Shaun Rawson, Richard M. Walsh, Benjamin Velez, Helena M. Schnell, Fenglong Jiao, Marie Blickling, Jessie Ang, Meera K. Bhanu, Lan Huang, and John Hanna. 8/4/2022. “Yeast PI31 inhibits the proteasome by a direct multisite mechanism.” Nature Structural & Molecular Biology. Publisher's VersionAbstract
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 30þinspace}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'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.
Matthew C. J. Yip, Samantha F. Sedor, and Sichen Shao. 8/1/2022. “Mechanism of client selection by the protein quality-control factor UBE2O.” Nature Structural & Molecular Biology. Publisher's VersionAbstract
The E2/E3 enzyme UBE2O ubiquitylates diverse clients to mediate important processes, including targeting unassembled `orphan' 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.
Benjamin R. Morehouse, Matthew C. J. Yip, Alexander F. A. Keszei, Nora K. McNamara-Bordewick, Sichen Shao, and Philip J. Kranzusch. 7/20/2022. “Cryo-EM structure of an active bacterial TIR–STING filament complex.” Nature. Publisher's VersionAbstract
Stimulator of interferon genes (STING) is an antiviral signalling protein that is broadly conserved in both innate immunity in animals and phage defence in prokaryotes1–4. Activation of STING requires its assembly into an oligomeric filament structure through binding of a cyclic dinucleotide4–13, 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)–STING filament complex from a Sphingobacterium faecium cyclic-oligonucleotide-based antiphage signalling system (CBASS) defence operon. Bacterial TIR–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–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.
Jacob Ronald Anderson, Jing Li, Timothy A. Springer, and Alan Brown. 7/1/2022. “Structures of VWF tubules before and after concatemerization reveal a mechanism of disulfide bond exchange.” Blood. Publisher's VersionAbstract
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.
Martin Filipovski, Jelly H. M. Soffers, Seychelle M. Vos, and Lucas Farnung. 6/16/2022. “Structural basis of nucleosome retention during transcription elongation.” Science, 376, 6599, Pp. 1313-1316. Publisher's VersionAbstract
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–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–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–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. —DJ A high-resolution cryo–electron microscopy structure explains how nucleosomes are retained to prevent disruption of chromatin across actively transcribed genes.
Nils Marechal, Banyuhay P. Serrano, Xinyan Zhang, and Charles J. Weitz. 5/2/2022. “Formation of thyroid hormone revealed by a cryo-EM structure of native bovine thyroglobulin.” Nature Communications, 13, 1, Pp. 2380. Publisher's VersionAbstract
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.
Di Liu, François A. Thélot, Joseph A. Piccirilli, Maofu Liao, and Peng Yin. 5/2/2022. “Sub-3-Å cryo-EM structure of RNA enabled by engineered homomeric self-assembly.” Nature Methods. Publisher's VersionAbstract
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–-ROCK (RNA oligomerization-enabled cryo-EM via installing kissing loops)–-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 (2.85þinspace}\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–-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.
Wang Zheng, Chen Shen, Longfei Wang, Shaun Rawson, Wen Jun Xie, Carl Nist-Lund, Jason Wu, Zhangfei Shen, Shiyu Xia, Jeffrey R. Holt, Hao Wu, and Tian-Min Fu. 3/25/2022. “pH regulates potassium conductance and drives a constitutive proton current in human TMEM175.” Science Advances, 8, 12, Pp. eabm1568. Publisher's VersionAbstract
Human TMEM175, a noncanonical potassium (K+) channel in endolysosomes, contributes to their pH stability and is implicated in the pathogenesis of Parkinson’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.
Xiaozhe Xiong, Songhai Tian, Pan Yang, Francois Lebreton, Huan Bao, Kuanwei Sheng, Linxiang Yin, Pengsheng Chen, Jie Zhang, Wanshu Qi, Jianbin Ruan, Hao Wu, Hong Chen, David T. Breault, Ashlee M. Earl, Michael S. Gilmore, Jonathan Abraham, and Min Dong. 3/7/2022. “Emerging enterococcus pore-forming toxins with MHC/HLA-I as receptors.” Cell. Publisher's Version
Blake M. Hauser, Maya Sangesland, Kerri J. St. Denis, Evan C. Lam, James Brett Case, Ian W. Windsor, Jared Feldman, Timothy M. Caradonna, Ty Kannegieter, Michael S. Diamond, Alejandro B. Balazs, Daniel Lingwood, and Aaron G. Schmidt. 3/7/2022. “Rationally designed immunogens enable immune focusing following SARS-CoV-2 spike imprinting.” Cell Reports, 38, 12, Pp. 110561. Publisher's VersionAbstract
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.
