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1.
J Med Virol ; 94(4): 1641-1649, 2022 04.
Article in English | MEDLINE | ID: covidwho-1718410

ABSTRACT

Emerging severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) variants, especially those of concern, may have an impact on the virus's transmissibility and pathogenicity, as well as diagnostic equipment performance and vaccine effectiveness. Even though the SARS-CoV-2 Delta variant (B.1.617.2) emerged during India's second wave of infections, Delta variants have grown dominant internationally and are still evolving. On November 26, 2021, World Health Organization identified the variant B.1.1.529 as a variant of concern, naming it Omicron, based on evidence that Omicron contains numerous mutations that may influence its behavior. However, the mode of transmission and severity of the Omicron variant remains unknown. We used computational studies to examine the Delta and Omicron variants in this study and found that the Omicron variant had a higher affinity for human angiotensin-converting enzyme 2 (ACE2) than the Delta variant due to a significant number of mutations in the SARS-CoV-2 receptor-binding domain (RBD), indicating a higher potential for transmission. Based on docking studies, the Q493R, N501Y, S371L, S373P, S375F, Q498R, and T478K mutations contribute significantly to high binding affinity with human ACE2. In comparison to the Delta variant, both the entire spike protein and the RBD in Omicron include a high proportion of hydrophobic amino acids such as leucine and phenylalanine. These amino acids are located within the protein's core and are required for structural stability. We observed a disorder-order transition in the Omicron variant between spike protein RBD regions 468-473, and it may be significant in the influence of disordered residues/regions on spike protein stability and binding to ACE2. A future study might investigate the epidemiological and biological consequences of the Omicron variant.


Subject(s)
SARS-CoV-2/chemistry , Spike Glycoprotein, Coronavirus/chemistry , Angiotensin-Converting Enzyme 2/chemistry , Binding Sites , COVID-19/virology , Humans , Hydrophobic and Hydrophilic Interactions , Molecular Docking Simulation , Mutation , Protein Binding , Protein Stability , Protein Structure, Secondary , SARS-CoV-2/genetics , Spike Glycoprotein, Coronavirus/genetics
2.
Viruses ; 14(2)2022 02 10.
Article in English | MEDLINE | ID: covidwho-1715772

ABSTRACT

Enterovirus genus has over one hundred genotypes and could cause several kinds of severe animal and human diseases. Understanding the role of conserved residues in the VP1 capsid protein among the enterovirus genus may lead to anti-enteroviral drug development. The highly conserved residues were found to be located at the loop and ß-barrel intersections. To elucidate the role of these VP1 residues among the enterovirus genus, alanine substitution reverse genetics (rg) variants were generated, and virus properties were investigated for their impact. Six highly conserved residues were identified as located near the inside of the canyon, and four of them were close to the ß-barrel and loop intersection. The variants rgVP1-R86A, rgVP1-P193A, rgVP1-G231A, and rgVP1-K256A were unable to be obtained, which may be due to disruption in the virus replication process. In contrast, rgVP1-E134A and rgVP1-P157A replicated well and rgVP1-P157A showed smaller plaque size, lower viral growth kinetics, and thermal instability at 39.5°C when compared to the rg wild type virus. These findings showed that the conserved residues located at the ß-barrel and loop junction play roles in modulating viral replication, which may provide a pivotal role for pan-enteroviral inhibitor candidate.


Subject(s)
Capsid Proteins/chemistry , Enterovirus/physiology , Virus Replication , Amino Acid Sequence , Antiviral Agents/chemistry , Capsid Proteins/genetics , Cell Line, Tumor , Conserved Sequence , Humans , Mutation , Protein Conformation , Protein Stability , RNA, Viral/metabolism , Small Molecule Libraries/chemistry , Temperature , Viral Load
3.
Int J Mol Sci ; 23(4)2022 Feb 16.
Article in English | MEDLINE | ID: covidwho-1700048

