, issn = {1476-4687}, doi = {10.1038/s41586-022-05566-4}, url = {https://doi.org/10.1038/s41586-022-05566-4}, author = {Li, Jiazhi and Wang, Longfei and Hahn, Quentin and Nowak, Rados{\l}aw P. and Viennet, Thibault and Orellana, Esteban A. and Roy Burman, Shourya S. and Yue, Hong and Hunkeler, Moritz and Fontana, Pietro and Wu, Hao and Arthanari, Haribabu and Fischer, Eric S. and Gregory, Richard I.} } @article {Meleppattu2022, title = {Mechanism of IFT-A polymerization into trains for ciliary transport}, journal = {Cell}, volume = {185}, number = {26}, year = {2022}, month = {Dec}, pages = {4986-4998.e12}, publisher = {Elsevier}, issn = {0092-8674}, doi = {10.1016/j.cell.2022.11.033}, url = {https://doi.org/10.1016/j.cell.2022.11.033}, author = {Meleppattu, Shimi and Zhou, Haixia and Dai, Jin and Gui, Miao and Brown, Alan} } @article {Xiao2022, title = {Cryo-EM structures of the active NLRP3 inflammasome disk}, journal = {Nature}, year = {2022}, month = {Nov}, abstract = {Inflammasomes are cytosolic innate immune complexes that activate caspase-1 upon detection of pathogenic and endogenous dangers1-5, and NLRP3 is an inflammasome sensor of membrane damage highly important in inducing inflammation2,6,7. Here we report cryo-EM structures of disk-shaped active NLRP3 oligomers in complex with ATP\&\#x1D6FE;S, the centrosomal kinase NEK7, and the adaptor protein ASC which recruits caspase-1. In these NLRP3-NEK7-ASC complexes, the central NACHT domain of NLRP3 assumes an ATP-bound conformation in which two of its subdomains rotate by \textasciitilde85 {\textdegree} relative to the ADP-bound inactive conformation8-12. The FISNA domain conserved in NLRP3 but absent in most NLRPs13 becomes ordered in its key regions to stabilize the active NACHT conformation and mediate most interactions in the disk. Mutations on these interactions compromise NLRP3-mediated caspase-1 activation. The N-terminal PYDs from all the NLRP3 subunits gather together to form a PYD filament that recruits ASC PYD to elicit downstream signalling. Surprisingly, the C-terminal LRR domain and the LRR-bound NEK7 do not participate in disk interfaces. Together with previous structures of inactive NLRP3 cage in which LRR-LRR interactions play an important role8-11, we propose that the role of NEK7 is to break the inactive cage to transform NLRP3 into the active NLRP3 inflammasome disk.}, issn = {1476-4687}, doi = {10.1038/s41586-022-05570-8}, url = {https://doi.org/10.1038/s41586-022-05570-8}, author = {Xiao, Le and Magupalli, Venkat Giri and Wu, Hao} } @article {Velilla2022, title = {Structural basis of colibactin activation by the ClbP peptidase}, journal = {Nature Chemical Biology}, year = {2022}, month = {Oct}, abstract = {Colibactin, a DNA cross-linking agent produced by gut bacteria, is implicated in colorectal cancer. Its biosynthesis uses a prodrug resistance mechanism: a non-toxic precursor assembled in the cytoplasm is activated after export to the periplasm. This activation is mediated by ClbP, an inner-membrane peptidase with an N-terminal periplasmic catalytic domain and a C-terminal three-helix transmembrane domain. Although the transmembrane domain is required for colibactin activation, its role in catalysis is unclear. Our structure of full-length ClbP bound to a product analog reveals an interdomain interface important for substrate binding and enzyme stability and interactions that explain the selectivity of ClbP for the N-acyl-d-asparagine prodrug motif. Based on structural and biochemical evidence, we propose that ClbP dimerizes to form an extended substrate-binding site that can accommodate a pseudodimeric precolibactin with its two terminal prodrug motifs in the two ClbP active sites, thus enabling the coordinated activation of both electrophilic warheads.}, issn = {1552-4469}, doi = {10.1038/s41589-022-01142-z}, url = {https://doi.org/10.1038/s41589-022-01142-z}, author = {Velilla, Jos{\'e} A. and Volpe, Matthew R. and Kenney, Grace E. and Walsh, Richard M. and Balskus, Emily P. and Gaudet, Rachelle} } @article {Dong2022, title = {Structural principles of B-cell antigen receptor assembly}, journal = {Nature}, year = {2022}, month = {Oct}, abstract = {The B-cell antigen receptor (BCR) is composed of a membrane-bound immunoglobulin (mIg) of class M, D, G, E or A for antigen recognition1{\textendash}3 and a disulfide-linked Ig$\alpha$ and Ig$\beta$ heterodimer (Ig$\alpha$/$\beta$) that functions as the signalling entity through their intracellular immunoreceptor tyrosine-based activation motifs (ITAMs)4,5. The organizing principle of the BCR remains elusive. Here we report cryogenic electron microscopy structures of mouse full-length IgM BCR at 8.2 \AA resolution and its Fab-deleted form at 3.3 \AA resolution. At the ectodomain (ECD), the Ig$\alpha$/$\beta$ heterodimer mainly uses Ig$\alpha$ to associate with C{\textmu}3-C{\textmu}4 domains of one heavy chain ({\textmu}HC) while leaving the other heavy chain ({\textmu}HC{\textquoteright}) empty. The transmembrane domain (TMD) helices of the two {\textmu}HCs interact with those of the Ig$\alpha$/$\beta$ heterodimer to form a tight 4-helix bundle. The asymmetry at the TMD prevents the recruitment of two Ig$\alpha$/$\beta$ heterodimers. Surprisingly, the connecting peptide between the ECD and TMD of {\textmu}HC intervenes in between those of Ig$\alpha$ and Ig$\beta$ to guide the TMD assembly through striking charge complementarity. Weaker but distinct density for the Ig$\beta$ ITAMs nestles next to the TMD, suggesting potential autoinhibition of ITAM phosphorylation. Interfacial analyses suggest that all BCR classes utilize a general organizational architecture. Our studies provide a structural platform for understanding B-cell signalling and designing rational therapies against BCR-mediated diseases.}, issn = {1476-4687}, doi = {10.1038/s41586-022-05412-7}, url = {https://doi.org/10.1038/s41586-022-05412-7}, author = {Dong, Ying and Pi, Xiong and Bartels-Burgahn, Frauke and Saltukoglu, Deniz and Liang, Zhuoyi and Yang, Jianying and Frederick W. Alt and Reth, Michael and Wu, Hao} } @article { doi:10.1073/pnas.2207605119, title = {SPACA9 is a lumenal protein of human ciliary singlet and doublet microtubules}, journal = {Proceedings of the National Academy of Sciences}, volume = {119}, number = {41}, year = {2022}, pages = {e2207605119}, abstract = {The cilium-centrosome complex contains triplet, doublet, and singlet microtubules. The lumenal\ surfaces of each microtubule within this diverse array are decorated by microtubule inner proteins (MIPs). Here, we used single-particle cryo-electron microscopy methods to build atomic models of two types of human ciliary microtubule: the doublet microtubules of multiciliated respiratory cells and the distal singlet microtubules of monoflagellated human spermatozoa. We discover that SPACA9 is a polyspecific MIP capable of binding both microtubule types. SPACA9 forms intralumenal striations in the B tubule of respiratory doublet microtubules and noncontinuous spirals in sperm singlet microtubules. By acquiring new and reanalyzing previous cryo-electron tomography data, we show that SPACA9-like intralumenal striations are common features of different microtubule types in animal cilia. Our structures provide detailed references to help rationalize ciliopathy-causing mutations and position cryo-EM as a tool for the analysis of samples obtained directly from ciliopathy patients.}, doi = {10.1073/pnas.2207605119}, url = {https://www.pnas.org/doi/abs/10.1073/pnas.2207605119}, author = {Gui, Miao and Jacob T. Croft and Davide Zabeo and Vajradhar Acharya and Justin M. Kollman and Thomas Burgoyne and Johanna L. H{\"o}{\"o}g and Brown, Alan} } @article {Navarro2022, title = {Cell wall synthesis and remodelling dynamics determine division site architecture and cell shape in Escherichia coli}, journal = {Nature Microbiology}, year = {2022}, month = {Sep}, abstract = {The bacterial division apparatus catalyses the synthesis and remodelling of septal peptidoglycan (sPG) to build the cell wall layer that fortifies the daughter cell poles. Understanding of this essential process has been limited by the lack of native three-dimensional views of developing septa. Here, we apply state-of-the-art cryogenic electron tomography (cryo-ET) and fluorescence microscopy to visualize the division site architecture and sPG biogenesis dynamics of the Gram-negative bacterium Escherichia coli. We identify a wedge-like sPG structure that fortifies the ingrowing septum. Experiments with strains defective in sPG biogenesis revealed that the septal architecture and mode of division can be modified to more closely resemble that of other Gram-negative (Caulobacter crescentus) or Gram-positive (Staphylococcus aureus) bacteria, suggesting that a conserved mechanism underlies the formation of different septal morphologies. Finally, analysis of mutants impaired in amidase activation ($Δ$envC $Δ$nlpD) showed that cell wall remodelling affects the placement and stability of the cytokinetic ring. Taken together, our results support a model in which competition between the cell elongation and division machineries determines the shape of cell constrictions and the poles they form. They also highlight how the activity of the division system can be modulated to help generate the diverse array of shapes observed in the bacterial domain.}, issn = {2058-5276}, doi = {10.1038/s41564-022-01210-z}, url = {https://doi.org/10.1038/s41564-022-01210-z}, author = {Navarro, Paula P. and Vettiger, Andrea and Ananda, Virly Y. and Llopis, Paula Montero and Allolio, Christoph and Bernhardt, Thomas G. and Chao, Luke H.} } @article {Jenni2022, title = {Visualizing molecular interactions that determine assembly of a bullet-shaped vesicular stomatitis virus particle}, journal = {Nature Communications}, volume = {13}, number = {1}, year = {2022}, month = {Aug}, pages = {4802}, abstract = {Vesicular stomatitis virus (VSV) is a negative-strand RNA virus with a non-segmented genome, closely related to rabies virus. Both have characteristic bullet-like shapes. We report the structure of intact, infectious VSV particles determined by cryogenic electron microscopy. By compensating for polymorphism among viral particles with computational classification, we obtained a reconstruction of the shaft ({\textquoteleft}{\textquoteleft}trunk{\textquoteright}{\textquoteright}) at 3.5{\th}inspace}\AA resolution, with lower resolution for the rounded tip. The ribonucleoprotein (RNP), genomic RNA complexed with nucleoprotein (N), curls into a dome-like structure with about eight gradually expanding turns before transitioning into the regular helical trunk. Two layers of matrix (M) protein link the RNP with the membrane. Radial inter-layer subunit contacts are fixed within single RNA-N-M1-M2 modules, but flexible lateral and axial interactions allow assembly of polymorphic virions. Together with published structures of recombinant N in various states, our results suggest a mechanism for membrane-coupled self-assembly of VSV and its relatives.}, issn = {2041-1723}, doi = {10.1038/s41467-022-32223-1}, url = {https://doi.org/10.1038/s41467-022-32223-1}, author = {Jenni, Simon and Horwitz, Joshua A. and Bloyet, Louis-Marie and Whelan, Sean P. J. and Harrison, Stephen C.} } @article { doi:10.1126/sciimmunol.add5446, title = {An Antibody from Single Human VH-rearranging Mouse Neutralizes All SARS-CoV-2 Variants Through BA.5 by Inhibiting Membrane Fusion}, journal = {Science Immunology}, year = {2022}, pages = {eadd5446}, abstract = {SARS-CoV-2 Omicron sub-variants have generated a world-wide health crisis due to resistance to most approved SARS-CoV-2 neutralizing antibodies and evasion of vaccination-induced antibodies. To manage Omicron sub-variants and prepare for potential new variants, additional means of isolating broad and potent humanized SARS-CoV-2-neutralizing antibodies are desirable. Here, we describe a mouse model in which the primary B cell receptor (BCR) repertoire is generated solely through V(D)J recombination of a human VH1-2 heavy chain (HC) and, substantially, a human Vκ1-33 light chain (LC). Thus, primary humanized BCR repertoire diversity in these mice derives from immensely diverse HC and LC antigen-contact complementarity-region-3 (CDR3) sequences generated by non-templated junctional modifications during V(D)J recombination. Immunizing the human VH1-2/Vκ1-33-rearranging mouse model with SARS-CoV-2 (Wuhan-Hu-1) spike protein immunogens elicited several VH1-2/Vκ1-33-based neutralizing antibodies that bound RBD in a different mode from each other and from those of many prior human patient-derived VH1-2-based neutralizing antibodies. Of these, SP1-77 potently and broadly neutralized all SARS-CoV-2 variants through BA.5. Cryo-EM studies revealed that SP1-77 bound RBD away from the receptor-binding-motif via a CDR3-dominated recognition mode. Lattice-light-sheet-microscopy-based studies showed that SP1-77 did not block ACE2-mediated viral attachment or endocytosis, but rather blocked viral-host membrane fusion. The broad and potent SP1-77 neutralization activity and non-traditonal mechanism of action suggest this antibody might have therapeutic potential. Likewise, the SP1-77 binding epitope may further inform on vacccine strategies. Finally, the general class of humanized mouse models we have described may contribute to identifying therapeutic antibodies against future SARS-CoV-2 variants and other pathogens. A humanized antibody from a recently-developed mouse model potently neutralizes SARS-CoV-2 variants by inhibiting membrane fusion.}, doi = {10.1126/sciimmunol.add5446}, url = {https://www.science.org/doi/abs/10.1126/sciimmunol.add5446}, author = {Sai Luo and Zhang, Jun and Alex J.B. Kreutzberger and Amanda Eaton and Robert J. Edwards and Changbin Jing and Hai-Qiang Dai and Gregory D. Sempowski and Kenneth Cronin and Robert Parks and Ye, Adam Yongxin and Katayoun Mansouri and Maggie Barr and Pishesha, Novalia and Aimee Chapdelaine Williams and Lucas Vieira Francisco and Anand Saminathan and Peng, Hanqin and Himanshu Batra and Lorenza Bellusci and Surender Khurana and S. Munir Alam and David C. Montefiori and Kevin O. Saunders and Tian, Ming and Ploegh, Hidde and Tom Kirchhausen and Chen, Bing and Barton F. Haynes and Frederick W. Alt} } @article {Rawson2022, title = {Yeast PI31 inhibits the proteasome by a direct multisite mechanism}, journal = {Nature Structural \& Molecular Biology}, year = {2022}, month = {Aug}, abstract = {Proteasome inhibitors are widely used as therapeutics and research tools, and typically target one of the three active sites, each present twice in the proteasome complex. An endogeneous proteasome inhibitor, PI31, was identified 30{\th}inspace}years ago, but its inhibitory mechanism has remained unclear. Here, we identify the mechanism of Saccharomyces cerevisiae PI31, also known as Fub1. Using cryo-electron microscopy (cryo-EM), we show that the conserved carboxy-terminal domain of Fub1 is present inside the proteasome{\textquoteright}s barrel-shaped core particle (CP), where it simultaneously interacts with all six active sites. Targeted mutations of Fub1 disrupt proteasome inhibition at one active site, while leaving the other sites unaffected. Fub1 itself evades degradation through distinct mechanisms at each active site. The gate that allows substrates to access the CP is constitutively closed, and Fub1 is enriched in mutant CPs with an abnormally open gate, suggesting that Fub1 may function to neutralize aberrant proteasomes, thereby ensuring the fidelity of proteasome-mediated protein degradation.