Publications

2021
Moritz Hunkeler, Cyrus Y. Jin, Michelle W. Ma, Julie K. Monda, Daan Overwijn, Eric J. Bennett, and Eric S. Fischer. 7/26/2021. “Solenoid architecture of HUWE1 contributes to ligase activity and substrate recognition.” Molecular Cell. Publisher's Version
Pei Tong, Avneesh Gautam, Ian W. Windsor, Meghan Travers, Yuezhou Chen, Nicholas Garcia, Noah B. Whiteman, Lindsay G. A. McKay, Nadia Storm, Lauren E. Malsick, Anna N. Honko, Felipe J.N. Lelis, Shaghayegh Habibi, Simon Jenni, Yongfei Cai, Linda J. Rennick, W. Paul Duprex, Kevin R. McCarthy, Christy L. Lavine, Teng Zuo, Junrui Lin, Adam Zuiani, Jared Feldman, Elizabeth A. MacDonald, Blake M. Hauser, Anthony Griffths, Michael S. Seaman, Aaron G. Schmidt, Bing Chen, Donna Neuberg, Goran Bajic, Stephen C. Harrison, and Duane R. Wesemann. 7/22/2021. “Memory B Cell Repertoire for Recognition of Evolving SARS-CoV-2 Spike.” Cell. Publisher's Version
Yongfei Cai, Jun Zhang, Tianshu Xiao, Christy L. Lavine, Shaun Rawson, Hanqin Peng, Haisun Zhu, Krishna Anand, Pei Tong, Avneesh Gautam, Shen Lu, Sarah M. Sterling, Richard M. Walsh, Sophia Rits-Volloch, Jianming Lu, Duane R. Wesemann, Wei Yang, Michael S. Seaman, and Bing Chen. 6/24/2021. “Structural basis for enhanced infectivity and immune evasion of SARS-CoV-2 variants.” Science. Publisher's VersionAbstract
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.
Yun Quan, Stephen M. Hinshaw, Pang-Che Wang, Stephen C. Harrison, and Huilin Zhou. 6/3/2021. “Ctf3/CENP-I provides a docking site for the desumoylase Ulp2 at the kinetochore.” Journal of Cell Biology, 220, 8. Publisher's VersionAbstract
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.
Humayun Sharif, L. Robert Hollingsworth, Andrew R. Griswold, Jeffrey C. Hsiao, Qinghui Wang, Daniel A. Bachovchin, and Hao Wu. 5/20/2021. “Dipeptidyl peptidase 9 sets a threshold for CARD8 inflammasome formation by sequestering its active C-terminal fragment.” Immunity. Publisher's VersionAbstract
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.
Shiyu Xia, Zhibin Zhang, Venkat Giri Magupalli, Juan Lorenzo Pablo, Ying Dong, Setu M. Vora, Longfei Wang, Tian-Min Fu, Matthew P. Jacobson, Anna Greka, Judy Lieberman, Jianbin Ruan, and Hao Wu. 4/21/2021. “Gasdermin D pore structure reveals preferential release of mature interleukin-1.” Nature. Publisher's VersionAbstract
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.
Helena M. Schnell, Richard M. Walsh, Shaun Rawson, Mandeep Kaur, Meera K. Bhanu, Geng Tian, Miguel A. Prado, Angel Guerra-Moreno, Joao A. Paulo, Steven P. Gygi, Jeroen Roelofs, Daniel Finley, and John Hanna. 4/12/2021. “Structures of chaperone-associated assembly intermediates reveal coordinated mechanisms of proteasome biogenesis.” Nature Structural & Molecular Biology. Publisher's VersionAbstract
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.
L. Robert Hollingsworth, Humayun Sharif, Andrew R. Griswold, Pietro Fontana, Julian Mintseris, Kevin B. Dagbay, Joao A. Paulo, Steven P. Gygi, Daniel A. Bachovchin, and Hao Wu. 3/18/2021. “DPP9 sequesters the C terminus of NLRP1 to repress inflammasome activation.” Nature. Publisher's VersionAbstract
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.
Jun Zhang, Yongfei Cai, Tianshu Xiao, Jianming Lu, Hanqin Peng, Sarah M. Sterling, Richard M. Walsh, Sophia Rits-Volloch, Haisun Zhu, Alec N. Woosley, Wei Yang, Piotr Sliz, and Bing Chen. 3/16/2021. “Structural impact on SARS-CoV-2 spike protein by D614G substitution.” Science. Publisher's VersionAbstract
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.