ABSTRACT

Structural and biochemical studies have recently revealed a range of rationally engineered nanobodies with efficient neutralizing capacity against the SARS-CoV-2 virus and resilience against mutational escape. In this study, we performed a comprehensive computational analysis of the SARS-CoV-2 spike trimer complexes with single nanobodies Nb6, VHH E, and complex with VHH E/VHH V nanobody combination. We combined coarse-grained and all-atom molecular simulations and collective dynamics analysis with binding free energy scanning, perturbation-response scanning, and network centrality analysis to examine mechanisms of nanobody-induced allosteric modulation and cooperativity in the SARS-CoV-2 spike trimer complexes with these nanobodies. By quantifying energetic and allosteric determinants of the SARS-CoV-2 spike protein binding with nanobodies, we also examined nanobody-induced modulation of escaping mutations and the effect of the Omicron variant on nanobody binding. The mutational scanning analysis supported the notion that E484A mutation can have a significant detrimental effect on nanobody binding and result in Omicron-induced escape from nanobody neutralization. Our findings showed that SARS-CoV-2 spike protein might exploit the plasticity of specific allosteric hotspots to generate escape mutants that alter response to binding without compromising activity. The network analysis supported these findings showing that VHH E/VHH V nanobody binding can induce long-range couplings between the cryptic binding epitope and ACE2-binding site through a broader ensemble of communication paths that is less dependent on specific mediating centers and therefore may be less sensitive to mutational perturbations of functional residues. The results suggest that binding affinity and long-range communications of the SARS-CoV-2 complexes with nanobodies can be determined by structurally stable regulatory centers and conformationally adaptable hotspots that are allosterically coupled and collectively control resilience to mutational escape.


Subject(s)
SARS-CoV-2/genetics , Single-Domain Antibodies/chemistry , Single-Domain Antibodies/metabolism , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/metabolism , Allosteric Regulation , Cryoelectron Microscopy , Molecular Conformation , Molecular Dynamics Simulation , Protein Stability , Spike Glycoprotein, Coronavirus/genetics
4.
FASEB J ; 36(3): e22199, 2022 03.
Article in English | MEDLINE | ID: covidwho-1684809

ABSTRACT

Spike trimer plays a key role in SARS-CoV-2 infection and vaccine development. It consists of a globular head and a flexible stalk domain that anchors the protein into the viral membrane. While the head domain has been extensively studied, the properties of the adjoining stalk are poorly understood. Here, we characterize the coiled-coil formation and thermodynamic stability of the stalk domain and its segments. We find that the N-terminal segment of the stalk does not form coiled-coils and remains disordered in solution. The C-terminal stalk segment forms a trimeric coiled-coil in solution, which becomes significantly stabilized in the context of the full-length stalk. Its crystal structure reveals a novel antiparallel tetramer coiled-coil with an unusual combination of a-d and e-a-d hydrophobic core packing. Structural analysis shows that a subset of hydrophobic residues stabilizes different coiled-coil structures: trimer, tetramer, and heterohexamer, underscoring a highly polymorphic nature of the SARS-CoV-2 stalk sequence.


Subject(s)
COVID-19/virology , Models, Molecular , Protein Domains , SARS-CoV-2/chemistry , Spike Glycoprotein, Coronavirus/chemistry , Amino Acid Sequence , Crystallization , Crystallography, X-Ray , Humans , Hydrophobic and Hydrophilic Interactions , Protein Stability , Protein Structure, Secondary , Scattering, Small Angle , Temperature , X-Ray Diffraction
5.
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
6.
Nat Commun ; 12(1): 6977, 2021 11 30.
Article in English | MEDLINE | ID: covidwho-1545609

ABSTRACT

Despite an unprecedented global gain in knowledge since the emergence of SARS-CoV-2, almost all mechanistic knowledge related to the molecular and cellular details of viral replication, pathology and virulence has been generated using early prototypic isolates of SARS-CoV-2. Here, using atomic force microscopy and molecular dynamics, we investigated how these mutations quantitatively affected the kinetic, thermodynamic and structural properties of RBD-ACE2 complex formation. We observed for several variants of concern a significant increase in the RBD-ACE2 complex stability. While the N501Y and E484Q mutations are particularly important for the greater stability, the N501Y mutation is unlikely to significantly affect antibody neutralization. This work provides unprecedented atomistic detail on the binding of SARS-CoV-2 variants and provides insight into the impact of viral mutations on infection-induced immunity.


