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1.
Biophys J ; 120(14): 2766-2770, 2021 07 20.
Article in English | MEDLINE | ID: covidwho-1603998

ABSTRACT

Understanding the structure of messenger RNA (mRNA) lipid nanoparticles, and specifically the microenvironment of the mRNA molecules within these entities, is fundamental to advancing their biomedical potential. Here, we show that a permeating cationic dye, thionine, can serve as a cryogenic electron microscopy contrasting agent by binding selectively to encapsulated mRNA without disturbing lipid nanoparticle morphology. Cryo-electron microscopy images identify the mRNA location, revealing that mRNA may exist within solvent-filled cavities or may be substantially lipid associated.


Subject(s)
Lipids , Nanoparticles , Cryoelectron Microscopy , RNA, Messenger/genetics
2.
Nat Commun ; 12(1): 7345, 2021 12 20.
Article in English | MEDLINE | ID: covidwho-1585860

ABSTRACT

The emergence of SARS-CoV-2 Kappa and Beta variants with enhanced transmissibility and resistance to neutralizing antibodies has created new challenges for the control of the ongoing COVID-19 pandemic. Understanding the structural nature of Kappa and Beta spike (S) proteins and their association with ACE2 is of significant importance. Here we present two cryo-EM structures for each of the Kappa and Beta spikes in the open and open-prone transition states. Compared with wild-type (WT) or G614 spikes, the two variant spikes appear more untwisted/open especially for Beta, and display a considerable population shift towards the open state as well as more pronounced conformational dynamics. Moreover, we capture four conformational states of the S-trimer/ACE2 complex for each of the two variants, revealing an enlarged conformational landscape for the Kappa and Beta S-ACE2 complexes and pronounced population shift towards the three RBDs up conformation. These results implicate that the mutations in Kappa and Beta may modify the kinetics of receptor binding and viral fusion to improve virus fitness. Combined with biochemical analysis, our structural study shows that the two variants are enabled to efficiently interact with ACE2 receptor despite their sensitive ACE2 binding surface is modified to escape recognition by some potent neutralizing MAbs. Our findings shed new light on the pathogenicity and immune evasion mechanism of the Beta and Kappa variants.


Subject(s)
Angiotensin-Converting Enzyme 2/chemistry , Cryoelectron Microscopy , SARS-CoV-2 , Spike Glycoprotein, Coronavirus/chemistry , Antibodies, Neutralizing/immunology , Binding Sites , COVID-19 , Humans , Kinetics , Molecular Conformation , Mutation , Protein Binding
3.
J Chem Inf Model ; 62(1): 176-186, 2022 01 10.
Article in English | MEDLINE | ID: covidwho-1575860

ABSTRACT

The coronavirus disease 19 (COVID-19) pandemic is causing a global health crisis and has already caused a devastating societal and economic burden. The pathogen, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has a high sequence and architecture identity with SARS-CoV, but far more people have been infected by SARS-CoV-2. Here, combining the structural data from cryo-electron microscopy and structure prediction, we constructed bottom-up Martini coarse-grained models of intact SARS-CoV and SARS-CoV-2 envelopes. Microsecond molecular dynamics simulations were performed, allowing us to explore their dynamics and supramolecular organization. Both SARS-CoV and SARS-CoV-2 envelopes present a spherical morphology, with structural proteins forming multiple string-like islands in the membrane and clusters between the heads of spike proteins. Critical differences between the SARS-CoV and SARS-CoV-2 envelopes are the interaction pattern between the spike proteins and the flexibility of the spike proteins. Our models provide structural and dynamic insights into the SARS virus envelopes and could be used for further investigation, such as drug design and membrane fusion and fission processes.


Subject(s)
COVID-19 , SARS Virus , Cryoelectron Microscopy , Humans , SARS-CoV-2 , Spike Glycoprotein, Coronavirus , Viral Envelope , Virion
4.
Nat Struct Mol Biol ; 28(7): 614-625, 2021 07.
Article in English | MEDLINE | ID: covidwho-1550333

ABSTRACT

p97 processes ubiquitinated substrates and plays a central role in cellular protein homeostasis. Here, we report a series of cryo-EM structures of the substrate-engaged human p97 complex with resolutions ranging from 2.9 to 3.8 Å that captured 'power-stroke'-like motions of both the D1 and D2 ATPase rings of p97. A key feature of these structures is the critical conformational changes of the intersubunit signaling (ISS) motifs, which tighten the binding of nucleotides and neighboring subunits and contribute to the spiral staircase conformation of the D1 and D2 rings. In addition, we determined the cryo-EM structure of human p97 in complex with NMS-873, a potent p97 inhibitor, at a resolution of 2.4 Å. The structures showed that NMS-873 binds at a cryptic groove in the D2 domain and interacts with the ISS motif, preventing its conformational change and thus blocking substrate translocation allosterically.


