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1.
Int J Mol Sci ; 23(10)2022 May 18.
Article in English | MEDLINE | ID: covidwho-1862812

ABSTRACT

Animal coronaviruses (CoVs) have been identified to be the origin of Severe Acute Respiratory Syndrome (SARS)-CoV, Middle East respiratory syndrome (MERS)-CoV, and probably SARS-CoV-2 that cause severe to fatal diseases in humans. Variations of zoonotic coronaviruses pose potential threats to global human beings. To overcome this problem, we focused on the main protease (Mpro), which is an evolutionary conserved viral protein among different coronaviruses. The broad-spectrum anti-coronaviral drug, GC376, was repurposed to target canine coronavirus (CCoV), which causes gastrointestinal infections in dogs. We found that GC376 can efficiently block the protease activity of CCoV Mpro and can thermodynamically stabilize its folding. The structure of CCoV Mpro in complex with GC376 was subsequently determined at 2.75 Å. GC376 reacts with the catalytic residue C144 of CCoV Mpro and forms an (R)- or (S)-configuration of hemithioacetal. A structural comparison of CCoV Mpro and other animal CoV Mpros with SARS-CoV-2 Mpro revealed three important structural determinants in a substrate-binding pocket that dictate entry and release of substrates. As compared with the conserved A141 of the S1 site and P188 of the S4 site in animal coronaviral Mpros, SARS-CoV-2 Mpro contains N142 and Q189 at equivalent positions which are considered to be more catalytically compatible. Furthermore, the conserved loop with residues 46-49 in animal coronaviral Mpros has been replaced by a stable α-helix in SARS-CoV-2 Mpro. In addition, the species-specific dimerization interface also influences the catalytic efficiency of CoV Mpros. Conclusively, the structural information of this study provides mechanistic insights into the ligand binding and dimerization of CoV Mpros among different species.


Subject(s)
COVID-19 , Peptide Hydrolases , Animals , Coronavirus 3C Proteases , Dimerization , Dogs , Endopeptidases , Ligands , Peptide Hydrolases/chemistry , SARS-CoV-2
2.
Int J Mol Sci ; 23(9)2022 May 09.
Article in English | MEDLINE | ID: covidwho-1847347

ABSTRACT

3CLpro of SARS-CoV-2 is a promising target for developing anti-COVID19 agents. In order to evaluate the catalytic activity of 3CLpros according to the presence or absence of the dimerization domain, two forms had been purified and tested. Enzyme kinetic studies with a FRET method revealed that the catalytic domain alone presents enzymatic activity, despite it being approximately 8.6 times less than that in the full domain. The catalytic domain was crystallized and its X-ray crystal structure has been determined to 2.3 Å resolution. There are four protomers in the asymmetric unit. Intriguingly, they were packed as a dimer though the dimerization domain was absent. The RMSD of superimposed two catalytic domains was 0.190 for 182 Cα atoms. A part of the long hinge loop (LH-loop) from Gln189 to Asp197 was not built in the model due to its flexibility. The crystal structure indicates that the decreased proteolytic activity of the catalytic domain was due to the incomplete construction of the substrate binding part built by the LH-loop. A structural survey with other 3CLpros showed that SARS-CoV families do not have interactions between DM-loop due to the conformational difference at the last turn of helix α7 compared with others. Therefore, we can conclude that the monomeric form contains nascent enzyme activity and that its efficiency increases by dimerization. This new insight may contribute to understanding the behavior of SARS-CoV-2 3CLpro and thus be useful in developing anti-COVID-19 agents.


Subject(s)
COVID-19 , SARS-CoV-2 , Catalytic Domain , Coronavirus 3C Proteases , Dimerization , Humans , Kinetics , X-Rays
3.
Phys Chem Chem Phys ; 24(16): 9141-9145, 2022 Apr 20.
Article in English | MEDLINE | ID: covidwho-1784055

ABSTRACT

Dimerization of SARS-CoV-2 main protease (Mpro) is a prerequisite for its processing activity. With >2000 mutations already reported in Mpro, SARS-CoV-2 may accumulate mutations in the Mpro dimeric interface to stabilize it further. We employed high-throughput protein design strategies to design the symmetrical dimeric interface of Mpro (300 000 designs) to identify mutational hotspots that render the Mpro more stable. We found that ∼22% of designed mutations that yield stable Mpro dimers already exist in SARS-CoV-2 genomes and are currently circulating. Our multi-parametric analyses highlight potential Mpro mutations that SARS-CoV-2 may develop, providing a foundation for assessing viral adaptation and mutational surveillance.


