scholarly journals Characterization and structural basis of a lethal mouse-adapted SARS-CoV-2

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Shihui Sun ◽  
Hongjing Gu ◽  
Lei Cao ◽  
Qi Chen ◽  
Qing Ye ◽  
...  
Keyword(s):  
Binding Affinity ◽  
Receptor Binding ◽  
Structural Basis ◽  
Rapid Adaptation ◽  
Cryo Electron Microscopy ◽  
Electron Microscopy Analysis ◽  
Microscopy Analysis ◽  
And Gender ◽  
Related Mortality

AbstractThere is an urgent need for animal models to study SARS-CoV-2 pathogenicity. Here, we generate and characterize a novel mouse-adapted SARS-CoV-2 strain, MASCp36, that causes severe respiratory symptoms, and mortality. Our model exhibits age- and gender-related mortality akin to severe COVID-19. Deep sequencing identified three amino acid substitutions, N501Y, Q493H, and K417N, at the receptor binding domain (RBD) of MASCp36, during in vivo passaging. All three RBD mutations significantly enhance binding affinity to its endogenous receptor, ACE2. Cryo-electron microscopy analysis of human ACE2 (hACE2), or mouse ACE2 (mACE2), in complex with the RBD of MASCp36, at 3.1 to 3.7 Å resolution, reveals the molecular basis for the receptor-binding switch. N501Y and Q493H enhance the binding affinity to hACE2, whereas triple mutations at N501Y/Q493H/K417N decrease affinity and reduce infectivity of MASCp36. Our study provides a platform for studying SARS-CoV-2 pathogenesis, and unveils the molecular mechanism for its rapid adaptation and evolution.

scholarly journals Structural basis of ER-associated protein degradation mediated by the Hrd1 ubiquitin ligase complex

Science ◽  
2020 ◽  
Vol 368 (6489) ◽  
pp. eaaz2449 ◽  
Author(s):  
Xudong Wu ◽  
Marc Siggel ◽  
Sergey Ovchinnikov ◽  
Wei Mi ◽  
Vladimir Svetlov ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Ubiquitin Ligase ◽  
Dynamics Simulation ◽  
Structural Basis ◽  
Cryo Electron Microscopy ◽  
Ubiquitin Ligase Complex ◽  
Electron Microscopy Analysis ◽  
Microscopy Analysis ◽  
Simulation Results ◽  
Membrane Region

Misfolded luminal endoplasmic reticulum (ER) proteins undergo ER-associated degradation (ERAD-L): They are retrotranslocated into the cytosol, polyubiquitinated, and degraded by the proteasome. ERAD-L is mediated by the Hrd1 complex (composed of Hrd1, Hrd3, Der1, Usa1, and Yos9), but the mechanism of retrotranslocation remains mysterious. Here, we report a structure of the active Hrd1 complex, as determined by cryo–electron microscopy analysis of two subcomplexes. Hrd3 and Yos9 jointly create a luminal binding site that recognizes glycosylated substrates. Hrd1 and the rhomboid-like Der1 protein form two “half-channels” with cytosolic and luminal cavities, respectively, and lateral gates facing one another in a thinned membrane region. These structures, along with crosslinking and molecular dynamics simulation results, suggest how a polypeptide loop of an ERAD-L substrate moves through the ER membrane.

scholarly journals Characterization and structural basis of a lethal mouse-adapted SARS-CoV-2

2020 ◽  
Author(s):  
Shihui Sun ◽  
Hongjing Gu ◽  
Lei Cao ◽  
Qi Chen ◽  
Guan Yang ◽  
...  
Keyword(s):  
Amino Acid ◽  
Receptor Binding ◽  
Lethal Dose ◽  
Skewed Distribution ◽  
Structural Basis ◽  
Laboratory Mice ◽  
Age And Gender ◽  
Cryo Electron Microscopy ◽  
Amino Acid Mutations ◽  
And Gender

The ongoing SARS-CoV-2 pandemic has brought an urgent need for animal models to study the pathogenicity of the virus. Herein, we generated and characterized a novel mouse-adapted SARS-CoV-2 strain named MASCp36 that causes acute respiratory symptoms and mortality in standard laboratory mice. Particularly, this model exhibits age and gender related skewed distribution of mortality akin to severe COVID-19, and the 50% lethal dose (LD50) of MASCp36 was ~100 PFU in aged, male BALB/c mice. Deep sequencing identified three amino acid mutations, N501Y, Q493H, and K417N, subsequently emerged at the receptor binding domain (RBD) of MASCp36, which significantly enhanced the binding affinity to its endogenous receptor, mouse ACE2 (mACE2). Cryo-electron microscopy (cryo-EM) analysis of mACE2 in complex with the RBD of MASCp36 at 3.7-angstrom resolution elucidates molecular basis for the receptor-binding switch driven by amino acid substitutions. Our study not only provides a robust platform for studying the pathogenesis of severe COVID-19 and rapid evaluation of coutermeasures against SARS-CoV-2, but also unveils the molecular mechanism for the rapid adaption and evolution of SARS-CoV-2 in mice.

