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1.
Nat Commun ; 13(1): 1214, 2022 03 03.
Article in English | MEDLINE | ID: covidwho-1730288

ABSTRACT

The omicron variant of SARS-CoV-2 has been spreading rapidly across the globe. The virus-surface spike protein plays a critical role in the cell entry and immune evasion of SARS-CoV-2. Here we determined the 3.0 Å cryo-EM structure of the omicron spike protein ectodomain. In contrast to the original strain of SARS-CoV-2 where the receptor-binding domain (RBD) of the spike protein takes a mixture of open ("standing up") and closed ("lying down") conformations, the omicron spike molecules are predominantly in the open conformation, with one upright RBD ready for receptor binding. The open conformation of the omicron spike is stabilized by enhanced inter-domain and inter-subunit packing, which involves new mutations in the omicron strain. Moreover, the omicron spike has undergone extensive mutations in RBD regions where known neutralizing antibodies target, allowing the omicron variant to escape immune surveillance aimed at the original viral strain. The stable open conformation of the omicron spike sheds light on the cell entry and immune evasion mechanisms of the omicron variant.


Subject(s)
COVID-19/virology , SARS-CoV-2/chemistry , SARS-CoV-2/ultrastructure , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/ultrastructure , Antibodies, Neutralizing/immunology , Antibodies, Viral/immunology , COVID-19/immunology , Cryoelectron Microscopy , Humans , Immune Evasion/genetics , Models, Molecular , Mutation , Pandemics , Protein Conformation , Protein Domains/genetics , Protein Domains/immunology , Protein Interaction Domains and Motifs/genetics , Protein Interaction Domains and Motifs/immunology , SARS-CoV-2/genetics , Spike Glycoprotein, Coronavirus/genetics , Virus Internalization
2.
Int J Mol Sci ; 23(3)2022 Jan 31.
Article in English | MEDLINE | ID: covidwho-1667196

ABSTRACT

Structural and functional studies of the SARS-CoV-2 spike proteins have recently determined distinct functional states of the B.1.1.7 and B.1.351 spike variants, providing a molecular framework for understanding the mechanisms that link the effect of mutations with the enhanced virus infectivity and transmissibility. A detailed dynamic and energetic analysis of these variants was undertaken in the present work to quantify the effects of different mutations on functional conformational changes and stability of the SARS-CoV-2 spike protein. We employed the efficient and accurate coarse-grained (CG) simulations of multiple functional states of the D614G mutant, B.1.1.7 and B.1.351 spike variants to characterize conformational dynamics of the SARS-CoV-2 spike proteins and identify dynamic signatures of the functional regions that regulate transitions between the closed and open forms. By combining molecular simulations with full atomistic reconstruction of the trajectories and the ensemble-based mutational frustration analysis, we characterized how the intrinsic flexibility of specific spike regions can control functional conformational changes required for binding with the host-cell receptor. Using the residue-based mutational scanning of protein stability, we determined protein stability hotspots and identified potential energetic drivers favoring the receptor-accessible open spike states for the B.1.1.7 and B.1.351 spike variants. The results suggested that modulation of the energetic frustration at the inter-protomer interfaces can serve as a mechanism for allosteric couplings between mutational sites and the inter-protomer hinges of functional motions. The proposed mechanism of mutation-induced energetic frustration may result in greater adaptability and the emergence of multiple conformational states in the open form. This study suggested that SARS-CoV-2 B.1.1.7 and B.1.351 variants may leverage the intrinsic plasticity of functional regions in the spike protein for mutation-induced modulation of protein dynamics and allosteric regulation to control binding with the host cell receptor.