}, issn = {1545-9985}, doi = {10.1038/s41594-022-00808-5}, url = {https://doi.org/10.1038/s41594-022-00808-5}, author = {Rawson, Shaun and Walsh, Richard M. and Velez, Benjamin and Schnell, Helena M. and Jiao, Fenglong and Blickling, Marie and Ang, Jessie and Bhanu, Meera K. and Huang, Lan and Hanna, John} } @article {Yip2022, title = {Mechanism of client selection by the protein quality-control factor UBE2O}, journal = {Nature Structural \& Molecular Biology}, year = {2022}, month = {Aug}, abstract = {The E2/E3 enzyme UBE2O ubiquitylates diverse clients to mediate important processes, including targeting unassembled {\textquoteleft}orphan{\textquoteright} proteins for quality control and clearing ribosomes during erythropoiesis. How quality-control factors, such as UBE2O, select clients on the basis of heterogeneous features is largely unknown. Here, we show that UBE2O client selection is regulated by ubiquitin binding and a cofactor, NAP1L1. Attaching a single ubiquitin onto a client enhances UBE2O binding and multi-mono-ubiquitylation. UBE2O also repurposes the histone chaperone NAP1L1 as an adapter to recruit a subset of clients. Cryo-EM structures of human UBE2O in complex with NAP1L1 reveal a malleable client recruitment interface that is autoinhibited by the intrinsically reactive UBC domain. Adding a ubiquitylated client identifies a distinct ubiquitin-binding SH3-like domain required for client selection. Our findings reveal how multivalency and a feed-forward mechanism drive the selection of protein quality-control clients.}, issn = {1545-9985}, doi = {10.1038/s41594-022-00807-6}, url = {https://doi.org/10.1038/s41594-022-00807-6}, author = {Yip, Matthew C. J. and Sedor, Samantha F. and Shao, Sichen} } @article {Morehouse2022, title = {Cryo-EM structure of an active bacterial TIR{\textendash}STING filament complex}, journal = {Nature}, year = {2022}, month = {Jul}, abstract = {Stimulator of interferon genes (STING) is an antiviral signalling protein that is broadly conserved in both innate immunity in animals and phage defence in prokaryotes1{\textendash}4. Activation of STING requires its assembly into an oligomeric filament structure through binding of a cyclic dinucleotide4{\textendash}13, but the molecular basis of STING filament assembly and extension remains unknown. Here we use cryogenic electron microscopy to determine the structure of the active Toll/interleukin-1 receptor (TIR){\textendash}STING filament complex from a Sphingobacterium faecium cyclic-oligonucleotide-based antiphage signalling system (CBASS) defence operon. Bacterial TIR{\textendash}STING filament formation is driven by STING interfaces that become exposed on high-affinity recognition of the cognate cyclic dinucleotide signal c-di-GMP. Repeating dimeric STING units stack laterally head-to-head through surface interfaces, which are also essential for human STING tetramer formation and downstream immune signalling in mammals5. The active bacterial TIR{\textendash}STING structure reveals further cross-filament contacts that brace the assembly and coordinate packing of the associated TIR NADase effector domains at the base of the filament to drive NAD+ hydrolysis. STING interface and cross-filament contacts are essential for cell growth arrest in vivo and reveal a stepwise mechanism of activation whereby STING filament assembly is required for subsequent effector activation. Our results define the structural basis of STING filament formation in prokaryotic antiviral signalling.}, issn = {1476-4687}, doi = {10.1038/s41586-022-04999-1}, url = {https://doi.org/10.1038/s41586-022-04999-1}, author = {Morehouse, Benjamin R. and Yip, Matthew C. J. and Keszei, Alexander F. A. and McNamara-Bordewick, Nora K. and Shao, Sichen and Kranzusch, Philip J.} } @article {10.1182/blood.2022016467, title = {Structures of VWF tubules before and after concatemerization reveal a mechanism of disulfide bond exchange}, journal = {Blood}, year = {2022}, note = {blood.2022016467}, month = {07}, abstract = {von Willebrand Factor (VWF) is an adhesive glycoprotein that circulates in the blood as disulfide-linked concatemers and functions in primary hemostasis. The loss of long VWF concatemers is associated with the excess bleeding of type 2A von Willebrand (VW) disease. Formation of the disulfide bonds that concatemerize VWF requires VWF to self-associate into helical tubules, yet how the helical tubules template intermolecular disulfide bonds is not known. Here, we report cryo-EM structures of complete VWF tubules before and after intermolecular disulfide-bond formation. The structures provide evidence that VWF tubulates through a charge-neutralization mechanism and that the A1 domain enhances tubule length by crosslinking successive helical turns. In addition, the structures reveal disulfide states prior to and after disulfide bond-mediated concatemerization. The structures and proposed assembly mechanism provide a foundation to rationalize VW disease-causing mutations.}, issn = {0006-4971}, doi = {10.1182/blood.2022016467}, url = {https://doi.org/10.1182/blood.2022016467}, author = {Anderson, Jacob Ronald and Li, Jing and Springer, Timothy A. and Brown, Alan} } @article { doi:10.1126/science.abo3851, title = {Structural basis of nucleosome retention during transcription elongation}, journal = {Science}, volume = {376}, number = {6599}, year = {2022}, pages = {1313-1316}, abstract = {In eukaryotes, RNA polymerase (Pol) II transcribes chromatin and must move past nucleosomes, often resulting in nucleosome displacement. How Pol II unwraps the DNA from nucleosomes to allow transcription and how DNA rewraps to retain nucleosomes has been unclear. Here, we report the 3.0-angstrom cryo{\textendash}electron microscopy structure of a mammalian Pol II-DSIF-SPT6-PAF1c-TFIIS-nucleosome complex stalled 54 base pairs within the nucleosome. The structure provides a mechanistic basis for nucleosome retention during transcription elongation where upstream DNA emerging from the Pol II cleft has rewrapped the proximal side of the nucleosome. The structure uncovers a direct role for Pol II and transcription elongation factors in nucleosome retention and explains how nucleosomes are retained to prevent the disruption of chromatin structure across actively transcribed genes. Eukaryotic cells organize their large genomes into a compacted structure called chromatin. The condensed structure of chromatin, with its fundamental unit the nucleosome, represents a challenge to nucleic acid{\textendash}transacting machines including RNA polymerase II (Pol II), the enzyme responsible for the transcription of most protein-coding genes. How RNA Pol II overcomes nucleosomes without disrupting chromatin organization remains unknown. Using cryo{\textendash}electron microscopy, Filipovski et al. provided structural snapshots of a complex between mammalian RNA Pol II and a nucleosome that show how previously transcribed DNA rewraps the nucleosome. The finding provides a structural basis of how nucleosomes, and consequently epigenetic marks, are retained during transcription. {\textemdash}DJ A high-resolution cryo{\textendash}electron microscopy structure explains how nucleosomes are retained to prevent disruption of chromatin across actively transcribed genes.}, doi = {10.1126/science.abo3851}, url = {https://www.science.org/doi/abs/10.1126/science.abo3851}, author = {Martin Filipovski and Jelly H. M. Soffers and Seychelle M. Vos and Lucas Farnung} } @article { doi:10.1126/sciimmunol.abo3425, title = {Antibodies induced by an ancestral SARS-CoV-2 strain that cross-neutralize variants from Alpha to Omicron BA.1}, journal = {Science Immunology}, volume = {7}, number = {74}, year = {2022}, pages = {eabo3425}, abstract = {Neutralizing antibodies that recognize the SARS-CoV-2 spike glycoprotein are the principal host defense against viral invasion. Variants of SARS-CoV-2 bear mutations that allow escape from neutralization by many human antibodies, especially those in widely distributed ({\textquotedblleft}public{\textquotedblright}) classes. Identifying antibodies that neutralize these variants of concern and determining their prevalence are important goals for understanding immune protection. To determine the Delta and Omicron BA.1 variant specificity of B cell repertoires established by an initial Wuhan strain infection, we measured neutralization potencies of 73 antibodies from an unbiased survey of the early memory B cell response. Antibodies recognizing each of three previously defined epitopic regions on the spike receptor binding domain (RBD) varied in neutralization potency and variant-escape resistance. The ACE2 binding surface ({\textquotedblleft}RBD-2{\textquotedblright}) harbored the binding sites of neutralizing antibodies with the highest potency but with the greatest sensitivity to viral escape; two other epitopic regions on the RBD ({\textquotedblleft}RBD-1{\textquotedblright} and {\textquotedblleft}RBD-3{\textquotedblright}) bound antibodies of more modest potency but greater breadth. The structures of several Fab:spike complexes that neutralized all five variants of concern tested, including one Fab each from the RBD-1, -2, and -3 clusters, illustrated the determinants of broad neutralization and showed that B cell repertoires can have specificities that avoid immune escape driven by public antibodies. The structure of the RBD-2 binding, broad neutralizer shows why it retains neutralizing activity for Omicron BA.1, unlike most others in the same public class. Our results correlate with real-world data on vaccine efficacy, which indicate mitigation of disease caused by Omicron BA.1. Structures of SARS-CoV-2 spike-bound antibodies from Wuhan strain infection show features needed to neutralize variants of concern. As new SARS-CoV-2 variants of concern (VOCs) emerge, it is crucial to determine whether immune responses to previous iterations of the virus protect against VOCs. Because antibodies are critical for protection against SARS-CoV-2, a key issue is reactivity for VOCs of antibodies generated by ancestral SARS-CoV-2 exposure. Here, Windsor et al. evaluated VOC binding, cross-inhibition, and neutralization potency of 73 monoclonal antibodies from donors infected in early 2020 with ancestral SARS-CoV-2. They identified three antibodies that neutralized all VOCs tested (including Omicron BA.1) and used cryo-EM of these antibodies bound with SARS-CoV-2 spike to suggest ways in which somatic mutation might restore VOC recognition by other antibodies. This study thus yields better understanding of the reactivity for VOCs of humoral immune responses to ancestral SARS-CoV-2.}, doi = {10.1126/sciimmunol.abo3425}, url = {https://www.science.org/doi/abs/10.1126/sciimmunol.abo3425}, author = {Windsor, Ian W. and Tong, Pei and Olivia Lavidor and Ali Sanjari Moghaddam and McKay, Lindsay G. A. and Gautam, Avneesh and Chen, Yuezhou and MacDonald, Elizabeth A. and Duck Kyun Yoo and Griffths, Anthony and Wesemann, Duane R. and Harrison, Stephen C.} } @article {Marechal2022, title = {Formation of thyroid hormone revealed by a cryo-EM structure of native bovine thyroglobulin}, journal = {Nature Communications}, volume = {13}, number = {1}, year = {2022}, month = {May}, pages = {2380}, abstract = {Thyroid hormones are essential regulators of metabolism, development, and growth. They are formed from pairs of iodinated tyrosine residues within the precursor thyroglobulin (TG), a 660-kDa homodimer of the thyroid gland, by an oxidative coupling reaction. Tyrosine pairs that give rise to thyroid hormones have been assigned within the structure of human TG, but the process of hormone formation is poorly understood. Here we report a \textasciitilde3.3-\AA cryo-EM structure of native bovine TG with nascent thyroid hormone formed at one of the predicted hormonogenic sites. Local structural rearrangements provide insight into mechanisms underlying thyroid hormone formation and stabilization.}, issn = {2041-1723}, doi = {10.1038/s41467-022-30082-4}, url = {https://doi.org/10.1038/s41467-022-30082-4}, author = {Marechal, Nils and Serrano, Banyuhay P. and Zhang, Xinyan and Weitz, Charles J.} } @article {Liu2022, title = {Sub-3-{\r A} cryo-EM structure of RNA enabled by engineered homomeric self-assembly}, journal = {Nature Methods}, year = {2022}, month = {May}, abstract = {High-resolution structural studies are essential for understanding the folding and function of diverse RNAs. Herein, we present a nanoarchitectural engineering strategy for efficient structural determination of RNA-only structures using single-particle cryogenic electron microscopy (cryo-EM). This strategy{\textendash}-ROCK (RNA oligomerization-enabled cryo-EM via installing kissing loops){\textendash}-involves installing kissing-loop sequences onto the functionally nonessential stems of RNAs for homomeric self-assembly into closed rings with multiplied molecular weights and mitigated structural flexibility. ROCK enables cryo-EM reconstruction of the Tetrahymena group I intron at 2.98-\AA resolution overall (2.85{\th}inspace}\AA for the core), allowing de novo model building of the complete RNA, including the previously unknown peripheral domains. ROCK is further applied to two smaller RNAs{\textendash}-the Azoarcus group I intron and the FMN riboswitch, revealing the conformational change of the former and the bound ligand in the latter. ROCK holds promise to greatly facilitate the use of cryo-EM in RNA structural studies.