scholarly journals Structural basis of mismatch recognition by a SARS-CoV-2 proofreading enzyme

Science ◽  
2021 ◽  
pp. eabi9310
Author(s):  
Chang Liu ◽  
Wei Shi ◽  
Scott T. Becker ◽  
David G. Schatz ◽  
Bin Liu ◽  
...  
Keyword(s):  
Rational Design ◽  
Rna Synthesis ◽  
Molecular Mechanisms ◽  
Nonstructural Protein ◽  
Structural Basis ◽  
Mismatch Correction ◽  
Cryo Electron Microscopy ◽  
Virus Life Cycle ◽  
Nucleotide Analog ◽  
Molecular Determinants

Coronavirus 3′–5′ exoribonuclease (ExoN), residing in the nonstructural protein (nsp) 10-nsp14 complex, boosts replication fidelity by proofreading RNA synthesis and is critical for the virus life cycle. ExoN also recognizes and excises nucleotide analog inhibitors incorporated into the nascent RNA, undermining the effectiveness of nucleotide analog-based antivirals. Here, we present cryo-electron microscopy structures of both wild-type and mutant SARS-CoV-2 nsp10-nsp14 in complex with an RNA substrate bearing a 3′-end mismatch at resolutions ranging from 2.5 Å to 3.9 Å. The structures reveal the molecular determinants of ExoN substrate specificity and give insight into the molecular mechanisms of mismatch correction during coronavirus RNA synthesis. Our findings provide guidance for rational design of improved anti-coronavirus therapies.

scholarly journals Structural basis for GTP-induced dimerization and antiviral function of guanylate-binding proteins

2021 ◽  
Vol 118 (15) ◽  
pp. e2022269118
Author(s):  
Wen Cui ◽  
Elisabeth Braun ◽  
Wei Wang ◽  
Jinhong Tang ◽  
Yanyan Zheng ◽  
...  
Keyword(s):  
Binding Proteins ◽  
Molecular Mechanisms ◽  
Innate Immune ◽  
Structural Basis ◽  
Immune Functions ◽  
Face To Face ◽  
Molecular Determinants ◽  
Antiviral Function ◽  
Large Gtpases ◽  
Hiv 1

Guanylate-binding proteins (GBPs) form a family of dynamin-related large GTPases which mediate important innate immune functions. They were proposed to form oligomers upon GTP binding/hydrolysis, but the molecular mechanisms remain elusive. Here, we present crystal structures of C-terminally truncated human GBP5 (hGBP51–486), comprising the large GTPase (LG) and middle (MD) domains, in both its nucleotide-free monomeric and nucleotide-bound dimeric states, together with nucleotide-free full-length human GBP2. Upon GTP-loading, hGBP51–486 forms a closed face-to-face dimer. The MD of hGBP5 undergoes a drastic movement relative to its LG domain and forms extensive interactions with the LG domain and MD of the pairing molecule. Disrupting the MD interface (for hGBP5) or mutating the hinge region (for hGBP2/5) impairs their ability to inhibit HIV-1. Our results point to a GTP-induced dimerization mode that is likely conserved among all GBP members and provide insights into the molecular determinants of their antiviral function.

scholarly journals The cryo-EM structure of a 12-subunit variant of RNA polymerase I reveals dissociation of the A49-A34.5 heterodimer and rearrangement of subunit A12.2

eLife ◽  
2019 ◽  
Vol 8 ◽  
Author(s):  
Lucas Tafur ◽  
Yashar Sadian ◽  
Jonas Hanske ◽  
Rene Wetzel ◽  
Felix Weis ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Ribosomal Rna ◽  
Transcription Initiation ◽  
Molecular Mechanisms ◽  
Reversible Binding ◽  
Cryo Electron Microscopy ◽  
Nucleotide Analog ◽  
Pol I ◽  
Polymerase I ◽  
Insight Into

RNA polymerase (Pol) I is a 14-subunit enzyme that solely transcribes pre-ribosomal RNA. Cryo-electron microscopy (EM) structures of Pol I initiation and elongation complexes have given first insights into the molecular mechanisms of Pol I transcription. Here, we present cryo-EM structures of yeast Pol I elongation complexes (ECs) bound to the nucleotide analog GMPCPP at 3.2 to 3.4 Å resolution that provide additional insight into the functional interplay between the Pol I-specific transcription-like factors A49-A34.5 and A12.2. Strikingly, most of the nucleotide-bound ECs lack the A49-A34.5 heterodimer and adopt a Pol II-like conformation, in which the A12.2 C-terminal domain is bound in a previously unobserved position at the A135 surface. Our structural and biochemical data suggest a mechanism where reversible binding of the A49-A34.5 heterodimer could contribute to the regulation of Pol I transcription initiation and elongation.

