%0 Journal Article %J Nature Structural & Molecular Biology %D 2021 %T Structural insights into metazoan pretargeting GET complexes %A Keszei, Alexander F. A. %A Yip, Matthew C. J. %A Hsieh, Ta-Chien %A Shao, Sichen %X 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 3.3–3.6þinspace}\AA. The structures reveal that Get3 helixþinspace}8 and the Get4 Cþinspace}terminus form a composite lid over the Get3 substrate-binding chamber that is opened by SGTA. Another interaction with Get4 prevents formation of Get3 helixþinspace}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. %B Nature Structural & Molecular Biology %V 28 %P 1029-1037 %8 Dec %G eng %U https://doi.org/10.1038/s41594-021-00690-7 %N 12 %R 10.1038/s41594-021-00690-7 %0 Journal Article %J Cell %D 2021 %T NLRP3 cages revealed by full-length mouse NLRP3 structure control pathway activation %A Andreeva, Liudmila %A David, Liron %A Rawson, Shaun %A Shen, Chen %A Pasricha, Teerithveen %A Pelegrin, Pablo %A Wu, Hao %B Cell %I Elsevier %8 2021/12/03 %G eng %U https://doi.org/10.1016/j.cell.2021.11.011 %R 10.1016/j.cell.2021.11.011 %0 Journal Article %J Science %D 2021 %T Structural basis for continued antibody evasion by the SARS-CoV-2 receptor binding domain %A Katherine G. Nabel %A Sarah A. Clark %A Sundaresh Shankar %A Junhua Pan %A Lars E. Clark %A Pan Yang %A Adrian Coscia %A McKay, Lindsay G. A. %A Haley H. Varnum %A Vesna Brusic %A Nicole V. Tolan %A Guohai Zhou %A Michaël Desjardins %A Sarah E. Turbett %A Sanjat Kanjilal %A Amy C. Sherman %A Anand Dighe %A Regina C. LaRocque %A Edward T. Ryan %A Casey Tylek %A Joel F. Cohen-Solal %A Anhdao T. Darcy %A Davide Tavella %A Anca Clabbers %A Yao Fan %A Griffiths, Anthony %A Ivan R. Correia %A Jane Seagal %A Lindsey R. Baden %A Richelle C. Charles %A Jonathan Abraham %X 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. %B Science %P eabl6251 %G eng %R 10.1126/science.abl6251} URL = ttps://www.science.org/doi/abs/10.1126/science.abl6251 %0 Journal Article %J Nucleic Acids Research %D 2021 %T Structure of CRL2Lrr1, the E3 ubiquitin ligase that promotes DNA replication termination in vertebrates %A Zhou, Haixia %A Zaher, Manal S %A Walter, Johannes C %A Brown, Alan %X 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’s catalytic module is positioned next to the ubiquitylation site on Mcm7, and why CRL2Lrr1 binds CMG only after replisomes converge. %B Nucleic Acids Research %8 12 %G eng %U https://doi.org/10.1093/nar/gkab1174 %R 10.1093/nar/gkab1174 %0 Journal Article %J Cell %D 2021 %T De novo identification of mammalian ciliary motility proteins using cryo-EM %A Gui, Miao %A Farley, Hannah %A Anujan, Priyanka %A Anderson, Jacob R. %A Maxwell, Dale W. %A Whitchurch, Jonathan B. %A Botsch, J. Josephine %A Qiu, Tao %A Meleppattu, Shimi %A Singh, Sandeep K. %A Zhang, Qi %A Thompson, James %A Lucas, Jane S. %A Bingle, Colin D. %A Norris, Dominic P. %A Roy, Sudipto %A Brown, Alan %B Cell %I Elsevier %8 2021/10/28 %G eng %U https://doi.org/10.1016/j.cell.2021.10.007 %R 10.1016/j.cell.2021.10.007 %0 Journal Article %J Science %D 2021 %T Membrane fusion and immune evasion by the spike protein of SARS-CoV-2 Delta variant %A Zhang, Jun %A Xiao, Tianshu %A Cai, Yongfei %A Lavine, Christy L. %A Peng, Hanqin %A Zhu, Haisun %A Anand, Krishna %A Tong, Pei %A Gautam, Avneesh %A Megan L. Mayer %A Walsh Jr., Richard M. %A Rits-Volloch, Sophia %A Wesemann, Duane R. %A Yang, Wei %A Seaman, Michael S. %A Lu, Jianming %A Chen, Bing %X 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. %B Science %P eabl9463 %G eng %R 10.1126/science.abl9463} URL = ttps://www.science.org/doi/abs/10.1126/science.abl9463 %0 Journal Article %J Antibody Therapeutics %D 2021 %T Development of an integrated structural biology