}, issn = {1548-7105}, doi = {10.1038/s41592-022-01455-w}, url = {https://doi.org/10.1038/s41592-022-01455-w}, author = {Liu, Di and Fran{\c c}ois A. Th{\'e}lot and Piccirilli, Joseph A. and Maofu Liao and Peng Yin} } @article { doi:10.1126/sciadv.abm1568, title = {pH regulates potassium conductance and drives a constitutive proton current in human TMEM175}, journal = {Science Advances}, volume = {8}, number = {12}, year = {2022}, pages = {eabm1568}, abstract = {Human TMEM175, a noncanonical potassium (K+) channel in endolysosomes, contributes to their pH stability and is implicated in the pathogenesis of Parkinson{\textquoteright}s disease (PD). Structurally, the TMEM175 family exhibits an architecture distinct from canonical potassium channels, as it lacks the typical TVGYG selectivity filter. Here, we show that human TMEM175 not only exhibits pH-dependent structural changes that reduce K+ permeation at acidic pH but also displays proton permeation. TMEM175 constitutively conducts K+ at pH 7.4 but displays reduced K+ permeation at lower pH. In contrast, proton current through TMEM175 increases with decreasing pH because of the increased proton gradient. Molecular dynamics simulation, structure-based mutagenesis, and electrophysiological analysis suggest that K+ ions and protons share the same permeation pathway. The M393T variant of human TMEM175 associated with PD shows reduced function in both K+ and proton permeation. Together, our structural and electrophysiological analysis reveals a mechanism of TMEM175 regulation by pH. Human TMEM175, a noncanonical potassium channel, conducts protons but displays reduced K+ permeation at lower pH.}, doi = {10.1126/sciadv.abm1568}, url = {https://www.science.org/doi/abs/10.1126/sciadv.abm1568}, author = {Wang Zheng and Shen, Chen and Wang, Longfei and Rawson, Shaun and Wen Jun Xie and Nist-Lund, Carl and Jason Wu and Shen, Zhangfei and Xia, Shiyu and Jeffrey R. Holt and Wu, Hao and Tian-Min Fu} } @article {ref1, title = {Emerging enterococcus pore-forming toxins with MHC/HLA-I as receptors}, journal = {Cell}, year = {2022}, month = {2022/03/07}, publisher = {Elsevier}, issn = {0092-8674}, doi = {10.1016/j.cell.2022.02.002}, url = {https://doi.org/10.1016/j.cell.2022.02.002}, author = {Xiong, Xiaozhe and Tian, Songhai and Pan Yang and Lebreton, Francois and Bao, Huan and Sheng, Kuanwei and Yin, Linxiang and Chen, Pengsheng and Zhang, Jie and Qi, Wanshu and Ruan, Jianbin and Wu, Hao and Chen, Hong and Breault, David T. and Earl, Ashlee M. and Gilmore, Michael S. and Jonathan Abraham and Dong, Min} } @article {HAUSER2022110561, title = {Rationally designed immunogens enable immune focusing following SARS-CoV-2 spike imprinting}, journal = {Cell Reports}, volume = {38}, number = {12}, year = {2022}, pages = {110561}, abstract = {Summary Eliciting antibodies to surface-exposed viral glycoproteins can generate protective responses that control and prevent future infections. Targeting conserved sites may reduce the likelihood of viral escape and limit the spread of related viruses with pandemic potential. Here we leverage rational immunogen design to focus humoral responses on conserved epitopes. Using glycan engineering and epitope scaffolding in boosting immunogens, we focus murine serum antibody responses to conserved receptor binding motif (RBM) and receptor binding domain (RBD) epitopes following severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike imprinting. Although all engineered immunogens elicit a robust SARS-CoV-2-neutralizing serum response, RBM-focusing immunogens exhibit increased potency against related sarbecoviruses, SARS-CoV, WIV1-CoV, RaTG13-CoV, and SHC014-CoV; structural characterization of representative antibodies defines a conserved epitope. RBM-focused sera confer protection against SARS-CoV-2 challenge. Thus, RBM focusing is a promising strategy to elicit breadth across emerging sarbecoviruses without compromising SARS-CoV-2 protection. These engineering strategies are adaptable to other viral glycoproteins for targeting conserved epitopes.}, keywords = {Coronavirus, glycan, immune focusing, immunogen design, SARS-CoV-2}, issn = {2211-1247}, doi = {https://doi.org/10.1016/j.celrep.2022.110561}, url = {https://www.sciencedirect.com/science/article/pii/S2211124722003059}, author = {Hauser, Blake M. and Maya Sangesland and Kerri J. St. Denis and Evan C. Lam and James Brett Case and Windsor, Ian W. and Feldman, Jared and Timothy M. Caradonna and Ty Kannegieter and Michael S. Diamond and Alejandro B. Balazs and Daniel Lingwood and Schmidt, Aaron G.} } @article {Arlt2022, title = {Seipin forms a flexible cage at lipid droplet formation sites}, journal = {Nature Structural \& Molecular Biology}, year = {2022}, month = {Feb}, abstract = {Lipid droplets (LDs) form in the endoplasmic reticulum by phase separation of neutral lipids. This process is facilitated by the seipin protein complex, which consists of a ring of seipin monomers, with a yet unclear function. Here, we report a structure of S. cerevisiae seipin based on cryogenic-electron microscopy and structural modeling data. Seipin forms a decameric, cage-like structure with the lumenal domains forming a stable ring at the cage floor and transmembrane segments forming the cage sides and top. The transmembrane segments interact with adjacent monomers in two distinct, alternating conformations. These conformations result from changes in switch regions, located between the lumenal domains and the transmembrane segments, that are required for seipin function. Our data indicate a model for LD formation in which a closed seipin cage enables triacylglycerol phase separation and subsequently switches to an open conformation to allow LD growth and budding.}, issn = {1545-9985}, author = {Arlt, Henning and Sui, Xuewu and Folger, Brayden and Adams, Carson and Chen, Xiao and Remme, Roman and Hamprecht, Fred A. and Frank DiMaio and Maofu Liao and Goodman, Joel M. and Farese, Robert V. and Walther, Tobias C.} } @article {doi:10.1126/science.abj8432, title = {Bacterial gasdermins reveal an ancient mechanism of cell death}, journal = {Science}, volume = {375}, number = {6577}, year = {2022}, pages = {221-225}, abstract = {Gasdermins are cell death proteins in mammals that form membrane pores in response to pathogen infection. Johnson et al. report that diverse bacteria encode structural and functional homologs of mammalian gasdermins. Like their mammalian counterparts, bacterial gasdermins are activated by caspase-like proteases, oligomerize into large membrane pores, and defend against pathogen{\textemdash}in this case, bacteriophage{\textemdash}infection. Proteolytic activation occurs through the release of a short inhibitory peptide, and many bacterial gasdermins are lipidated to facilitate membrane pore formation. Pyroptotic cell death, a central component of mammalian innate immunity, thus has a shared origin with an ancient antibacteriophage defense system. {\textemdash}SMH Bacteria encode gasdermins that are activated by dedicated proteases, defend from phage, and induce cell death. Gasdermin proteins form large membrane pores in human cells that release immune cytokines and induce lytic cell death. Gasdermin pore formation is triggered by caspase-mediated cleavage during inflammasome signaling and is critical for defense against pathogens and cancer. We discovered gasdermin homologs encoded in bacteria that defended against phages and executed cell death. Structures of bacterial gasdermins revealed a conserved pore-forming domain that was stabilized in the inactive state with a buried lipid modification. Bacterial gasdermins were activated by dedicated caspase-like proteases that catalyzed site-specific cleavage and the removal of an inhibitory C-terminal peptide. Release of autoinhibition induced the assembly of large and heterogeneous pores that disrupted membrane integrity. Thus, pyroptosis is an ancient form of regulated cell death shared between bacteria and animals.}, doi = {10.1126/science.abj8432}, url = {https://www.science.org/doi/abs/10.1126/science.abj8432}, author = {Alex G. Johnson and Tanita Wein and Megan L. Mayer and Brianna Duncan-Lowey and Erez Yirmiya and Yaara Oppenheimer-Shaanan and Gil Amitai and Sorek, Rotem and Kranzusch, Philip J.} } @article {Keszei2021, title = {Structural insights into metazoan pretargeting GET complexes}, journal = {Nature Structural \& Molecular Biology}, volume = {28}, number = {12}, year = {2021}, month = {Dec}, pages = {1029-1037}, abstract = {Close coordination between chaperones is essential for protein biosynthesis, including the delivery of tail-anchored (TA) proteins containing a single C-terminal transmembrane domain to the endoplasmic reticulum (ER) by the conserved GET pathway. For successful targeting, nascent TA proteins must be promptly chaperoned and loaded onto the cytosolic ATPase Get3 through a transfer reaction involving the chaperone SGTA and bridging factors Get4, Ubl4a and Bag6. Here, we report cryo-electron microscopy structures of metazoan pretargeting GET complexes at 3.3{\textendash}3.6{\th}inspace}\AA. The structures reveal that Get3 helix{\th}inspace}8 and the Get4 C{\th}inspace}terminus form a composite lid over the Get3 substrate-binding chamber that is opened by SGTA. Another interaction with Get4 prevents formation of Get3 helix{\th}inspace}4, which links the substrate chamber and ATPase domain. Both interactions facilitate TA protein transfer from SGTA to Get3. Our findings show how the pretargeting complex primes Get3 for coordinated client loading and ER targeting.}, issn = {1545-9985}, doi = {10.1038/s41594-021-00690-7}, url = {https://doi.org/10.1038/s41594-021-00690-7}, author = {Keszei, Alexander F. A. and Yip, Matthew C. J. and Hsieh, Ta-Chien and Shao, Sichen} } @article {ref1, title = {NLRP3 cages revealed by full-length mouse NLRP3 structure control pathway activation}, journal = {Cell}, year = {2021}, month = {2021/12/03}, publisher = {Elsevier}, issn = {0092-8674}, doi = {10.1016/j.cell.2021.11.011}, url = {https://doi.org/10.1016/j.cell.2021.11.011}, author = {Andreeva, Liudmila and David, Liron and Rawson, Shaun and Shen, Chen and Pasricha, Teerithveen and Pelegrin, Pablo and Wu, Hao} } @article {doi:10.1126/science.abl6251, title = {Structural basis for continued antibody evasion by the SARS-CoV-2 receptor binding domain}, journal = {Science}, year = {2021}, pages = {eabl6251}, abstract = {Many studies have examined the impact of SARS-CoV-2 variants on neutralizing antibody activity after they have become dominant strains. Here, we evaluate the consequences of further viral evolution. We demonstrate mechanisms through which the SARS-CoV-2 receptor binding domain (RBD) can tolerate large numbers of simultaneous antibody escape mutations and show that pseudotypes containing up to seven mutations, as opposed to the one to three found in previously studied variants of concern, are more resistant to neutralization by therapeutic antibodies and serum from vaccine recipients. We identify an antibody that binds the RBD core to neutralize pseudotypes for all tested variants but show that the RBD can acquire an N-linked glycan to escape neutralization. Our findings portend continued emergence of escape variants as SARS-CoV-2 adapts to humans.}, doi = {10.1126/science.abl6251} URL = ttps://www.science.org/doi/abs/10.1126/science.abl6251}, author = {Katherine G. Nabel and Sarah A. Clark and Sundaresh Shankar and Junhua Pan and Lars E. Clark and Pan Yang and Adrian Coscia and McKay, Lindsay G. A. and Haley H. Varnum and Vesna Brusic and Nicole V. Tolan and Guohai Zhou and Micha{\"e}l Desjardins and Sarah E. Turbett and Sanjat Kanjilal and Amy C. Sherman and Anand Dighe and Regina C. LaRocque and Edward T. Ryan and Casey Tylek and Joel F. Cohen-Solal and Anhdao T. Darcy and Davide Tavella and Anca Clabbers and Yao Fan and Griffiths, Anthony and Ivan R. Correia and Jane Seagal and Lindsey R. Baden and Richelle C. Charles and Jonathan Abraham} } @article {10.1093/nar/gkab1174, title = {Structure of CRL2Lrr1, the E3 ubiquitin ligase that promotes DNA replication termination in vertebrates}, journal = {Nucleic Acids Research}, year = {2021}, note = {gkab1174}, month = {12}, abstract = {When vertebrate replisomes from neighboring origins converge, the Mcm7 subunit of the replicative helicase, CMG, is ubiquitylated by the E3 ubiquitin ligase, CRL2Lrr1. Polyubiquitylated CMG is then disassembled by the p97 ATPase, leading to replication termination. To avoid premature replisome disassembly, CRL2Lrr1 is only recruited to CMGs after they converge, but the underlying mechanism is unclear. Here, we use cryogenic electron microscopy to determine structures of recombinant Xenopus laevis CRL2Lrr1 with and without neddylation. The structures reveal that CRL2Lrr1 adopts an unusually open architecture, in which the putative substrate-recognition subunit, Lrr1, is located far from the catalytic module that catalyzes ubiquitin transfer. We further demonstrate that a predicted, flexible pleckstrin homology domain at the N-terminus of Lrr1 is essential to target CRL2Lrr1 to terminated CMGs. We propose a hypothetical model that explains how CRL2Lrr1{\textquoteright}s catalytic module is positioned next to the ubiquitylation site on Mcm7, and why CRL2Lrr1 binds CMG only after replisomes converge.}, issn = {0305-1048}, doi = {10.1093/nar/gkab1174}, url = {https://doi.org/10.1093/nar/gkab1174}, author = {Zhou, Haixia and Zaher, Manal S and Walter, Johannes C and Brown, Alan} } @article {ref1, title = {De novo identification of mammalian ciliary motility proteins using cryo-EM}, journal = {Cell}, year = {2021}, month = {2021/10/28}, publisher = {Elsevier}, issn = {0092-8674}, doi = {10.1016/j.cell.2021.10.007}, url = {https://doi.org/10.1016/j.cell.2021.10.007}, author = {Gui, Miao and Farley, Hannah and Anujan, Priyanka and Anderson, Jacob R. and Maxwell, Dale W. and Whitchurch, Jonathan B. and Botsch, J. Josephine and Qiu, Tao and Meleppattu, Shimi and Singh, Sandeep K. and Zhang, Qi and Thompson, James and Lucas, Jane S. and Bingle, Colin D. and Norris, Dominic P. and Roy, Sudipto and Brown, Alan} } @article {doi:10.1126/science.abl9463, title = {Membrane fusion and immune evasion by the spike protein of SARS-CoV-2 Delta variant}, journal = {Science}, year = {2021}, pages = {eabl9463}, abstract = {The Delta variant of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has outcompeted previously prevalent variants and become a dominant strain worldwide. We report the structure, function, and antigenicity of its full-length spike (S) trimer and those of the Gamma and Kappa variants and compare their characteristics with the G614, Alpha, and Beta variants. Delta S can fuse membranes more efficiently at low levels of cellular receptor ACE2, and its pseudotyped viruses infect target cells substantially faster than the other five variants, possibly accounting for its heightened transmissibility. Each variant shows different rearrangement of the antigenic surface of the N-terminal domain of the S protein, but only causes local changes in the receptor-binding domain (RBD), making the RBD a better target for therapeutic antibodies.