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1.
J Med Chem ; 64(19): 14887-14894, 2021 10 14.
Article in English | MEDLINE | ID: covidwho-1428719

ABSTRACT

Antiviral treatments of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have been extensively pursued to conquer the pandemic. To inhibit the viral entry to the host cell, we designed and obtained three peptide sequences via quartz crystal microbalance measurement screening, which showed high affinity at nanomole to the S1 subunit of the spike protein and wild-type SARS-CoV-2 pseudovirus. Circular dichroism spectroscopy measurements revealed significant conformation changes of the S1 protein upon encounter with the three peptides. The peptides were able to effectively block the infection of a pseudovirus to 50% by inhibiting the host cell lines binding with the S1 protein, evidenced by the results from Western blotting and pseudovirus luciferase assay. Moreover, the combination of the three peptides could increase the inhibitory rate to 75%. In conclusion, the three chemically synthetic neutralizing peptides and their combinations hold promising potential as effective therapeutics in the prevention and treatment of COVID-19.


Subject(s)
Peptides/metabolism , Spike Glycoprotein, Coronavirus/metabolism , A549 Cells , Angiotensin-Converting Enzyme 2/chemistry , Angiotensin-Converting Enzyme 2/metabolism , COVID-19/pathology , COVID-19/virology , Cell Survival/drug effects , Circular Dichroism , Humans , Neutralization Tests , Peptides/chemistry , Peptides/pharmacology , Protein Binding , Protein Subunits/chemistry , Protein Subunits/metabolism , SARS-CoV-2/isolation & purification , Spike Glycoprotein, Coronavirus/chemistry , Virus Internalization/drug effects
2.
Signal Transduct Target Ther ; 5(1): 220, 2020 10 06.
Article in English | MEDLINE | ID: covidwho-1387194
3.
Cell Res ; 31(10): 1047-1060, 2021 10.
Article in English | MEDLINE | ID: covidwho-1380899

ABSTRACT

The outbreak of SARS-CoV-2 (SARS2) has caused a global COVID-19 pandemic. The spike protein of SARS2 (SARS2-S) recognizes host receptors, including ACE2, to initiate viral entry in a complex biomechanical environment. Here, we reveal that tensile force, generated by bending of the host cell membrane, strengthens spike recognition of ACE2 and accelerates the detachment of spike's S1 subunit from the S2 subunit to rapidly prime the viral fusion machinery. Mechanistically, such mechano-activation is fulfilled by force-induced opening and rotation of spike's receptor-binding domain to prolong the bond lifetime of spike/ACE2 binding, up to 4 times longer than that of SARS-S binding with ACE2 under 10 pN force application, and subsequently by force-accelerated S1/S2 detachment which is up to ~103 times faster than that in the no-force condition. Interestingly, the SARS2-S D614G mutant, a more infectious variant, shows 3-time stronger force-dependent ACE2 binding and 35-time faster force-induced S1/S2 detachment. We also reveal that an anti-S1/S2 non-RBD-blocking antibody that was derived from convalescent COVID-19 patients with potent neutralizing capability can reduce S1/S2 detachment by 3 × 106 times under force. Our study sheds light on the mechano-chemistry of spike activation and on developing a non-RBD-blocking but S1/S2-locking therapeutic strategy to prevent SARS2 invasion.


Subject(s)
COVID-19/diagnosis , SARS-CoV-2/metabolism , Spike Glycoprotein, Coronavirus/metabolism , Tensile Strength , Angiotensin-Converting Enzyme 2/chemistry , Angiotensin-Converting Enzyme 2/metabolism , Antibodies, Neutralizing/immunology , Binding Sites , COVID-19/therapy , COVID-19/virology , Humans , Hydrogen-Ion Concentration , Immunization, Passive , Molecular Dynamics Simulation , Protein Binding , Protein Domains/immunology , Protein Subunits/chemistry , Protein Subunits/immunology , Protein Subunits/metabolism , SARS-CoV-2/isolation & purification , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/immunology , Virus Internalization
4.
J Am Chem Soc ; 143(33): 12930-12934, 2021 08 25.
Article in English | MEDLINE | ID: covidwho-1358340