Meng Zhang, Miao Gui, Zi-Fu Wang, Christoph Gorgulla, James J. Yu, Hao Wu, Zhen-yu J. Sun, Christoph Klenk, Lisa Merklinger, Lena Morstein, Franz Hagn, Andreas Plückthun, Alan Brown, Mahmoud L. Nasr, and Gerhard Wagner. 2/25/2021. “Cryo-EM structure of an activated GPCR–G protein complex in lipid nanodiscs.” Nature Structural & Molecular Biology, 28, 3, Pp. 258-267. Publisher's VersionAbstract
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.
Chen Shen, Mihir Vohra, Pengfei Zhang, Xianrong Mao, Matthew D. Figley, Jian Zhu, Yo Sasaki, Hao Wu, Aaron DiAntonio, and Jeffrey Milbrandt. 1/26/2021. “Multiple domain interfaces mediate SARM1 autoinhibition.” Proceedings of the National Academy of Sciences, 118, 4. Publisher's VersionAbstract
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).
Travis Walton, Hao Wu, and Alan Brown. 1/20/2021. “Structure of a microtubule-bound axonemal dynein.” Nature Communications, 12, 1, Pp. 477. Publisher's VersionAbstract
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.
Katherine J. Susa, Shaun Rawson, Andrew C. Kruse, and Stephen C. Blacklow. 1/15/2021. “Cryo-EM structure of the B cell co-receptor CD19 bound to the tetraspanin CD81.” Science, 371, 6526, Pp. 300–305. Publisher's VersionAbstract
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.
Tobias Herrmann, Raúl Torres, Eric N. Salgado, Cristina Berciu, Daniel Stoddard, Daniela Nicastro, Simon Jenni, and Stephen C. Harrison. 1/13/2021. “Functional refolding of the penetration protein on a non-enveloped virus.” Nature, 590, 7847, Pp. 666-670. Publisher's VersionAbstract
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.
Tianshu Xiao, Jianming Lu, Jun Zhang, Rebecca I. Johnson, Lindsay G. A. McKay, Nadia Storm, Christy L. Lavine, Hanqin Peng, Yongfei Cai, Sophia Rits-Volloch, Shen Lu, Brian D. Quinlan, Michael Farzan, Michael S. Seaman, Anthony Griffiths, and Bing Chen. 1/11/2021. “A trimeric human angiotensin-converting enzyme 2 as an anti-SARS-CoV-2 agent.” Nature Structural & Molecular Biology. Publisher's VersionAbstract
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.
L. Robert Hollingsworth, Liron David, Yang Li, Andrew R. Griswold, Jianbin Ruan, Humayun Sharif, Pietro Fontana, Elizabeth L. Orth-He, Tian-Min Fu, Daniel A. Bachovchin, and Hao Wu. 1/8/2021. “Mechanism of filament formation in UPA-promoted CARD8 and NLRP1 inflammasomes.” Nature Communications, 12, 1, Pp. 189. Publisher's VersionAbstract
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.
2020
Kazuki Kato, Sadeem Ahmad, Zixiang Zhu, Janet M. Young, Xin Mu, Se Hoon Park, Harmit S. Malik, and Sun Hur. 12/16/2020. “Structural analysis of RIG-I-like receptors reveals ancient rules of engagement between diverse RNA helicases and TRIM ubiquitin ligases.” Molecular Cell. Publisher's VersionAbstract
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.
Miao Gui, Meisheng Ma, Erica Sze-Tu, Xiangli Wang, Fujiet Koh, Ellen D. Zhong, Bonnie Berger, Joseph H. Davis, Susan K. Dutcher, Rui Zhang, and Alan Brown. 12/14/2020. “Structures of radial spokes and associated complexes important for ciliary motility.” Nature Structural & Molecular Biology, 28, 1, Pp. 29-37. Publisher's VersionAbstract
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.
Longfei Wang, Di Wu, Carol V. Robinson, Hao Wu, and Tian-Min Fu. 10/15/2020. “Structures of a Complete Human V-ATPase Reveal Mechanisms of Its Assembly.” Molecular Cell. Publisher's Version
Pranav N. M. Shah, David J. Filman, Krishanthi S. Karunatilaka, Emma L. Hesketh, Elisabetta Groppelli, Mike Strauss, and James M. Hogle. 10/2/2020. “Cryo-EM structures reveal two distinct conformational states in a picornavirus cell entry intermediate.” PLOS Pathogens, 16, 9, Pp. 1-26. Publisher's VersionAbstract
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.

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