}, doi = {10.1126/science.abl9463} URL = ttps://www.science.org/doi/abs/10.1126/science.abl9463}, author = {Zhang, Jun and Xiao, Tianshu and Cai, Yongfei and Lavine, Christy L. and Peng, Hanqin and Zhu, Haisun and Anand, Krishna and Tong, Pei and Gautam, Avneesh and Megan L. Mayer and Walsh Jr., Richard M. and Rits-Volloch, Sophia and Wesemann, Duane R. and Yang, Wei and Seaman, Michael S. and Lu, Jianming and Chen, Bing} } @article {10.1093/abt/tbab025, title = {Development of an integrated structural biology platform specialized for sub-100 kDa protein complexes to support biologics discovery and rational engineering}, journal = {Antibody Therapeutics}, volume = {4}, number = {4}, year = {2021}, month = {10}, pages = {242-251}, abstract = {Developing a biologic medicine requires successful decision making at early stages. Knowing structural information for discovery candidates greatly increases the probability of success.We have evaluated and integrated various structural biology and computation tools and established a cost-effective platform that allows us to obtain fast and accurate structural information for nearly all our biologics projects.We report four case studies selected from 38 projects and share how we integrate cryo-EM structure determination, computational modeling, and molecular dynamics simulation. With proper decision making and strategic planning, the platform allows to obtain results within days to weeks, including sub-100 kDa complexes.Our utilization of this differential approach and multiple software packages allows to manage priorities and resources to achieve goals efficiently. We demonstrate how to overcome particle orientation bias by altering complex composition. In several of our examples, we use glycan density and protein information to facilitate interpretation of low-resolution 3D maps.}, issn = {2516-4236}, doi = {10.1093/abt/tbab025}, url = {https://doi.org/10.1093/abt/tbab025}, author = {Iozzo (Egor Svidritskiy), Yuri and Qiu, Yu and Xu, Albert and Park, Anna and Wendt, Maria and Zhou, Yanfeng} } @article {doi:10.1126/science.abi9009, title = {Distinct allosteric mechanisms of first-generation MsbA inhibitors}, journal = {Science}, year = {2021}, month = {29 Oct 2021}, abstract = {ATP-binding cassette (ABC) transporters couple ATP hydrolysis to substrate transport across biological membranes. Although many are promising drug targets, their mechanisms of modulation by small molecule inhibitors remain largely unknown. Intriguingly, two first-generation inhibitors of the MsbA transporter, TBT1 and G247, induce opposite effects on ATP hydrolysis. Using single-particle cryo-electron microscopy and functional assays, we show that TBT1 and G247 bind adjacent yet separate pockets in the MsbA transmembrane domains. Two TBT1 molecules asymmetrically occupy the substrate binding site, leading to a collapsed inward-facing conformation with decreased distance between the nucleotide-binding domains (NBDs). In contrast, two G247 molecules symmetrically increases NBD distance in a wide inward-open state of MsbA. The divergent mechanisms of action of these MsbA inhibitors provide important insights into ABC transporter pharmacology.}, doi = {10.1126/science.abi9009} URL = ttps://www.science.org/doi/abs/10.1126/science.abi9009}, author = {Fran{\c c}ois A. Th{\'e}lot and Wenyi Zhang and Song, KangKang and Xu, Chen and Jing Huang and Maofu Liao} } @article {ref1, title = {Solenoid architecture of HUWE1 contributes to ligase activity and substrate recognition}, journal = {Molecular Cell}, year = {2021}, month = {2021/07/27}, publisher = {Elsevier}, issn = {1097-2765}, doi = {10.1016/j.molcel.2021.06.032}, url = {https://doi.org/10.1016/j.molcel.2021.06.032}, author = {Hunkeler, Moritz and Jin, Cyrus Y. and Ma, Michelle W. and Monda, Julie K. and Overwijn, Daan and Bennett, Eric J. and Fischer, Eric S.} } @article {ref1, title = {Memory B Cell Repertoire for Recognition of Evolving SARS-CoV-2 Spike}, journal = {Cell}, year = {2021}, month = {2021/07/27}, publisher = {Elsevier}, issn = {0092-8674}, doi = {10.1016/j.cell.2021.07.025}, url = {https://doi.org/10.1016/j.cell.2021.07.025}, author = {Tong, Pei and Gautam, Avneesh and Windsor, Ian W. and Travers, Meghan and Chen, Yuezhou and Garcia, Nicholas and Whiteman, Noah B. and McKay, Lindsay G. A. and Storm, Nadia and Malsick, Lauren E. and Honko, Anna N. and Lelis, Felipe J.N. and Habibi, Shaghayegh and Jenni, Simon and Cai, Yongfei and Rennick, Linda J. and Duprex, W. Paul and McCarthy, Kevin R. and Lavine, Christy L. and Zuo, Teng and Lin, Junrui and Zuiani, Adam and Feldman, Jared and MacDonald, Elizabeth A. and Hauser, Blake M. and Griffths, Anthony and Seaman, Michael S. and Schmidt, Aaron G. and Chen, Bing and Neuberg, Donna and Bajic, Goran and Harrison, Stephen C. and Wesemann, Duane R.} } @article {Caieabi9745, title = {Structural basis for enhanced infectivity and immune evasion of SARS-CoV-2 variants}, journal = {Science}, year = {2021}, publisher = {American Association for the Advancement of Science}, abstract = {Several fast-spreading variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have become the dominant circulating strains in the COVID-19 pandemic. We report here cryo-EM structures of the full-length spike (S) trimers of the B.1.1.7 and B.1.351 variants, as well as their biochemical and antigenic properties. Amino acid substitutions in the B.1.1.7 protein increase the accessibility of its receptor binding domain and also the binding affinity for receptor angiotensin-converting enzyme 2 (ACE2). The enhanced receptor engagement may account for the increased transmissibility. The B.1.351 variant has evolved to reshape antigenic surfaces of the major neutralizing sites on the S protein, making it resistant to some potent neutralizing antibodies. These findings provide structural details on how SARS-CoV-2 has evolved to enhance viral fitness and immune evasion.}, issn = {0036-8075}, doi = {10.1126/science.abi9745}, url = {https://science.sciencemag.org/content/early/2021/06/23/science.abi9745}, author = {Cai, Yongfei and Zhang, Jun and Xiao, Tianshu and Lavine, Christy L. and Rawson, Shaun and Peng, Hanqin and Zhu, Haisun and Anand, Krishna and Tong, Pei and Gautam, Avneesh and Lu, Shen and Sterling, Sarah M. and Walsh, Richard M. and Rits-Volloch, Sophia and Lu, Jianming and Wesemann, Duane R. and Yang, Wei and Seaman, Michael S. and Chen, Bing} } @article {10.1083/jcb.202012149, title = {Ctf3/CENP-I provides a docking site for the desumoylase Ulp2 at the kinetochore}, journal = {Journal of Cell Biology}, volume = {220}, number = {8}, year = {2021}, note = {e202012149}, month = {06}, abstract = {The step-by-step process of chromosome segregation defines the stages of the cell cycle. In eukaryotes, signals controlling these steps converge upon the kinetochore, a multiprotein assembly that connects spindle microtubules to chromosomal centromeres. Kinetochores control and adapt to major chromosomal transactions, including replication of centromeric DNA, biorientation of sister centromeres on the metaphase spindle, and transit of sister chromatids into daughter cells during anaphase. Although the mechanisms that ensure tight microtubule coupling at anaphase are at least partly understood, kinetochore adaptations that support other cell cycle transitions are not. We report here a mechanism that enables regulated control of kinetochore sumoylation. A conserved surface of the Ctf3/CENP-I kinetochore protein provides a binding site for Ulp2, the nuclear enzyme that removes SUMO chains from modified substrates. Ctf3 mutations that disable Ulp2 recruitment cause elevated inner kinetochore sumoylation and defective chromosome segregation. The location of the site within the assembled kinetochore suggests coordination between sumoylation and other cell cycle{\textendash}regulated processes.}, issn = {0021-9525}, doi = {10.1083/jcb.202012149}, url = {https://doi.org/10.1083/jcb.202012149}, author = {Quan, Yun and Stephen M. Hinshaw and Wang, Pang-Che and Harrison, Stephen C. and Zhou, Huilin} } @article {SHARIF2021, title = {Dipeptidyl peptidase 9 sets a threshold for CARD8 inflammasome formation by sequestering its active C-terminal fragment}, journal = {Immunity}, year = {2021}, abstract = {Summary CARD8 detects intracellular danger signals and forms a caspase-1 activating inflammasome. Like the related inflammasome sensor NLRP1, CARD8 autoprocesses into noncovalently associated N-terminal (NT) and C-terminal (CT) fragments and binds the cellular dipeptidyl peptidases DPP8 and 9 (DPP8/9). Certain danger-associated signals, including the DPP8/9 inhibitor Val-boroPro (VbP) and HIV protease, induce proteasome-mediated NT degradation and thereby liberate the inflammasome-forming CT. Here, we report cryoelectron microscopy (cryo-EM) structures of CARD8 bound to DPP9, revealing a repressive ternary complex consisting of DPP9, full-length CARD8, and CARD8-CT. Unlike NLRP1-CT, CARD8-CT does not interact with the DPP8/9 active site and is not directly displaced by VbP. However, larger DPP8/9 active-site probes can directly weaken this complex in\ vitro, and VbP itself nevertheless appears to disrupt this complex, perhaps indirectly, in cells. Thus, DPP8/9 inhibitors can activate the CARD8 inflammasome by promoting CARD8 NT degradation and by weakening ternary complex stability.}, keywords = {CARD8, cryo-EM, DPP9, inflammasome, NLRP1, pyroptosis, targeted degradation, Val-boroPro (VbP)}, issn = {1074-7613}, doi = {https://doi.org/10.1016/j.immuni.2021.04.024}, url = {https://www.sciencedirect.com/science/article/pii/S1074761321001862}, author = {Sharif, Humayun and Robert Hollingsworth, L. and Griswold, Andrew R. and Jeffrey C. Hsiao and Qinghui Wang and Bachovchin, Daniel A. and Wu, Hao} } @article {Xia2021, title = {Gasdermin D pore structure reveals preferential release of mature interleukin-1}, journal = {Nature}, year = {2021}, month = {Apr}, abstract = {As organelles of the innate immune system, inflammasomes activate caspase-1 and other inflammatory caspases that cleave gasdermin D (GSDMD). Caspase-1 also cleaves inactive precursors of the interleukin (IL)-1 family to generate mature cytokines such as IL-1$\beta$ and IL-18. Cleaved GSDMD forms transmembrane pores to enable the release of IL-1 and to drive cell lysis through pyroptosis1{\textendash}9. Here we report cryo-electron microscopy structures of the pore and the prepore of GSDMD. These structures reveal the different conformations of the two states, as well as extensive membrane-binding elements including a hydrophobic anchor and three positively charged patches. The GSDMD pore conduit is predominantly negatively charged. By contrast, IL-1 precursors have an acidic domain that is proteolytically removed by caspase-110. When permeabilized by GSDMD pores, unlysed liposomes release positively charged and neutral cargoes faster than negatively charged cargoes of similar sizes, and the pores favour the passage of IL-1$\beta$ and IL-18 over that of their precursors. Consistent with these findings, living{\textendash}-but not pyroptotic{\textendash}-macrophages preferentially release mature IL-1$\beta$ upon perforation by GSDMD. Mutation of the acidic residues of GSDMD compromises this preference, hindering intracellular retention of the precursor and secretion of the mature cytokine. The GSDMD pore therefore mediates IL-1 release by electrostatic filtering, which suggests the importance of charge in addition to size in the transport of cargoes across this large channel.}, issn = {1476-4687}, doi = {10.1038/s41586-021-03478-3}, url = {https://doi.org/10.1038/s41586-021-03478-3}, author = {Xia, Shiyu and Zhang, Zhibin and Magupalli, Venkat Giri and Pablo, Juan Lorenzo and Dong, Ying and Vora, Setu M. and Wang, Longfei and Tian-Min Fu and Jacobson, Matthew P. and Greka, Anna and Lieberman, Judy and Ruan, Jianbin and Wu, Hao} } @article {Schnell2021, title = {Structures of chaperone-associated assembly intermediates reveal coordinated mechanisms of proteasome biogenesis}, journal = {Nature Structural \& Molecular Biology}, year = {2021}, month = {Apr}, abstract = {The proteasome mediates most selective protein degradation. Proteolysis occurs within the 20S core particle (CP), a barrel-shaped chamber with an $\alpha$7$\beta$7$\beta$7$\alpha$7 configuration. CP biogenesis proceeds through an ordered multistep pathway requiring five chaperones, Pba1{\textendash}4 and Ump1. Using Saccharomyces cerevisiae, we report high-resolution structures of CP assembly intermediates by cryogenic-electron microscopy. The first structure corresponds to the 13S particle, which consists of a complete $\alpha$-ring, partial $\beta$-ring ($\beta$2{\textendash}4), Ump1 and Pba1/2. The second structure contains two additional subunits ($\beta$5{\textendash}6) and represents a later pre-15S intermediate. These structures reveal the architecture and positions of Ump1 and $\beta$2/$\beta$5 propeptides, with important implications for their functions. Unexpectedly, Pba1{\textquoteright}s N terminus extends through an open CP pore, accessing the CP interior to contact Ump1 and the $\beta$5 propeptide. These results reveal how the coordinated activity of Ump1, Pba1 and the active site propeptides orchestrate key aspects of CP assembly.}, issn = {1545-9985}, doi = {10.1038/s41594-021-00583-9}, url = {https://doi.org/10.1038/s41594-021-00583-9}, author = {Schnell, Helena M. and Walsh, Richard M. and Rawson, Shaun and Kaur, Mandeep and Bhanu, Meera K. and Tian, Geng and Miguel A. Prado and Guerra-Moreno, Angel and Paulo, Joao A. and Steven P. Gygi and Roelofs, Jeroen and Finley, Daniel and Hanna, John} } @article {Hollingsworth2021, title = {DPP9 sequesters the C terminus of NLRP1 to repress inflammasome activation}, journal = {Nature}, year = {2021}, month = {Mar}, abstract = {Nucleotide-binding domain and leucine-rich repeat pyrin-domain containing protein\ 1 (NLRP1) is an inflammasome sensor that mediates the activation of caspase-1 to induce cytokine maturation and pyroptosis1{\textendash}4. Gain-of-function mutations of NLRP1 cause severe inflammatory diseases of the skin4{\textendash}6. NLRP1 contains a function-to-find domain that auto-proteolyses into noncovalently associated subdomains7{\textendash}9, and proteasomal degradation of the repressive N-terminal fragment of NLRP1 releases its inflammatory C-terminal fragment (NLRP1\ CT)10,11. Cytosolic dipeptidyl peptidases 8 and 9 (hereafter, DPP8/DPP9) both interact with NLRP1, and small-molecule inhibitors of DPP8/DPP9 activate NLRP1 by mechanisms that are currently unclear10,12{\textendash}14. Here we report cryo-electron microscopy structures of the human NLRP1{\textendash}DPP9 complex alone and with Val-boroPro (VbP), an inhibitor of DPP8/DPP9. The structures reveal a ternary complex that comprises DPP9, full-length NLRP1 and the NLRPT\ CT. The binding of the NLRP1\ CT to DPP9 requires full-length NLRP1, which suggests that NLRP1 activation is regulated by the ratio of NLRP1\ CT to full-length NLRP1. Activation of the inflammasome by ectopic expression of the NLRP1\ CT is consistently rescued by co-expression of autoproteolysis-deficient full-length NLRP1. The N\ terminus of the NLRP1\ CT inserts into the DPP9 active site, and VbP disrupts this interaction. Thus, VbP weakens the NLRP1{\textendash}DPP9 interaction and accelerates degradation of the N-terminal fragment10 to induce inflammasome activation. Overall, these data demonstrate that DPP9 quenches low levels of NLRP1\ CT and thus serves as a checkpoint for activation of the NLRP1\ inflammasome.