Subject(s)
Coronavirus 3C Proteases , Protein Engineering , SARS-CoV-2 , COVID-19 , Coronavirus 3C Proteases/genetics , Dimerization , Humans , SARS-CoV-2/enzymology , SARS-CoV-2/genetics
4.
Int J Biol Macromol ; 200: 428-437, 2022 Mar 01.
Article in English | MEDLINE | ID: covidwho-1633983

ABSTRACT

Nucleocapsid protein (N protein) is the primary antigen of the virus for development of sensitive diagnostic assays of COVID-19. In this paper, we demonstrate the significant impact of dimerization of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) N-protein on sensitivity of enzyme-linked immunosorbent assay (ELISA) based diagnostics. The expressed purified protein from E. coli is composed of dimeric and monomeric forms, which have been further characterized using biophysical and immunological techniques. Indirect ELISA indicated elevated susceptibility of the dimeric form of the nucleocapsid protein for identification of protein-specific monoclonal antibody as compared to the monomeric form. This finding also confirmed with the modelled structure of monomeric and dimeric nucleocapsid protein via HHPred software and its solvent accessible surface area, which indicates higher stability and antigenicity of the dimeric type as compared to the monomeric form. The sensitivity and specificity of the ELISA at 95% CI are 99.0% (94.5-99.9) and 95.0% (83.0-99.4), respectively, for the highest purified dimeric form of the N protein. As a result, using the highest purified dimeric form will improve the sensitivity of the current nucleocapsid-dependent ELISA for COVID-19 diagnosis, and manufacturers should monitor and maintain the monomer-dimer composition for accurate and robust diagnostics.


Subject(s)
COVID-19 Testing/methods , Coronavirus Nucleocapsid Proteins/chemistry , Enzyme-Linked Immunosorbent Assay/methods , SARS-CoV-2/immunology , Antibodies, Viral/immunology , Circular Dichroism , Coronavirus Nucleocapsid Proteins/biosynthesis , Coronavirus Nucleocapsid Proteins/immunology , Coronavirus Nucleocapsid Proteins/isolation & purification , Dimerization , Epitopes/chemistry , Escherichia coli/genetics , Humans , Immunoglobulin G/immunology , Models, Molecular , Phosphoproteins/biosynthesis , Phosphoproteins/chemistry , Phosphoproteins/immunology , Phosphoproteins/isolation & purification , Recombinant Proteins/biosynthesis , Recombinant Proteins/chemistry , Recombinant Proteins/immunology , Recombinant Proteins/isolation & purification , Sensitivity and Specificity
5.
Int J Biol Macromol ; 197: 68-76, 2022 Feb 01.
Article in English | MEDLINE | ID: covidwho-1587673

ABSTRACT

The C-terminal domain of SARS-CoV main protease (Mpro-C) can form 3D domain-swapped dimer by exchanging the α1-helices fully buried inside the protein hydrophobic core, under non-denaturing conditions. Here, we report that Mpro-C can also form amyloid fibrils under the 3D domain-swappable conditions in vitro, and the fibrils are not formed through runaway/propagated domain swapping. It is found that there are positive correlations between the rates of domain swapping dimerization and amyloid fibrillation at different temperatures, and for different mutants. However, some Mpro-C mutants incapable of 3D domain swapping can still form amyloid fibrils, indicating that 3D domain swapping is not essential for amyloid fibrillation. Furthermore, NMR H/D exchange data and molecular dynamics simulation results suggest that the protofibril core region tends to unpack at the early stage of 3D domain swapping, so that the amyloid fibrillation can proceed during the 3D domain swapping process. We propose that 3D domain swapping makes it possible for the unpacking of the amyloidogenic fragment of the protein and thus accelerates the amyloid fibrillation process kinetically, which explains the well-documented correlations between amyloid fibrillation and 3D domain swapping observed in many proteins.


Subject(s)
Amyloid/chemistry , Amyloid/metabolism , Amyloidosis/metabolism , Coronavirus 3C Proteases/chemistry , Coronavirus 3C Proteases/metabolism , Protein Domains/physiology , Amyloidosis/genetics , Coronavirus 3C Proteases/genetics , Dimerization , Disulfides/chemistry , Disulfides/metabolism , Kinetics , Models, Molecular , Molecular Dynamics Simulation , Mutation , Polymerization , Protein Conformation, alpha-Helical , Protein Domains/genetics , Protein Folding , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Temperature
6.
Nucleic Acids Res ; 50(2): 1017-1032, 2022 01 25.
Article in English | MEDLINE | ID: covidwho-1574599