ABSTRACT

The main protease from SARS-CoV-2 is a homodimer. Yet, a recent 0.1-ms-long molecular dynamics simulation performed by D. E. Shaw's research group shows that it readily undergoes a symmetry-breaking event on passing from the solid state to aqueous solution. As a result, the subunits present distinct conformations of the binding pocket. By analyzing this long simulation, we uncover a previously unrecognized role of water molecules in triggering the transition. Interestingly, each subunit presents a different collection of long-lived water molecules. Enhanced sampling simulations performed here, along with machine learning approaches, further establish that the transition to the asymmetric state is essentially irreversible.


Subject(s)
SARS-CoV-2/enzymology , Viral Matrix Proteins/chemistry , Water/chemistry , COVID-19/pathology , COVID-19/virology , Crystallography, X-Ray , Humans , Hydrogen Bonding , Molecular Dynamics Simulation , Protein Structure, Quaternary , Protein Subunits/chemistry , Protein Subunits/metabolism , SARS-CoV-2/isolation & purification , Viral Matrix Proteins/metabolism
5.
Viruses ; 13(8)2021 08 15.
Article in English | MEDLINE | ID: covidwho-1355053

ABSTRACT

We compared the electrostatic properties of the spike proteins (S-proteins) of three coronaviruses, SARS-CoV, MERS-CoV, and SARS-CoV-2, and their interactions with photosensitizers (PSs), octacationic octakis(cholinyl)zinc phthalocyanine (Zn-PcChol8+) and monocationic methylene blue (MB). We found a major common PS binding site at the connection of the S-protein stalk and head. The molecules of Zn-PcChol8+ and MB also form electrostatic encounter complexes with large area of negative electrostatic potential at the head of the S-protein of SARS-CoV-2, between fusion protein and heptad repeat 1 domain. The top of the SARS-CoV spike head demonstrates a notable area of electrostatic contacts with Zn-PcChol8+ and MB that corresponds to the N-terminal domain. The S-protein protomers of SARS-CoV-2 in "open" and "closed" conformations demonstrate different ability to attract PS molecules. In contrast with Zn-PcChol8+, MB possesses the ability to penetrate inside the pocket formed as a result of SARS-CoV-2 receptor binding domain transition into the "open" state. The existence of binding site for cationic PSs common to the S-proteins of SARS-CoV, SARS-CoV-2, and MERS-CoV creates prospects for the wide use of this type of PSs to combat the spread of coronaviruses.


Subject(s)
Indoles/metabolism , Middle East Respiratory Syndrome Coronavirus/chemistry , Organometallic Compounds/metabolism , Photosensitizing Agents/metabolism , Spike Glycoprotein, Coronavirus/metabolism , Binding Sites , Indoles/chemistry , Isoindoles , Methylene Blue/metabolism , Models, Molecular , Molecular Dynamics Simulation , Organometallic Compounds/chemistry , Protein Conformation , Protein Domains , Protein Subunits/chemistry , SARS Virus/chemistry , SARS-CoV-2/chemistry , Spike Glycoprotein, Coronavirus/chemistry , Static Electricity , Zinc Compounds
6.
Int J Mol Sci ; 22(15)2021 Jul 30.
Article in English | MEDLINE | ID: covidwho-1350316