Subject(s)
COVID-19/metabolism , SARS-CoV-2/ultrastructure , Spike Glycoprotein, Coronavirus/ultrastructure , Allosteric Regulation , Binding Sites , COVID-19/pathology , Humans , Molecular Conformation , Molecular Dynamics Simulation , Mutation , Protein Binding , Protein Stability , SARS-CoV-2/genetics , SARS-CoV-2/pathogenicity , Spike Glycoprotein, Coronavirus/genetics
3.
Molecules ; 27(3)2022 Jan 26.
Article in English | MEDLINE | ID: covidwho-1650788

ABSTRACT

The entry of the SARS-CoV-2, a causative agent of COVID-19, into human host cells is mediated by the SARS-CoV-2 spike (S) glycoprotein, which critically depends on the formation of complexes involving the spike protein receptor-binding domain (RBD) and the human cellular membrane receptor angiotensin-converting enzyme 2 (hACE2). Using classical site density functional theory (SDFT) and structural bioinformatics methods, we investigate binding and conformational properties of these complexes and study the overlooked role of water-mediated interactions. Analysis of the three-dimensional reference interaction site model (3DRISM) of SDFT indicates that water mediated interactions in the form of additional water bridges strongly increases the binding between SARS-CoV-2 spike protein and hACE2 compared to SARS-CoV-1-hACE2 complex. By analyzing structures of SARS-CoV-2 and SARS-CoV-1, we find that the homotrimer SARS-CoV-2 S receptor-binding domain (RBD) has expanded in size, indicating large conformational change relative to SARS-CoV-1 S protein. Protomer with the up-conformational form of RBD, which binds with hACE2, exhibits stronger intermolecular interactions at the RBD-ACE2 interface, with differential distributions and the inclusion of specific H-bonds in the CoV-2 complex. Further interface analysis has shown that interfacial water promotes and stabilizes the formation of CoV-2/hACE2 complex. This interaction causes a significant structural rigidification of the spike protein, favoring proteolytic processing of the S protein for the fusion of the viral and cellular membrane. Moreover, conformational dynamics simulations of RBD motions in SARS-CoV-2 and SARS-CoV-1 point to the role in modification of the RBD dynamics and their impact on infectivity.


Subject(s)
Angiotensin-Converting Enzyme 2/ultrastructure , SARS-CoV-2/metabolism , Spike Glycoprotein, Coronavirus/ultrastructure , Angiotensin-Converting Enzyme 2/metabolism , COVID-19/metabolism , COVID-19/physiopathology , Computational Biology/methods , Density Functional Theory , Humans , Models, Theoretical , Protein Binding , Protein Domains , SARS-CoV-2/pathogenicity , Spike Glycoprotein, Coronavirus/metabolism , Spike Glycoprotein, Coronavirus/physiology , Structure-Activity Relationship
4.
Cell ; 185(4): 630-640.e10, 2022 02 17.
Article in English | MEDLINE | ID: covidwho-1611650

ABSTRACT

The coronavirus disease 2019 (COVID-19) pandemic continues worldwide with many variants arising, some of which are variants of concern (VOCs). A recent VOC, omicron (B.1.1.529), which obtains a large number of mutations in the receptor-binding domain (RBD) of the spike protein, has risen to intense scientific and public attention. Here, we studied the binding properties between the human receptor ACE2 (hACE2) and the VOC RBDs and resolved the crystal and cryoelectron microscopy structures of the omicron RBD-hACE2 complex as well as the crystal structure of the delta RBD-hACE2 complex. We found that, unlike alpha, beta, and gamma, omicron RBD binds to hACE2 at a similar affinity to that of the prototype RBD, which might be due to compensation of multiple mutations for both immune escape and transmissibility. The complex structures of omicron RBD-hACE2 and delta RBD-hACE2 reveal the structural basis of how RBD-specific mutations bind to hACE2.


Subject(s)
Angiotensin-Converting Enzyme 2/chemistry , Receptors, Virus/chemistry , SARS-CoV-2/chemistry , Amino Acid Sequence , Cryoelectron Microscopy , Humans , Models, Molecular , Mutation/genetics , Phylogeny , Protein Binding , Protein Domains , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/ultrastructure , Static Electricity , Structural Homology, Protein
5.
Int Immunopharmacol ; 102: 108424, 2022 Jan.
Article in English | MEDLINE | ID: covidwho-1549851

ABSTRACT

SARS-CoV2 mutants B.1.1.7, B.1.351, and P.1 contain a key mutation N501Y. B.1.135 and P.1 lineages have another mutation, E484K. Here, we decode the effect of these two mutations on the host receptor, ACE2, and neutralizing antibody (B38) recognition. The N501Y RBD mutant binds to ACE2 with higher affinity due to improved π-π stacking and π-cation interactions. The higher binding affinity of the E484K mutant is caused due to the formation of additional hydrogen bond and salt-bridge interactions with ACE2. Both the mutants bind to the B38 antibody with reduced affinity due to the loss of several hydrogen-bonding interactions. The insights obtained from the study are crucial to interpret the increased transmissibility and reduced neutralization efficacy of rapidly emerging SARS-CoV2 VOCs.