ABSTRACT

The ongoing COVID-19 pandemic highlights the necessity for a more fundamental understanding of the coronavirus life cycle. The causative agent of the disease, SARS-CoV-2, is being studied extensively from a structural standpoint in order to gain insight into key molecular mechanisms required for its survival. Contained within the untranslated regions of the SARS-CoV-2 genome are various conserved stem-loop elements that are believed to function in RNA replication, viral protein translation, and discontinuous transcription. While the majority of these regions are variable in sequence, a 41-nucleotide s2m element within the genome 3' untranslated region is highly conserved among coronaviruses and three other viral families. In this study, we demonstrate that the SARS-CoV-2 s2m element dimerizes by forming an intermediate homodimeric kissing complex structure that is subsequently converted to a thermodynamically stable duplex conformation. This process is aided by the viral nucleocapsid protein, potentially indicating a role in mediating genome dimerization. Furthermore, we demonstrate that the s2m element interacts with multiple copies of host cellular microRNA (miRNA) 1307-3p. Taken together, our results highlight the potential significance of the dimer structures formed by the s2m element in key biological processes and implicate the motif as a possible therapeutic drug target for COVID-19 and other coronavirus-related diseases.


Subject(s)
3' Untranslated Regions/genetics , COVID-19/genetics , MicroRNAs/genetics , Nucleotide Motifs/genetics , RNA, Viral/genetics , SARS-CoV-2/genetics , Base Sequence , Binding Sites/genetics , COVID-19/metabolism , COVID-19/virology , Conserved Sequence/genetics , Dimerization , Genome, Viral/genetics , Host-Pathogen Interactions/genetics , Humans , MicroRNAs/metabolism , Nucleic Acid Conformation , Proton Magnetic Resonance Spectroscopy/methods , RNA, Viral/chemistry , RNA, Viral/metabolism , SARS-CoV-2/metabolism , SARS-CoV-2/physiology
7.
J Biol Chem ; 297(4): 101218, 2021 10.
Article in English | MEDLINE | ID: covidwho-1433454

ABSTRACT

The SARS-CoV-2 replication-transcription complex is an assembly of nonstructural viral proteins that collectively act to reproduce the viral genome and generate mRNA transcripts. While the structures of the individual proteins involved are known, how they assemble into a functioning superstructure is not. Applying molecular modeling tools, including protein-protein docking, to the available structures of nsp7-nsp16 and the nucleocapsid, we have constructed an atomistic model of how these proteins associate. Our principal finding is that the complex is hexameric, centered on nsp15. The nsp15 hexamer is capped on two faces by trimers of nsp14/nsp16/(nsp10)2, which then recruit six nsp12/nsp7/(nsp8)2 polymerase subunits to the complex. To this, six subunits of nsp13 are arranged around the superstructure, but not evenly distributed. Polymerase subunits that coordinate dimers of nsp13 are capable of binding the nucleocapsid, which positions the 5'-UTR TRS-L RNA over the polymerase active site, a state distinguishing transcription from replication. Analysis of the viral RNA path through the complex indicates the dsRNA that exits the polymerase passes over the nsp14 exonuclease and nsp15 endonuclease sites before being unwound by a convergence of zinc fingers from nsp10 and nsp14. The template strand is then directed away from the complex, while the nascent strand is directed to the sites responsible for mRNA capping. The model presents a cohesive picture of the multiple functions of the coronavirus replication-transcription complex and addresses fundamental questions related to proofreading, template switching, mRNA capping, and the role of the endonuclease.


Subject(s)
Endoribonucleases/metabolism , Models, Molecular , SARS-CoV-2/metabolism , Viral Nonstructural Proteins/metabolism , Binding Sites , COVID-19/pathology , COVID-19/virology , Dimerization , Endoribonucleases/chemistry , Endoribonucleases/genetics , Humans , Molecular Docking Simulation , Protein Structure, Quaternary , RNA, Double-Stranded/chemistry , RNA, Double-Stranded/metabolism , SARS-CoV-2/isolation & purification , Transcription, Genetic , Viral Nonstructural Proteins/chemistry , Viral Nonstructural Proteins/genetics , Virus Replication
8.
Nat Commun ; 12(1): 1936, 2021 03 29.
Article in English | MEDLINE | ID: covidwho-1387331

ABSTRACT

The SARS-CoV-2 nucleocapsid (N) protein is an abundant RNA-binding protein critical for viral genome packaging, yet the molecular details that underlie this process are poorly understood. Here we combine single-molecule spectroscopy with all-atom simulations to uncover the molecular details that contribute to N protein function. N protein contains three dynamic disordered regions that house putative transiently-helical binding motifs. The two folded domains interact minimally such that full-length N protein is a flexible and multivalent RNA-binding protein. N protein also undergoes liquid-liquid phase separation when mixed with RNA, and polymer theory predicts that the same multivalent interactions that drive phase separation also engender RNA compaction. We offer a simple symmetry-breaking model that provides a plausible route through which single-genome condensation preferentially occurs over phase separation, suggesting that phase separation offers a convenient macroscopic readout of a key nanoscopic interaction.