Subject(s)
Adenosine Triphosphate/chemistry , Protein Folding , Proteostasis/physiology , Signal Transduction/physiology , Valosin Containing Protein/metabolism , Acetanilides/pharmacology , Animals , Benzothiazoles/pharmacology , Cryoelectron Microscopy , Endoplasmic Reticulum/metabolism , Endoplasmic Reticulum-Associated Degradation/physiology , Humans , Intracellular Signaling Peptides and Proteins/metabolism , Nuclear Proteins/metabolism , Proteasome Endopeptidase Complex/metabolism , Protein Conformation , Ubiquitinated Proteins/metabolism , Valosin Containing Protein/antagonists & inhibitors
5.
Nat Commun ; 12(1): 3172, 2021 05 26.
Article in English | MEDLINE | ID: covidwho-1550281

ABSTRACT

Secreted class 3 semaphorins (Sema3s) form tripartite complexes with the plexin receptor and neuropilin coreceptor, which are both transmembrane proteins that together mediate semaphorin signal for neuronal axon guidance and other processes. Despite extensive investigations, the overall architecture of and the molecular interactions in the Sema3/plexin/neuropilin complex are incompletely understood. Here we present the cryo-EM structure of a near intact extracellular region complex of Sema3A, PlexinA4 and Neuropilin 1 (Nrp1) at 3.7 Å resolution. The structure shows a large symmetric 2:2:2 assembly in which each subunit makes multiple interactions with others. The two PlexinA4 molecules in the complex do not interact directly, but their membrane proximal regions are close to each other and poised to promote the formation of the intracellular active dimer for signaling. The structure reveals a previously unknown interface between the a2b1b2 module in Nrp1 and the Sema domain of Sema3A. This interaction places the a2b1b2 module at the top of the complex, far away from the plasma membrane where the transmembrane regions of Nrp1 and PlexinA4 embed. As a result, the region following the a2b1b2 module in Nrp1 must span a large distance to allow the connection to the transmembrane region, suggesting an essential role for the long non-conserved linkers and the MAM domain in neuropilin in the semaphorin/plexin/neuropilin complex.


Subject(s)
Nerve Tissue Proteins/ultrastructure , Neuropilin-1/ultrastructure , Receptors, Cell Surface/ultrastructure , Semaphorin-3A/ultrastructure , Animals , COS Cells , Chlorocebus aethiops , Cryoelectron Microscopy , HEK293 Cells , Humans , Mutation , Nerve Tissue Proteins/genetics , Nerve Tissue Proteins/isolation & purification , Nerve Tissue Proteins/metabolism , Neuropilin-1/genetics , Neuropilin-1/isolation & purification , Neuropilin-1/metabolism , Protein Binding/genetics , Protein Domains/genetics , Protein Multimerization/genetics , Receptors, Cell Surface/genetics , Receptors, Cell Surface/isolation & purification , Receptors, Cell Surface/metabolism , Recombinant Proteins/genetics , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolism , Recombinant Proteins/ultrastructure , Semaphorin-3A/genetics , Semaphorin-3A/isolation & purification , Semaphorin-3A/metabolism
6.
Cell Rep ; 37(12): 110156, 2021 12 21.
Article in English | MEDLINE | ID: covidwho-1549680

ABSTRACT

The recently emerged severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) Beta (B.1.351) and Gamma (P.1) variants of concern (VoCs) include a key mutation (N501Y) found in the Alpha (B.1.1.7) variant that enhances affinity of the spike protein for its receptor, angiotensin-converting enzyme 2 (ACE2). Additional mutations are found in these variants at residues 417 and 484 that appear to promote antibody evasion. In contrast, the Epsilon variants (B.1.427/429) lack the N501Y mutation yet exhibit antibody evasion. We have engineered spike proteins to express these receptor binding domain (RBD) VoC mutations either in isolation or in different combinations and analyze the effects using biochemical assays and cryoelectron microscopy (cryo-EM) structural analyses. Overall, our findings suggest that the emergence of new SARS-CoV-2 variant spikes can be rationalized as the result of mutations that confer increased ACE2 affinity, increased antibody evasion, or both, providing a framework to dissect the molecular factors that drive VoC evolution.