}, issn = {1476-4687}, doi = {10.1038/s41586-021-03350-4}, url = {https://doi.org/10.1038/s41586-021-03350-4}, author = {Robert Hollingsworth, L. and Sharif, Humayun and Griswold, Andrew R. and Fontana, Pietro and Mintseris, Julian and Dagbay, Kevin B. and Paulo, Joao A. and Steven P. Gygi and Bachovchin, Daniel A. and Wu, Hao} } @article {Zhangeabf2303, title = {Structural impact on SARS-CoV-2 spike protein by D614G substitution}, journal = {Science}, year = {2021}, publisher = {American Association for the Advancement of Science}, abstract = {Substitution for aspartic acid by glycine at position 614 in the spike (S) protein of severe acute respiratory syndrome coronavirus 2 appears to facilitate rapid viral spread. The G614 strain and its recent variants are now the dominant circulating forms. We report here cryo-EM structures of a full-length G614 S trimer, which adopts three distinct prefusion conformations differing primarily by the position of one receptor-binding domain. A loop disordered in the D614 S trimer wedges between domains within a protomer in the G614 spike. This added interaction appears to prevent premature dissociation of the G614 trimer, effectively increasing the number of functional spikes and enhancing infectivity, and to modulate structural rearrangements for membrane fusion. These findings extend our understanding of viral entry and suggest an improved immunogen for vaccine development.}, issn = {0036-8075}, doi = {10.1126/science.abf2303}, url = {https://science.sciencemag.org/content/early/2021/03/16/science.abf2303}, author = {Zhang, Jun and Cai, Yongfei and Xiao, Tianshu and Lu, Jianming and Peng, Hanqin and Sterling, Sarah M. and Walsh, Richard M. and Rits-Volloch, Sophia and Zhu, Haisun and Woosley, Alec N. and Yang, Wei and Sliz, Piotr and Chen, Bing} } @article {Zhang2021, title = {Cryo-EM structure of an activated GPCR{\textendash}G protein complex in lipid nanodiscs}, journal = {Nature Structural \& Molecular Biology}, volume = {28}, number = {3}, year = {2021}, month = {Mar}, pages = {258-267}, abstract = {G-protein-coupled receptors (GPCRs) are the largest superfamily of transmembrane proteins and the targets of over 30\% of currently marketed pharmaceuticals. Although several structures have been solved for GPCR{\textendash}G protein complexes, few are in a lipid membrane environment. Here, we report cryo-EM structures of complexes of neurotensin, neurotensin receptor 1 and G$\alpha$i1$\beta$1$\gamma$1 in two conformational states, resolved to resolutions of 4.1 and 4.2{\th}inspace}\AA. The structures, determined in a lipid bilayer without any stabilizing antibodies or nanobodies, reveal an extended network of protein{\textendash}protein interactions at the GPCR{\textendash}G protein interface as compared to structures obtained in detergent micelles. The findings show that the lipid membrane modulates the structure and dynamics of complex formation and provide a molecular explanation for the stronger interaction between GPCRs and G proteins in lipid bilayers. We propose an allosteric mechanism for GDP release, providing new insights into the activation of G proteins for downstream signaling.}, issn = {1545-9985}, doi = {10.1038/s41594-020-00554-6}, url = {https://doi.org/10.1038/s41594-020-00554-6}, author = {Zhang, Meng and Gui, Miao and Wang, Zi-Fu and Gorgulla, Christoph and Yu, James J. and Wu, Hao and Sun, Zhen-yu J. and Klenk, Christoph and Merklinger, Lisa and Morstein, Lena and Hagn, Franz and Pl{\"u}ckthun, Andreas and Brown, Alan and Nasr, Mahmoud L. and Wagner, Gerhard} } @article {Shene2023151118, title = {Multiple domain interfaces mediate SARM1 autoinhibition}, journal = {Proceedings of the National Academy of Sciences}, volume = {118}, number = {4}, year = {2021}, publisher = {National Academy of Sciences}, abstract = {Axon degeneration is an active program of subcellular self-destruction that drives pathology in the injured and diseased nervous system. SARM1 is an inducible NAD+ hydrolase and the central executioner of axon loss. In healthy axons, the SARM1 NADase is autoinhibited. With injury or disease, this autoinhibition is relieved and SARM1 depletes NAD+, inducing a metabolic crisis and subsequent axon loss. Here we combine peptide screening, cryo-electron microscopy, and site-directed mutagenesis with analysis of axonal metabolomics and axon degeneration to define five domain interactions within and across SARM1 protomers that are required to maintain an inactive SARM1 octamer. These structural insights may enable the development of SARM1 inhibitors that stabilize this autoinhibited conformation and thereby block axon degeneration.Axon degeneration is an active program of self-destruction mediated by the protein SARM1. In healthy neurons, SARM1 is autoinhibited and, upon injury autoinhibition is relieved, activating the SARM1 enzyme to deplete NAD+ and induce axon degeneration. SARM1 forms a homomultimeric octamer with each monomer composed of an N-terminal autoinhibitory ARM domain, tandem SAM domains that mediate multimerization, and a C-terminal TIR domain encoding the NADase enzyme. Here we discovered multiple intramolecular and intermolecular domain interfaces required for SARM1 autoinhibition using peptide mapping and cryo-electron microscopy (cryo-EM). We identified a candidate autoinhibitory region by screening a panel of peptides derived from the SARM1 ARM domain, identifying a peptide mediating high-affinity inhibition of the SARM1 NADase. Mutation of residues in full-length SARM1 within the region encompassed by the peptide led to loss of autoinhibition, rendering SARM1 constitutively active and inducing spontaneous NAD+ and axon loss. The cryo-EM structure of SARM1 revealed 1) a compact autoinhibited SARM1 octamer in which the TIR domains are isolated and prevented from oligomerization and enzymatic activation and 2) multiple candidate autoinhibitory interfaces among the domains. Mutational analysis demonstrated that five distinct interfaces are required for autoinhibition, including intramolecular and intermolecular ARM-SAM interfaces, an intermolecular ARM-ARM interface, and two ARM-TIR interfaces formed between a single TIR and two distinct ARM domains. These autoinhibitory regions are not redundant, as point mutants in each led to constitutively active SARM1. These studies define the structural basis for SARM1 autoinhibition and may enable the development of SARM1 inhibitors that stabilize the autoinhibited state.All study data are included in the article and/or supporting information. The atomic coordinates and cryo-EM map have been deposited in the Protein Data Bank (PDB), http://www.rcsb.org/ (PDB ID code 7KNQ) (46), and the EM Data Resource, https://www.emdataresource.org/ (ID code EMD-22954) (47).}, issn = {0027-8424}, doi = {10.1073/pnas.2023151118}, url = {https://www.pnas.org/content/118/4/e2023151118}, author = {Shen, Chen and Vohra, Mihir and Zhang, Pengfei and Mao, Xianrong and Figley, Matthew D. and Jian Zhu and Sasaki, Yo and Wu, Hao and DiAntonio, Aaron and Milbrandt, Jeffrey} } @article {Walton2021, title = {Structure of a microtubule-bound axonemal dynein}, journal = {Nature Communications}, volume = {12}, number = {1}, year = {2021}, month = {Jan}, pages = {477}, abstract = {Axonemal dyneins are tethered to doublet microtubules inside cilia to drive ciliary beating, a process critical for cellular motility and extracellular fluid flow. Axonemal dyneins are evolutionarily and biochemically distinct from cytoplasmic dyneins that transport cargo, and the mechanisms regulating their localization and function are poorly understood. Here, we report a single-particle cryo-EM reconstruction of a three-headed axonemal dynein natively bound to doublet microtubules isolated from cilia. The slanted conformation of the axonemal dynein causes interaction of its motor domains with the neighboring dynein complex. Our structure shows how a heterotrimeric docking complex specifically localizes the linear array of axonemal dyneins to the doublet microtubule by directly interacting with the heavy chains. Our structural analysis establishes the arrangement of conserved heavy, intermediate and light chain subunits, and provides a framework to understand the roles of individual subunits and the interactions between dyneins during ciliary waveform generation.}, issn = {2041-1723}, doi = {10.1038/s41467-020-20735-7}, url = {https://doi.org/10.1038/s41467-020-20735-7}, author = {Walton, Travis and Wu, Hao and Brown, Alan} } @article {Susa300, title = {Cryo-EM structure of the B cell co-receptor CD19 bound to the tetraspanin CD81}, journal = {Science}, volume = {371}, number = {6526}, year = {2021}, pages = {300{\textendash}305}, publisher = {American Association for the Advancement of Science}, abstract = {A core component of the immune system are B cells, which are activated by infection and then mature to provide long-lived immunity. Activation is initiated when a cell surface B cell receptor, in association with its coreceptor, recognizes an antigen. Susa et al. report a structure of the B cell receptor CD81 in complex with its co receptor, CD19. CD81 alone binds to cholesterol, but the conformational changes associated with binding to CD19 occlude the cholesterol-binding pocket. Regulating cholesterol binding could play a role in the activation mechanism. The structure also provides a basis for the design of immunotherapies.Science, this issue p. 300Signaling through the CD19-CD81 co-receptor complex, in combination with the B cell receptor, is a critical determinant of B cell development and activation. It is unknown how CD81 engages CD19 to enable co-receptor function. Here, we report a 3.8-angstrom structure of the CD19-CD81 complex bound to a therapeutic antigen-binding fragment, determined by cryo{\textendash}electron microscopy (cryo-EM). The structure includes both the extracellular domains and the transmembrane helices of the complex, revealing a contact interface between the ectodomains that drives complex formation. Upon binding to CD19, CD81 opens its ectodomain to expose a hydrophobic CD19-binding surface and reorganizes its transmembrane helices to occlude a cholesterol binding pocket present in the apoprotein. Our data reveal the structural basis for CD19-CD81 complex assembly, providing a foundation for rational design of therapies for B cell dysfunction.}, issn = {0036-8075}, doi = {10.1126/science.abd9836}, url = {https://science.sciencemag.org/content/371/6526/300}, author = {Susa, Katherine J. and Rawson, Shaun and Kruse, Andrew C. and Blacklow, Stephen C.} } @article {Herrmann2021, title = {Functional refolding of the penetration protein on a non-enveloped virus}, journal = {Nature}, volume = {590}, number = {7847}, year = {2021}, month = {Feb}, pages = {666-670}, abstract = {A non-enveloped virus requires a membrane lesion to deliver its genome into a target cell1. For rotaviruses, membrane perforation is a principal function of the viral outer-layer protein, VP42,3. Here we describe the\ use of electron cryomicroscopy to determine how VP4 performs this function and show that when activated by cleavage to VP8* and VP5*, VP4 can rearrange on the virion surface from an {\textquoteleft}upright{\textquoteright} to a {\textquoteleft}reversed{\textquoteright} conformation. The reversed structure projects a previously buried {\textquoteleft}foot{\textquoteright} domain outwards into the membrane of the host cell to which the virion has attached. Electron cryotomograms of virus particles entering cells are consistent with this picture. Using a disulfide mutant of VP4, we have also stabilized a probable intermediate in the transition between the two conformations. Our results define molecular mechanisms for the first steps of the penetration of rotaviruses into the membranes of target cells and suggest similarities with mechanisms postulated for other viruses.}, issn = {1476-4687}, doi = {10.1038/s41586-020-03124-4}, url = {https://doi.org/10.1038/s41586-020-03124-4}, author = {Herrmann, Tobias and Torres, Ra{\'u}l and Salgado, Eric N. and Berciu, Cristina and Stoddard, Daniel and Nicastro, Daniela and Jenni, Simon and Harrison, Stephen C.} } @article {Xiao2021, title = {A trimeric human angiotensin-converting enzyme 2 as an anti-SARS-CoV-2 agent}, journal = {Nature Structural \& Molecular Biology}, year = {2021}, month = {Jan}, abstract = {Effective intervention strategies are urgently needed to control the COVID-19 pandemic. Human angiotensin-converting enzyme 2 (ACE2) is a membrane-bound carboxypeptidase that forms a dimer and serves as the cellular receptor for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). ACE2 is also a key negative regulator of the renin{\textendash}angiotensin system that modulates vascular functions. We report here the properties of a trimeric ACE2 ectodomain variant, engineered using a structure-based approach. The trimeric ACE2 variant has a binding affinity of \textasciitilde60{\th}inspace}pM for the spike protein of SARS-CoV-2 (compared with 77{\th}inspace}nM for monomeric ACE2 and 12{\textendash}22{\th}inspace}nM for dimeric ACE2 constructs), and its peptidase activity and the ability to block activation of angiotensin II receptor type 1 in the renin{\textendash}angiotensin system are preserved. Moreover, the engineered ACE2 potently inhibits SARS-CoV-2 infection in cell culture. These results suggest that engineered, trimeric ACE2 may be a promising anti-SARS-CoV-2 agent for treating COVID-19.