Subject(s)
Angiotensin-Converting Enzyme 2/metabolism , Antibodies, Neutralizing/metabolism , SARS-CoV-2/pathogenicity , Spike Glycoprotein, Coronavirus/genetics , Angiotensin-Converting Enzyme 2/ultrastructure , Antibody Affinity/genetics , Binding Sites/genetics , Crystallography, X-Ray , Humans , Mutation , SARS-CoV-2/genetics , SARS-CoV-2/immunology , Spike Glycoprotein, Coronavirus/immunology , Spike Glycoprotein, Coronavirus/metabolism , Spike Glycoprotein, Coronavirus/ultrastructure , Virus Internalization
6.
Nat Commun ; 12(1): 6103, 2021 10 20.
Article in English | MEDLINE | ID: covidwho-1475296

ABSTRACT

Multiple SARS-CoV-2 variants of concern (VOCs) have been emerging and some have been linked to an increase in case numbers globally. However, there is yet a lack of understanding of the molecular basis for the interactions between the human ACE2 (hACE2) receptor and these VOCs. Here we examined several VOCs including Alpha, Beta, and Gamma, and demonstrate that five variants receptor-binding domain (RBD) increased binding affinity for hACE2, and four variants pseudoviruses increased entry into susceptible cells. Crystal structures of hACE2-RBD complexes help identify the key residues facilitating changes in hACE2 binding affinity. Additionally, soluble hACE2 protein efficiently prevent most of the variants pseudoviruses. Our findings provide important molecular information and may help the development of novel therapeutic and prophylactic agents targeting these emerging mutants.


Subject(s)
Angiotensin-Converting Enzyme 2/metabolism , COVID-19/virology , Protein Interaction Domains and Motifs/genetics , SARS-CoV-2/metabolism , Spike Glycoprotein, Coronavirus/metabolism , Amino Acid Sequence , Angiotensin-Converting Enzyme 2/genetics , Angiotensin-Converting Enzyme 2/isolation & purification , Angiotensin-Converting Enzyme 2/ultrastructure , Animals , Cell Line, Tumor , Crystallography, X-Ray , HEK293 Cells , Humans , Molecular Dynamics Simulation , Mutation , Recombinant Proteins/genetics , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolism , Recombinant Proteins/ultrastructure , SARS-CoV-2/genetics , Sf9 Cells , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/isolation & purification , Spike Glycoprotein, Coronavirus/ultrastructure , Spodoptera , Surface Plasmon Resonance , Virus Attachment , Virus Internalization
7.
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
8.
Viruses ; 13(10)2021 09 25.
Article in English | MEDLINE | ID: covidwho-1438747

ABSTRACT

Recently, two cases of complete remission of classical Hodgkin lymphoma (cHL) and follicular lymphoma (FL) after SARS-CoV-2 infection were reported. However, the precise molecular mechanism of this rare event is yet to be understood. Here, we hypothesize a potential anti-tumor immune response of SARS-CoV-2 and based on a computational approach show that: (i) SARS-CoV-2 Spike-RBD may bind to the extracellular domains of CD15, CD27, CD45, and CD152 receptors of cHL or FL and may directly inhibit cell proliferation. (ii) Alternately, upon internalization after binding to these CD molecules, the SARS-CoV-2 membrane (M) protein and ORF3a may bind to gamma-tubulin complex component 3 (GCP3) at its tubulin gamma-1 chain (TUBG1) binding site. (iii) The M protein may also interact with TUBG1, blocking its binding to GCP3. (iv) Both the M and ORF3a proteins may render the GCP2-GCP3 lateral binding where the M protein possibly interacts with GCP2 at its GCP3 binding site and the ORF3a protein to GCP3 at its GCP2 interacting residues. (v) Interactions of the M and ORF3a proteins with these gamma-tubulin ring complex components potentially block the initial process of microtubule nucleation, leading to cell-cycle arrest and apoptosis. (vi) The Spike-RBD may also interact with and block PD-1 signaling similar to pembrolizumab and nivolumab- like monoclonal antibodies and may induce B-cell apoptosis and remission. (vii) Finally, the TRADD interacting "PVQLSY" motif of Epstein-Barr virus LMP-1, that is responsible for NF-kB mediated oncogenesis, potentially interacts with SARS-CoV-2 Mpro, NSP7, NSP10, and spike (S) proteins, and may inhibit the LMP-1 mediated cell proliferation. Taken together, our results suggest a possible therapeutic potential of SARS-CoV-2 in lymphoproliferative disorders.