scholarly journals Structural basis for IFN antagonism by human respiratory syncytial virus nonstructural protein 2

2021 ◽  
Vol 118 (10) ◽  
pp. e2020587118
Author(s):  
Jingjing Pei ◽  
Nicole D. Wagner ◽  
Angela J. Zou ◽  
Srirupa Chatterjee ◽  
Dominika Borek ◽  
...  
Keyword(s):  
Respiratory Syncytial Virus ◽  
Messenger Rna ◽  
Molecular Mechanisms ◽  
Nonstructural Protein ◽  
Human Respiratory Syncytial Virus ◽  
Host Responses ◽  
Structural Basis ◽  
Downstream Signaling ◽  
Nonstructural Protein 2 ◽  
Syncytial Virus

Human respiratory syncytial virus (RSV) nonstructural protein 2 (NS2) inhibits host interferon (IFN) responses stimulated by RSV infection by targeting early steps in the IFN-signaling pathway. But the molecular mechanisms related to how NS2 regulates these processes remain incompletely understood. To address this gap, here we solved the X-ray crystal structure of NS2. This structure revealed a unique fold that is distinct from other known viral IFN antagonists, including RSV NS1. We also show that NS2 directly interacts with an inactive conformation of the RIG-I–like receptors (RLRs) RIG-I and MDA5. NS2 binding prevents RLR ubiquitination, a process critical for prolonged activation of downstream signaling. Structural analysis, including by hydrogen-deuterium exchange coupled to mass spectrometry, revealed that the N terminus of NS2 is essential for binding to the RIG-I caspase activation and recruitment domains. N-terminal mutations significantly diminish RIG-I interactions and result in increased IFNβ messenger RNA levels. Collectively, our studies uncover a previously unappreciated regulatory mechanism by which NS2 further modulates host responses and define an approach for targeting host responses.

scholarly journals Structural basis for channel conduction in the pump-like channelrhodopsin ChRmine

2021 ◽  
Author(s):  
Koichiro E. Kishi ◽  
Yoon Seok Kim ◽  
Masahiro Fukuda ◽  
Tsukasa Kusakizako ◽  
Elina Thadhani ◽  
...  
Keyword(s):  
Molecular Mechanisms ◽  
Transmembrane Helix ◽  
Action Spectrum ◽  
Light Sensitivity ◽  
Structural Basis ◽  
Ion Pump ◽  
Cryo Electron Microscopy ◽  
Architectural Features ◽  
Computational Analyses ◽  
Insight Into

ChRmine, a recently-discovered bacteriorhodopsin-like cation-conducting channelrhodopsin, exhibits puzzling properties (unusually-large photocurrents, exceptional red-shift in action spectrum, and extreme light-sensitivity) that have opened up new opportunities in optogenetics. ChRmine and its homologs function as light-gated ion channels, but by primary sequence more closely resemble ion pump rhodopsins; the molecular mechanisms for passive channel conduction in this family of proteins, as well as the unusual properties of ChRmine itself, have remained mysterious. Here we present the cryo-electron microscopy structure of ChRmine at 2.0 Å resolution. The structure reveals striking architectural features never seen before in channelrhodopsins including trimeric assembly, a short transmembrane-helix 3 unwound in the middle of the membrane, a prominently-twisting extracellular-loop 1, remarkably-large intracellular cavities and extracellular vestibule, and an unprecedented hydrophilic pore that extends through the center of the trimer, separate from the three individual monomer pores. Electrophysiological, spectroscopic, and computational analyses provide insight into conduction and gating of light-gated channels with these distinct design features, and point the way toward structure-guided creation of novel channelrhodopsins for optogenetic applications in biology.

scholarly journals Structural basis for translational shutdown and immune evasion by the Nsp1 protein of SARS-CoV-2

2020 ◽  
Author(s):  
Matthias Thoms ◽  
Robert Buschauer ◽  
Michael Ameismeier ◽  
Lennart Koepke ◽  
Timo Denk ◽  
...  
Keyword(s):  
Nonstructural Protein ◽  
Mrna Translation ◽  
Structural Basis ◽  
Innate Immune Responses ◽  
Structure Based Drug Design ◽  
Nonstructural Protein 1 ◽  
Cryo Electron Microscopy ◽  
C Terminus