Subject(s)
Angiotensin-Converting Enzyme 2/chemistry , SARS-CoV-2/genetics , Spike Glycoprotein, Coronavirus/chemistry , Angiotensin-Converting Enzyme 2/metabolism , Binding Sites , Cryoelectron Microscopy , Humans , Molecular Dynamics Simulation , Mutation , Protein Interaction Domains and Motifs , SARS-CoV-2/chemistry , SARS-CoV-2/classification , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/metabolism
8.
Cell Rep ; 37(4): 109882, 2021 10 26.
Article in English | MEDLINE | ID: covidwho-1525720

ABSTRACT

Remdesivir (RDV), a nucleotide analog with broad-spectrum features, has exhibited effectiveness in COVID-19 treatment. However, the precise working mechanism of RDV when targeting the viral RNA-dependent RNA polymerase (RdRP) has not been fully elucidated. Here, we solve a 3.0-Å structure of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RdRP elongation complex (EC) and assess RDV intervention in polymerase elongation phase. Although RDV could induce an "i+3" delayed termination in meta-stable complexes, only pausing and subsequent elongation are observed in the EC. A comparative investigation using an enterovirus RdRP further confirms similar delayed intervention and demonstrates that steric hindrance of the RDV-characteristic 1'-cyano at the -4 position is responsible for the "i+3" intervention, although two representative Flaviviridae RdRPs do not exhibit similar behavior. A comparison of representative viral RdRP catalytic complex structures indicates that the product RNA backbone encounters highly conserved structural elements, highlighting the broad-spectrum intervention potential of 1'-modified nucleotide analogs in anti-RNA virus drug development.


Subject(s)
Adenosine Monophosphate/analogs & derivatives , Alanine/analogs & derivatives , Antiviral Agents/pharmacology , RNA-Dependent RNA Polymerase/drug effects , SARS-CoV-2/drug effects , Viral Proteins/drug effects , Adenosine Monophosphate/pharmacology , Alanine/pharmacology , COVID-19/drug therapy , Cryoelectron Microscopy , Humans , RNA, Viral/chemistry , RNA, Viral/drug effects , RNA-Dependent RNA Polymerase/chemistry , SARS-CoV-2/chemistry , Viral Proteins/chemistry , Virus Replication/drug effects
9.
Proc Natl Acad Sci U S A ; 118(48)2021 11 30.
Article in English | MEDLINE | ID: covidwho-1517667

ABSTRACT

The spike protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) mediates membrane fusion to allow entry of the viral genome into host cells. To understand its detailed entry mechanism and develop a specific entry inhibitor, in situ structural information on the SARS-CoV-2 spike protein in different states is urgent. Here, by using cryo-electron tomography, we observed both prefusion and postfusion spikes in ß-propiolactone-inactivated SARS-CoV-2 virions and solved the in situ structure of the postfusion spike at nanometer resolution. Compared to previous reports, the six-helix bundle fusion core, the glycosylation sites, and the location of the transmembrane domain were clearly resolved. We observed oligomerization patterns of the spikes on the viral membrane, likely suggesting a mechanism of fusion pore formation.


Subject(s)
SARS-CoV-2/ultrastructure , Spike Glycoprotein, Coronavirus/chemistry , Amino Acid Motifs , Animals , Chlorocebus aethiops , Cryoelectron Microscopy , Electron Microscope Tomography , Glycosylation , Protein Domains , Protein Multimerization , Spike Glycoprotein, Coronavirus/metabolism , Vero Cells
10.
Signal Transduct Target Ther ; 6(1): 396, 2021 11 15.
Article in English | MEDLINE | ID: covidwho-1517609

ABSTRACT

Coronavirus disease 2019 (COVID-19), a highly infectious disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has infected more than 235 million individuals and led to more than 4.8 million deaths worldwide as of October 5 2021. Cryo-electron microscopy and topology show that the SARS-CoV-2 genome encodes lots of highly glycosylated proteins, such as spike (S), envelope (E), membrane (M), and ORF3a proteins, which are responsible for host recognition, penetration, binding, recycling and pathogenesis. Here we reviewed the detections, substrates, biological functions of the glycosylation in SARS-CoV-2 proteins as well as the human receptor ACE2, and also summarized the approved and undergoing SARS-CoV-2 therapeutics associated with glycosylation. This review may not only broad the understanding of viral glycobiology, but also provide key clues for the development of new preventive and therapeutic methodologies against SARS-CoV-2 and its variants.


