MEMS Publications

2022
Simon Jenni, Joshua A. Horwitz, Louis-Marie Bloyet, Sean P. J. Whelan, and Stephen C. Harrison. 8/15/2022. “Visualizing molecular interactions that determine assembly of a bullet-shaped vesicular stomatitis virus particle.” Nature Communications, 13, 1, Pp. 4802. Publisher's VersionAbstract
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 (``trunk'') at 3.5þinspace}\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.
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.
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
2021
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.
2020
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.
Yongfei Cai, Jun Zhang, Tianshu Xiao, Hanqin Peng, Sarah M. Sterling, Richard M. Walsh, Shaun Rawson, Sophia Rits-Volloch, and Bing Chen. 7/21/2020. “Distinct conformational states of SARS-CoV-2 spike protein.” Science. Publisher's VersionAbstract
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 (2.9Å resolution) and postfusion (3.0Å 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.
Xuewu Sui, Kun Wang, Nina L. Gluchowski, Shane D. Elliott, Maofu Liao, Tobias C. Walther, and Robert V. Farese. 5/13/2020. “Structure and catalytic mechanism of a human triacylglycerol-synthesis enzyme.” Nature. Publisher's VersionAbstract
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) enzymes2–4, 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–-possibly representing an acyl-acceptor molecule–-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.
Benjamin J Orlando and Maofu Liao. 2020. “ABCG2 transports anticancer drugs via a closed-to-open switch.” Nat Commun, 11, 1, Pp. 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.
Brianna Lowey, Aaron T. Whiteley, Alexander F. A. Keszei, Benjamin R. Morehouse, Ian T. Mathews, Sadie P. Antine, Victor J. Cabrera, Dmitry Kashin, Percy Niemann, Mohit Jain, Frank Schwede, John J. Mekalanos, Sichen Shao, Amy S.Y. Lee, and Philip J. Kranzusch. 2020. “CBASS Immunity Uses CARF-Related Effectors to Sense 3′–5′- and 2′–5′-Linked Cyclic Oligonucleotide Signals and Protect Bacteria from Phage Infection.” Cell, 182, 1, Pp. 38-49.e17. Publisher's VersionAbstract
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′–5′- and 3′–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.
Benjamin R. Morehouse, Apurva A. Govande, Adi Millman, Alexander F. A. Keszei, Brianna Lowey, Gal Ofir, Sichen Shao, Rotem Sorek, and Philip J. Kranzusch. 2020. “STING cyclic dinucleotide sensing originated in bacteria.” Nature, 586, 7829, Pp. 429-433. Publisher's VersionAbstract
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–AMP signalling during viral infection and anti-tumour immunity1–5. STING shares no structural homology with other known signalling proteins6–9, 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–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–STING pathway in prokaryotic defence against bacteriophages.
Joshua A. Horwitz, Simon Jenni, Stephen C. Harrison, and Sean P. J. Whelan. 2020. “Structure of a rabies virus polymerase complex from electron cryo-microscopy.” Proceedings of the National Academy of Sciences, 117, 4, Pp. 2099–2107. Publisher's VersionAbstract
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 Å 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.
2018
Chao Fan, Minrui Fan, Benjamin J. Orlando, Nathan M. Fastman, Jinru Zhang, Yan Xu, Melissa G. Chambers, Xiaofang Xu, Kay Perry, Maofu Liao, and Liang Feng. 7/11/2018. “X-ray and cryo-EM structures of the mitochondrial calcium uniporter.” Nature, 559, Pp. 575-579.
Yibei Xiao, Min Luo, Adam E. Dolan, Maofu Liao, and Ailong Ke. 7/6/2018. “Structure basis for RNA-guided DNA degradation by Cascade and Cas3.” Science, 361, 6397.
Anura P. Srivastava, Min Luo, Wenchang Zhou, Jindrich Symersky, Dongyang Bai, Melissa G. Chambers, Jose D. Faraldo-Gomez, Maofu Liao, and David M. Mueller. 5/11/2018. “High-resolution cryo-EM analysis of the yeast ATP synthase in a lipid membrane.” Science, 360, 6389.
2017
Wei Mi, Yanyan Li, Sung Hwan Yoon, Robert K. Ernst, Thomas Walz, and Maofu Liao. 9/14/2017. “Structural basis of MsbA-mediated lipopolysaccharide transport.” Nature, 549, Pp. 233-237.
Bart Alewijnse, Alun W. Ashton, Melissa G. Chambers, Songye Chen, Anchi Cheng, Mark Ebrahim, Edward T. Eng, Win J.H. Hagen, Abraham J. Koster, Claudia S. Lopez, Natalya Lukoyanova, Joaquin Ortega, Ludovic Renault, Steve Reyntjens, William J. Rice, Giovanna Scapin, Raymond Schrijver, Alistair Siebert, Scott M. Stagg, Valerie Grum-Tokars, Elizabeth R. Wright, Shenping Wu, Zhiheng Yu, Hong Zhou, Bridget Carragher, and Clinton S. Potter. 9/2017. “Best practices for managing large CryoEM facilities.” Journal of Structural Biology, 199, 3, Pp. 225-236.
Yibei Xiao, Min Luo, Robert B. Hayes, Jonathan Kim, Sherwin Ng, Fang Ding, Maofu Liao, and Ailong Ke. 6/29/2017. “Structure Basis for Directional R-loop Formation and Substrate Handover Mechanisms in Type I CRISPR-Cas System.” Cell, 170, 1, Pp. 48-60.
2015
Heng Ru, Melissa G. Chambers, Tian-Min Fu, Alexander B. Tong, Maofu Liao, and Hao Wu. 11/5/2015. “Molecular Mechanism of V(D)J Recombination from Synaptic RAG1-RAG2 Complex Structures.” Cell, 163, 5, Pp. 1138-1152.