Subject(s)
COVID-19/metabolism , Lymphoma/immunology , SARS-CoV-2/immunology , Antibodies, Monoclonal/immunology , Antineoplastic Agents/pharmacology , Binding Sites , COVID-19/complications , Glycoproteins/metabolism , Glycoproteins/ultrastructure , Humans , Immunity/immunology , Lymphoma/therapy , Lymphoma/virology , Models, Theoretical , Molecular Docking Simulation , Protein Binding , Protein Domains , Spike Glycoprotein, Coronavirus/immunology , Spike Glycoprotein, Coronavirus/ultrastructure , Viroporin Proteins/metabolism , Viroporin Proteins/ultrastructure
9.
Cell Rep ; 37(2): 109814, 2021 10 12.
Article in English | MEDLINE | ID: covidwho-1433045

ABSTRACT

Control of the ongoing SARS-CoV-2 pandemic is endangered by the emergence of viral variants with increased transmission efficiency, resistance to marketed therapeutic antibodies, and reduced sensitivity to vaccine-induced immunity. Here, we screen B cells from COVID-19 donors and identify P5C3, a highly potent and broadly neutralizing monoclonal antibody with picomolar neutralizing activity against all SARS-CoV-2 variants of concern (VOCs) identified to date. Structural characterization of P5C3 Fab in complex with the spike demonstrates a neutralizing activity defined by a large buried surface area, highly overlapping with the receptor-binding domain (RBD) surface necessary for ACE2 interaction. We further demonstrate that P5C3 shows complete prophylactic protection in the SARS-CoV-2-infected hamster challenge model. These results indicate that P5C3 opens exciting perspectives either as a prophylactic agent in immunocompromised individuals with poor response to vaccination or as combination therapy in SARS-CoV-2-infected individuals.


Subject(s)
Broadly Neutralizing Antibodies/therapeutic use , COVID-19/drug therapy , SARS-CoV-2/immunology , Animals , Antibodies, Neutralizing/immunology , Antibodies, Viral/immunology , B-Lymphocytes/immunology , Broadly Neutralizing Antibodies/immunology , COVID-19/immunology , Cell Line , Cricetinae , Disease Models, Animal , Epitopes/immunology , Humans , Immunoglobulin Fab Fragments/immunology , Immunoglobulin Fab Fragments/metabolism , Neutralization Tests , Protein Binding/immunology , SARS-CoV-2/pathogenicity , Spike Glycoprotein, Coronavirus/immunology , Spike Glycoprotein, Coronavirus/ultrastructure , Structure-Activity Relationship , Vaccination
10.
Nat Commun ; 12(1): 141, 2021 01 08.
Article in English | MEDLINE | ID: covidwho-1387322

ABSTRACT

Coronaviruses spike (S) glycoproteins mediate viral entry into host cells by binding to host receptors. However, how the S1 subunit undergoes conformational changes for receptor recognition has not been elucidated in Alphacoronavirus. Here, we report the cryo-EM structures of the HCoV-229E S trimer in prefusion state with two conformations. The activated conformation may pose the potential exposure of the S1-RBDs by decreasing of the interaction area between the S1-RBDs and the surrounding S1-NTDs and S1-RBDs compared to the closed conformation. Furthermore, structural comparison of our structures with the previously reported HCoV-229E S structure showed that the S trimers trended to open the S2 subunit from the closed conformation to open conformation, which could promote the transition from pre- to postfusion. Our results provide insights into the mechanisms involved in S glycoprotein-mediated Alphacoronavirus entry and have implications for vaccine and therapeutic antibody design.


