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1.
Structure ; 30(4): 590-607.e4, 2022 Apr 07.
Article in English | MEDLINE | ID: covidwho-1638219

ABSTRACT

Recent developments in the SARS-CoV-2 pandemic point to its inevitable transformation into an endemic disease, urging both refinement of diagnostics for emerging variants of concern (VOCs) and design of variant-specific drugs in addition to vaccine adjustments. Exploring the structure and dynamics of the SARS-CoV-2 Spike protein, we argue that the high-mutability characteristic of RNA viruses coupled with the remarkable flexibility and dynamics of viral proteins result in a substantial involvement of allosteric mechanisms. While allosteric effects of mutations should be considered in predictions and diagnostics of new VOCs, allosteric drugs advantageously avoid escape mutations via non-competitive inhibition originating from alternative distal locations. The exhaustive allosteric signaling and probing maps presented herein provide a comprehensive picture of allostery in the spike protein, making it possible to locate potential mutations that could work as new VOC "drivers" and to determine binding patches that may be targeted by newly developed allosteric drugs.


Subject(s)
COVID-19 , Spike Glycoprotein, Coronavirus , COVID-19/drug therapy , Humans , Mutation , Pandemics , SARS-CoV-2/genetics , Spike Glycoprotein, Coronavirus/genetics
2.
J Mol Biol ; 434(2): 167332, 2022 01 30.
Article in English | MEDLINE | ID: covidwho-1492301

ABSTRACT

Extensive glycosylation of viral glycoproteins is a key feature of the antigenic surface of viruses and yet glycan processing can also be influenced by the manner of their recombinant production. The low yields of the soluble form of the trimeric spike (S) glycoprotein from SARS-CoV-2 has prompted advances in protein engineering that have greatly enhanced the stability and yields of the glycoprotein. The latest expression-enhanced version of the spike incorporates six proline substitutions to stabilize the prefusion conformation (termed SARS-CoV-2 S HexaPro). Although the substitutions greatly enhanced expression whilst not compromising protein structure, the influence of these substitutions on glycan processing has not been explored. Here, we show that the site-specific N-linked glycosylation of the expression-enhanced HexaPro resembles that of an earlier version containing two proline substitutions (2P), and that both capture features of native viral glycosylation. However, there are site-specific differences in glycosylation of HexaPro when compared to 2P. Despite these discrepancies, analysis of the serological reactivity of clinical samples from infected individuals confirmed that both HexaPro and 2P protein are equally able to detect IgG, IgA, and IgM responses in all sera analysed. Moreover, we extend this observation to include an analysis of glycan engineered S protein, whereby all N-linked glycans were converted to oligomannose-type and conclude that serological activity is not impacted by large scale changes in glycosylation. These observations suggest that variations in glycan processing will not impact the serological assessments currently being performed across the globe.


Subject(s)
Antibodies, Viral/immunology , COVID-19/immunology , Mutation, Missense/immunology , SARS-CoV-2/immunology , Spike Glycoprotein, Coronavirus/immunology , Antibodies, Viral/blood , Binding Sites/genetics , COVID-19/virology , Glycosylation , Humans , Immunoglobulin A/blood , Immunoglobulin A/immunology , Immunoglobulin G/blood , Immunoglobulin G/immunology , Immunoglobulin M/blood , Immunoglobulin M/immunology , Mannose/metabolism , Mutation, Missense/genetics , Oligosaccharides/metabolism , Polysaccharides/metabolism , Proline/genetics , Proline/immunology , Proline/metabolism , Recombinant Proteins/chemistry , Recombinant Proteins/immunology , Recombinant Proteins/metabolism , SARS-CoV-2/metabolism , SARS-CoV-2/physiology , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/metabolism
3.
Biochemistry ; 60(27): 2153-2169, 2021 07 13.
Article in English | MEDLINE | ID: covidwho-1387101

ABSTRACT

A central tenet in the design of vaccines is the display of native-like antigens in the elicitation of protective immunity. The abundance of N-linked glycans across the SARS-CoV-2 spike protein is a potential source of heterogeneity among the many different vaccine candidates under investigation. Here, we investigate the glycosylation of recombinant SARS-CoV-2 spike proteins from five different laboratories and compare them against S protein from infectious virus, cultured in Vero cells. We find patterns that are conserved across all samples, and this can be associated with site-specific stalling of glycan maturation that acts as a highly sensitive reporter of protein structure. Molecular dynamics simulations of a fully glycosylated spike support a model of steric restrictions that shape enzymatic processing of the glycans. These results suggest that recombinant spike-based SARS-CoV-2 immunogen glycosylation reproducibly recapitulates signatures of viral glycosylation.


Subject(s)
COVID-19/genetics , Protein Conformation , SARS-CoV-2/ultrastructure , Spike Glycoprotein, Coronavirus/ultrastructure , Animals , COVID-19/immunology , COVID-19/virology , COVID-19 Vaccines/genetics , COVID-19 Vaccines/immunology , Chlorocebus aethiops , Glycosylation , Humans , Molecular Dynamics Simulation , Protein Binding/genetics , SARS-CoV-2/genetics , SARS-CoV-2/pathogenicity , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/immunology , Vero Cells
4.
Biochemistry ; 60(27): 2153-2169, 2021 07 13.
Article in English | MEDLINE | ID: covidwho-1294429

ABSTRACT

A central tenet in the design of vaccines is the display of native-like antigens in the elicitation of protective immunity. The abundance of N-linked glycans across the SARS-CoV-2 spike protein is a potential source of heterogeneity among the many different vaccine candidates under investigation. Here, we investigate the glycosylation of recombinant SARS-CoV-2 spike proteins from five different laboratories and compare them against S protein from infectious virus, cultured in Vero cells. We find patterns that are conserved across all samples, and this can be associated with site-specific stalling of glycan maturation that acts as a highly sensitive reporter of protein structure. Molecular dynamics simulations of a fully glycosylated spike support a model of steric restrictions that shape enzymatic processing of the glycans. These results suggest that recombinant spike-based SARS-CoV-2 immunogen glycosylation reproducibly recapitulates signatures of viral glycosylation.