}, issn = {1545-9985}, doi = {10.1038/s41594-020-00549-3}, url = {https://doi.org/10.1038/s41594-020-00549-3}, author = {Xiao, Tianshu and Lu, Jianming and Zhang, Jun and Johnson, Rebecca I. and McKay, Lindsay G. A. and Storm, Nadia and Lavine, Christy L. and Peng, Hanqin and Cai, Yongfei and Rits-Volloch, Sophia and Lu, Shen and Quinlan, Brian D. and Farzan, Michael and Seaman, Michael S. and Griffiths, Anthony and Chen, Bing} } @article {RobertHollingsworth2021, title = {Mechanism of filament formation in UPA-promoted CARD8 and NLRP1 inflammasomes}, journal = {Nature Communications}, volume = {12}, number = {1}, year = {2021}, month = {Jan}, pages = {189}, abstract = {NLRP1 and CARD8 are related cytosolic sensors that upon activation form supramolecular signalling complexes known as canonical inflammasomes, resulting in caspase-1 activation, cytokine maturation and/or pyroptotic cell death. NLRP1 and CARD8 use their C-terminal (CT) fragments containing a caspase recruitment domain (CARD) and the UPA (conserved in UNC5, PIDD, and ankyrins) subdomain for self-oligomerization, which in turn form the platform to recruit the inflammasome adaptor ASC (apoptosis-associated speck-like protein containing a CARD) or caspase-1, respectively. Here, we report cryo-EM structures of NLRP1-CT and CARD8-CT assemblies, in which the respective CARDs form central helical filaments that are promoted by oligomerized, but flexibly linked, UPAs surrounding the filaments. Through biochemical and cellular approaches, we demonstrate that the UPA itself reduces the threshold needed for NLRP1-CT and CARD8-CT filament formation and signalling. Structural analyses provide insights on the mode of ASC recruitment by NLRP1-CT and the contrasting direct recruitment of caspase-1 by CARD8-CT. We also discover that subunits in the central NLRP1CARD filament dimerize with additional exterior CARDs, which roughly doubles its thickness and is unique among all known CARD filaments. Finally, we engineer and determine the structure of an ASCCARD{\textendash}caspase-1CARD octamer, which suggests that ASC uses opposing surfaces for NLRP1, versus caspase-1, recruitment. Together these structures capture the architecture and specificity of the active NLRP1 and CARD8 inflammasomes in addition to key heteromeric CARD-CARD interactions governing inflammasome signalling.}, issn = {2041-1723}, doi = {10.1038/s41467-020-20320-y}, url = {https://doi.org/10.1038/s41467-020-20320-y}, author = {Robert Hollingsworth, L. and David, Liron and Li, Yang and Griswold, Andrew R. and Ruan, Jianbin and Sharif, Humayun and Fontana, Pietro and Orth-He, Elizabeth L. and Tian-Min Fu and Bachovchin, Daniel A. and Wu, Hao} } @article {KATO2020, title = {Structural analysis of RIG-I-like receptors reveals ancient rules of engagement between diverse RNA helicases and TRIM ubiquitin ligases}, journal = {Molecular Cell}, year = {2020}, abstract = {Summary RNA helicases and E3 ubiquitin ligases mediate many critical functions in cells, but their actions have largely been studied in distinct biological contexts. Here, we uncover evolutionarily conserved rules of engagement between RNA helicases and tripartite motif (TRIM) E3 ligases that lead to their functional coordination in vertebrate innate immunity. Using cryoelectron microscopy and biochemistry, we show that RIG-I-like receptors (RLRs), viral RNA receptors with helicase domains, interact with their cognate TRIM/TRIM-like E3 ligases through similar epitopes in the helicase domains. Their interactions are avidity driven, restricting the actions of TRIM/TRIM-like proteins and consequent immune activation to RLR multimers. Mass spectrometry and phylogeny-guided biochemical analyses further reveal that similar rules of engagement may apply to diverse RNA helicases and TRIM/TRIM-like proteins. Our analyses suggest not only conserved substrates for TRIM proteins but also, unexpectedly, deep evolutionary connections between TRIM proteins and RNA helicases, linking ubiquitin and RNA biology throughout animal evolution.}, keywords = {Dicer, E3 ubiquitin ligase, innate immunity, LGP2, MDA5, RIG-I, RIPLET, TRIM, TRIM25, TRIM65}, issn = {1097-2765}, doi = {https://doi.org/10.1016/j.molcel.2020.11.047}, url = {http://www.sciencedirect.com/science/article/pii/S1097276520308893}, author = {Kazuki Kato and Sadeem Ahmad and Zixiang Zhu and Janet M. Young and Xin Mu and Park, Se Hoon and Harmit S. Malik and Sun Hur} } @article {Gui2021, title = {Structures of radial spokes and associated complexes important for ciliary motility}, journal = {Nature Structural \& Molecular Biology}, volume = {28}, number = {1}, year = {2020}, month = {Jan}, pages = {29-37}, abstract = {In motile cilia, a mechanoregulatory network is responsible for converting the action of thousands of dynein motors bound to doublet microtubules into a single propulsive waveform. Here, we use two complementary cryo-EM strategies to determine structures of the major mechanoregulators that bind ciliary doublet microtubules in Chlamydomonas reinhardtii. We determine structures of isolated radial spoke RS1 and the microtubule-bound RS1, RS2 and the nexin-dynein regulatory complex (N-DRC). From these structures, we identify and build atomic models for 30 proteins, including 23 radial-spoke subunits. We reveal how mechanoregulatory complexes dock to doublet microtubules with regular 96-nm periodicity and communicate with one another. Additionally, we observe a direct and dynamically coupled association between RS2 and the dynein motor inner dynein arm subform c (IDAc), providing a molecular basis for the control of motor activity by mechanical signals. These structures advance our understanding of the role of mechanoregulation in defining the ciliary waveform.}, issn = {1545-9985}, doi = {10.1038/s41594-020-00530-0}, url = {https://doi.org/10.1038/s41594-020-00530-0}, author = {Gui, Miao and Ma, Meisheng and Sze-Tu, Erica and Wang, Xiangli and Koh, Fujiet and Zhong, Ellen D. and Berger, Bonnie and Davis, Joseph H. and Dutcher, Susan K. and Zhang, Rui and Brown, Alan} } @article {ref1, title = {Structures of a Complete Human V-ATPase Reveal Mechanisms of Its Assembly}, journal = {Molecular Cell}, year = {2020}, month = {2020/10/21}, publisher = {Elsevier}, issn = {1097-2765}, doi = {10.1016/j.molcel.2020.09.029}, url = {https://doi.org/10.1016/j.molcel.2020.09.029}, author = {Wang, Longfei and Wu, Di and Robinson, Carol V. and Wu, Hao and Tian-Min Fu} } @article {10.1371/journal.ppat.1008920, title = {Cryo-EM structures reveal two distinct conformational states in a picornavirus cell entry intermediate}, journal = {PLOS Pathogens}, volume = {16}, number = {9}, year = {2020}, month = {09}, pages = {1-26}, publisher = {Public Library of Science}, abstract = {Author summary Nonenveloped viruses need to provide mechanisms that allow their genomes to be delivered across membranes. This process remains poorly understood. For enteroviruses such as poliovirus, genome delivery involves a program of conformational changes that include expansion of the particle and externalization of two normally internal peptides, VP4 and the VP1 N-terminus, which then insert into the cell membrane, triggering endocytosis and the creation of pores that facilitate the transfer of the viral RNA genome across the endosomal membrane. This manuscript describes five high-resolution cryo-EM structures of altered poliovirus particles that represent a number of intermediates along this pathway. The structures reveal several surprising findings, including the discovery of a new intermediate that is expanded, but has not yet externalized the membrane interactive peptides; the clear identification of a unique exit site for the VP1 N-terminus; the demonstration that the externalized VP1 N-terminus partitions between two different sites in a temperature-dependent fashion; direct visualization of an amphipathic helix at the N-terminus of VP1 in an ideal position for interaction with cellular membranes; and the observation that a significant portion of VP4 remains inside the particle and accounts for a density feature that had previously been ascribed to part of the viral RNA. These findings represent significant additions to our understanding of the cell entry process of an important class of human pathogens.}, doi = {10.1371/journal.ppat.1008920}, url = {https://doi.org/10.1371/journal.ppat.1008920}, author = {Shah, Pranav N. M. and Filman, David J. and Karunatilaka, Krishanthi S. and Hesketh, Emma L. and Groppelli, Elisabetta and Strauss, Mike and Hogle, James M.} } @article {Kim2020, title = {Shared structural mechanisms of general anaesthetics and benzodiazepines}, journal = {Nature}, year = {2020}, month = {Sep}, abstract = {Most general anaesthetics and classical benzodiazepine drugs act through positive modulation of $\gamma$-aminobutyric acid type A (GABAA) receptors to dampen neuronal activity in the brain1{\textendash}5. However, direct structural information on the mechanisms of general anaesthetics at their physiological receptor sites is lacking. Here we present cryo-electron microscopy structures of GABAA receptors bound to intravenous anaesthetics, benzodiazepines and inhibitory modulators. These structures were solved in a lipidic environment and are complemented by electrophysiology and molecular dynamics simulations. Structures of GABAA receptors in complex with the anaesthetics phenobarbital, etomidate and propofol reveal both distinct and common transmembrane binding sites, which are shared in part by the benzodiazepine drug diazepam. Structures in which GABAA receptors are bound by benzodiazepine-site ligands identify an additional membrane binding site for diazepam and suggest an allosteric mechanism for anaesthetic reversal by flumazenil. This study provides a foundation for understanding how pharmacologically diverse and clinically essential drugs act through overlapping and distinct mechanisms to potentiate inhibitory signalling in the brain.}, issn = {1476-4687}, doi = {10.1038/s41586-020-2654-5}, url = {https://doi.org/10.1038/s41586-020-2654-5}, author = {Kim, Jeong Joo and Gharpure, Anant and Teng, Jinfeng and Zhuang, Yuxuan and Howard, Rebecca J. and Zhu, Shaotong and Noviello, Colleen M. and Walsh, Richard M. and Lindahl, Erik and Hibbs, Ryan E.} } @article {Caieabd4251, title = {Distinct conformational states of SARS-CoV-2 spike protein}, journal = {Science}, year = {2020}, publisher = {American Association for the Advancement of Science}, abstract = {Intervention strategies are urgently needed to control the SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) pandemic. The trimeric viral spike (S) protein catalyzes fusion between viral and target cell membranes to initiate infection. Here we report two cryo-EM structures, derived from a preparation of the full-length S protein, representing its prefusion (2.9{\r A} resolution) and postfusion (3.0{\r A} resolution) conformations, respectively. The spontaneous transition to the postfusion state is independent of target cells. The prefusion trimer has three receptor-binding domains clamped down by a segment adjacent to the fusion peptide. The postfusion structure is strategically decorated by N-linked glycans, suggesting possible protective roles against host immune responses and harsh external conditions. These findings advance our understanding of SARS-CoV-2 entry and may guide development of vaccines and therapeutics.}, issn = {0036-8075}, doi = {10.1126/science.abd4251}, url = {https://science.sciencemag.org/content/early/2020/07/20/science.abd4251}, author = {Cai, Yongfei and Zhang, Jun and Xiao, Tianshu and Peng, Hanqin and Sterling, Sarah M. and Walsh, Richard M. and Rawson, Shaun and Rits-Volloch, Sophia and Chen, Bing} } @article {HINSHAW2020, title = {The Structural Basis for Kinetochore Stabilization by Cnn1/CENP-T}, journal = {Current Biology}, year = {2020}, abstract = {Summary Chromosome segregation depends on a regulated connection between spindle microtubules and centromeric DNA. The kinetochore mediates this connection and ensures it persists during anaphase, when sister chromatids must transit into daughter cells uninterrupted. The Ctf19 complex (Ctf19c) forms the centromeric base of the kinetochore in budding yeast. Biochemical experiments show that Ctf19c members associate hierarchically when purified from cell extract [1], an observation that is mostly explained by the structure of the complex [2]. The Ctf3 complex (Ctf3c), which is not required for the assembly of most other Ctf19c factors, disobeys the biochemical assembly hierarchy when observed in dividing cells that lack more basal components [3]. Thus, the biochemical experiments do not completely recapitulate the logic of centromeric Ctf19c assembly. We now present a high-resolution structure of the Ctf3c bound to the Cnn1-Wip1 heterodimer. Associated live-cell imaging experiments provide a mechanism for Ctf3c and Cnn1-Wip1 recruitment to the kinetochore. The mechanism suggests feedback regulation of Ctf19c assembly and unanticipated similarities in kinetochore organization between yeast and vertebrates.}, keywords = {Cell Cycle, Centromere, Chromosome Segregation, cryo-EM, kinetochore, Mitosis, spindle}, issn = {0960-9822}, doi = {https://doi.org/10.1016/j.cub.2020.06.024}, url = {http://www.sciencedirect.com/science/article/pii/S096098222030840X}, author = {Stephen M. Hinshaw and Harrison, Stephen C.} } @article {Tomasek2020, title = {Structure of a nascent membrane protein as it folds on the BAM complex}, journal = {Nature}, year = {2020}, month = {Jun}, abstract = {Mitochondria, chloroplasts and Gram-negative bacteria are encased in a double layer of membranes. The outer membrane contains proteins with a $\beta$-barrel structure1,2. $\beta$-Barrels are sheets of $\beta$-strands wrapped into a cylinder, in which the first strand is hydrogen-bonded to the final strand. Conserved multi-subunit molecular machines fold and insert these proteins into the outer membrane3{\textendash}5. One subunit of the machines is itself a $\beta$-barrel protein that has a central role in folding other $\beta$-barrels. In Gram-negative bacteria, the $\beta$-barrel assembly machine (BAM) consists of the $\beta$-barrel protein BamA, and four lipoproteins5{\textendash}8. To understand how the BAM complex accelerates folding without using exogenous energy (for example, ATP)9, we trapped folding intermediates on this machine. Here we report the structure of the BAM complex of Escherichia coli folding BamA itself. The BamA catalyst forms an asymmetric hybrid $\beta$-barrel with the BamA substrate. The N-terminal edge of the BamA catalyst has an antiparallel hydrogen-bonded interface with the C-terminal edge of the BamA substrate, consistent with previous crosslinking studies10{\textendash}12; the other edges of the BamA catalyst and substrate are close to each other, but curl inward and do not pair. Six hydrogen bonds in a membrane environment make the interface between the two proteins very stable. This stability allows folding, but creates a high kinetic barrier to substrate release after folding has finished. Features at each end of the substrate overcome this barrier and promote release by stepwise exchange of hydrogen bonds. This mechanism of substrate-assisted product release explains how the BAM complex can stably associate with the substrate during folding and then turn over rapidly when folding is complete.