Subject(s)
CD13 Antigens/metabolism , Coronavirus 229E, Human/physiology , Spike Glycoprotein, Coronavirus/metabolism , Virus Internalization , Cell Line, Tumor , Coronavirus Infections/prevention & control , Coronavirus Infections/virology , Cryoelectron Microscopy , Humans , Models, Molecular , Protein Conformation, alpha-Helical , Protein Multimerization , Protein Structure, Quaternary , Protein Subunits/metabolism , Spike Glycoprotein, Coronavirus/ultrastructure
11.
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
12.
Cell Host Microbe ; 28(6): 867-879.e5, 2020 12 09.
Article in English | MEDLINE | ID: covidwho-1385264

ABSTRACT

The SARS-CoV-2 spike employs mobile receptor-binding domains (RBDs) to engage the human ACE2 receptor and to facilitate virus entry, which can occur through low-pH-endosomal pathways. To understand how ACE2 binding and low pH affect spike conformation, we determined cryo-electron microscopy structures-at serological and endosomal pH-delineating spike recognition of up to three ACE2 molecules. RBDs freely adopted "up" conformations required for ACE2 interaction, primarily through RBD movement combined with smaller alterations in neighboring domains. In the absence of ACE2, single-RBD-up conformations dominated at pH 5.5, resolving into a solitary all-down conformation at lower pH. Notably, a pH-dependent refolding region (residues 824-858) at the spike-interdomain interface displayed dramatic structural rearrangements and mediated RBD positioning through coordinated movements of the entire trimer apex. These structures provide a foundation for understanding prefusion-spike mechanics governing endosomal entry; we suggest that the low pH all-down conformation potentially facilitates immune evasion from RBD-up binding antibody.


Subject(s)
Angiotensin-Converting Enzyme 2/genetics , COVID-19/genetics , Pandemics , Spike Glycoprotein, Coronavirus/ultrastructure , Amino Acid Sequence/genetics , Angiotensin-Converting Enzyme 2/ultrastructure , Antibodies, Neutralizing/genetics , Antibodies, Neutralizing/immunology , Binding Sites , COVID-19/pathology , COVID-19/virology , Cryoelectron Microscopy , Endosomes/ultrastructure , Humans , Hydrogen-Ion Concentration , Protein Binding , Protein Domains , Receptors, Virus/genetics , Receptors, Virus/ultrastructure , SARS-CoV-2/genetics , SARS-CoV-2/ultrastructure , Spike Glycoprotein, Coronavirus/genetics
13.
Molecules ; 26(17)2021 Aug 24.
Article in English | MEDLINE | ID: covidwho-1374471

ABSTRACT

The emergence of COVID-19 continues to pose severe threats to global public health. The pandemic has infected over 171 million people and claimed more than 3.5 million lives to date. We investigated the binding potential of antiviral cyanobacterial proteins including cyanovirin-N, scytovirin and phycocyanin with fundamental proteins involved in attachment and replication of SARS-CoV-2. Cyanovirin-N displayed the highest binding energy scores (-16.8 ± 0.02 kcal/mol, -12.3 ± 0.03 kcal/mol and -13.4 ± 0.02 kcal/mol, respectively) with the spike protein, the main protease (Mpro) and the papainlike protease (PLpro) of SARS-CoV-2. Cyanovirin-N was observed to interact with the crucial residues involved in the attachment of the human ACE2 receptor. Analysis of the binding affinities calculated employing the molecular mechanics-Poisson-Boltzmann surface area (MM-PBSA) approach revealed that all forms of energy, except the polar solvation energy, favourably contributed to the interactions of cyanovirin-N with the viral proteins. With particular emphasis on cyanovirin-N, the current work presents evidence for the potential inhibition of SARS-CoV-2 by cyanobacterial proteins, and offers the opportunity for in vitro and in vivo experiments to deploy the cyanobacterial proteins as valuable therapeutics against COVID-19.