AbstractSARS-CoV-2 is the causative agent of the current COVID-19 pandemic. A major virulence factor of SARS-CoVs is the nonstructural protein 1 (Nsp1) which suppresses host gene expression by ribosome association via an unknown mechanism. Here, we show that Nsp1 from SARS-CoV-2 binds to 40S and 80S ribosomes, resulting in shutdown of capped mRNA translation both in vitro and in cells. Structural analysis by cryo-electron microscopy (cryo-EM) of in vitro reconstituted Nsp1-40S and of native human Nsp1-ribosome complexes revealed that the Nsp1 C-terminus binds to and obstructs the mRNA entry tunnel. Thereby, Nsp1 effectively blocks RIG-I-dependent innate immune responses that would otherwise facilitate clearance of the infection. Thus, the structural characterization of the inhibitory mechanism of Nsp1 may aid structure-based drug design against SARS-CoV-2.

scholarly journals Phosphorylation of Serine 225 in Hepatitis C Virus NS5A Regulates Protein-Protein Interactions

2017 ◽  
Vol 91 (17) ◽  
Author(s):  
Niluka Goonawardane ◽  
Anna Gebhardt ◽  
Christopher Bartlett ◽  
Andreas Pichlmair ◽  
Mark Harris
Keyword(s):  
Life Cycle ◽  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Molecular Mechanisms ◽  
Nonstructural Protein ◽  
Antiviral Drugs ◽  
Cellular Proteins ◽  
Proximity Ligation Assay ◽  
Genome Replication ◽  
Virus Life Cycle

ABSTRACT Hepatitis C virus (HCV) nonstructural protein 5A (NS5A) is a phosphoprotein that plays key, yet poorly defined, roles in both virus genome replication and virion assembly/release. It has been proposed that differential phosphorylation could act as a switch to regulate the various functions of NS5A; however, the mechanistic details of the role of this posttranslational modification in the virus life cycle remain obscure. We previously reported (D. Ross-Thriepland, J. Mankouri, and M. Harris, J Virol 89:3123–3135, 2015, doi:10.1128/JVI.02995-14) a role for phosphorylation at serine 225 (S225) of NS5A in the regulation of JFH-1 (genotype 2a) genome replication. A phosphoablatant (S225A) mutation resulted in a 10-fold reduction in replication and a perinuclear restricted distribution of NS5A, whereas the corresponding phosphomimetic mutation (S225D) had no phenotype. To determine the molecular mechanisms underpinning this phenotype we conducted a label-free proteomics approach to identify cellular NS5A interaction partners. This analysis revealed that the S225A mutation disrupted the interactions of NS5A with a number of cellular proteins, in particular the nucleosome assembly protein 1-like protein 1 (NAP1L1), bridging integrator 1 (Bin1, also known as amphiphysin II), and vesicle-associated membrane protein-associated protein A (VAP-A). These interactions were validated by immunoprecipitation/Western blotting, immunofluorescence, and proximity ligation assay. Importantly, small interfering RNA (siRNA)-mediated knockdown of NAP1L1, Bin1 or VAP-A impaired viral genome replication and recapitulated the perinuclear redistribution of NS5A seen in the S225A mutant. These results demonstrate that S225 phosphorylation regulates the interactions of NS5A with a defined subset of cellular proteins. Furthermore, these interactions regulate both HCV genome replication and the subcellular localization of replication complexes. IMPORTANCE Hepatitis C virus is an important human pathogen. The viral nonstructural 5A protein (NS5A) is the target for new antiviral drugs. NS5A has multiple functions during the virus life cycle, but the biochemical details of these roles remain obscure. NS5A is known to be phosphorylated by cellular protein kinases, and in this study, we set out to determine whether this modification is required for the binding of NS5A to other cellular proteins. We identified 3 such proteins and show that they interacted only with NS5A that was phosphorylated on a specific residue. Furthermore, these proteins were required for efficient virus replication and the ability of NS5A to spread throughout the cytoplasm of the cell. Our results help to define the function of NS5A and may contribute to an understanding of the mode of action of the highly potent antiviral drugs that are targeted to NS5A.

scholarly journals Cryo-EM structure of the B cell co-receptor CD19 bound to the tetraspanin CD81

Science ◽  
2021 ◽  
Vol 371 (6526) ◽  
pp. 300-305
Author(s):  
Katherine J. Susa ◽  
Shaun Rawson ◽  
Andrew C. Kruse ◽  
Stephen C. Blacklow
Keyword(s):  
B Cell ◽  
Rational Design ◽  
Cell Receptor ◽  
Binding Pocket ◽  
Structural Basis ◽  
Transmembrane Helices ◽  
Critical Determinant ◽  
Cell Dysfunction ◽  
Cryo Electron Microscopy ◽  
Binding Surface

Signaling 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.

scholarly journals Structural basis for the therapeutic advantage of dual and triple agonists at the human GIP, GLP-1 or GCG receptors