Henning Arlt, Xuewu Sui, Brayden Folger, Carson Adams, Xiao Chen, Roman Remme, Fred A. Hamprecht, Frank DiMaio, Maofu Liao, Joel M. Goodman, Robert V. Farese, and Tobias C. Walther. 2/24/2022. “Seipin forms a flexible cage at lipid droplet formation sites.” Nature Structural & Molecular Biology.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.
Alex G. Johnson, Tanita Wein, Megan L. Mayer, Brianna Duncan-Lowey, Erez Yirmiya, Yaara Oppenheimer-Shaanan, Gil Amitai, Rotem Sorek, and Philip J. Kranzusch. 1/13/2022. “Bacterial gasdermins reveal an ancient mechanism of cell death.” Science, 375, 6577, Pp. 221-225. Publisher's VersionAbstract
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—in this case, bacteriophage—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. —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.
Alexander F. A. Keszei, Matthew C. J. Yip, Ta-Chien Hsieh, and Sichen Shao. 12/9/2021. “Structural insights into metazoan pretargeting GET complexes.” Nature Structural & Molecular Biology, 28, 12, Pp. 1029-1037. Publisher's VersionAbstract
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.
Liudmila Andreeva, Liron David, Shaun Rawson, Chen Shen, Teerithveen Pasricha, Pablo Pelegrin, and Hao Wu. 12/3/2021. “NLRP3 cages revealed by full-length mouse NLRP3 structure control pathway activation.” Cell. Publisher's Version
Katherine G. Nabel, Sarah A. Clark, Sundaresh Shankar, Junhua Pan, Lars E. Clark, Pan Yang, Adrian Coscia, Lindsay G. A. McKay, Haley H. Varnum, Vesna Brusic, Nicole V. Tolan, Guohai Zhou, Michaël Desjardins, Sarah E. Turbett, Sanjat Kanjilal, Amy C. Sherman, Anand Dighe, Regina C. LaRocque, Edward T. Ryan, Casey Tylek, Joel F. Cohen-Solal, Anhdao T. Darcy, Davide Tavella, Anca Clabbers, Yao Fan, Anthony Griffiths, Ivan R. Correia, Jane Seagal, Lindsey R. Baden, Richelle C. Charles, and Jonathan Abraham. 12/2/2021. “Structural basis for continued antibody evasion by the SARS-CoV-2 receptor binding domain.” Science, Pp. 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.
Haixia Zhou, Manal S Zaher, Johannes C Walter, and Alan Brown. 12/1/2021. “Structure of CRL2Lrr1, the E3 ubiquitin ligase that promotes DNA replication termination in vertebrates.” Nucleic Acids Research. Publisher's VersionAbstract
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.
Miao Gui, Hannah Farley, Priyanka Anujan, Jacob R. Anderson, Dale W. Maxwell, Jonathan B. Whitchurch, J. Josephine Botsch, Tao Qiu, Shimi Meleppattu, Sandeep K. Singh, Qi Zhang, James Thompson, Jane S. Lucas, Colin D. Bingle, Dominic P. Norris, Sudipto Roy, and Alan Brown. 10/28/2021. “De novo identification of mammalian ciliary motility proteins using cryo-EM.” Cell. Publisher's Version
Jun Zhang, Tianshu Xiao, Yongfei Cai, Christy L. Lavine, Hanqin Peng, Haisun Zhu, Krishna Anand, Pei Tong, Avneesh Gautam, Megan L. Mayer, Richard M. Walsh Jr., Sophia Rits-Volloch, Duane R. Wesemann, Wei Yang, Michael S. Seaman, Jianming Lu, and Bing Chen. 10/26/2021. “Membrane fusion and immune evasion by the spike protein of SARS-CoV-2 Delta variant.” Science, Pp. 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.
Yuri Iozzo (Egor Svidritskiy), Yu Qiu, Albert Xu, Anna Park, Maria Wendt, and Yanfeng Zhou. 10/4/2021. “Development of an integrated structural biology platform specialized for sub-100 kDa protein complexes to support biologics discovery and rational engineering.” Antibody Therapeutics, 4, 4, Pp. 242-251. Publisher's VersionAbstract
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.
François A. Thélot, Wenyi Zhang, KangKang Song, Chen Xu, Jing Huang, and Maofu Liao. 9/23/2021. “Distinct allosteric mechanisms of first-generation MsbA inhibitors.” Science.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.