Subject(s)
Antibodies, Neutralizing/immunology , SARS-CoV-2/immunology , SARS-CoV-2/metabolism , Angiotensin-Converting Enzyme 2/chemistry , Angiotensin-Converting Enzyme 2/metabolism , Antibodies, Neutralizing/pharmacology , COVID-19/therapy , COVID-19/virology , Humans , Kinetics , Microscopy, Atomic Force , Molecular Dynamics Simulation , Mutation , Protein Binding/drug effects , Protein Interaction Domains and Motifs , Protein Stability , SARS-CoV-2/genetics , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/immunology , Spike Glycoprotein, Coronavirus/metabolism , Thermodynamics
7.
J Cell Biochem ; 123(2): 417-430, 2022 02.
Article in English | MEDLINE | ID: covidwho-1525444

ABSTRACT

Since the emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), a large number of mutations in its genome have been reported. Some of the mutations occur in noncoding regions without affecting the pathobiology of the virus, while mutations in coding regions are significant. One of the regions where a mutation can occur, affecting the function of the virus is at the receptor-binding domain (RBD) of the spike protein. RBD interacts with angiotensin-converting enzyme 2 (ACE2) and facilitates the entry of the virus into the host cells. There is a lot of focus on RBD mutations, especially the displacement of N501Y which is observed in the UK/Kent, South Africa, and Brazilian lineages of SARS-CoV-2. Our group utilizes computational biology approaches such as immunoinformatics, protein-protein interaction analysis, molecular dynamics, free energy computation, and tertiary structure analysis to disclose the consequences of N501Y mutation at the molecular level. Surprisingly, we discovered that this mutation reduces the immunogenicity of the spike protein; also, displacement of Asn with Tyr reduces protein compactness and significantly increases the stability of the spike protein and its affinity to ACE2. Moreover, following the N501Y mutation secondary structure and folding of the spike protein changed dramatically.


Subject(s)
COVID-19/virology , Mutation, Missense , Pandemics , Point Mutation , SARS-CoV-2/genetics , Spike Glycoprotein, Coronavirus/genetics , Amino Acid Substitution , Angiotensin-Converting Enzyme 2/metabolism , Antigens, Viral/chemistry , Antigens, Viral/immunology , Binding Sites , Computational Biology/methods , Energy Transfer , Epitopes/chemistry , Epitopes/immunology , Evolution, Molecular , Humans , Molecular Docking Simulation , Protein Binding , Protein Conformation , Protein Stability , Receptors, Virus/metabolism , SARS-CoV-2/immunology , SARS-CoV-2/physiology , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/immunology , Spike Glycoprotein, Coronavirus/metabolism , Structure-Activity Relationship
8.
J Biol Chem ; 297(6): 101371, 2021 12.
Article in English | MEDLINE | ID: covidwho-1487811

ABSTRACT

The emergence of severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) and the subsequent COVID-19 pandemic have visited a terrible cost on the world in the forms of disease, death, and economic turmoil. The rapid development and deployment of extremely effective vaccines against SARS-CoV-2 have seemingly brought within reach the end of the pandemic. However, the virus has acquired mutations. and emerging variants of concern are more infectious and reduce the efficacy of existing vaccines. Although promising efforts to combat these variants are underway, the evolutionary pressures leading to these variants are poorly understood. To that end, here we have studied the effects on the structure and function of the SARS-CoV-2 spike glycoprotein receptor-binding domain of three amino-acid substitutions found in several variants of concern, including alpha (B.1.1.7), beta (B.1.351), and gamma (P.1). We found that these substitutions alter the receptor-binding domain structure, stability, and ability to bind to angiotensin converting enzyme 2, in such a way as to possibly have opposing and compensatory effects. These findings provide new insights into how these variants of concern may have been selected for infectivity while maintaining the structure and stability of the receptor binding domain.