ABSTRACT

Increasing evidence suggests that elderly people with dementia are vulnerable to the development of severe coronavirus disease 2019 (COVID-19). In Alzheimer's disease (AD), the major form of dementia, ß-amyloid (Aß) levels in the blood are increased; however, the impact of elevated Aß levels on the progression of COVID-19 remains largely unknown. Here, our findings demonstrate that Aß1-42, but not Aß1-40, bound to various viral proteins with a preferentially high affinity for the spike protein S1 subunit (S1) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and the viral receptor, angiotensin-converting enzyme 2 (ACE2). These bindings were mainly through the C-terminal residues of Aß1-42. Furthermore, Aß1-42 strengthened the binding of the S1 of SARS-CoV-2 to ACE2 and increased the viral entry and production of IL-6 in a SARS-CoV-2 pseudovirus infection model. Intriguingly, data from a surrogate mouse model with intravenous inoculation of Aß1-42 show that the clearance of Aß1-42 in the blood was dampened in the presence of the extracellular domain of the spike protein trimers of SARS-CoV-2, whose effects can be prevented by a novel anti-Aß antibody. In conclusion, these findings suggest that the binding of Aß1-42 to the S1 of SARS-CoV-2 and ACE2 may have a negative impact on the course and severity of SARS-CoV-2 infection. Further investigations are warranted to elucidate the underlying mechanisms and examine whether reducing the level of Aß1-42 in the blood is beneficial to the fight against COVID-19 and AD.


Subject(s)
Amyloid beta-Peptides/metabolism , Angiotensin-Converting Enzyme 2/metabolism , Peptide Fragments/metabolism , SARS-CoV-2/enzymology , Spike Glycoprotein, Coronavirus/metabolism , A549 Cells , Alzheimer Disease/complications , Alzheimer Disease/metabolism , Amyloid beta-Peptides/chemistry , Animals , COVID-19/complications , COVID-19/metabolism , Chlorocebus aethiops , Humans , Interleukin-6/metabolism , Mice, Inbred C57BL , Mice, Transgenic , Peptide Fragments/chemistry , Protein Subunits/chemistry , Protein Subunits/metabolism , Spike Glycoprotein, Coronavirus/chemistry , Vero Cells , Virus Internalization
7.
J Am Chem Soc ; 143(33): 13205-13211, 2021 08 25.
Article in English | MEDLINE | ID: covidwho-1349637

ABSTRACT

The receptor binding and proteolysis of Spike of SARS-CoV-2 release its S2 subunit to rearrange and catalyze viral-cell fusion. This deploys the fusion peptide for insertion into the cell membranes targeted. We show that this fusion peptide transforms from intrinsic disorder in solution into a wedge-shaped structure inserted in bilayered micelles, according to chemical shifts, 15N NMR relaxation, and NOEs. The globular fold of three helices contrasts the open, extended forms of this region observed in the electron density of compact prefusion states. In the hydrophobic, narrow end of the wedge, helices 1 and 2 contact the fatty acyl chains of phospholipids, according to NOEs and proximity to a nitroxide spin label deep in the membrane mimic. The polar end of the wedge may engage and displace lipid head groups and bind Ca2+ ions for membrane fusion. Polar helix 3 protrudes from the bilayer where it might be accessible to antibodies.


Subject(s)
Micelles , Peptides/metabolism , SARS-CoV-2/metabolism , Spike Glycoprotein, Coronavirus/chemistry , COVID-19/pathology , COVID-19/virology , Humans , Hydrophobic and Hydrophilic Interactions , Peptides/chemistry , Phospholipids/chemistry , Phospholipids/metabolism , Protein Binding , Protein Conformation, alpha-Helical , Protein Subunits/chemistry , Protein Subunits/metabolism , SARS-CoV-2/isolation & purification , Spike Glycoprotein, Coronavirus/metabolism
8.
Int J Mol Sci ; 22(15)2021 Jul 30.
Article in English | MEDLINE | ID: covidwho-1335101