2021 ◽  
Author(s):  
Fenghui Zhao ◽  
Qingtong Zhou ◽  
Zhaotong Cong ◽  
Kaini Hang ◽  
Xinyu Zou ◽  
...  
Keyword(s):  
Molecular Mechanisms ◽  
Atomic Level ◽  
Structural Basis ◽  
Therapeutic Advantage ◽  
Cryo Electron Microscopy ◽  
Glucagon Like Peptide ◽  
Glucagon Like Peptide 1 ◽  
Diabetes And Obesity ◽  
Functional Versatility

Glucose homeostasis, regulated by glucose-dependent insulinotropic polypeptide (GIP), glucagon-like peptide-1 (GLP-1) and glucagon (GCG) is critical to human health. Several multi-targeting agonists at GIPR, GLP-1R or GCGR, developed to maximize metabolic benefits with reduced side-effects, are in clinical trials to treat type 2 diabetes and obesity. To elucidate the molecular mechanisms by which tirzepatide, a GIPR/GLP-1R dualagonist, and peptide 20, a GIPR/GLP-1R/GCGR triagonist, manifest their superior efficacies over monoagonist such as semaglutide, we determined cryo-electron microscopy structures of tirzepatide-bound GIPR and GLP-1R as well as peptide 20-bound GIPR, GLP-1R and GCGR The structures reveal both common and unique features for the dual and triple agonism by illustrating key interactions of clinical relevance at the atomic level. Retention of glucagon function is required to achieve such an advantage over GLP-1 monotherapy. Our findings provide valuable insights into the structural basis of functional versatility and therapeutic supremacy of tirzepatide and peptide 20.

Structural insights into immunoglobulin M

Science ◽  
2020 ◽  
Vol 367 (6481) ◽  
pp. 1014-1017 ◽  
Author(s):  
Yaxin Li ◽  
Guopeng Wang ◽  
Ningning Li ◽  
Yuxin Wang ◽  
Qinyu Zhu ◽  
...  
Keyword(s):  
Mucosal Immunity ◽  
Molecular Mechanisms ◽  
Immunoglobulin M ◽  
Structural Basis ◽  
Polymeric Immunoglobulin Receptor ◽  
Immunoglobulin Receptor ◽  
J Chain ◽  
Cryo Electron Microscopy ◽  
Large Conformational Change ◽  
Structural Insights

Immunoglobulin M (IgM) plays a pivotal role in both humoral and mucosal immunity. Its assembly and transport depend on the joining chain (J-chain) and the polymeric immunoglobulin receptor (pIgR), but the underlying molecular mechanisms of these processes are unclear. We report a cryo–electron microscopy structure of the Fc region of human IgM in complex with the J-chain and pIgR ectodomain. The IgM-Fc pentamer is formed asymmetrically, resembling a hexagon with a missing triangle. The tailpieces of IgM-Fc pack into an amyloid-like structure to stabilize the pentamer. The J-chain caps the tailpiece assembly and bridges the interaction between IgM-Fc and the polymeric immunoglobulin receptor, which undergoes a large conformational change to engage the IgM-J complex. These results provide a structural basis for the function of IgM.

scholarly journals Structural basis for activation and gating of IP3 receptors

2021 ◽  
Author(s):  
Emily A Schmitz ◽  
Hirohide Takahashi ◽  
Erkan Karakas
Keyword(s):  
Conformational Changes ◽  
Molecular Mechanisms ◽  
Receptor Activation ◽  
Structural Basis ◽  
Ip3 Receptors ◽  
Cellular Processes ◽  
Cryo Electron Microscopy ◽  
Channel Opening ◽  
High Concentrations ◽  
Type 3

Calcium (Ca2+) is a universal and versatile cellular messenger used to regulate numerous cellular processes in response to external or internal stimuli. A pivotal component of the Ca2+ signaling toolbox in cells is the inositol 1,4,5-triphosphate (IP3) receptors (IP3Rs), which mediate Ca2+ release from the endoplasmic reticulum (ER), controlling cytoplasmic and organellar Ca2+ concentrations. IP3Rs are activated by IP3 and Ca2+, inhibited by Ca2+ at high concentrations, and potentiated by ATP1-3. However, the underlying molecular mechanisms are unclear due to the lack of structures in the active conformation. Here we report cryo-electron microscopy (cryo-EM) structures of human type-3 IP3R in multiple gating conformations; IP3-ATP bound pre-active states with closed channels, IP3-ATP-Ca2+ bound active state with an open channel, and IP3-ATP-Ca2+ bound inactive state with a closed channel. The structures demonstrate how IP3-induced conformational changes prime the receptor for activation by Ca2+, how Ca2+ binding leads to channel opening, and how ATP modulates the activity, providing insights into the long-sought questions regarding the molecular mechanism of the receptor activation and gating.

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