Subject(s)
Amino Acid Substitution , COVID-19/virology , SARS-CoV-2/genetics , Spike Glycoprotein, Coronavirus/genetics , Angiotensin-Converting Enzyme 2/metabolism , COVID-19/metabolism , Humans , Models, Molecular , Point Mutation , Protein Binding , Protein Domains , Protein Stability , SARS-CoV-2/chemistry , SARS-CoV-2/physiology , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/metabolism
9.
Biomed Res Int ; 2021: 9982729, 2021.
Article in English | MEDLINE | ID: covidwho-1476892

ABSTRACT

The human transmembrane protease serine 2 (TMPRSS2) protein plays an important role in prostate cancer progression. It also facilitates viral entry into target cells by proteolytically cleaving and activating the S protein of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). In the current study, we used different available tools like SIFT, PolyPhen2.0, PROVEAN, SNAP2, PMut, MutPred2, I-Mutant Suite, MUpro, iStable, ConSurf, ModPred, SwissModel, PROCHECK, Verify3D, and TM-align to identify the most deleterious variants and to explore possible effects on the TMPRSS2 stability, structure, and function. The six missense variants tested were evaluated to have deleterious effects on the protein by SIFT, PolyPhen2.0, PROVEAN, SNAP2, and PMut. Additionally, V160M, G181R, R240C, P335L, G432A, and D435Y variants showed a decrease in stability by at least 2 servers; G181R, G432A, and D435Y are highly conserved and identified posttranslational modifications sites (PTMs) for proteolytic cleavage and ADP-ribosylation using ConSurf and ModPred servers. The 3D structure of TMPRSS2 native and mutants was generated using 7 meq as a template from the SwissModeller group, refined by ModRefiner, and validated using the Ramachandran plot. Hence, this paper can be advantageous to understand the association between these missense variants rs12329760, rs781089181, rs762108701, rs1185182900, rs570454392, and rs867186402 and susceptibility to SARS-CoV-2.


Subject(s)
COVID-19/genetics , Mutation, Missense , Serine Endopeptidases/chemistry , Serine Endopeptidases/genetics , Binding Sites , Computational Biology/methods , Evolution, Molecular , Genetic Predisposition to Disease , Humans , Models, Molecular , Phylogeny , Polymorphism, Single Nucleotide , Protein Conformation , Protein Stability , Serine Endopeptidases/metabolism
10.
Viruses ; 13(10)2021 10 18.
Article in English | MEDLINE | ID: covidwho-1471002

ABSTRACT

West Java Health Laboratory (WJHL) is one of the many institutions in Indonesia that have sequenced SARS-CoV-2 genome. Although having submitted a large number of sequences since September 2020, however, these submitted data lack advanced analyses. Therefore, in this study, we analyze the variant distribution, hotspot mutation, and its impact on protein structure and function of SARS-CoV-2 from the collected samples from WJHL. As many as one hundred sixty-three SARS-CoV-2 genome sequences submitted by West Java Health Laboratory (WJHL), with collection dates between September 2020 and June 2021, were retrieved from GISAID. Subsequently, the frequency and distribution of non-synonymous mutations across different cities and regencies from these samples were analyzed. The effect of the most prevalent mutations from dominant variants on the stability of their corresponding proteins was examined. The samples mostly consisted of people of working-age, and were distributed between female and male equally. All of the sample sequences showed varying levels of diversity, especially samples from West Bandung which carried the highest diversity. Dominant variants are the VOC B.1.617.2 (Delta) variant, B.1.466.2 variant, and B.1.470 variant. The genomic regions with the highest number of mutations are the spike, NSP3, nucleocapsid, NSP12, and ORF3a protein. Mutation analysis showed that mutations in structural protein might increase the stability of the protein. Oppositely, mutations in non-structural protein might lead to a decrease in protein stability. However, further research to study the impact of mutations on the function of SARS-CoV-2 proteins are required.


Subject(s)
Genome, Viral/genetics , SARS-CoV-2/genetics , Viral Proteins/genetics , Viral Proteins/metabolism , COVID-19/pathology , Coronavirus Nucleocapsid Proteins/genetics , Coronavirus Papain-Like Proteases/genetics , Coronavirus RNA-Dependent RNA Polymerase/genetics , Disease Hotspot , Female , Humans , Indonesia , Male , Molecular Docking Simulation , Mutation/genetics , Phosphoproteins/genetics , Protein Stability , Spike Glycoprotein, Coronavirus/genetics , Viroporin Proteins/genetics , Whole Genome Sequencing
11.
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
12.
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
13.
J Biol Chem ; 297(4): 101208, 2021 10.
Article in English | MEDLINE | ID: covidwho-1415532