ABSTRACT

Increasing evidence suggests that elderly people with dementia are vulnerable to the development of severe coronavirus disease 2019 (COVID-19). In Alzheimer's disease (AD), the major form of dementia, ß-amyloid (Aß) levels in the blood are increased; however, the impact of elevated Aß levels on the progression of COVID-19 remains largely unknown. Here, our findings demonstrate that Aß1-42, but not Aß1-40, bound to various viral proteins with a preferentially high affinity for the spike protein S1 subunit (S1) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and the viral receptor, angiotensin-converting enzyme 2 (ACE2). These bindings were mainly through the C-terminal residues of Aß1-42. Furthermore, Aß1-42 strengthened the binding of the S1 of SARS-CoV-2 to ACE2 and increased the viral entry and production of IL-6 in a SARS-CoV-2 pseudovirus infection model. Intriguingly, data from a surrogate mouse model with intravenous inoculation of Aß1-42 show that the clearance of Aß1-42 in the blood was dampened in the presence of the extracellular domain of the spike protein trimers of SARS-CoV-2, whose effects can be prevented by a novel anti-Aß antibody. In conclusion, these findings suggest that the binding of Aß1-42 to the S1 of SARS-CoV-2 and ACE2 may have a negative impact on the course and severity of SARS-CoV-2 infection. Further investigations are warranted to elucidate the underlying mechanisms and examine whether reducing the level of Aß1-42 in the blood is beneficial to the fight against COVID-19 and AD.


Subject(s)
Amyloid beta-Peptides/metabolism , Angiotensin-Converting Enzyme 2/metabolism , Peptide Fragments/metabolism , SARS-CoV-2/enzymology , Spike Glycoprotein, Coronavirus/metabolism , A549 Cells , Alzheimer Disease/complications , Alzheimer Disease/metabolism , Amyloid beta-Peptides/chemistry , Animals , COVID-19/complications , COVID-19/metabolism , Chlorocebus aethiops , Humans , Interleukin-6/metabolism , Mice, Inbred C57BL , Mice, Transgenic , Peptide Fragments/chemistry , Protein Subunits/chemistry , Protein Subunits/metabolism , Spike Glycoprotein, Coronavirus/chemistry , Vero Cells , Virus Internalization
9.
Int J Mol Sci ; 21(20)2020 Oct 09.
Article in English | MEDLINE | ID: covidwho-1298152

ABSTRACT

Nicotinic acetylcholine receptors (nAChRs) are pentameric ligand-gated ion channels responsible for rapid neural and neuromuscular signal transmission. Although it is well documented that 16 subunits are encoded by the human genome, their presence in airway epithelial cells (AECs) remains poorly understood, and contribution to pathology is mainly discussed in the context of cancer. We analysed nAChR subunit expression in the human lungs of smokers and non-smokers using transcriptomic data for whole-lung tissues, isolated large AECs, and isolated small AECs. We identified differential expressions of nAChRs in terms of detection and repartition in the three modalities. Smoking-associated alterations were also unveiled. Then, we identified an nAChR transcriptomic print at the single-cell level. Finally, we reported the localizations of detectable nAChRs in bronchi and large bronchioles. Thus, we compiled the first complete atlas of pulmonary nAChR subunits to open new avenues to further unravel the involvement of these receptors in lung homeostasis and respiratory diseases.


Subject(s)
Lung/metabolism , Protein Subunits/metabolism , Receptors, Nicotinic/metabolism , Adult , Age Factors , Cell Cycle , Epithelial Cells/metabolism , Gene Expression Regulation , Humans , Protein Subunits/chemistry , Protein Subunits/genetics , Receptors, Nicotinic/chemistry , Receptors, Nicotinic/genetics , Respiratory Mucosa/metabolism , Respiratory Mucosa/pathology , Signal Detection, Psychological , Smoking , Transcription, Genetic
10.
Science ; 373(6555): 648-654, 2021 08 06.
Article in English | MEDLINE | ID: covidwho-1295161

ABSTRACT

A novel variant of concern (VOC) named CAL.20C (B.1.427/B.1.429), which was originally detected in California, carries spike glycoprotein mutations S13I in the signal peptide, W152C in the N-terminal domain (NTD), and L452R in the receptor-binding domain (RBD). Plasma from individuals vaccinated with a Wuhan-1 isolate-based messenger RNA vaccine or from convalescent individuals exhibited neutralizing titers that were reduced 2- to 3.5-fold against the B.1.427/B.1.429 variant relative to wild-type pseudoviruses. The L452R mutation reduced neutralizing activity in 14 of 34 RBD-specific monoclonal antibodies (mAbs). The S13I and W152C mutations resulted in total loss of neutralization for 10 of 10 NTD-specific mAbs because the NTD antigenic supersite was remodeled by a shift of the signal peptide cleavage site and the formation of a new disulfide bond, as revealed by mass spectrometry and structural studies.