scholarly journals Isolation of Disease-relevant p53 for Cryo-Electron Microscopy Analysis

2021 ◽  
Author(s):  
Maria J. Solares ◽  
GM Jonaid ◽  
William Y. Luqiu ◽  
Yanping Liang ◽  
Madison C. Evans ◽  
...  
Keyword(s):  
P53 Protein ◽  
P53 Gene ◽  
Tp53 Gene ◽  
Tumor Suppressor Protein ◽  
Protein Assemblies ◽  
Cryo Electron Microscopy ◽  
Naturally Occurring ◽  
Imaging Protocols ◽  
Electron Microscopy Analysis ◽  
Microscopy Analysis

Abstract Tumor suppressor protein TP53 (p53) plays a multi-faceted role in all cells of thehuman body. Sadly, mutations in the TP53 gene are involved in nearly ~50% of tumors,spurring erratic cell growth and disease progression. Until recently, structural informationfor p53 remained incomplete and there are limited studies on native p53 produced inhuman tumors. Here, we present a highly reproducible and effective protocol to extract,enrich, and purify native p53 protein assemblies from cancer cells for downstreamstructural studies. This method does not introduce purification tags into the p53 gene andmaintains naturally occurring modifications. In conjunction with cryo-Electron Microscopytechniques, we determined new structures for p53 monomers (~50 kDa) and tetramers(~200 kDa) at spatial resolutions of ~4.8 Å and ~7 Å, respectively.1 These modelsrevealed new insights for flexible regions of p53 along with biologically-relevantubiquitination sites. Combining biochemical and structural imaging protocols, we aim tobuild a better understanding of native p53’s impact in cancer formation.

Abstract 530: Hemostasis Revisited: a Combined Light and Electron Microscopy Analysis of Hemostatic Plug Formation in vivo

2018 ◽  
Vol 38 (Suppl_1) ◽  
Author(s):  
Maurizio Tomaiuolo ◽  
Chelsea N Matzko ◽  
Izmarie Poventud-Fuentes ◽  
John W Weisel ◽  
Lawrence F Brass ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Light And Electron Microscopy ◽  
Electron Microscopy Analysis ◽  
Microscopy Analysis ◽  
Plug Formation

In Vitro Androgen Receptor Binding Affinity and in Vivo Inhibitory Activity of 5?-Pregnane-3, 20-Dione

1988 ◽  
Vol 529 (1 Fourth Colloq) ◽  
pp. 239-241
Author(s):  
SAUDHAMINI PARTHASARATHY ◽  
ANDREA CHIN ◽  
VIRGINIA MALLOY ◽  
JONATHAN MATIAS
Keyword(s):  
Androgen Receptor ◽  
Binding Affinity ◽  
Receptor Binding ◽  
Inhibitory Activity ◽  
Receptor Binding Affinity

scholarly journals Structural investigation of ACE2 dependent disassembly of the trimeric SARS-CoV-2 Spike glycoprotein

2020 ◽  
Author(s):  
Dongchun Ni ◽  
Kelvin Lau ◽  
Frank Lehmann ◽  
Andri Fränkl ◽  
David Hacker ◽  
...  
Keyword(s):  
Structural Rearrangement ◽  
Host Cells ◽  
Surface Glycoprotein ◽  
Angiotensin Converting Enzyme 2 ◽  
Cryo Electron Microscopy ◽  
Monomeric Complex ◽  
Electron Microscopy Analysis ◽  
Microscopy Analysis ◽  
Viral Surface

AbstractThe human membrane protein Angiotensin-converting enzyme 2 (hACE2) acts as the main receptor for host cells invasion of the new coronavirus SARS-CoV-2. The viral surface glycoprotein Spike binds to hACE2, which triggers virus entry into cells. As of today, the role of hACE2 for virus fusion is not well understood. Blocking the transition of Spike from its prefusion to post-fusion state might be a strategy to prevent or treat COVID-19. Here we report a single particle cryo-electron microscopy analysis of SARS-CoV-2 trimeric Spike in presence of the human ACE2 ectodomain. The binding of purified hACE2 ectodomain to Spike induces the disassembly of the trimeric form of Spike and a structural rearrangement of its S1 domain to form a stable, monomeric complex with hACE2. This observed hACE2 dependent dissociation of the Spike trimer suggests a mechanism for the therapeutic role of recombinant soluble hACE2 for treatment of COVID-19.