Subject(s)
Antiviral Agents/pharmacology , Bacterial Proteins/pharmacology , COVID-19/drug therapy , Coronavirus Protease Inhibitors/pharmacology , Antiviral Agents/therapeutic use , Bacterial Proteins/therapeutic use , Bacterial Proteins/ultrastructure , COVID-19/virology , Coronavirus 3C Proteases/antagonists & inhibitors , Coronavirus 3C Proteases/metabolism , Coronavirus 3C Proteases/ultrastructure , Coronavirus Papain-Like Proteases/antagonists & inhibitors , Coronavirus Papain-Like Proteases/metabolism , Coronavirus Papain-Like Proteases/ultrastructure , Coronavirus Protease Inhibitors/therapeutic use , Coronavirus Protease Inhibitors/ultrastructure , Humans , Molecular Docking Simulation , Molecular Dynamics Simulation , Protein Binding , Protein Interaction Mapping , Spike Glycoprotein, Coronavirus/antagonists & inhibitors , Spike Glycoprotein, Coronavirus/metabolism , Spike Glycoprotein, Coronavirus/ultrastructure , X-Ray Diffraction
14.
Int J Mol Sci ; 22(17)2021 Aug 24.
Article in English | MEDLINE | ID: covidwho-1374424

ABSTRACT

The Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) outbreak in December 2019 has caused a global pandemic. The rapid mutation rate in the virus has created alarming situations worldwide and is being attributed to the false negativity in RT-PCR tests. It has also increased the chances of reinfection and immune escape. Recently various lineages namely, B.1.1.7 (Alpha), B.1.617.1 (Kappa), B.1.617.2 (Delta) and B.1.617.3 have caused rapid infection around the globe. To understand the biophysical perspective, we have performed molecular dynamic simulations of four different spikes (receptor binding domain)-hACE2 complexes, namely wildtype (WT), Alpha variant (N501Y spike mutant), Kappa (L452R, E484Q) and Delta (L452R, T478K), and compared their dynamics, binding energy and molecular interactions. Our results show that mutation has caused significant increase in the binding energy between the spike and hACE2 in Alpha and Kappa variants. In the case of Kappa and Delta variants, the mutations at L452R, T478K and E484Q increased the stability and intra-chain interactions in the spike protein, which may change the interaction ability of neutralizing antibodies to these spike variants. Further, we found that the Alpha variant had increased hydrogen interaction with Lys353 of hACE2 and more binding affinity in comparison to WT. The current study provides the biophysical basis for understanding the molecular mechanism and rationale behind the increase in the transmissivity and infectivity of the mutants compared to wild-type SARS-CoV-2.


Subject(s)
Angiotensin-Converting Enzyme 2/metabolism , COVID-19/transmission , SARS-CoV-2/pathogenicity , Spike Glycoprotein, Coronavirus/metabolism , Angiotensin-Converting Enzyme 2/ultrastructure , Antibodies, Neutralizing/immunology , Antibodies, Neutralizing/metabolism , Antibodies, Viral/immunology , Antibodies, Viral/metabolism , COVID-19/virology , Crystallography, X-Ray , Humans , Molecular Dynamics Simulation , Mutation , Protein Stability , SARS-CoV-2/genetics , SARS-CoV-2/immunology , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/immunology , Spike Glycoprotein, Coronavirus/ultrastructure , Thermodynamics
15.
Nature ; 588(7838): 498-502, 2020 12.
Article in English | MEDLINE | ID: covidwho-1343462

ABSTRACT

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virions are surrounded by a lipid bilayer from which spike (S) protein trimers protrude1. Heavily glycosylated S trimers bind to the angiotensin-converting enzyme 2 receptor and mediate entry of virions into target cells2-6. S exhibits extensive conformational flexibility: it modulates exposure of its receptor-binding site and subsequently undergoes complete structural rearrangement to drive fusion of viral and cellular membranes2,7,8. The structures and conformations of soluble, overexpressed, purified S proteins have been studied in detail using cryo-electron microscopy2,7,9-12, but the structure and distribution of S on the virion surface remain unknown. Here we applied cryo-electron microscopy and tomography to image intact SARS-CoV-2 virions and determine the high-resolution structure, conformational flexibility and distribution of S trimers in situ on the virion surface. These results reveal the conformations of S on the virion, and provide a basis from which to understand interactions between S and neutralizing antibodies during infection or vaccination.