Subject(s)
Coronavirus Nucleocapsid Proteins/chemistry , Coronavirus Nucleocapsid Proteins/metabolism , RNA, Viral/chemistry , RNA, Viral/metabolism , SARS-CoV-2/chemistry , SARS-CoV-2/metabolism , Binding Sites , COVID-19/virology , Dimerization , Molecular Dynamics Simulation , Phosphoproteins/chemistry , Phosphoproteins/metabolism , Protein Conformation , Protein Domains
9.
J Phys Chem Lett ; 12(26): 6218-6226, 2021 Jul 08.
Article in English | MEDLINE | ID: covidwho-1387122

ABSTRACT

Following our previous work ( Chem. Sci. 2021, 12, 4889-4907), we study the structural dynamics of the SARS-CoV-2 Main Protease dimerization interface (apo dimer) by means of microsecond adaptive sampling molecular dynamics simulations (50 µs) using the AMOEBA polarizable force field (PFF). This interface is structured by a complex H-bond network that is stable only at physiological pH. Structural correlations analysis between its residues and the catalytic site confirms the presence of a buried allosteric site. However, noticeable differences in allosteric connectivity are observed between PFFs and non-PFFs. Interfacial polarizable water molecules are shown to appear at the heart of this discrepancy because they are connected to the global interface H-bond network and able to adapt their dipole moment (and dynamics) to their diverse local physicochemical microenvironments. The water-interface many-body interactions appear to drive the interface volume fluctuations and to therefore mediate the allosteric interactions with the catalytic cavity.


Subject(s)
Molecular Dynamics Simulation , SARS-CoV-2/metabolism , Viral Matrix Proteins/chemistry , Water/chemistry , Allosteric Site , COVID-19/pathology , COVID-19/virology , Catalytic Domain , Dimerization , Humans , Hydrogen Bonding , Hydrogen-Ion Concentration , SARS-CoV-2/isolation & purification , Viral Matrix Proteins/metabolism
10.
J Phys Chem Lett ; 11(14): 5661-5667, 2020 Jul 16.
Article in English | MEDLINE | ID: covidwho-1387115

ABSTRACT

Coronaviruses may produce severe acute respiratory syndrome (SARS). As a matter of fact, a new SARS-type virus, SARS-CoV-2, is responsible for the global pandemic in 2020 with unprecedented sanitary and economic consequences for most countries. In the present contribution we study, by all-atom equilibrium and enhanced sampling molecular dynamics simulations, the interaction between the SARS Unique Domain and RNA guanine quadruplexes, a process involved in eluding the defensive response of the host thus favoring viral infection of human cells. Our results evidence two stable binding modes involving an interaction site spanning either the protein dimer interface or only one monomer. The free energy profile unequivocally points to the dimer mode as the thermodynamically favored one. The effect of these binding modes in stabilizing the protein dimer was also assessed, being related to its biological role in assisting the SARS viruses to bypass the host protective response. This work also constitutes a first step in the possible rational design of efficient therapeutic agents aiming at perturbing the interaction between SARS Unique Domain and guanine quadruplexes, hence enhancing the host defenses against the virus.


Subject(s)
Betacoronavirus/chemistry , Betacoronavirus/genetics , Coronavirus Infections/virology , G-Quadruplexes/drug effects , Pneumonia, Viral/virology , RNA, Viral/chemistry , RNA, Viral/genetics , Betacoronavirus/drug effects , COVID-19 , Dimerization , Humans , Models, Molecular , Molecular Dynamics Simulation , Pandemics , Protein Binding , SARS-CoV-2 , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/genetics
11.
J Chem Inf Model ; 60(12): 5815-5831, 2020 12 28.
Article in English | MEDLINE | ID: covidwho-1387107