Subject(s)
COVID-19/virology , Immune Evasion , SARS-CoV-2/immunology , SARS-CoV-2/pathogenicity , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/immunology , Amino Acid Substitution , Antibodies, Monoclonal/immunology , Antibodies, Neutralizing/blood , Antibodies, Neutralizing/immunology , Antibodies, Viral/blood , Antibodies, Viral/immunology , Antigens, Viral/immunology , COVID-19/immunology , COVID-19 Vaccines/immunology , Cryoelectron Microscopy , Humans , Models, Molecular , Mutation , Neutralization Tests , Protein Conformation , Protein Domains , Protein Interaction Domains and Motifs , Protein Subunits/chemistry , SARS-CoV-2/genetics , Spike Glycoprotein, Coronavirus/chemistry
11.
Science ; 373(6555): 642-648, 2021 08 06.
Article in English | MEDLINE | ID: covidwho-1282051

ABSTRACT

Several fast-spreading variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have become the dominant circulating strains in the COVID-19 pandemic. We report here cryo-electron microscopy structures of the full-length spike (S) trimers of the B.1.1.7 and B.1.351 variants, as well as their biochemical and antigenic properties. Amino acid substitutions in the B.1.1.7 protein increase both the accessibility of its receptor binding domain and the binding affinity for receptor angiotensin-converting enzyme 2 (ACE2). The enhanced receptor engagement may account for the increased transmissibility. The B.1.351 variant has evolved to reshape antigenic surfaces of the major neutralizing sites on the S protein, making it resistant to some potent neutralizing antibodies. These findings provide structural details on how SARS-CoV-2 has evolved to enhance viral fitness and immune evasion.


Subject(s)
COVID-19/virology , Immune Evasion , SARS-CoV-2/chemistry , SARS-CoV-2/pathogenicity , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/immunology , Amino Acid Substitution , Angiotensin-Converting Enzyme 2/metabolism , Antibodies, Viral/immunology , Antigens, Viral/immunology , Cryoelectron Microscopy , HEK293 Cells , Humans , Models, Molecular , Mutation , Protein Binding , Protein Conformation , Protein Domains , Protein Interaction Domains and Motifs , Protein Subunits/chemistry , Receptors, Coronavirus/metabolism , SARS-CoV-2/genetics , SARS-CoV-2/immunology , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/metabolism
12.
Science ; 373(6555)2021 08 06.
Article in English | MEDLINE | ID: covidwho-1282050

ABSTRACT

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants with multiple spike mutations enable increased transmission and antibody resistance. We combined cryo-electron microscopy (cryo-EM), binding, and computational analyses to study variant spikes, including one that was involved in transmission between minks and humans, and others that originated and spread in human populations. All variants showed increased angiotensin-converting enzyme 2 (ACE2) receptor binding and increased propensity for receptor binding domain (RBD)-up states. While adaptation to mink resulted in spike destabilization, the B.1.1.7 (UK) spike balanced stabilizing and destabilizing mutations. A local destabilizing effect of the RBD E484K mutation was implicated in resistance of the B.1.1.28/P.1 (Brazil) and B.1.351 (South Africa) variants to neutralizing antibodies. Our studies revealed allosteric effects of mutations and mechanistic differences that drive either interspecies transmission or escape from antibody neutralization.