Subject(s)
Angiotensin-Converting Enzyme 2/genetics , COVID-19/genetics , SARS-CoV-2/genetics , Spike Glycoprotein, Coronavirus/genetics , Angiotensin-Converting Enzyme 2/metabolism , COVID-19/metabolism , COVID-19/pathology , COVID-19/virology , Cryoelectron Microscopy , Glycosylation , Humans , Peptidyl-Dipeptidase A/genetics , Protein Binding/genetics , SARS-CoV-2/metabolism , SARS-CoV-2/pathogenicity , Viral Envelope Proteins/genetics , Viral Matrix Proteins/genetics
11.
Theranostics ; 12(1): 1-17, 2022.
Article in English | MEDLINE | ID: covidwho-1512993

ABSTRACT

Background: Administration of potent anti-receptor-binding domain (RBD) monoclonal antibodies has been shown to curtail viral shedding and reduce hospitalization in patients with SARS-CoV-2 infection. However, the structure-function analysis of potent human anti-RBD monoclonal antibodies and its links to the formulation of antibody cocktails remains largely elusive. Methods: Previously, we isolated a panel of neutralizing anti-RBD monoclonal antibodies from convalescent patients and showed their neutralization efficacy in vitro. Here, we elucidate the mechanism of action of antibodies and dissect antibodies at the epitope level, which leads to a formation of a potent antibody cocktail. Results: We found that representative antibodies which target non-overlapping epitopes are effective against wild type virus and recently emerging variants of concern, whilst being encoded by antibody genes with few somatic mutations. Neutralization is associated with the inhibition of binding of viral RBD to ACE2 and possibly of the subsequent fusion process. Structural analysis of representative antibodies, by cryo-electron microscopy and crystallography, reveals that they have some unique aspects that are of potential value while sharing some features in common with previously reported neutralizing monoclonal antibodies. For instance, one has a common VH 3-53 public variable region yet is unusually resilient to mutation at residue 501 of the RBD. We evaluate the in vivo efficacy of an antibody cocktail consisting of two potent non-competing anti-RBD antibodies in a Syrian hamster model. We demonstrate that the cocktail prevents weight loss, reduces lung viral load and attenuates pulmonary inflammation in hamsters in both prophylactic and therapeutic settings. Although neutralization of one of these antibodies is abrogated by the mutations of variant B.1.351, it is also possible to produce a bi-valent cocktail of antibodies both of which are resilient to variants B.1.1.7, B.1.351 and B.1.617.2. Conclusions: These findings support the up-to-date and rational design of an anti-RBD antibody cocktail as a therapeutic candidate against COVID-19.


Subject(s)
Antibodies, Monoclonal/chemistry , Antibodies, Monoclonal/pharmacology , COVID-19/drug therapy , SARS-CoV-2/metabolism , Angiotensin-Converting Enzyme 2/genetics , Angiotensin-Converting Enzyme 2/metabolism , Animals , Antibodies, Monoclonal/metabolism , Antibodies, Neutralizing/chemistry , Antibodies, Neutralizing/immunology , Antibodies, Neutralizing/pharmacology , Binding Sites , Binding, Competitive , COVID-19/virology , Cricetinae , Cryoelectron Microscopy , Crystallography, X-Ray , Dogs , Epitopes , Female , Humans , Madin Darby Canine Kidney Cells , Neutralization Tests , Protein Domains , SARS-CoV-2/genetics , SARS-CoV-2/immunology , SARS-CoV-2/pathogenicity , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/immunology , Spike Glycoprotein, Coronavirus/metabolism
12.
Proc Natl Acad Sci U S A ; 118(41)2021 10 12.
Article in English | MEDLINE | ID: covidwho-1462069