platform specialized for sub-100 kDa protein complexes to support biologics discovery and rational engineering %A Iozzo (Egor Svidritskiy), Yuri %A Qiu, Yu %A Xu, Albert %A Park, Anna %A Wendt, Maria %A Zhou, Yanfeng %X 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. %B Antibody Therapeutics %V 4 %P 242-251 %8 10 %G eng %U https://doi.org/10.1093/abt/tbab025 %N 4 %R 10.1093/abt/tbab025 %0 Journal Article %J Science %D 2021 %T Distinct allosteric mechanisms of first-generation MsbA inhibitors %A François A. Thélot %A Wenyi Zhang %A Song, KangKang %A Xu, Chen %A Jing Huang %A Maofu Liao %X 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. %B Science %8 29 Oct 2021 %G eng %R 10.1126/science.abi9009} URL = ttps://www.science.org/doi/abs/10.1126/science.abi9009 %0 Journal Article %J Molecular Cell %D 2021 %T Solenoid architecture of HUWE1 contributes to ligase activity and substrate recognition %A Hunkeler, Moritz %A Jin, Cyrus Y. %A Ma, Michelle W. %A Monda, Julie K. %A Overwijn, Daan %A Bennett, Eric J. %A Fischer, Eric S. %B Molecular Cell %I Elsevier %8 2021/07/27 %G eng %U https://doi.org/10.1016/j.molcel.2021.06.032 %R 10.1016/j.molcel.2021.06.032 %0 Journal Article %J Cell %D 2021 %T Memory B Cell Repertoire for Recognition of Evolving SARS-CoV-2 Spike %A Tong, Pei %A Gautam, Avneesh %A Windsor, Ian W. %A Travers, Meghan %A Chen, Yuezhou %A Garcia, Nicholas %A Whiteman, Noah B. %A McKay, Lindsay G. A. %A Storm, Nadia %A Malsick, Lauren E. %A Honko, Anna N. %A Lelis, Felipe J.N. %A Habibi, Shaghayegh %A Jenni, Simon %A Cai, Yongfei %A Rennick, Linda J. %A Duprex, W. Paul %A McCarthy, Kevin R. %A Lavine, Christy L. %A Zuo, Teng %A Lin, Junrui %A Zuiani, Adam %A Feldman, Jared %A MacDonald, Elizabeth A. %A Hauser, Blake M. %A Griffths, Anthony %A Seaman, Michael S. %A Schmidt, Aaron G. %A Chen, Bing %A Neuberg, Donna %A Bajic, Goran %A Harrison, Stephen C. %A Wesemann, Duane R. %B Cell %I Elsevier %8 2021/07/27 %G eng %U https://doi.org/10.1016/j.cell.2021.07.025 %R 10.1016/j.cell.2021.07.025 %0 Journal Article %J Science %D 2021 %T Structural basis for enhanced infectivity and immune evasion of SARS-CoV-2 variants %A Cai, Yongfei %A Zhang, Jun %A Xiao, Tianshu %A Lavine, Christy L. %A Rawson, Shaun %A Peng, Hanqin %A Zhu, Haisun %A Anand, Krishna %A Tong, Pei %A Gautam, Avneesh %A Lu, Shen %A Sterling, Sarah M. %A Walsh, Richard M. %A Rits-Volloch, Sophia %A Lu, Jianming %A Wesemann, Duane R. %A Yang, Wei %A Seaman, Michael S. %A Chen, Bing %X 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. %B Science %I American Association for the Advancement of Science %G eng %U https://science.sciencemag.org/content/early/2021/06/23/science.abi9745 %R 10.1126/science.abi9745 %0 Journal Article %J Journal of Cell Biology %D 2021 %T Ctf3/CENP-I provides a docking site for the desumoylase Ulp2 at the kinetochore %A Quan, Yun %A Stephen M. Hinshaw %A Wang, Pang-Che %A Harrison, Stephen C. %A Zhou, Huilin %X 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–regulated processes. %B Journal of Cell Biology %V 220 %8 06 %G eng %U https://doi.org/10.1083/jcb.202012149 %N 8 %R 10.1083/jcb.202012149 %0 Journal Article %J Immunity %D 2021 %T Dipeptidyl peptidase 9 sets a threshold for CARD8 inflammasome formation by sequestering its active C-terminal fragment %A Sharif, Humayun %A Robert Hollingsworth, L. %A Griswold, Andrew R. %A Jeffrey C. Hsiao %A Qinghui Wang %A Bachovchin, Daniel A. %A Wu, Hao %K CARD8 %K cryo-EM %K DPP9 %K inflammasome %K NLRP1 %K pyroptosis %K targeted degradation %K Val-boroPro (VbP) %X 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. %B Immunity %G eng %U https://www.sciencedirect.com/science/article/pii/S1074761321001862 %R https://doi.org/10.1016/j.immuni.2021.04.024 %0 Journal Article %J Nature %D 2021 %T Gasdermin D pore structure reveals preferential release of mature interleukin-1 %A Xia, Shiyu %A Zhang, Zhibin %A Magupalli, Venkat Giri %A Pablo, Juan Lorenzo %A Dong, Ying %A Vora, Setu M. %A Wang, Longfei %A Tian-Min Fu %A Jacobson, Matthew P. %A Greka, Anna %A Lieberman, Judy %A Ruan, Jianbin %A Wu, Hao %X 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 pyroptosis1–9. 