Subject(s)
Cryoelectron Microscopy , SARS-CoV-2/metabolism , SARS-CoV-2/ultrastructure , Spike Glycoprotein, Coronavirus/analysis , Spike Glycoprotein, Coronavirus/ultrastructure , Virion/chemistry , Virion/ultrastructure , Antibodies, Neutralizing/immunology , COVID-19/immunology , COVID-19 Vaccines/immunology , Cell Line, Tumor , Humans , Models, Molecular , Pliability , Protein Conformation , Protein Multimerization , SARS-CoV-2/chemistry , SARS-CoV-2/isolation & purification , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/isolation & purification , Virion/isolation & purification , Virion/metabolism
16.
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
17.
Brief Bioinform ; 22(6)2021 11 05.
Article in English | MEDLINE | ID: covidwho-1276146

ABSTRACT

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), a causative agent of the coronavirus disease (COVID-19), is a part of the $\beta $-Coronaviridae family. The virus contains five major protein classes viz., four structural proteins [nucleocapsid (N), membrane (M), envelop (E) and spike glycoprotein (S)] and replicase polyproteins (R), synthesized as two polyproteins (ORF1a and ORF1ab). Due to the severity of the pandemic, most of the SARS-CoV-2-related research are focused on finding therapeutic solutions. However, studies on the sequences and structure space throughout the evolutionary time frame of viral proteins are limited. Besides, the structural malleability of viral proteins can be directly or indirectly associated with the dysfunctionality of the host cell proteins. This dysfunctionality may lead to comorbidities during the infection and may continue at the post-infection stage. In this regard, we conduct the evolutionary sequence-structure analysis of the viral proteins to evaluate their malleability. Subsequently, intrinsic disorder propensities of these viral proteins have been studied to confirm that the short intrinsically disordered regions play an important role in enhancing the likelihood of the host proteins interacting with the viral proteins. These interactions may result in molecular dysfunctionality, finally leading to different diseases. Based on the host cell proteins, the diseases are divided in two distinct classes: (i) proteins, directly associated with the set of diseases while showing similar activities, and (ii) cytokine storm-mediated pro-inflammation (e.g. acute respiratory distress syndrome, malignancies) and neuroinflammation (e.g. neurodegenerative and neuropsychiatric diseases). Finally, the study unveils that males and postmenopausal females can be more vulnerable to SARS-CoV-2 infection due to the androgen-mediated protein transmembrane serine protease 2.


Subject(s)
COVID-19/genetics , Genome, Viral/genetics , Protein Conformation , SARS-CoV-2/ultrastructure , COVID-19/virology , Coronavirus Envelope Proteins/genetics , Coronavirus Envelope Proteins/ultrastructure , Humans , Membrane Proteins/genetics , Membrane Proteins/ultrastructure , Nucleocapsid Proteins/genetics , Nucleocapsid Proteins/ultrastructure , SARS-CoV-2/genetics , SARS-CoV-2/pathogenicity , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/ultrastructure , Viral Replicase Complex Proteins/genetics , Viral Replicase Complex Proteins/ultrastructure , Viral Structural Proteins/genetics , Viral Structural Proteins/ultrastructure
18.
Viruses ; 13(5)2021 05 11.
Article in English | MEDLINE | ID: covidwho-1224261

ABSTRACT

The emergence of SARS-CoV-2 variants, as observed with the D614G spike protein mutant and, more recently, with B.1.1.7 (501Y.V1), B.1.351 (501Y.V2) and B.1.1.28.1 (P.1) lineages, represent a continuous threat and might lead to strains of higher infectivity and/or virulence. We report on the occurrence of a SARS-CoV-2 haplotype with nine mutations including D614G/T307I double-mutation of the spike. This variant expanded and completely replaced previous lineages within a short period in the subantarctic Magallanes Region, southern Chile. The rapid lineage shift was accompanied by a significant increase of cases, resulting in one of the highest incidence rates worldwide. Comparative coarse-grained molecular dynamic simulations indicated that T307I and D614G belong to a previously unrecognized dynamic domain, interfering with the mobility of the receptor binding domain of the spike. The T307I mutation showed a synergistic effect with the D614G. Continuous surveillance of new mutations and molecular analyses of such variations are important tools to understand the molecular mechanisms defining infectivity and virulence of current and future SARS-CoV-2 strains.