ABSTRACT

Herein, we investigate the structure and flexibility of the hydrated SARS-CoV-2 main protease by means of 2.0 µs molecular dynamics (MD) simulations in explicit solvent. After having performed electrostatic pKa calculations on several X-ray structures, we consider both the native (unbound) configuration of the enzyme and its noncovalent complex with a model peptide, Ace-Ala-Val-Leu-Gln∼Ser-Nme, which mimics the polyprotein sequence recognized at the active site. For each configuration, we also study their monomeric and homodimeric forms. The simulations of the unbound systems show that the relative orientation of domain III is not stable in the monomeric form and provide further details about interdomain motions, protomer-protomer interactions, inter-residue contacts, accessibility at the catalytic site, etc. In the presence of the peptide substrate, the monomeric protease exhibits a stable interdomain arrangement, but the relative orientation between the scissile peptide bond and the catalytic dyad is not favorable for catalysis. By means of comparative analysis, we further assess the catalytic impact of the enzyme dimerization, the actual flexibility of the active site region, and other structural effects induced by substrate binding. Overall, our computational results complement previous crystallographic studies on the SARS-CoV-2 enzyme and, together with other simulation studies, should contribute to outline useful structure-activity relationships.


Subject(s)
COVID-19/metabolism , Coronavirus 3C Proteases/metabolism , Peptides/chemistry , Peptides/metabolism , SARS-CoV-2/metabolism , Amino Acid Sequence , Catalytic Domain , Dimerization , Humans , Molecular Dynamics Simulation , Protein Conformation , Static Electricity , Structure-Activity Relationship , Substrate Specificity , Thermodynamics
12.
Commun Biol ; 4(1): 999, 2021 08 24.
Article in English | MEDLINE | ID: covidwho-1371605

ABSTRACT

The coronavirus SARS-CoV-2 uses an RNA-dependent RNA polymerase (RdRp) to replicate and transcribe its genome. Previous structures of the RdRp revealed a monomeric enzyme composed of the catalytic subunit nsp12, two copies of subunit nsp8, and one copy of subunit nsp7. Here we report an alternative, dimeric form of the enzyme and resolve its structure at 5.5 Å resolution. In this structure, the two RdRps contain only one copy of nsp8 each and dimerize via their nsp7 subunits to adopt an antiparallel arrangement. We speculate that the RdRp dimer facilitates template switching during production of sub-genomic RNAs.


Subject(s)
SARS-CoV-2/enzymology , Dimerization , Humans , RNA-Dependent RNA Polymerase/chemistry , RNA-Dependent RNA Polymerase/metabolism
13.
mBio ; 12(4): e0209421, 2021 08 31.
Article in English | MEDLINE | ID: covidwho-1360546

ABSTRACT

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent for coronavirus disease 2019 (COVID-19), encodes two proteases required for replication. The main protease (Mpro), encoded as part of two polyproteins, pp1a and pp1ab, is responsible for 11 different cleavages of these viral polyproteins to produce mature proteins required for viral replication. Mpro is therefore an attractive target for therapeutic interventions. Certain proteins in cells under oxidative stress undergo modification of reactive cysteines. We show Mpro is susceptible to glutathionylation, leading to inhibition of dimerization and activity. Activity of glutathionylated Mpro could be restored with reducing agents or glutaredoxin. Analytical studies demonstrated that glutathionylated Mpro primarily exists as a monomer and that modification of a single cysteine with glutathione is sufficient to block dimerization and inhibit its activity. Gel filtration studies as well as analytical ultracentrifugation confirmed that glutathionylated Mpro exists as a monomer. Tryptic and chymotryptic digestions of Mpro as well as experiments using a C300S Mpro mutant revealed that Cys300, which is located at the dimer interface, is a primary target of glutathionylation. Moreover, Cys300 is required for inhibition of activity upon Mpro glutathionylation. These findings indicate that Mpro dimerization and activity can be regulated through reversible glutathionylation of a non-active site cysteine, Cys300, which itself is not required for Mpro activity, and provides a novel target for the development of agents to block Mpro dimerization and activity. This feature of Mpro may have relevance to the pathophysiology of SARS-CoV-2 and related bat coronaviruses. IMPORTANCE SARS-CoV-2 is responsible for the devastating COVID-19 pandemic. Therefore, it is imperative that we learn as much as we can about the biochemistry of the coronavirus proteins to inform development of therapy. One attractive target is the main protease (Mpro), a dimeric enzyme necessary for viral replication. Most work thus far developing Mpro inhibitors has focused on the active site. Our work has revealed a regulatory mechanism for Mpro activity through glutathionylation of a cysteine (Cys300) at the dimer interface, which can occur in cells under oxidative stress. Cys300 glutathionylation inhibits Mpro activity by blocking its dimerization. This provides a novel accessible and reactive target for drug development. Moreover, this process may have implications for disease pathophysiology in humans and bats. It may be a mechanism by which SARS-CoV-2 has evolved to limit replication and avoid killing host bats when they are under oxidative stress during flight.