}, issn = {1476-4687}, doi = {10.1038/s41586-020-2370-1}, url = {https://doi.org/10.1038/s41586-020-2370-1}, author = {Tomasek, David and Rawson, Shaun and Lee, James and Wzorek, Joseph S. and Harrison, Stephen C. and Li, Zongli and Kahne, Daniel} } @article {Sui2020, title = {Structure and catalytic mechanism of a human triacylglycerol-synthesis enzyme}, journal = {Nature}, year = {2020}, month = {May}, abstract = {Triacylglycerols store metabolic energy in organisms and have industrial uses as foods and fuels. Excessive accumulation of triacylglycerols in humans causes obesity and is associated with metabolic diseases1. Triacylglycerol synthesis is catalysed by acyl-CoA diacylglycerol acyltransferase (DGAT) enzymes2{\textendash}4, the structures and catalytic mechanisms of which remain unknown. Here we determined the structure of dimeric human DGAT1, a member of the membrane-bound O-acyltransferase (MBOAT) family, by cryo-electron microscopy at approximately 3.0\ \AA resolution. DGAT1 forms a homodimer through N-terminal segments and a hydrophobic interface, with putative active sites within the membrane region. A structure obtained with oleoyl-CoA substrate resolved at approximately 3.2\ \AA shows that the CoA moiety binds DGAT1 on the cytosolic side and the acyl group lies deep within a hydrophobic channel, positioning the acyl-CoA thioester bond near an invariant catalytic histidine residue. The reaction centre is located inside a large cavity, which opens laterally to the membrane bilayer, providing lipid access to the active site. A lipid-like density{\textendash}-possibly representing an acyl-acceptor molecule{\textendash}-is located within the reaction centre, orthogonal to acyl-CoA. Insights provided by the DGAT1 structures, together with mutagenesis and functional studies, provide the basis for a model of the\ catalysis of triacylglycerol synthesis by DGAT.}, issn = {1476-4687}, doi = {10.1038/s41586-020-2289-6}, url = {https://doi.org/10.1038/s41586-020-2289-6}, author = {Sui, Xuewu and Wang, Kun and Gluchowski, Nina L. and Elliott, Shane D. and Maofu Liao and Walther, Tobias C. and Farese, Robert V.} } @article {1506510, title = {ABCG2 transports anticancer drugs via a closed-to-open switch}, journal = {Nat Commun}, volume = {11}, number = {1}, year = {2020}, month = {2020 May 08}, pages = {2264}, abstract = {ABCG2 is an ABC transporter that extrudes a variety of compounds from cells, and presents an obstacle in treating chemotherapy-resistant cancers. Despite recent structural insights, no anticancer drug bound to ABCG2 has been resolved, and the mechanisms of multidrug transport remain obscure. Such a~gap of knowledge limits the development of novel compounds that block or evade this critical molecular pump. Here we present single-particle cryo-EM studies of ABCG2 in the apo state, and bound to the three structurally distinct chemotherapeutics. Without the binding of conformation-selective antibody fragments or inhibitors, the resting ABCG2 adopts a closed conformation. Our cryo-EM, biochemical, and functional analyses reveal the binding mode of three chemotherapeutic compounds, demonstrate how these molecules open the closed conformation of the transporter, and establish that imatinib is particularly effective in stabilizing the inward facing conformation of ABCG2. Together these studies reveal the previously unrecognized conformational cycle of ABCG2.}, issn = {2041-1723}, doi = {10.1038/s41467-020-16155-2}, author = {Orlando, Benjamin J and Maofu Liao} } @article {Wueaaz2449, title = {Structural basis of ER-associated protein degradation mediated by the Hrd1 ubiquitin ligase complex}, journal = {Science}, volume = {368}, number = {6489}, year = {2020}, publisher = {American Association for the Advancement of Science}, abstract = {Misfolded endoplasmic reticulum (ER) proteins are retrotranslocated into the cytosol, polyubiquitinated, and degraded by the proteasome in a process known as ER-associated protein degradation (ERAD). ERAD of misfolded luminal ER proteins (ERAD-L) is mediated by the Hrd1 complex, composed of the ubiquitin ligase Hrd1 and four additional proteins (Hrd3, Der1, Usa1, and Yos9). Wu et al. report a cryo{\textendash}electron microscopy structure of the active Hrd1 complex from yeast and, based on this structure, developed a model for how substrates are recognized and retrotranslocated. They propose that Hrd3 and Yos9 jointly create a luminal binding site for misfolded glycoproteins. Hrd1 and Der1 form {\textquotedblleft}half-channels{\textquotedblright} juxtaposed in a thinned section of the ER membrane, which allows a polypeptide loop of an ERAD-L substrate to move through it.Science, this issue p. eaaz2449INTRODUCTIONProtein homeostasis in the endoplasmic reticulum (ER) is maintained by a quality control system. When a newly synthesized ER protein misfolds, it is ultimately retrotranslocated into the cytosol, polyubiquitinated, and degraded by the proteasome, a pathway referred to as ER-associated protein degradation (ERAD). ERAD alleviates cytotoxic stress imposed by protein misfolding and is implicated in numerous diseases. ERAD is found in all eukaryotic cells but is best studied for the ERAD-L pathway in Saccharomyces cerevisiae, which disposes of misfolded glycoproteins in the ER lumen. The glycan attached to these proteins is first trimmed by glycosidases to generate a terminal α1,6-mannose residue. This residue, together with an unfolded polypeptide segment, targets the substrate to the Hrd1 complex, which is composed of the multispanning ubiquitin ligase Hrd1 and four additional proteins (Hrd3, Der1, Usa1, and Yos9). The Hrd1 complex mediates the retrotranslocation of the polypeptide into the cytosol, where it is polyubiquitinated, extracted from the membrane by the Cdc48 adenosine triphosphatase complex, and, finally, degraded by the proteasome.RATIONALEThe mechanism of ERAD-L remains poorly understood. Arguably the most mysterious aspect is how misfolded proteins cross the ER membrane, which normally presents a barrier to macromolecules. How ERAD-L substrates are recognized and distinguished from properly folding intermediates is also unclear. Answers to these questions require structural information on the Hrd1 complex.RESULTSHere, we report a structure of the active Hrd1 complex from S. cerevisiae, as determined by cryo{\textendash}electron microscopy (cryo-EM) analysis of two subcomplexes. Our structures, biochemical data, and experiments in vivo indicate that the Hrd1 complex functions as a monomer in ERAD-L. Hrd3 and Yos9 jointly create a luminal binding site that recognizes misfolded glycoproteins. The α1,6-mannose residue binds to the mannose 6-phosphate receptor homology (MRH) domain of Yos9, and the polypeptide segment downstream of the glycan attachment site is likely accommodated in a groove of the luminal domain of Hrd3. Hrd1 and the rhomboid-like Der1 protein are linked by Usa1 on the cytosolic side of the membrane. Both Der1 and Hrd1 have lateral gates that face one another within the membrane and possess luminal and cytosolic cavities, respectively. Both proteins distort the membrane region between the lateral gates, making it much thinner than a normal phospholipid bilayer, an observation supported by molecular dynamics simulations. The structures and photocrosslinking experiments indicate that the retrotranslocation of an ERAD-L substrate is initiated by loop insertion of the polypeptide into the membrane, with one strand of the loop interacting with Der1 and the other with Hrd1.CONCLUSIONOur results lead to a model for the mechanism of retrotranslocation through the Hrd1 complex. The pathway across the membrane is formed by two {\textquotedblleft}half-channels{\textquotedblright} corresponding to the luminal and cytosolic cavities of Der1 and Hrd1, respectively. These half-channels are juxtaposed in a thinned membrane region. The substrate inserts into the retrotranslocon as a hairpin that is hydrophilic on both sides. These features contrast with the Sec61 channel, which accepts substrates with a hydrophobic signal or transmembrane segment forming one side of the loop. This segment exits the lateral gate into the lipid environment and is not translocated, while the other side of the loop moves through the membrane in an entirely hydrophilic environment. The structural features of the retrotranslocon can facilitate movement of a fully hydrophilic substrate through a thinned and thus distorted membrane, a paradigm that may be replicated in other protein translocation systems.Initiation of ERAD-L revealed by cryo-EM and photocrosslinking.(A) Side view of a space-filling model of the Hrd1 complex, based on structures of the Hrd1~Usa1-Der1-Hrd3 and Hrd3-Yos9 subcomplexes. (B) Hypothetical position of a glycosylated ERAD-L substrate in the Hrd1 complex (dashed blue line). Substrate-interacting amino acid residues in Hrd1 and Der1 (red and orange, respectively) were determined by photocrosslinking. N, N terminus; C, C terminus; DHFR, dihydrofolate reductase; TM, transmembrane helix. (C) Model for the first three stages of retrotranslocation.Misfolded luminal endoplasmic reticulum (ER) proteins undergo ER-associated degradation (ERAD-L): They are retrotranslocated into the cytosol, polyubiquitinated, and degraded by the proteasome. ERAD-L is mediated by the Hrd1 complex (composed of Hrd1, Hrd3, Der1, Usa1, and Yos9), but the mechanism of retrotranslocation remains mysterious. Here, we report a structure of the active Hrd1 complex, as determined by cryo{\textendash}electron microscopy analysis of two subcomplexes. Hrd3 and Yos9 jointly create a luminal binding site that recognizes glycosylated substrates. Hrd1 and the rhomboid-like Der1 protein form two {\textquotedblleft}half-channels{\textquotedblright} with cytosolic and luminal cavities, respectively, and lateral gates facing one another in a thinned membrane region. These structures, along with crosslinking and molecular dynamics simulation results, suggest how a polypeptide loop of an ERAD-L substrate moves through the ER membrane.}, issn = {0036-8075}, doi = {10.1126/science.aaz2449}, url = {https://science.sciencemag.org/content/368/6489/eaaz2449}, author = {Wu, Xudong and Siggel, Marc and Ovchinnikov, Sergey and Wei Mi and Svetlov, Vladimir and Nudler, Evgeny and Maofu Liao and Hummer, Gerhard and Rapoport, Tom A.} } @article {10.7554/eLife.53322, title = {Structure and activation mechanism of the BBSome membrane protein trafficking complex}, journal = {eLife}, volume = {9}, year = {2020}, month = {jan}, pages = {e53322}, publisher = {eLife Sciences Publications, Ltd}, abstract = {Bardet-Biedl syndrome (BBS) is a currently incurable ciliopathy caused by the failure to correctly establish or maintain cilia-dependent signaling pathways. Eight proteins associated with BBS assemble into the BBSome, a key regulator of the ciliary membrane proteome. We report the electron cryomicroscopy (cryo-EM) structures of the native bovine BBSome in inactive and active states at 3.1 -and 3.5 {\r A} resolution, respectively. In the active state, the BBSome is bound to an Arf-family GTPase (ARL6/BBS3) that recruits the BBSome to ciliary membranes. ARL6 recognizes a composite binding site formed by BBS1 and BBS7 that is occluded in the inactive state. Activation requires an unexpected swiveling of the b-propeller domain of BBS1, the subunit most frequently implicated in substrate recognition, which widens a central cavity of the BBSome. Structural mapping of disease-causing mutations suggests that pathogenesis results from folding defects and the disruption of autoinhibition and activation.}, issn = {2050-084X}, doi = {10.7554/eLife.53322}, url = {https://doi.org/10.7554/eLife.53322}, author = {Singh, Sandeep K and Gui, Miao and Koh, Fujiet and Yip, Matthew CJ and Brown, Alan}, editor = {Carter, Andrew P} } @article {LOWEY202038, title = {CBASS Immunity Uses CARF-Related Effectors to Sense 3'{\textendash}5'- and 2'{\textendash}5'-Linked Cyclic Oligonucleotide Signals and Protect Bacteria from Phage Infection}, journal = {Cell}, volume = {182}, number = {1}, year = {2020}, pages = {38-49.e17}, abstract = {Summary cGAS/DncV-like nucleotidyltransferase (CD-NTase) enzymes are immune sensors that synthesize nucleotide second messengers and initiate antiviral responses in bacterial and animal cells. Here, we discover Enterobacter cloacae CD-NTase-associated protein 4 (Cap4) as a founding member of a diverse family of \>2,000 bacterial receptors that respond to CD-NTase signals. Structures of Cap4 reveal a promiscuous DNA endonuclease domain activated through ligand-induced oligomerization. Oligonucleotide recognition occurs through an appended SAVED domain that is an unexpected fusion of two CRISPR-associated Rossman fold (CARF) subunits co-opted from type III CRISPR immunity. Like a lock and key, SAVED effectors exquisitely discriminate 2'{\textendash}5'- and 3'{\textendash}5'-linked bacterial cyclic oligonucleotide signals and enable specific recognition of at least 180 potential nucleotide second messenger species. Our results reveal SAVED CARF family proteins as major nucleotide second messenger receptors in CBASS and CRISPR immune defense and extend the importance of linkage specificity beyond mammalian cGAS-STING signaling.}, keywords = {CARF, CBASS antiphage immunity, CD-NTase, nucleotide second messenger, SAVED}, issn = {0092-8674}, doi = {https://doi.org/10.1016/j.cell.2020.05.019}, url = {https://www.sciencedirect.com/science/article/pii/S0092867420306140}, author = {Lowey, Brianna and Aaron T. Whiteley and Keszei, Alexander F. A. and Morehouse, Benjamin R. and Ian T. Mathews and Sadie P. Antine and Victor J. Cabrera and Kashin, Dmitry and Niemann, Percy and Jain, Mohit and Schwede, Frank and John J. Mekalanos and Shao, Sichen and Amy S.Y. Lee and Kranzusch, Philip J.