ABSTRACT

We describe a general method that allows structure determination of small proteins by single-particle cryo-electron microscopy (cryo-EM). The method is based on the availability of a target-binding nanobody, which is then rigidly attached to two scaffolds: 1) a Fab fragment of an antibody directed against the nanobody and 2) a nanobody-binding protein A fragment fused to maltose binding protein and Fab-binding domains. The overall ensemble of ∼120 kDa, called Legobody, does not perturb the nanobody-target interaction, is easily recognizable in EM images due to its unique shape, and facilitates particle alignment in cryo-EM image processing. The utility of the method is demonstrated for the KDEL receptor, a 23-kDa membrane protein, resulting in a map at 3.2-Šoverall resolution with density sufficient for de novo model building, and for the 22-kDa receptor-binding domain (RBD) of SARS-CoV-2 spike protein, resulting in a map at 3.6-Šresolution that allows analysis of the binding interface to the nanobody. The Legobody approach thus overcomes the current size limitations of cryo-EM analysis.


Subject(s)
Cryoelectron Microscopy/methods , SARS-CoV-2/metabolism , Single-Domain Antibodies/metabolism , Spike Glycoprotein, Coronavirus/metabolism , Binding Sites/immunology , COVID-19/virology , HEK293 Cells , Humans , Models, Molecular , Protein Binding , Protein Domains , SARS-CoV-2/immunology , SARS-CoV-2/physiology , Single-Domain Antibodies/immunology , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/ultrastructure
13.
Cell Rep ; 37(4): 109881, 2021 10 26.
Article in English | MEDLINE | ID: covidwho-1458602

ABSTRACT

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has necessitated the rapid development of antibody-based therapies and vaccines as countermeasures. Here, we use cryoelectron microscopy (cryo-EM) to characterize two protective anti-SARS-CoV-2 murine monoclonal antibodies (mAbs) in complex with the spike protein, revealing similarities between epitopes targeted by human and murine B cells. The more neutralizing mAb, 2B04, binds the receptor-binding motif (RBM) of the receptor-binding domain (RBD) and competes with angiotensin-converting enzyme 2 (ACE2). By contrast, 2H04 binds adjacent to the RBM and does not compete for ACE2 binding. Naturally occurring sequence variants of SARS-CoV-2 and corresponding neutralization escape variants selected in vitro map to our structurally defined epitopes, suggesting that SARS-CoV-2 might evade therapeutic antibodies with a limited set of mutations, underscoring the importance of combination mAb therapeutics. Finally, we show that 2B04 neutralizes SARS-CoV-2 infection by preventing ACE2 engagement, whereas 2H04 reduces host cell attachment without directly disrupting ACE2-RBM interactions, providing distinct inhibitory mechanisms used by RBD-specific mAbs.


Subject(s)
Antibodies, Neutralizing/immunology , Antibodies, Viral/immunology , COVID-19/immunology , SARS-CoV-2/immunology , Spike Glycoprotein, Coronavirus/immunology , Angiotensin-Converting Enzyme 2/metabolism , Animals , Antibodies, Monoclonal/chemistry , Antibodies, Monoclonal/immunology , Antibodies, Neutralizing/chemistry , Antibodies, Viral/chemistry , Cryoelectron Microscopy , Epitopes, B-Lymphocyte/chemistry , Epitopes, B-Lymphocyte/immunology , Humans , Mice , Protein Interaction Domains and Motifs/immunology , Protein Structure, Quaternary , Spike Glycoprotein, Coronavirus/chemistry
14.
Cell Rep ; 37(1): 109784, 2021 10 05.
Article in English | MEDLINE | ID: covidwho-1442299

ABSTRACT

The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) lineages that are more transmissible and resistant to currently approved antibody therapies poses a considerable challenge to the clinical treatment of coronavirus disease (COVID-19). Therefore, the need for ongoing discovery efforts to identify broadly reactive monoclonal antibodies to SARS-CoV-2 is of utmost importance. Here, we report a panel of SARS-CoV-2 antibodies isolated using the linking B cell receptor to antigen specificity through sequencing (LIBRA-seq) technology from an individual who recovered from COVID-19. Of these antibodies, 54042-4 shows potent neutralization against authentic SARS-CoV-2 viruses, including variants of concern (VOCs). A cryoelectron microscopy (cryo-EM) structure of 54042-4 in complex with the SARS-CoV-2 spike reveals an epitope composed of residues that are highly conserved in currently circulating SARS-CoV-2 lineages. Further, 54042-4 possesses uncommon genetic and structural characteristics that distinguish it from other potently neutralizing SARS-CoV-2 antibodies. Together, these findings provide motivation for the development of 54042-4 as a lead candidate to counteract current and future SARS-CoV-2 VOCs.