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–-but not pyroptotic–-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. %B Nature %8 Apr %G eng %U https://doi.org/10.1038/s41586-021-03478-3 %R 10.1038/s41586-021-03478-3 %0 Journal Article %J Nature Structural & Molecular Biology %D 2021 %T Structures of chaperone-associated assembly intermediates reveal coordinated mechanisms of proteasome biogenesis %A Schnell, Helena M. %A Walsh, Richard M. %A Rawson, Shaun %A Kaur, Mandeep %A Bhanu, Meera K. %A Tian, Geng %A Miguel A. Prado %A Guerra-Moreno, Angel %A Paulo, Joao A. %A Steven P. Gygi %A Roelofs, Jeroen %A Finley, Daniel %A Hanna, John %X 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, Pba1–4 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$2–4), Ump1 and Pba1/2. The second structure contains two additional subunits ($\beta$5–6) 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'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. %B Nature Structural & Molecular Biology %8 Apr %G eng %U https://doi.org/10.1038/s41594-021-00583-9 %R 10.1038/s41594-021-00583-9 %0 Journal Article %J Nature %D 2021 %T DPP9 sequesters the C terminus of NLRP1 to repress inflammasome activation %A Robert Hollingsworth, L. %A Sharif, Humayun %A Griswold, Andrew R. %A Fontana, Pietro %A Mintseris, Julian %A Dagbay, Kevin B. %A Paulo, Joao A. %A Steven P. Gygi %A Bachovchin, Daniel A. %A Wu, Hao %X 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 pyroptosis1–4. Gain-of-function mutations of NLRP1 cause severe inflammatory diseases of the skin4–6. NLRP1 contains a function-to-find domain that auto-proteolyses into noncovalently associated subdomains7–9, 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 unclear10,12–14. Here we report cryo-electron microscopy structures of the human NLRP1–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–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. %B Nature %8 Mar %G eng %U https://doi.org/10.1038/s41586-021-03350-4 %R 10.1038/s41586-021-03350-4 %0 Journal Article %J Science %D 2021 %T Structural impact on SARS-CoV-2 spike protein by D614G substitution %A Zhang, Jun %A Cai, Yongfei %A Xiao, Tianshu %A Lu, Jianming %A Peng, Hanqin %A Sterling, Sarah M. %A Walsh, Richard M. %A Rits-Volloch, Sophia %A Zhu, Haisun %A Woosley, Alec N. %A Yang, Wei %A Sliz, Piotr %A Chen, Bing %X 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. %B Science %I American Association for the Advancement of Science %G eng %U https://science.sciencemag.org/content/early/2021/03/16/science.abf2303 %R 10.1126/science.abf2303 %0 Journal Article %J Nature Structural & Molecular Biology %D 2021 %T Cryo-EM structure of an activated GPCR–G protein complex in lipid nanodiscs %A Zhang, Meng %A Gui, Miao %A Wang, Zi-Fu %A Gorgulla, Christoph %A Yu, James J. %A Wu, Hao %A Sun, Zhen-yu J. %A Klenk, Christoph %A Merklinger, Lisa %A Morstein, Lena %A Hagn, Franz %A Plückthun, Andreas %A Brown, Alan %A Nasr, Mahmoud L. %A Wagner, Gerhard %X 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–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 4.2þinspace}\AA. The structures, determined in a lipid bilayer without any stabilizing antibodies or nanobodies, reveal an extended network of protein–protein interactions at the GPCR–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. %B