} } @article {Sloutskyeaaz0240, title = {Heterogeneity in human hippocampal CaMKII transcripts reveals allosteric hub-dependent regulation}, journal = {Science Signaling}, volume = {13}, number = {641}, year = {2020}, publisher = {Science Signaling}, abstract = {Members of the calcium/calmodulin-dependent protein kinase II (CaMKII) family of oligomeric serine and threonine kinases mediate numerous processes, including long-term memory formation. Each CaMKII subunit contains a kinase domain, a linker region, and a hub domain, which mediates oligomerization. Sloutsky et al. sequenced at least 70 CaMKII-encoding transcripts expressed in the human hippocampus, a primary memory center in the brain. Structural and functional analyses revealed interactions between the kinase and hub domains that affected the sensitivity of CaMKII variants to activation by calcium/calmodulin. Together, these data suggest an additional role for the hub domain in regulating CaMKII activation, which may provide a therapeutic target.Calcium/calmodulin-dependent protein kinase II (CaMKII) plays a central role in Ca2+ signaling throughout the body. In the hippocampus, CaMKII is required for learning and memory. Vertebrate genomes encode four CaMKII homologs: CaMKIIα, CaMKIIβ, CaMKIIγ, and CaMKIIδ. All CaMKIIs consist of a kinase domain, a regulatory segment, a variable linker region, and a hub domain, which is responsible for oligomerization. The four proteins differ primarily in linker length and composition because of extensive alternative splicing. Here, we report the heterogeneity of CaMKII transcripts in three complex samples of human hippocampus using deep sequencing. We showed that hippocampal cells contain a diverse collection of over 70 CaMKII transcripts from all four CaMKII-encoding genes. We characterized the Ca2+/CaM sensitivity of hippocampal CaMKII variants spanning a broad range of linker lengths and compositions. The effect of the variable linker on Ca2+/CaM sensitivity depended on the kinase and hub domains. Moreover, we revealed a previously uncharacterized role for the hub domain as an allosteric regulator of kinase activity, which may provide a pharmacological target for modulating CaMKII activity. Using small-angle x-ray scattering and single-particle cryo{\textendash}electron microscopy (cryo-EM), we present evidence for extensive interactions between the kinase and the hub domains, even in the presence of a 30-residue linker. Together, these data suggest that Ca2+/CaM sensitivity in CaMKII is homolog dependent and includes substantial contributions from the hub domain. Our sequencing approach, combined with biochemistry, provides insights into understanding the complex pool of endogenous CaMKII splice variants.}, issn = {1945-0877}, doi = {10.1126/scisignal.aaz0240}, url = {https://stke.sciencemag.org/content/13/641/eaaz0240}, author = {Sloutsky, Roman and Dziedzic, Noelle and Dunn, Matthew J. and Bates, Rachel M. and Torres-Ocampo, Ana P. and Boopathy, Sivakumar and Page, Brendan and Weeks, John G. and Chao, Luke H. and Stratton, Margaret M.} } @article {Morehouse2020, title = {STING cyclic dinucleotide sensing originated in bacteria}, journal = {Nature}, volume = {586}, number = {7829}, year = {2020}, month = {Oct}, pages = {429-433}, abstract = {Stimulator of interferon genes (STING) is a receptor in human cells that senses foreign cyclic dinucleotides that are released during bacterial infection and in endogenous cyclic GMP{\textendash}AMP signalling during viral infection and anti-tumour immunity1{\textendash}5. STING shares no structural homology with other known signalling proteins6{\textendash}9, which has limited attempts at functional analysis and prevented explanation of the origin of cyclic dinucleotide signalling in mammalian innate immunity. Here we reveal functional STING homologues encoded within prokaryotic defence islands, as well as a conserved mechanism of signal activation. Crystal structures of bacterial STING define a minimal homodimeric scaffold that selectively responds to cyclic di-GMP synthesized by a neighbouring cGAS/DncV-like nucleotidyltransferase (CD-NTase) enzyme. Bacterial STING domains couple the recognition of cyclic dinucleotides with the formation of protein filaments to drive oligomerization of TIR effector domains and rapid NAD+ cleavage. We reconstruct the evolutionary events that followed the acquisition of STING into metazoan innate immunity, and determine the structure of a full-length TIR{\textendash}STING fusion from the Pacific oyster Crassostrea gigas. Comparative structural analysis demonstrates how metazoan-specific additions to the core STING scaffold enabled a switch from direct effector function to regulation of antiviral transcription. Together, our results explain the mechanism of STING-dependent signalling and reveal the conservation of a functional cGAS{\textendash}STING pathway in prokaryotic defence against bacteriophages.}, issn = {1476-4687}, doi = {10.1038/s41586-020-2719-5}, url = {https://doi.org/10.1038/s41586-020-2719-5}, author = {Morehouse, Benjamin R. and Govande, Apurva A. and Millman, Adi and Keszei, Alexander F. A. and Lowey, Brianna and Ofir, Gal and Shao, Sichen and Sorek, Rotem and Kranzusch, Philip J.} } @article {Horwitz2099, title = {Structure of a rabies virus polymerase complex from electron cryo-microscopy}, journal = {Proceedings of the National Academy of Sciences}, volume = {117}, number = {4}, year = {2020}, pages = {2099{\textendash}2107}, publisher = {National Academy of Sciences}, abstract = {Rabies virus (RABV) and other viruses with single-segment, negative-sense, RNA genomes have a multi-functional polymerase protein (L) that carries out the various reactions required for transcription and replication. Many of these viruses are serious human pathogens, and L is a potential target for antiviral therapeutics. Drugs that inhibit polymerases of HCV and HIV-1 provide successful precedents. The structure described here of the RABV L protein in complex with its P-protein cofactor shows a conformation poised for initiation of transcription or replication. Channels in the molecule and the relative positions of catalytic sites suggest that L couples a distinctive capping reaction with priming and initiation of transcription, and that replication and transcription have different priming configurations and different product exit sites.Nonsegmented negative-stranded (NNS) RNA viruses, among them the virus that causes rabies (RABV), include many deadly human pathogens. The large polymerase (L) proteins of NNS RNA viruses carry all of the enzymatic functions required for viral messenger RNA (mRNA) transcription and replication: RNA polymerization, mRNA capping, and cap methylation. We describe here a complete structure of RABV L bound with its phosphoprotein cofactor (P), determined by electron cryo-microscopy at 3.3 {\r A} resolution. The complex closely resembles the vesicular stomatitis virus (VSV) L-P, the one other known full-length NNS-RNA L-protein structure, with key local differences (e.g., in L-P interactions). Like the VSV L-P structure, the RABV complex analyzed here represents a preinitiation conformation. Comparison with the likely elongation state, seen in two structures of pneumovirus L-P complexes, suggests differences between priming/initiation and elongation complexes. Analysis of internal cavities within RABV L suggests distinct template and product entry and exit pathways during transcription and replication.}, issn = {0027-8424}, doi = {10.1073/pnas.1918809117}, url = {https://www.pnas.org/content/117/4/2099}, author = {Horwitz, Joshua A. and Jenni, Simon and Harrison, Stephen C. and Whelan, Sean P. J.} } @article {Park2019, title = {Architecture of autoinhibited and active BRAF-MEK1-14-3-3 complexes}, journal = {Nature}, year = {2019}, abstract = {RAF family kinases are RAS-activated switches that initiate signaling through the MAP kinase cascade to control cellular proliferation, differentiation and survival1-3. RAF activity is tightly regulated, and inappropriate activation is a frequent cause of cancer4-6. At present, the structural basis of RAF regulation is poorly understood. Here we describe autoinhibited and active state structures of full-length BRAF in complexes with MEK1 and a 14-3-3 dimer, determined using cryo-electron microscopy (cryo-EM). A 4.1?\AA resolution cryo-EM reconstruction reveals an inactive BRAF-MEK1 complex restrained in a cradle formed by the 14-3-3 dimer, which binds the phosphorylated S365 and S729 sites that flank the BRAF kinase domain. The BRAF cysteine-rich domain (CRD) occupies a central position that stabilizes this assembly, but the adjacent RAS-binding domain (RBD) is poorly ordered and peripheral. The 14-3-3 cradle maintains autoinhibition by sequestering the membrane-binding CRD and blocking dimerization of the BRAF kinase domain. In the active state, these inhibitory interactions are released and a single 14-3-3 dimer rearranges to bridge the C-terminal pS729 binding sites of two BRAFs, driving formation of an active, back-to-back BRAF dimer. Our structural snapshots provide a foundation for understanding normal RAF regulation and its mutational disruption in cancer and developmental syndromes.}, issn = {1476-4687}, doi = {10.1038/s41586-019-1660-y}, url = {https://doi.org/10.1038/s41586-019-1660-y}, author = {Park, Eun Young and Rawson, Shaun and Li, Kunhua and Kim, Byeong-Won and Ficarro, Scott B. and Pino, Gonzalo Gonzalez-Del and Sharif, Humayun and Marto, Jarrod A. and Jeon, Hyesung and Eck, Michael J.} } @article {10.7554/eLife.48215, title = {The structure of the yeast Ctf3 complex}, journal = {eLife}, volume = {8}, year = {2019}, month = {jun}, pages = {e48215}, publisher = {eLife Sciences Publications, Ltd}, abstract = {Kinetochores are the chromosomal attachment points for spindle microtubules. They are also signaling hubs that control major cell cycle transitions and coordinate chromosome folding. Most well-studied eukaryotes rely on a conserved set of factors, which are divided among two loosely-defined groups, for these functions. Outer kinetochore proteins contact microtubules or regulate this contact directly. Inner kinetochore proteins designate the kinetochore assembly site by recognizing a specialized nucleosome containing the H3 variant Cse4/CENP-A. We previously determined the structure, resolved by cryo-electron microscopy (cryo-EM), of the yeast Ctf19 complex (Ctf19c, homologous to the vertebrate CCAN), providing a high-resolution view of inner kinetochore architecture (Hinshaw and Harrison, 2019). We now extend these observations by reporting a near-atomic model of the Ctf3 complex, the outermost Ctf19c sub-assembly seen in our original cryo-EM density. The model is sufficiently well-determined by the new data to enable molecular interpretation of Ctf3 recruitment and function.}, issn = {2050-084X}, doi = {10.7554/eLife.48215}, url = {https://doi.org/10.7554/eLife.48215}, author = {Hinshaw, Stephen M and Dates, Andrew N and Harrison, Stephen C}, editor = {Musacchio, Andrea} } @article {Twomeyeaax1033, title = {Substrate processing by the Cdc48 ATPase complex is initiated by ubiquitin unfolding}, journal = {Science}, year = {2019}, publisher = {American Association for the Advancement of Science}, abstract = {The Cdc48 ATPase (p97 or VCP in mammals) and its cofactor Ufd1/Npl4 extract poly-ubiquitinated proteins from membranes or macromolecular complexes for subsequent degradation by the proteasome. How Cdc48 processes its diverse and often well-folded substrates is unclear. Here, we report cryo-EM structures of the Cdc48 ATPase in complex with Ufd1/Npl4 and poly-ubiquitinated substrate. The structures show that the Cdc48 complex initiates substrate processing by unfolding a ubiquitin molecule. The unfolded ubiquitin molecule binds to Npl4 and projects its N-terminal segment through both hexameric ATPase rings. Pore loops of the second ring form a staircase that acts as a conveyer belt to move the polypeptide through the central pore. Inducing the unfolding of ubiquitin allows the Cdc48 ATPase complex to process a broad range of substrates.}, issn = {0036-8075}, doi = {10.1126/science.aax1033}, url = {https://science.sciencemag.org/content/early/2019/06/26/science.aax1033}, author = {Twomey, Edward C. and Ji, Zhejian and Wales, Thomas E. and Bodnar, Nicholas O. and Ficarro, Scott B. and Marto, Jarrod A. and Engen, John R. and Rapoport, Tom A.} } @article {1324269, title = {X-ray and cryo-EM structures of the mitochondrial calcium uniporter}, journal = {Nature}, volume = {559}, year = {2018}, pages = {575-579}, author = {Chao Fan and Minrui Fan and Benjamin J. Orlando and Nathan M. Fastman and Jinru Zhang and Xu, Yan and Melissa G. Chambers and Xiaofang Xu and Kay Perry and Maofu Liao and Feng, Liang} } @article {1324268, title = {Structure basis for RNA-guided DNA degradation by Cascade and Cas3}, journal = {Science}, volume = {361}, number = {6397}, year = {2018}, author = {Yibei Xiao and Luo, Min and Adam E. Dolan and Maofu Liao and Ailong Ke} } @article {1324267, title = {High-resolution cryo-EM analysis of the yeast ATP synthase in a lipid membrane}, journal = {Science}, volume = {360}, number = {6389}, year = {2018}, author = {Anura P. Srivastava and Luo, Min and Wenchang Zhou and Jindrich Symersky and Dongyang Bai and Melissa G. Chambers and Jose D. Faraldo-Gomez and Maofu Liao and David M. Mueller} } @article {1164641, title = {Structural basis of MsbA-mediated lipopolysaccharide transport}, journal = {Nature}, volume = {549}, year = {2017}, pages = {233-237}, author = {Wei Mi and Yanyan Li and Sung Hwan Yoon and Robert K. Ernst and Walz, Thomas and Maofu Liao} } @article {1196651, title = {Best practices for managing large CryoEM facilities}, journal = {Journal of Structural Biology}, volume = {199}, number = {3}, year = {2017}, pages = {225-236}, author = {Bart Alewijnse and Alun W. Ashton and Melissa G. Chambers and Songye Chen and Anchi Cheng and Mark Ebrahim and Eng, Edward T. and Win J.H. Hagen and Abraham J. Koster and Claudia S. Lopez and Natalya Lukoyanova and Joaquin Ortega and Ludovic Renault and Steve Reyntjens and William J. Rice and Giovanna Scapin and Raymond Schrijver and Alistair Siebert and Scott M. Stagg and Valerie Grum-Tokars and Elizabeth R. Wright and Shenping Wu and Zhiheng Yu and Hong Zhou and Carragher, Bridget and Clinton S. Potter} } @article {1159166, title = {Structure Basis for Directional R-loop Formation and Substrate Handover Mechanisms in Type I CRISPR-Cas System}, journal = {Cell}, volume = {170}, number = {1}, year = {2017}, pages = {48-60}, author = {Yibei Xiao and Luo, Min and Robert B. Hayes and Jonathan Kim and Sherwin Ng and Fang Ding and Maofu Liao and Ailong Ke} } @article {1159136, title = {Molecular Mechanism of V(D)J Recombination from Synaptic RAG1-RAG2 Complex Structures}, journal = {Cell}, volume = {163}, number = {5}, year = {2015}, pages = {1138-1152}, author = {Heng Ru and Melissa G. Chambers and Tian-Min Fu and Alexander B. Tong and Maofu Liao and Wu, Hao} }