Subject(s)
Angiotensin-Converting Enzyme 2/immunology , Antibodies, Monoclonal/immunology , Antibodies, Neutralizing/immunology , COVID-19/immunology , SARS-CoV-2/chemistry , SARS-CoV-2/immunology , Spike Glycoprotein, Coronavirus/immunology , Angiotensin-Converting Enzyme 2/chemistry , Animals , Antibodies, Viral/immunology , Antibody Formation , COVID-19/genetics , COVID-19/virology , Cell Line , Chlorocebus aethiops , Cryoelectron Microscopy , Epitope Mapping/methods , Epitopes/chemistry , Epitopes/immunology , High-Throughput Screening Assays/methods , Humans , Male , Middle Aged , Protein Binding , Protein Interaction Domains and Motifs , Receptors, Antigen, B-Cell/chemistry , Receptors, Antigen, B-Cell/immunology , SARS-CoV-2/genetics , Spike Glycoprotein, Coronavirus/chemistry , Vero Cells
15.
Cell Rep ; 37(1): 109771, 2021 10 05.
Article in English | MEDLINE | ID: covidwho-1439919

ABSTRACT

Understanding mechanisms of protective antibody recognition can inform vaccine and therapeutic strategies against SARS-CoV-2. We report a monoclonal antibody, 910-30, targeting the SARS-CoV-2 receptor-binding site for ACE2 as a member of a public antibody response encoded by IGHV3-53/IGHV3-66 genes. Sequence and structural analyses of 910-30 and related antibodies explore how class recognition features correlate with SARS-CoV-2 neutralization. Cryo-EM structures of 910-30 bound to the SARS-CoV-2 spike trimer reveal binding interactions and its ability to disassemble spike. Despite heavy-chain sequence similarity, biophysical analyses of IGHV3-53/3-66-encoded antibodies highlight the importance of native heavy:light pairings for ACE2-binding competition and SARS-CoV-2 neutralization. We develop paired heavy:light class sequence signatures and determine antibody precursor prevalence to be ∼1 in 44,000 human B cells, consistent with public antibody identification in several convalescent COVID-19 patients. These class signatures reveal genetic, structural, and functional immune features that are helpful in accelerating antibody-based medical interventions for SARS-CoV-2.


Subject(s)
Angiotensin-Converting Enzyme 2/immunology , Antibodies, Monoclonal/chemistry , Antibodies, Monoclonal/immunology , COVID-19/immunology , COVID-19/virology , SARS-CoV-2/immunology , Spike Glycoprotein, Coronavirus/immunology , Aged , Angiotensin-Converting Enzyme 2/chemistry , Animals , Antibodies, Monoclonal/genetics , Antibodies, Monoclonal/ultrastructure , Antibodies, Neutralizing/immunology , Antibodies, Viral/immunology , Antibody Formation , B-Lymphocytes/immunology , Binding Sites , Chlorocebus aethiops , Cryoelectron Microscopy , HEK293 Cells , Humans , Immunoglobulin Heavy Chains/chemistry , Immunoglobulin Heavy Chains/genetics , Immunoglobulin Heavy Chains/immunology , Immunoglobulin Heavy Chains/ultrastructure , Immunoglobulin Light Chains/chemistry , Immunoglobulin Light Chains/genetics , Immunoglobulin Light Chains/immunology , Immunoglobulin Light Chains/ultrastructure , Male , Protein Binding , Protein Interaction Domains and Motifs , SARS-CoV-2/chemistry , Spike Glycoprotein, Coronavirus/chemistry , Vero Cells
16.
Nat Commun ; 12(1): 5469, 2021 09 22.
Article in English | MEDLINE | ID: covidwho-1434103