ABSTRACT

Emergence of new severe acute respiratory syndrome coronavirus 2 variants has raised concerns related to the effectiveness of vaccines and antibody therapeutics developed against the unmutated wildtype virus. Here, we examined the effect of the 12 most commonly occurring mutations in the receptor-binding domain of the spike protein on its expression, stability, activity, and antibody escape potential. Stability was measured using thermal denaturation, and the activity and antibody escape potential were measured using isothermal titration calorimetry in terms of binding to the human angiotensin-converting enzyme 2 and to neutralizing human antibody CC12.1, respectively. Our results show that mutants differ in their expression levels. Of the eight best-expressed mutants, two (N501Y and K417T/E484K/N501Y) showed stronger affinity to angiotensin-converting enzyme 2 compared with the wildtype, whereas four (Y453F, S477N, T478I, and S494P) had similar affinity and two (K417N and E484K) had weaker affinity than the wildtype. Compared with the wildtype, four mutants (K417N, Y453F, N501Y, and K417T/E484K/N501Y) had weaker affinity for the CC12.1 antibody, whereas two (S477N and S494P) had similar affinity, and two (T478I and E484K) had stronger affinity than the wildtype. Mutants also differ in their thermal stability, with the two least stable mutants showing reduced expression. Taken together, these results indicate that multiple factors contribute toward the natural selection of variants, and all these factors need to be considered to understand the evolution of the virus. In addition, since not all variants can escape a given neutralizing antibody, antibodies to treat new variants can be chosen based on the specific mutations in that variant.


Subject(s)
Angiotensin-Converting Enzyme 2/metabolism , Antibodies, Neutralizing/immunology , SARS-CoV-2/metabolism , Spike Glycoprotein, Coronavirus/metabolism , Angiotensin-Converting Enzyme 2/chemistry , Angiotensin-Converting Enzyme 2/genetics , Antigen-Antibody Reactions , COVID-19/pathology , COVID-19/virology , HEK293 Cells , Humans , Polymorphism, Single Nucleotide , Protein Binding , Protein Domains/genetics , Protein Stability , Protein Structure, Tertiary , Recombinant Proteins/biosynthesis , Recombinant Proteins/chemistry , Recombinant Proteins/isolation & purification , SARS-CoV-2/isolation & purification , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/immunology , Transition Temperature
14.
J Chem Inf Model ; 61(9): 4152-4155, 2021 09 27.
Article in English | MEDLINE | ID: covidwho-1392764

ABSTRACT

The emergence of variants of SARS-CoV-2 with mutations in their spike protein are a major cause for concern for the efficacy of vaccines and control of the pandemic. We show that mutations in the spike protein of SARS-CoV-2 are selecting for amino acid changes that result in a more thermodynamically stable protein than expected from background. We suggest that the computationally efficient analysis of mutational stability may aid in early screening of variants.


Subject(s)
COVID-19 , SARS-CoV-2 , Humans , Mutation , Protein Stability , Spike Glycoprotein, Coronavirus/genetics
15.
Sci Rep ; 11(1): 9166, 2021 04 28.
Article in English | MEDLINE | ID: covidwho-1387472

ABSTRACT

The interaction between the receptor-binding domain (RBD) of the SARS-CoV-2 spike glycoprotein and the ACE2 enzyme is believed to be the entry point of the virus into various cells in the body, including the lungs, heart, liver, and kidneys. The current focus of several therapeutic design efforts explores attempts at affecting the binding potential between the two proteins to limit the activity of the virus and disease progression. In this work, we analyze the stability of the spike protein under all possible single-point mutations in the RBD and computationally explore mutations that can affect the binding with the ACE2 enzyme. We unravel the mutation landscape of the receptor region and assess the toxicity potential of single and multi-point mutations, generating insights for future vaccine efforts on mutations that might further stabilize the spike protein and increase its infectivity. We developed a tool, called SpikeMutator, to construct full atomic protein structures of the mutant spike proteins and shared a database of 3800 single-point mutant structures. We analyzed the recent 65,000 reported spike sequences across the globe and observed the emergence of stable multi-point mutant structures. Using the landscape, we searched through 7.5 million possible 2-point mutation combinations and report that the (R355D K424E) mutation produces one of the strongest spike proteins that therapeutic efforts should investigate for the sake of developing effective vaccines.