Subject(s)
COVID-19/genetics , Protein Conformation , SARS-CoV-2/ultrastructure , Spike Glycoprotein, Coronavirus/ultrastructure , Animals , COVID-19/immunology , COVID-19/virology , COVID-19 Vaccines/genetics , COVID-19 Vaccines/immunology , Chlorocebus aethiops , Glycosylation , Humans , Molecular Dynamics Simulation , Protein Binding/genetics , SARS-CoV-2/genetics , SARS-CoV-2/pathogenicity , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/immunology , Vero Cells
5.
Elife ; 102021 02 08.
Article in English | MEDLINE | ID: covidwho-1069944

ABSTRACT

The spike (S) protein is the main handle for SARS-CoV-2 to enter host cells via surface angiotensin-converting enzyme 2 (ACE2) receptors. How ACE2 binding activates proteolysis of S protein is unknown. Here, using amide hydrogen-deuterium exchange mass spectrometry and molecular dynamics simulations, we have mapped the S:ACE2 interaction interface and uncovered long-range allosteric propagation of ACE2 binding to sites necessary for host-mediated proteolysis of S protein, critical for viral host entry. Unexpectedly, ACE2 binding enhances dynamics at a distal S1/S2 cleavage site and flanking protease docking site ~27 Å away while dampening dynamics of the stalk hinge (central helix and heptad repeat [HR]) regions ~130 Å away. This highlights that the stalk and proteolysis sites of the S protein are dynamic hotspots in the prefusion state. Our findings provide a dynamics map of the S:ACE2 interface in solution and also offer mechanistic insights into how ACE2 binding is allosterically coupled to distal proteolytic processing sites and viral-host membrane fusion. Thus, protease docking sites flanking the S1/S2 cleavage site represent alternate allosteric hotspot targets for potential therapeutic development.


Subject(s)
Angiotensin-Converting Enzyme 2/metabolism , COVID-19/virology , SARS-CoV-2/physiology , Spike Glycoprotein, Coronavirus/metabolism , Allosteric Site , Amino Acid Sequence , Angiotensin-Converting Enzyme 2/chemistry , Binding Sites , COVID-19/metabolism , Humans , Mass Spectrometry/methods , Molecular Dynamics Simulation , Protein Binding , Protein Processing, Post-Translational , Proteolysis , Receptors, Virus/chemistry , Receptors, Virus/metabolism , SARS-CoV-2/metabolism , Spike Glycoprotein, Coronavirus/chemistry , Virus Internalization
6.
J Mol Cell Biol ; 12(12): 916-932, 2020 10 12.
Article in English | MEDLINE | ID: covidwho-968717

ABSTRACT

There is a link between high lipopolysaccharide (LPS) levels in the blood and the metabolic syndrome, and metabolic syndrome predisposes patients to severe COVID-19. Here, we define an interaction between SARS-CoV-2 spike (S) protein and LPS, leading to aggravated inflammation in vitro and in vivo. Native gel electrophoresis demonstrated that SARS-CoV-2 S protein binds to LPS. Microscale thermophoresis yielded a KD of ∼47 nM for the interaction. Computational modeling and all-atom molecular dynamics simulations further substantiated the experimental results, identifying a main LPS-binding site in SARS-CoV-2 S protein. S protein, when combined with low levels of LPS, boosted nuclear factor-kappa B (NF-κB) activation in monocytic THP-1 cells and cytokine responses in human blood and peripheral blood mononuclear cells, respectively. The in vitro inflammatory response was further validated by employing NF-κB reporter mice and in vivo bioimaging. Dynamic light scattering, transmission electron microscopy, and LPS-FITC analyses demonstrated that S protein modulated the aggregation state of LPS, providing a molecular explanation for the observed boosting effect. Taken together, our results provide an interesting molecular link between excessive inflammation during infection with SARS-CoV-2 and comorbidities involving increased levels of bacterial endotoxins.


Subject(s)
COVID-19/complications , Inflammation/etiology , Lipopolysaccharides/metabolism , SARS-CoV-2 , Spike Glycoprotein, Coronavirus/metabolism , Animals , Binding Sites , COVID-19/immunology , COVID-19/virology , Cytokine Release Syndrome/etiology , Cytokine Release Syndrome/immunology , Disease Models, Animal , Gram-Negative Bacterial Infections/complications , Gram-Negative Bacterial Infections/immunology , Humans , In Vitro Techniques , Lipid A/chemistry , Lipid A/immunology , Lipid A/metabolism , Lipopolysaccharides/chemistry , Lipopolysaccharides/immunology , Mice , Mice, Inbred BALB C , Mice, Transgenic , Models, Immunological , Models, Molecular , Molecular Docking Simulation , Protein Binding , Protein Interaction Domains and Motifs , Respiratory Distress Syndrome/etiology , Risk Factors , SARS-CoV-2/immunology , SARS-CoV-2/pathogenicity , SARS-CoV-2/physiology , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/immunology
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