Nature Structural & Molecular Biology %V 28 %P 258-267 %8 Mar %G eng %U https://doi.org/10.1038/s41594-020-00554-6 %N 3 %R 10.1038/s41594-020-00554-6 %0 Journal Article %J Proceedings of the National Academy of Sciences %D 2021 %T Multiple domain interfaces mediate SARM1 autoinhibition %A Shen, Chen %A Vohra, Mihir %A Zhang, Pengfei %A Mao, Xianrong %A Figley, Matthew D. %A Jian Zhu %A Sasaki, Yo %A Wu, Hao %A DiAntonio, Aaron %A Milbrandt, Jeffrey %X 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). %B Proceedings of the National Academy of Sciences %I National Academy of Sciences %V 118 %G eng %U https://www.pnas.org/content/118/4/e2023151118 %N 4 %R 10.1073/pnas.2023151118 %0 Journal Article %J Nature Communications %D 2021 %T Structure of a microtubule-bound axonemal dynein %A Walton, Travis %A Wu, Hao %A Brown, Alan %X 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. %B Nature Communications %V 12 %P 477 %8 Jan %G eng %U https://doi.org/10.1038/s41467-020-20735-7 %N 1 %R 10.1038/s41467-020-20735-7 %0 Journal Article %J Science %D 2021 %T Cryo-EM structure of the B cell co-receptor CD19 bound to the tetraspanin CD81 %A Susa, Katherine J. %A Rawson, Shaun %A Kruse, Andrew C. %A Blacklow, Stephen C. %X 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–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. %B Science %I American Association for the Advancement of Science %V 371 %P 300–305 %G eng %U https://science.sciencemag.org/content/371/6526/300 %N 6526 %R 10.1126/science.abd9836 %0 Journal Article %J Nature %D 2021 %T Functional refolding of the penetration protein on a non-enveloped virus %A Herrmann, Tobias %A Torres, Raúl %A Salgado, Eric N. %A Berciu, Cristina %A Stoddard, Daniel %A Nicastro, Daniela %A Jenni, Simon %A Harrison, Stephen C. %X 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 `upright' to a `reversed' conformation. The reversed structure projects a previously buried `foot' 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. %B Nature %V 590 %P 666-670 %8 Feb %G eng %U https://doi.org/10.1038/s41586-020-03124-4 %N 7847 %R 10.1038/s41586-020-03124-4 %0 Journal Article %J Nature Structural & Molecular Biology %D 2021 %T A trimeric human angiotensin-converting enzyme 2 as an anti-SARS-CoV-2 agent %A Xiao, Tianshu %A Lu, Jianming %A Zhang, Jun %A Johnson, Rebecca I. %A McKay, Lindsay G. A. %A Storm, Nadia %A Lavine, Christy L. %A Peng, Hanqin %A Cai, Yongfei %A Rits-Volloch, Sophia %A Lu, Shen %A Quinlan, Brian D. %A Farzan, Michael %A Seaman, Michael S. %A Griffiths, Anthony %A Chen, Bing %X 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–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 \textasciitilde60þinspace}pM for the spike protein of SARS‑CoV‑2 (compared with 77þinspace}nM for monomeric ACE2 and 12–22þinspace}nM for dimeric ACE2 constructs), and its peptidase activity and the ability to block activation of angiotensin II receptor type 1 in the renin–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. %B Nature Structural & Molecular Biology %8 Jan %G eng %U https://doi.org/10.1038/s41594-020-00549-3 %R 10.1038/s41594-020-00549-3 %0 Journal Article %J Nature Communications %D 2021 %T Mechanism of filament formation in UPA-promoted CARD8 and NLRP1 inflammasomes %A Robert Hollingsworth, L. %A David, Liron %A Li, Yang %A Griswold, Andrew R. %A Ruan, Jianbin %A Sharif, Humayun %A Fontana, Pietro %A Orth-He, Elizabeth L. %A Tian-Min Fu %A Bachovchin, Daniel A. %A Wu, Hao %X 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–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. %B Nature Communications %V 12 %P 189 %8 Jan %G eng %U https://doi.org/10.1038/s41467-020-20320-y %N 1 %R 10.1038/s41467-020-20320-y