Subject(s)
Mutation , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/metabolism , Angiotensin-Converting Enzyme 2/metabolism , Binding Sites , Computational Biology/methods , Humans , Models, Molecular , Molecular Dynamics Simulation , Protein Conformation , Protein Stability , SARS-CoV-2/genetics , SARS-CoV-2/isolation & purification , Spike Glycoprotein, Coronavirus/chemistry
16.
J Phys Chem B ; 125(3): 850-873, 2021 01 28.
Article in English | MEDLINE | ID: covidwho-1387111

ABSTRACT

The rapidly growing body of structural and biochemical studies of the SARS-CoV-2 spike glycoprotein has revealed a variety of distinct functional states with radically different arrangements of the receptor-binding domain, highlighting a remarkable function-driven conformational plasticity and adaptability of the spike proteins. In this study, we examined molecular mechanisms underlying conformational and dynamic changes in the SARS-CoV-2 spike mutant trimers through the lens of dynamic analysis of allosteric interaction networks and atomistic modeling of signal transmission. Using an integrated approach that combined coarse-grained molecular simulations, protein stability analysis, and perturbation-based modeling of residue interaction networks, we examined how mutations in the regulatory regions of the SARS-CoV-2 spike protein can differentially affect dynamics and allosteric signaling in distinct functional states. The results of this study revealed key functional regions and regulatory centers that govern collective dynamics, allosteric interactions, and control signal transmission in the SARS-CoV-2 spike proteins. We found that the experimentally confirmed regulatory hotspots that dictate dynamic switching between conformational states of the SARS-CoV-2 spike protein correspond to the key hinge sites and global mediating centers of the allosteric interaction networks. The results of this study provide a novel insight into allosteric regulatory mechanisms of SARS-CoV-2 spike proteins showing that mutations at the key regulatory positions can differentially modulate distribution of states and determine topography of signal communication pathways operating through state-specific cascades of control switch points. This analysis provides a plausible strategy for allosteric probing of the conformational equilibrium and therapeutic intervention by targeting specific hotspots of allosteric interactions and communications in the SARS-CoV-2 spike proteins.


Subject(s)
Models, Biological , Mutation , SARS-CoV-2/chemistry , Spike Glycoprotein, Coronavirus/chemistry , Allosteric Regulation , Binding Sites , Cysteine/genetics , Molecular Dynamics Simulation , Protein Binding , Protein Conformation , Protein Stability , Protein Subunits , SARS-CoV-2/genetics , Signal Transduction/genetics , Spike Glycoprotein, Coronavirus/genetics
17.
Sci Rep ; 10(1): 21393, 2020 12 07.
Article in English | MEDLINE | ID: covidwho-1387456

ABSTRACT

Antibodies are essential tools for therapy and diagnostics. Yet, production remains expensive as it is mostly done in mammalian expression systems. As most therapeutic IgG require mammalian glycosylation to interact with the human immune system, other expression systems are rarely used for production. However, for neutralizing antibodies that are not required to activate the human immune system as well as antibodies used in diagnostics, a cheaper production system would be advantageous. In our study, we show cost-efficient, easy and high yield production of antibodies as well as various secreted antigens including Interleukins and SARS-CoV-2 related proteins in a baculovirus-free insect cell expression system. To improve yields, we optimized the expression vector, media and feeding strategies. In addition, we showed the feasibility of lyophilization of the insect cell produced antibodies. Furthermore, stability and activity of the antibodies was compared to antibodies produced by Expi293F cells revealing a lower aggregation of antibodies originating from High Five cell production. Finally, the newly established High Five expression system was compared to the Expi293F mammalian expression system in regard of yield and costs. Most interestingly, all tested proteins were producible in our High Five cell expression system what was not the case in the Expi293F system, hinting that the High Five cell system is especially suited to produce difficult-to-express target proteins.