Subject(s)
Coronavirus 3C Proteases/metabolism , Cysteine/chemistry , Glutathione/chemistry , Protein Multimerization , SARS-CoV-2/metabolism , Animals , COVID-19/pathology , Chiroptera/virology , Coronavirus 3C Proteases/antagonists & inhibitors , Dimerization , Glutaredoxins/metabolism , Humans , SARS-CoV-2/enzymology
14.
Structure ; 29(12): 1382-1396.e6, 2021 12 02.
Article in English | MEDLINE | ID: covidwho-1356461

ABSTRACT

The COVID-19 pandemic has resulted in 198 million reported infections and more than 4 million deaths as of July 2021 (covid19.who.int). Research to identify effective therapies for COVID-19 includes: (1) designing a vaccine as future protection; (2) de novo drug discovery; and (3) identifying existing drugs to repurpose them as effective and immediate treatments. To assist in drug repurposing and design, we determine two apo structures of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) main protease at ambient temperature by serial femtosecond X-ray crystallography. We employ detailed molecular simulations of selected known main protease inhibitors with the structures and compare binding modes and energies. The combined structural and molecular modeling studies not only reveal the dynamics of small molecules targeting the main protease but also provide invaluable opportunities for drug repurposing and structure-based drug design strategies against SARS-CoV-2.


Subject(s)
COVID-19/drug therapy , Coronavirus 3C Proteases/chemistry , Drug Design , Drug Repositioning , SARS-CoV-2 , Catalytic Domain , Computer Simulation , Crystallography, X-Ray , Dimerization , Molecular Conformation , Molecular Docking Simulation , Principal Component Analysis , Protein Conformation , Recombinant Proteins/chemistry , Temperature
15.
Anal Bioanal Chem ; 413(29): 7205-7214, 2021 Dec.
Article in English | MEDLINE | ID: covidwho-1356000

ABSTRACT

Native mass spectrometry (MS) enjoyed tremendous success in the past two decades in a wide range of studies aiming at understanding the molecular mechanisms of physiological processes underlying a variety of pathologies and accelerating the drug discovery process. However, the success record of native MS has been surprisingly modest with respect to the most recent challenge facing the biomedical community-the novel coronavirus infection (COVID-19). The major reason for the paucity of successful studies that use native MS to target various aspects of SARS-CoV-2 interaction with its host is the extreme degree of heterogeneity of the viral protein playing a key role in the host cell invasion. Indeed, the SARS-CoV-2 spike protein (S-protein) is extensively glycosylated, presenting a formidable challenge for native MS as a means of characterizing its interactions with both the host cell-surface receptor ACE2 and the drug candidates capable of disrupting this interaction. In this work, we evaluate the utility of native MS complemented with the experimental methods using gas-phase chemistry (limited charge reduction) to obtain meaningful information on the association of the S1 domain of the S-protein with the ACE2 ectodomain, and the influence of a small synthetic heparinoid on this interaction. Native MS reveals the presence of several different S1 oligomers in solution and allows the stoichiometry of the most prominent S1/ACE2 complexes to be determined. This enables meaningful interpretation of the changes in native MS that are observed upon addition of a small synthetic heparinoid (the pentasaccharide fondaparinux) to the S1/ACE2 solution, confirming that the small polyanion destabilizes the protein/receptor binding.


Subject(s)
Receptors, Virus/metabolism , Spectrometry, Mass, Electrospray Ionization/methods , Spike Glycoprotein, Coronavirus/metabolism , Dimerization , Humans , Protein Binding
16.
Molecules ; 26(16)2021 Aug 12.
Article in English | MEDLINE | ID: covidwho-1355016

ABSTRACT

The COVID-19 outbreak has rapidly spread on a global scale, affecting the economy and public health systems throughout the world. In recent years, peptide-based therapeutics have been widely studied and developed to treat infectious diseases, including viral infections. Herein, the antiviral effects of the lysine linked dimer des-Cys11, Lys12,Lys13-(pBthTX-I)2K ((pBthTX-I)2K)) and derivatives against SARS-CoV-2 are reported. The lead peptide (pBthTX-I)2K and derivatives showed attractive inhibitory activities against SARS-CoV-2 (EC50 = 28-65 µM) and mostly low cytotoxic effect (CC50 > 100 µM). To shed light on the mechanism of action underlying the peptides' antiviral activity, the Main Protease (Mpro) and Papain-Like protease (PLpro) inhibitory activities of the peptides were assessed. The synthetic peptides showed PLpro inhibition potencies (IC50s = 1.0-3.5 µM) and binding affinities (Kd = 0.9-7 µM) at the low micromolar range but poor inhibitory activity against Mpro (IC50 > 10 µM). The modeled binding mode of a representative peptide of the series indicated that the compound blocked the entry of the PLpro substrate toward the protease catalytic cleft. Our findings indicated that non-toxic dimeric peptides derived from the Bothropstoxin-I have attractive cellular and enzymatic inhibitory activities, thereby suggesting that they are promising prototypes for the discovery and development of new drugs against SARS-CoV-2 infection.