Subject(s)
SARS-CoV-2/chemistry , SARS-CoV-2/genetics , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/genetics , Amino Acid Substitution , Angiotensin-Converting Enzyme 2/metabolism , Animals , Antibodies, Neutralizing/immunology , Antibodies, Viral/immunology , Antigens, Viral/immunology , COVID-19/transmission , COVID-19/veterinary , COVID-19/virology , Cryoelectron Microscopy , Host Adaptation , Humans , Immune Evasion , Mink/virology , Models, Molecular , Mutation , Protein Binding , Protein Conformation , Protein Interaction Domains and Motifs , Protein Structure, Quaternary , Protein Subunits/chemistry , Receptors, Coronavirus/metabolism , SARS-CoV-2/immunology , Spike Glycoprotein, Coronavirus/immunology , Spike Glycoprotein, Coronavirus/metabolism
13.
Nat Commun ; 12(1): 3661, 2021 06 16.
Article in English | MEDLINE | ID: covidwho-1275912

ABSTRACT

SARS-CoV-2, the virus responsible for COVID-19, has caused a global pandemic. Antibodies can be powerful biotherapeutics to fight viral infections. Here, we use the human apoferritin protomer as a modular subunit to drive oligomerization of antibody fragments and transform antibodies targeting SARS-CoV-2 into exceptionally potent neutralizers. Using this platform, half-maximal inhibitory concentration (IC50) values as low as 9 × 10-14 M are achieved as a result of up to 10,000-fold potency enhancements compared to corresponding IgGs. Combination of three different antibody specificities and the fragment crystallizable (Fc) domain on a single multivalent molecule conferred the ability to overcome viral sequence variability together with outstanding potency and IgG-like bioavailability. The MULTi-specific, multi-Affinity antiBODY (Multabody or MB) platform thus uniquely leverages binding avidity together with multi-specificity to deliver ultrapotent and broad neutralizers against SARS-CoV-2. The modularity of the platform also makes it relevant for rapid evaluation against other infectious diseases of global health importance. Neutralizing antibodies are a promising therapeutic for SARS-CoV-2.


Subject(s)
Antibodies, Monoclonal/pharmacology , Antibodies, Neutralizing/immunology , Antibodies, Viral/chemistry , SARS-CoV-2/immunology , Animals , Antibodies, Monoclonal/chemistry , Antibodies, Monoclonal/genetics , Antibodies, Monoclonal/immunology , Antibodies, Neutralizing/chemistry , Antibodies, Viral/immunology , Antibody Specificity , Apoferritins/chemistry , Biological Availability , Epitope Mapping , Humans , Immunoglobulin G/immunology , Male , Mice, Inbred BALB C , Mice, Inbred C57BL , Protein Engineering/methods , Protein Subunits/chemistry , Spike Glycoprotein, Coronavirus/immunology , Tissue Distribution
14.
Sci Adv ; 7(16)2021 04.
Article in English | MEDLINE | ID: covidwho-1189804

ABSTRACT

The COVID-19 (coronavirus disease 2019) pandemic underwent a rapid transition with the emergence of a dominant viral variant (from the "D-form" to the "G-form") that carried an amino acid substitution D614G in its "Spike" protein. The G-form is more infectious in vitro and is associated with increased viral loads in the upper airways. To gain insight into the molecular-level underpinnings of these characteristics, we used microsecond all-atom simulations. We show that changes in the protein energetics favor a higher population of infection-capable states in the G-form through release of asymmetry present in the D-form inter-protomer interactions. Thus, the increased infectivity of the G-form is likely due to a higher rate of profitable binding encounters with the host receptor. It is also predicted to be more neutralization sensitive owing to enhanced exposure of the receptor binding domain, a key target region for neutralizing antibodies. These results are critical for vaccine design.