Subject(s)
Antibodies, Monoclonal/biosynthesis , Antibodies, Neutralizing/biosynthesis , Antigens, Viral/biosynthesis , Cloning, Molecular , Recombinant Proteins/biosynthesis , SARS-CoV-2/immunology , Animals , HEK293 Cells , Humans , Protein Stability , Spodoptera
19.
Proc Natl Acad Sci U S A ; 118(37)2021 09 14.
Article in English | MEDLINE | ID: covidwho-1376237

ABSTRACT

COVID-19, caused by severe acute respiratory coronavirus 2 (SARS-CoV-2), has presented a serious risk to global public health. The viral main protease Mpro (also called 3Clpro) encoded by NSP5 is an enzyme essential for viral replication. However, very few host proteins have been experimentally validated as targets of 3Clpro. Here, through bioinformatics analysis of 300 interferon stimulatory genes (ISGs) based on the prediction method NetCorona, we identify RNF20 (Ring Finger Protein 20) as a novel target of 3Clpro. We have also provided evidence that 3Clpro, but not the mutant 3ClproC145A without catalytic activity, cleaves RNF20 at a conserved Gln521 across species, which subsequently prevents SREBP1 from RNF20-mediated degradation and promotes SARS-CoV-2 replication. We show that RNA interference (RNAi)-mediated depletion of either RNF20 or RNF40 significantly enhances viral replication, indicating the antiviral role of RNF20/RNF40 complex against SARS-CoV-2. The involvement of SREBP1 in SARS-CoV-2 infection is evidenced by a decrease of viral replication in the cells with SREBP1 knockdown and inhibitor AM580. Taken together, our findings reveal RNF20 as a novel host target for SARS-CoV-2 main protease and indicate that 3Clpro inhibitors may treat COVID-19 through not only blocking viral polyprotein cleavage but also enhancing host antiviral response.


Subject(s)
Coronavirus 3C Proteases/metabolism , Protein Stability , SARS-CoV-2/pathogenicity , Sterol Regulatory Element Binding Protein 1/metabolism , Ubiquitin-Protein Ligases/metabolism , Virus Replication , Animals , Antiviral Agents/pharmacology , Cell Line , Chlorocebus aethiops , Gene Expression Regulation , Interferons/physiology , SARS-CoV-2/immunology , Sterol Regulatory Element Binding Protein 1/antagonists & inhibitors , Vero Cells
20.
Mol Genet Genomic Med ; 9(8): e1740, 2021 08.
Article in English | MEDLINE | ID: covidwho-1375613

ABSTRACT

BACKGROUND: Diarrhea is a common occurrence in children below the age of 5 years. In chronic cases, it induces malnutrition that severely stunts growth. Bile acid diarrhea (BAD), caused by malabsorption of bile acid (BA), is a rare form of chronic diarrhea seldom observed in pediatric patients. Here, we present a clinical report on a novel case of chronic BAD, with severe stunting in an infant, induced by a homozygous mutation of SLC10A2. METHODS: We performed DNA extraction, whole-exome sequencing analysis, and mutation analysis of SLC10A2 to obtain genetic data on the patient. We subsequently analyzed the patient's clinical and genetic data. RESULTS: The patient's clinical manifestations were chronic diarrhea with increased BAs in the feces and extreme stunting, which was diagnosed as BAD. A homozygous mutation of SLC10A2 at the c.313T>C (rs201206937) site was detected. CONCLUSION: Our report reveals the youngest case illustrating the characteristics of BAD induced by genetic variant at 313T>C, and the second case entailing a clear association between a SLC10A2 genetic mutation and the onset of BAD. Our findings expand the mutant spectrum of the SLC10A2 gene and contribute to the refinement of the genotype-phenotype mapping of severe stunting induced by pediatric BAD. Moreover, they highlight the value of molecular genetic screening for diagnosing BAD in young patients.


Subject(s)
Diarrhea/genetics , Growth Disorders/genetics , Organic Anion Transporters, Sodium-Dependent/genetics , Symporters/genetics , Bile Acids and Salts/metabolism , Diarrhea/pathology , Female , Growth Disorders/pathology , Homozygote , Humans , Infant , Mutation , Organic Anion Transporters, Sodium-Dependent/chemistry , Organic Anion Transporters, Sodium-Dependent/metabolism , Protein Stability , Symporters/chemistry , Symporters/metabolism
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