Subject(s)
SARS-CoV-2/genetics , Spike Glycoprotein, Coronavirus/genetics , Antarctic Regions , Antibodies, Neutralizing/metabolism , Antibodies, Viral/genetics , COVID-19/epidemiology , COVID-19/genetics , COVID-19/metabolism , Chile , Haplotypes/genetics , Humans , Mutant Proteins/genetics , Mutation , Protein Binding , SARS-CoV-2/pathogenicity , Spike Glycoprotein, Coronavirus/ultrastructure
19.
Cell Res ; 31(5): 517-525, 2021 05.
Article in English | MEDLINE | ID: covidwho-1139736

ABSTRACT

Neutralizing monoclonal antibodies (nAbs) to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) represent promising candidates for clinical intervention against coronavirus disease 2019 (COVID-19). We isolated a large number of nAbs from SARS-CoV-2-infected individuals capable of disrupting proper interaction between the receptor binding domain (RBD) of the viral spike (S) protein and the receptor angiotensin converting enzyme 2 (ACE2). However, the structural basis for their potent neutralizing activity remains unclear. Here, we report cryo-EM structures of the ten most potent nAbs in their native full-length IgG-form or in both IgG-form and Fab-form bound to the trimeric S protein of SARS-CoV-2. The bivalent binding of the full-length IgG is found to associate with more RBDs in the "up" conformation than the monovalent binding of Fab, perhaps contributing to the enhanced neutralizing activity of IgG and triggering more shedding of the S1 subunit from the S protein. Comparison of a large number of nAbs identified common and unique structural features associated with their potent neutralizing activities. This work provides a structural basis for further understanding the mechanism of nAbs, especially through revealing the bivalent binding and its correlation with more potent neutralization and the shedding of S1 subunit.


Subject(s)
Antibodies, Neutralizing/immunology , COVID-19/immunology , Immunoglobulin G/immunology , SARS-CoV-2/immunology , Spike Glycoprotein, Coronavirus/immunology , Angiotensin-Converting Enzyme 2/immunology , Antibodies, Neutralizing/chemistry , Antibodies, Neutralizing/ultrastructure , Antibodies, Viral/chemistry , Antibodies, Viral/immunology , Antibodies, Viral/ultrastructure , Host-Pathogen Interactions , Humans , Immunoglobulin G/chemistry , Immunoglobulin G/ultrastructure , Models, Molecular , Protein Conformation , Protein Multimerization , SARS-CoV-2/physiology , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/ultrastructure
20.
Nat Commun ; 12(1): 1607, 2021 03 11.
Article in English | MEDLINE | ID: covidwho-1132069

ABSTRACT

In recognizing the host cellular receptor and mediating fusion of virus and cell membranes, the spike (S) glycoprotein of coronaviruses is the most critical viral protein for cross-species transmission and infection. Here we determined the cryo-EM structures of the spikes from bat (RaTG13) and pangolin (PCoV_GX) coronaviruses, which are closely related to SARS-CoV-2. All three receptor-binding domains (RBDs) of these two spike trimers are in the "down" conformation, indicating they are more prone to adopt the receptor-binding inactive state. However, we found that the PCoV_GX, but not the RaTG13, spike is comparable to the SARS-CoV-2 spike in binding the human ACE2 receptor and supporting pseudovirus cell entry. We further identified critical residues in the RBD underlying different activities of the RaTG13 and PCoV_GX/SARS-CoV-2 spikes. These results collectively indicate that tight RBD-ACE2 binding and efficient RBD conformational sampling are required for the evolution of SARS-CoV-2 to gain highly efficient infection.


Subject(s)
COVID-19/virology , Chiroptera/virology , Coronavirus/chemistry , Coronavirus/genetics , Pangolins/virology , SARS-CoV-2/chemistry , SARS-CoV-2/genetics , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/genetics , Amino Acid Sequence , Angiotensin-Converting Enzyme 2/chemistry , Animals , COVID-19/epidemiology , COVID-19/transmission , Cryoelectron Microscopy , Evolution, Molecular , Host Microbial Interactions , Humans , Models, Molecular , Pandemics , Protein Domains , Sequence Homology, Amino Acid , Species Specificity , Spike Glycoprotein, Coronavirus/ultrastructure
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