Subject(s)
Crotalid Venoms/chemistry , Dimerization , Papain/antagonists & inhibitors , Peptides/chemistry , Peptides/pharmacology , SARS-CoV-2/enzymology , Antiviral Agents/chemistry , Antiviral Agents/metabolism , Antiviral Agents/pharmacology , Molecular Docking Simulation , Papain/chemistry , Papain/metabolism , Peptides/metabolism , Protease Inhibitors/chemistry , Protease Inhibitors/metabolism , Protease Inhibitors/pharmacology , Protein Conformation , SARS-CoV-2/drug effects
17.
J Mol Biol ; 433(18): 167118, 2021 09 03.
Article in English | MEDLINE | ID: covidwho-1303602

ABSTRACT

SARS-CoV-2 is the causative agent of COVID-19. The dimeric form of the viral Mpro is responsible for the cleavage of the viral polyprotein in 11 sites, including its own N and C-terminus. The lack of structural information for intermediary forms of Mpro is a setback for the understanding its self-maturation process. Herein, we used X-ray crystallography combined with biochemical data to characterize multiple forms of SARS-CoV-2 Mpro. For the immature form, we show that extra N-terminal residues caused conformational changes in the positioning of domain-three over the active site, hampering the dimerization and diminishing its activity. We propose that this form preludes the cis and trans-cleavage of N-terminal residues. Using fragment screening, we probe new cavities in this form which can be used to guide therapeutic development. Furthermore, we characterized a serine site-directed mutant of the Mpro bound to its endogenous N and C-terminal residues during dimeric association stage of the maturation process. We suggest this form is a transitional state during the C-terminal trans-cleavage. This data sheds light in the structural modifications of the SARS-CoV-2 main protease during its self-maturation process.


Subject(s)
Peptide Hydrolases/chemistry , Peptide Hydrolases/metabolism , SARS-CoV-2/metabolism , Viral Proteins/chemistry , Viral Proteins/metabolism , Catalytic Domain/physiology , Crystallography, X-Ray/methods , Dimerization , Humans
18.
Cell ; 184(11): 2955-2972.e25, 2021 05 27.
Article in English | MEDLINE | ID: covidwho-1237636

ABSTRACT

Natural antibodies (Abs) can target host glycans on the surface of pathogens. We studied the evolution of glycan-reactive B cells of rhesus macaques and humans using glycosylated HIV-1 envelope (Env) as a model antigen. 2G12 is a broadly neutralizing Ab (bnAb) that targets a conserved glycan patch on Env of geographically diverse HIV-1 strains using a unique heavy-chain (VH) domain-swapped architecture that results in fragment antigen-binding (Fab) dimerization. Here, we describe HIV-1 Env Fab-dimerized glycan (FDG)-reactive bnAbs without VH-swapped domains from simian-human immunodeficiency virus (SHIV)-infected macaques. FDG Abs also recognized cell-surface glycans on diverse pathogens, including yeast and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike. FDG precursors were expanded by glycan-bearing immunogens in macaques and were abundant in HIV-1-naive humans. Moreover, FDG precursors were predominately mutated IgM+IgD+CD27+, thus suggesting that they originated from a pool of antigen-experienced IgM+ or marginal zone B cells.


Subject(s)
Antibodies, Neutralizing/immunology , HIV-1/immunology , Immunoglobulin Fab Fragments/immunology , Polysaccharides/immunology , SARS-CoV-2/immunology , Simian Immunodeficiency Virus/immunology , Spike Glycoprotein, Coronavirus/immunology , env Gene Products, Human Immunodeficiency Virus/immunology , Animals , B-Lymphocytes/immunology , Broadly Neutralizing Antibodies/immunology , COVID-19/immunology , Dimerization , Epitopes/immunology , Glycosylation , HIV Antibodies/immunology , HIV Infections/immunology , Humans , Immunoglobulin Fab Fragments/chemistry , Macaca mulatta , Polysaccharides/chemistry , Receptors, Antigen, B-Cell/chemistry , Simian Immunodeficiency Virus/genetics , Vaccines/immunology , env Gene Products, Human Immunodeficiency Virus/chemistry , env Gene Products, Human Immunodeficiency Virus/genetics
19.
Biomolecules ; 11(5)2021 05 17.
Article in English | MEDLINE | ID: covidwho-1234665