scholarly journals Structural basis for VIPP1 oligomerization and maintenance of thylakoid membrane integrity

2020 ◽  
Author(s):  
Tilak Kumar Gupta ◽  
Sven Klumpe ◽  
Karin Gries ◽  
Steffen Heinz ◽  
Wojciech Wietrzynski ◽  
...  
Keyword(s):  
Membrane Integrity ◽  
Point Mutations ◽  
Binding Pocket ◽  
Thylakoid Membranes ◽  
Lipid Binding ◽  
Structural Basis ◽  
Amphipathic Helices ◽  
Cryo Electron Microscopy

AbstractVesicle-inducing protein in plastids (VIPP1) is essential for the biogenesis and maintenance of thylakoid membranes, which transform light into life. However, it is unknown how VIPP1 performs its vital membrane-shaping function. Here, we use cryo-electron microscopy to determine structures of cyanobacterial VIPP1 rings, revealing how VIPP1 monomers flex and interweave to form basket-like assemblies of different symmetries. Three VIPP1 monomers together coordinate a non-canonical nucleotide binding pocket that is required for VIPP1 oligomerization. Inside the ring’s lumen, amphipathic helices from each monomer align to form large hydrophobic columns, enabling VIPP1 to bind and curve membranes. In vivo point mutations in these hydrophobic surfaces cause extreme thylakoid swelling under high light, indicating an essential role of VIPP1 lipid binding in resisting stress-induced damage. Our study provides a structural basis for understanding how the oligomerization of VIPP1 drives the biogenesis of thylakoid membranes and protects these life-giving membranes from environmental stress.

scholarly journals Molecular analysis of the interaction of LCMV with its cellular receptor α-dystroglycan

2001 ◽  
Vol 155 (2) ◽  
pp. 301-310 ◽  
Author(s):  
Stefan Kunz ◽  
Noemí Sevilla ◽  
Dorian B. McGavern ◽  
Kevin P. Campbell ◽  
Michael B.A. Oldstone
Keyword(s):  
Binding Site ◽  
Binding Affinity ◽  
Receptor Binding ◽  
Divalent Cations ◽  
Terminal Region ◽  
Cellular Receptor ◽  
Globular Domain ◽  
Cell Surface Molecule ◽  
Laminin 1

α-Dystroglycan (DG) has been identified as the cellular receptor for lymphocytic choriomeningitis virus (LCMV) and Lassa fever virus (LFV). This subunit of DG is a highly versatile cell surface molecule that provides a molecular link between the extracellular matrix (ECM) and a β-DG transmembrane component, which interacts with the actin-based cytoskeleton. In addition, DG exhibits a complex pattern of interaction with a wide variety of ECM and cellular proteins. In the present study, we characterized the binding of LCMV to α-DG and addressed the role of α-DG–associated host-derived proteins in virus infection. We found that the COOH-terminal region of α-DG's first globular domain and the NH2-terminal region of the mucin-related structures of α-DG together form the binding site for LCMV. The virus–α-DG binding unlike ECM α-DG interactions was not dependent on divalent cations. Despite such differences in binding, LCMV and laminin-1 use, in part, an overlapping binding site on α-DG, and the ability of an LCMV isolate to compete with laminin-1 for receptor binding is determined by its binding affinity to α-DG. This competition of the virus with ECM molecules for receptor binding likely explains the recently found correlation between the affinity of LCMV binding to α-DG, tissue tropism, and pathological potential. LCMV strains and variants with high binding affinity to α-DG but not low affinity binders are able to infect CD11c+ dendritic cells, which express α-DG at their surface. Infection followed by dysfunction of these antigen-presenting cells contributes to immunosuppression and persistent viral infection in vivo.

scholarly journals Improved Glucose Metabolism In Vitro and In Vivo by an Allosteric Monoclonal Antibody That Increases Insulin Receptor Binding Affinity

PLoS ONE ◽  
2014 ◽  
Vol 9 (2) ◽  
pp. e88684 ◽  
Author(s):  
John A. Corbin ◽  
Vinay Bhaskar ◽  
Ira D. Goldfine ◽  
Daniel H. Bedinger ◽  
Angela Lau ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Glucose Metabolism ◽  
Insulin Receptor ◽  
Binding Affinity ◽  
Receptor Binding ◽  
Receptor Binding Affinity ◽  
Insulin Receptor Binding
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