ABSTRACT

SARS-CoV-2 remains a global threat to human health particularly as escape mutants emerge. There is an unmet need for effective treatments against COVID-19 for which neutralizing single domain antibodies (nanobodies) have significant potential. Their small size and stability mean that nanobodies are compatible with respiratory administration. We report four nanobodies (C5, H3, C1, F2) engineered as homotrimers with pmolar affinity for the receptor binding domain (RBD) of the SARS-CoV-2 spike protein. Crystal structures show C5 and H3 overlap the ACE2 epitope, whilst C1 and F2 bind to a different epitope. Cryo Electron Microscopy shows C5 binding results in an all down arrangement of the Spike protein. C1, H3 and C5 all neutralize the Victoria strain, and the highly transmissible Alpha (B.1.1.7 first identified in Kent, UK) strain and C1 also neutralizes the Beta (B.1.35, first identified in South Africa). Administration of C5-trimer via the respiratory route showed potent therapeutic efficacy in the Syrian hamster model of COVID-19 and separately, effective prophylaxis. The molecule was similarly potent by intraperitoneal injection.


Subject(s)
Antibodies, Neutralizing/pharmacology , COVID-19/drug therapy , Single-Domain Antibodies/pharmacology , Spike Glycoprotein, Coronavirus/metabolism , Administration, Intranasal , Animals , Antibodies, Neutralizing/administration & dosage , Antibodies, Neutralizing/genetics , Antibodies, Neutralizing/immunology , Cryoelectron Microscopy , Crystallography, X-Ray , Disease Models, Animal , Dose-Response Relationship, Immunologic , Epitopes/chemistry , Epitopes/metabolism , Female , Male , Mesocricetus , Neutralization Tests , SARS-CoV-2/drug effects , Single-Domain Antibodies/administration & dosage , Single-Domain Antibodies/immunology , Single-Domain Antibodies/metabolism , Spike Glycoprotein, Coronavirus/chemistry
17.
J Biol Chem ; 297(4): 101238, 2021 10.
Article in English | MEDLINE | ID: covidwho-1433455

ABSTRACT

The D614G mutation in the spike protein of SARS-CoV-2 alters the fitness of the virus, leading to the dominant form observed in the COVID-19 pandemic. However, the molecular basis of the mechanism by which this mutation enhances fitness is not clear. Here we demonstrated by cryo-electron microscopy that the D614G mutation resulted in increased propensity of multiple receptor-binding domains (RBDs) in an upward conformation poised for host receptor binding. Multiple substates within the one RBD-up or two RBD-up conformational space were determined. According to negative staining electron microscopy, differential scanning calorimetry, and differential scanning fluorimetry, the most significant impact of the mutation lies in its ability to eliminate the unusual cold-induced unfolding characteristics and to significantly increase the thermal stability under physiological pH. The D614G spike variant also exhibited exceptional long-term stability when stored at 37 °C for up to 2 months. Our findings shed light on how the D614G mutation enhances the infectivity of SARS-CoV-2 through a stabilizing mutation and suggest an approach for better design of spike protein-based conjugates for vaccine development.


Subject(s)
SARS-CoV-2/metabolism , Spike Glycoprotein, Coronavirus/metabolism , COVID-19/pathology , COVID-19/virology , Calorimetry, Differential Scanning , Cryoelectron Microscopy , Humans , Mutagenesis, Site-Directed , Protein Domains , Protein Stability , Protein Structure, Quaternary , Recombinant Proteins/biosynthesis , Recombinant Proteins/chemistry , Recombinant Proteins/isolation & purification , SARS-CoV-2/isolation & purification , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/genetics , Temperature
18.
Cell Rep ; 37(2): 109822, 2021 10 12.
Article in English | MEDLINE | ID: covidwho-1433046

ABSTRACT

Potent neutralizing monoclonal antibodies are one of the few agents currently available to treat COVID-19. SARS-CoV-2 variants of concern (VOCs) that carry multiple mutations in the viral spike protein can exhibit neutralization resistance, potentially affecting the effectiveness of some antibody-based therapeutics. Here, the generation of a diverse panel of 91 human, neutralizing monoclonal antibodies provides an in-depth structural and phenotypic definition of receptor binding domain (RBD) antigenic sites on the viral spike. These RBD antibodies ameliorate SARS-CoV-2 infection in mice and hamster models in a dose-dependent manner and in proportion to in vitro, neutralizing potency. Assessing the effect of mutations in the spike protein on antibody recognition and neutralization highlights both potent single antibodies and stereotypic classes of antibodies that are unaffected by currently circulating VOCs, such as B.1.351 and P.1. These neutralizing monoclonal antibodies and others that bind analogous epitopes represent potentially useful future anti-SARS-CoV-2 therapeutics.