ABSTRACT

Cm-p5 is a snail-derived antimicrobial peptide, which demonstrated antifungal activity against the pathogenic strains of Candida albicans. Previously we synthetized a cyclic monomer as well as a parallel and an antiparallel dimer of Cm-p5 with improved antifungal activity. Considering the alarming increase of microbial resistance to conventional antibiotics, here we evaluated the antimicrobial activity of these derivatives against multiresistant and problematic bacteria and against important viral agents. The three peptides showed a moderate activity against Pseudomonas aeruginosa, Klebsiella pneumoniae Extended Spectrum ß-Lactamase (ESBL), and Streptococcus agalactiae, with MIC values > 100 µg/mL. They exerted a considerable activity with MIC values between 25-50 µg/mL against Acinetobacter baumanii and Enterococcus faecium. In addition, the two dimers showed a moderate activity against Pseudomonas aeruginosa PA14. The three Cm-p5 derivatives inhibited a virulent extracellular strain of Mycobacterium tuberculosis, in a dose-dependent manner. Moreover, they inhibited Herpes Simplex Virus 2 (HSV-2) infection in a concentration-dependent manner, but had no effect on infection by the Zika Virus (ZIKV) or pseudoparticles of Severe Acute Respiratory Syndrome Corona Virus 2 (SARS-CoV-2). At concentrations of >100 µg/mL, the three new Cm-p5 derivatives showed toxicity on different eukaryotic cells tested. Considering a certain cell toxicity but a potential interesting activity against the multiresistant strains of bacteria and HSV-2, our compounds require future structural optimization.


Subject(s)
Anti-Bacterial Agents/pharmacology , Antimicrobial Cationic Peptides/chemistry , Antiviral Agents/pharmacology , Drug Resistance, Multiple, Bacterial/drug effects , Herpesvirus 2, Human/drug effects , Amino Acid Sequence , Animals , Anti-Bacterial Agents/chemistry , Antimicrobial Cationic Peptides/pharmacology , Antiviral Agents/chemistry , Candida albicans/drug effects , Cell Line , Cell Survival/drug effects , Dimerization , Gram-Negative Bacteria/drug effects , Gram-Positive Bacteria/drug effects , Humans , Microbial Sensitivity Tests , SARS-CoV-2/drug effects
20.
Nucleic Acids Res ; 49(10): 5956-5966, 2021 06 04.
Article in English | MEDLINE | ID: covidwho-1231040

ABSTRACT

Replication of the ∼30 kb-long coronavirus genome is mediated by a complex of non-structural proteins (NSP), in which NSP7 and NSP8 play a critical role in regulating the RNA-dependent RNA polymerase (RdRP) activity of NSP12. The assembly of NSP7, NSP8 and NSP12 proteins is highly dynamic in solution, yet the underlying mechanism remains elusive. We report the crystal structure of the complex between NSP7 and NSP8 of SARS-CoV-2, revealing a 2:2 heterotetrameric form. Formation of the NSP7-NSP8 complex is mediated by two distinct oligomer interfaces, with interface I responsible for heterodimeric NSP7-NSP8 assembly, and interface II mediating the heterotetrameric interaction between the two NSP7-NSP8 dimers. Structure-guided mutagenesis, combined with biochemical and enzymatic assays, further reveals a structural coupling between the two oligomer interfaces, as well as the importance of these interfaces for the RdRP activity of the NSP7-NSP8-NSP12 complex. Finally, we identify an NSP7 mutation that differentially affects the stability of the NSP7-NSP8 and NSP7-NSP8-NSP12 complexes leading to a selective impairment of the RdRP activity. Together, this study provides deep insights into the structure and mechanism for the dynamic assembly of NSP7 and NSP8 in regulating the replication of the SARS-CoV-2 genome, with important implications for antiviral drug development.


Subject(s)
COVID-19 , Coronavirus RNA-Dependent RNA Polymerase/chemistry , SARS-CoV-2/enzymology , Viral Nonstructural Proteins/chemistry , Chromatography, Gel , Coronavirus RNA-Dependent RNA Polymerase/biosynthesis , Coronavirus RNA-Dependent RNA Polymerase/genetics , Crystallography, X-Ray , Dimerization , Models, Molecular , Multiprotein Complexes , Mutagenesis , Mutation , Protein Conformation , Protein Domains , Protein Interaction Mapping , SARS-CoV-2/genetics , SARS-CoV-2/physiology , Structure-Activity Relationship , Viral Nonstructural Proteins/genetics , Virus Replication
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