Subject(s)
SARS-CoV-2/genetics , Spike Glycoprotein, Coronavirus/chemistry , Amino Acid Sequence , Angiotensin-Converting Enzyme 2/chemistry , Angiotensin-Converting Enzyme 2/metabolism , Antibodies, Neutralizing/immunology , COVID-19/pathology , COVID-19/virology , Glycosylation , Humans , Hydrogen Bonding , Molecular Dynamics Simulation , Mutation , Protein Binding , Protein Structure, Quaternary , Protein Subunits/chemistry , Protein Subunits/immunology , SARS-CoV-2/immunology , SARS-CoV-2/isolation & purification , Spike Glycoprotein, Coronavirus/metabolism , Virus Internalization
15.
Science ; 372(6541): 525-530, 2021 04 30.
Article in English | MEDLINE | ID: covidwho-1138286

ABSTRACT

Substitution for aspartic acid (D) by glycine (G) at position 614 in the spike (S) protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) appears to facilitate rapid viral spread. The G614 strain and its recent variants are now the dominant circulating forms. Here, we report cryo-electron microscopy structures of a full-length G614 S trimer, which adopts three distinct prefusion conformations that differ primarily by the position of one receptor-binding domain. A loop disordered in the D614 S trimer wedges between domains within a protomer in the G614 spike. This added interaction appears to prevent premature dissociation of the G614 trimer-effectively increasing the number of functional spikes and enhancing infectivity-and to modulate structural rearrangements for membrane fusion. These findings extend our understanding of viral entry and suggest an improved immunogen for vaccine development.


Subject(s)
SARS-CoV-2/chemistry , SARS-CoV-2/genetics , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/genetics , Amino Acid Substitution , Angiotensin-Converting Enzyme 2/chemistry , Angiotensin-Converting Enzyme 2/metabolism , Antibodies, Viral/immunology , Antibodies, Viral/metabolism , COVID-19/virology , Cryoelectron Microscopy , Humans , Hydrophobic and Hydrophilic Interactions , Models, Molecular , Mutant Proteins/chemistry , Mutant Proteins/metabolism , Protein Binding , Protein Conformation , Protein Domains , Protein Subunits/chemistry , Protein Subunits/metabolism , Receptors, Coronavirus/chemistry , Receptors, Coronavirus/metabolism , SARS-CoV-2/physiology , Spike Glycoprotein, Coronavirus/immunology , Spike Glycoprotein, Coronavirus/metabolism , Virus Internalization
16.
Electrophoresis ; 42(6): 687-692, 2021 03.
Article in English | MEDLINE | ID: covidwho-1059406

ABSTRACT

In order to contribute to the scientific research on the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), we have investigated the isoelectric points (pI) of several related proteins, which are commercially available: the receptor-binding domain (RBD) with His- and Fc-tag, the S1 subunit with His-tag, the S1/S2 subunits with His-tag and the human angiotensin-converting enzyme 2 (hACE2) with His-tag. First, the theoretical pI values, based on the amino acid (AA) sequences of the proteins, were calculated using the ProtParam tool from the Bioinformatics Resource Portal ExPASy. The proteins were then measured with the Maurice imaged CIEF system (native fluorescence detection), testing various measurement conditions, such as different ampholytes or ampholyte mixtures. Due to isoforms, we get sections with several peaks and not just one peak for each protein. The determined pI range for the RBD/Fc is 8.24-9.32 (theoretical pI: 8.55), for the RBD/His it is 7.36-9.88 (8.91) and for the S1/His it is 7.30-8.37 (7.80). The pI range of the S1/S2/His is 4.41-5.87 (no theoretical pI, AA sequence unknown) and for hACE2/His, the determined global range is 5.19-6.11 (5.60) for all experimental conditions chosen. All theoretically derived values were found within these ranges, usually close to the center. Therefore, we consider theoretical values as useful to make predictions about the isoelectric points of SARS-CoV-2 proteins. The experimental conditions had only a minor influence on the pI ranges obtained and mainly influenced the peak shapes.