Subject(s)
Angiotensin-Converting Enzyme 2/immunology , Antibodies, Neutralizing/immunology , SARS-CoV-2/immunology , Angiotensin-Converting Enzyme 2/metabolism , Angiotensin-Converting Enzyme 2/ultrastructure , Animals , Antibodies, Monoclonal/immunology , Antibodies, Neutralizing/therapeutic use , Antibodies, Neutralizing/ultrastructure , Antibodies, Viral/immunology , COVID-19/immunology , Cricetinae , Cryoelectron Microscopy/methods , Epitopes/immunology , Female , Humans , Male , Mice , Mice, Inbred C57BL , Middle Aged , Neutralization Tests , Protein Binding/physiology , Receptors, Virus/metabolism , SARS-CoV-2/pathogenicity , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/immunology
19.
Nat Commun ; 12(1): 5493, 2021 09 17.
Article in English | MEDLINE | ID: covidwho-1428816

ABSTRACT

Macromolecular dynamics manifest as disorder in structure determination, which is subsequently accounted for by displacement parameters (also called temperature factors, or B-factors) or alternate conformations. Though B-factors contain detailed information about structural dynamics, they are the total of multiple sources of disorder, making them difficult to interpret and thus little-used in structural analysis. We report here an analytical approach for decomposing molecular disorder into a parsimonious hierarchical series of contributions, providing an intuitive basis for quantitative structural-dynamics analysis. We demonstrate the decomposition of disorder on example SARS-CoV-2 and STEAP4 structures, from both crystallographic and cryo-electron microscopy data, and reveal how understanding of the macromolecular disorder leads to deeper understanding of molecular motions and flexibility, and suggests hypotheses for molecular mechanisms.


Subject(s)
Coronavirus 3C Proteases/chemistry , Macromolecular Substances/chemistry , Molecular Dynamics Simulation , SARS-CoV-2/enzymology , COVID-19 , Cryoelectron Microscopy , Humans , Membrane Proteins/chemistry , Oxidoreductases/chemistry , Protein Conformation
20.
J Biol Chem ; 297(4): 101202, 2021 10.
Article in English | MEDLINE | ID: covidwho-1428100

ABSTRACT

Combating the worldwide spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and the emergence of new variants demands understanding of the structural basis of the interaction of antibodies with the SARS-CoV-2 receptor-binding domain (RBD). Here, we report five X-ray crystal structures of sybodies (synthetic nanobodies) including those of binary and ternary complexes of Sb16-RBD, Sb45-RBD, Sb14-RBD-Sb68, and Sb45-RBD-Sb68, as well as unliganded Sb16. These structures reveal that Sb14, Sb16, and Sb45 bind the RBD at the angiotensin-converting enzyme 2 interface and that the Sb16 interaction is accompanied by a large conformational adjustment of complementarity-determining region 2. In contrast, Sb68 interacts at the periphery of the SARS-CoV-2 RBD-angiotensin-converting enzyme 2 interface. We also determined cryo-EM structures of Sb45 bound to the SARS-CoV-2 spike protein. Superposition of the X-ray structures of sybodies onto the trimeric spike protein cryo-EM map indicates that some sybodies may bind in both "up" and "down" configurations, but others may not. Differences in sybody recognition of several recently identified RBD variants are explained by these structures.


Subject(s)
Antigen-Antibody Complex , Single-Domain Antibodies/immunology , Spike Glycoprotein, Coronavirus/immunology , Amino Acid Sequence , Angiotensin-Converting Enzyme 2/chemistry , Angiotensin-Converting Enzyme 2/genetics , Angiotensin-Converting Enzyme 2/metabolism , COVID-19/pathology , COVID-19/virology , Cryoelectron Microscopy , Crystallography, X-Ray , Humans , Protein Binding , Protein Domains , Protein Stability , SARS-CoV-2/isolation & purification , SARS-CoV-2/metabolism , Sequence Alignment , Single-Domain Antibodies/chemistry , Single-Domain Antibodies/metabolism , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/metabolism
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