Subject(s)
Angiotensin-Converting Enzyme 2/chemistry , COVID-19/virology , Isoelectric Focusing/methods , SARS-CoV-2/chemistry , Spike Glycoprotein, Coronavirus/chemistry , Angiotensin-Converting Enzyme 2/metabolism , Binding Sites , COVID-19/metabolism , Humans , Isoelectric Point , Protein Interaction Domains and Motifs , Protein Subunits/chemistry , Protein Subunits/metabolism , SARS-CoV-2/metabolism , Spike Glycoprotein, Coronavirus/metabolism
17.
PLoS One ; 15(11): e0241168, 2020.
Article in English | MEDLINE | ID: covidwho-917991

ABSTRACT

The SARS-CoV-2 virion responsible for the current world-wide pandemic COVID-19 has a characteristic Spike protein (S) on its surface that embellishes both a prefusion state and fusion state. The prefusion Spike protein (S) is a large trimeric protein where each protomer may be in a so-called Up state or Down state, depending on the configuration of its receptor binding domain (RBD) within its distal, prefusion S1 domain. The Up state is believed to allow binding of the virion to ACE-2 receptors on human epithelial cells, whereas the Down state is believed to be relatively inactive or reduced in its binding behavior. We have performed detailed all-atom, dominant energy landscape mappings for noncovalent interactions (charge, partial charge, and van der Waals) of the SARS-CoV-2 Spike protein in its static prefusion state based on two recent and independent experimental structure publications. We included both interchain interactions and intrachain (domain) interactions in our mappings in order to determine any telling differences (different so-called "glue" points) between residues in the Up and Down state protomers. The S2 proximal, fusion domain demonstrated no appreciable energetic differences between Up and Down protomers, including interchain as well as each protomer's intrachain, S1-S2 interactions. However, the S1 domain interactions across neighboring protomers, which include the RBD-NTD cross chain interactions, showed significant energetic differences between Up-Down and Down-Down neighboring protomers. This included, for example, a key RBD residue ARG357 in the Up-Down interaction and a three residue sequence ALA520-PRO521-ALA522, associated with a turn structure in the RBD of the Up state protomer, acting as a stabilizing interaction with the NTD of its neighbor protomer. Additionally, our intra chain dominant energy mappings within each protomer, identified a significant "glue" point or possible "latch" for the Down state protomer between the S1 subdomain, SD1, and the RBD domain of the same protomer that was completely missing in the Up state protomer analysis. Ironically, this dominant energetic interaction in the Down state protomer involved the backbone atoms of the same three residue sequence ALA520-PRO521-ALA522 of the RBD with the amino acid R-group of GLN564 in the SD1 domain. Thus, this same three residue sequence acts as a stabilizer of the RBD in the Up conformation through its interactions with its neighboring NTD chain and a kind of latch in the Down state conformation through its interactions with its own SD1 domain. The dominant interaction energy residues identified here are also conserved across reported variations of SARS-CoV-2, as well as the closely related virions SARS-Cov and the bat corona virus RatG13. We conducted preliminary molecular dynamics simulations across 0.1 µ seconds to see if this latch provided structural stability and indeed found that a single point mutation (Q564G) resulted in the latch releasing transforming the protomer from the Down to the Up state conformation. Full trimeric Spike protein studies of the same mutation across all protomers, however, did not exhibit latch release demonstrating the critical importance of interchain interactions across the S1 domain, including RBD-NTD neighboring chain interactions. Therapies aimed at disrupting these noncovalent interactions could be a viable route for the physico-chemical mitigation of this deadly virion.


Subject(s)
Betacoronavirus/metabolism , Spike Glycoprotein, Coronavirus/metabolism , Angiotensin-Converting Enzyme 2 , Betacoronavirus/isolation & purification , COVID-19 , Coronavirus Infections/pathology , Coronavirus Infections/virology , Humans , Molecular Dynamics Simulation , Pandemics , Peptidyl-Dipeptidase A/chemistry , Peptidyl-Dipeptidase A/metabolism , Pneumonia, Viral/pathology , Pneumonia, Viral/virology , Point Mutation , Protein Binding , Protein Domains , Protein Stability , Protein Subunits/chemistry , Protein Subunits/genetics , Protein Subunits/metabolism , SARS-CoV-2 , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/genetics , Thermodynamics
18.
PLoS Biol ; 18(9): e3000827, 2020 09.
Article in English | MEDLINE | ID: covidwho-807960

ABSTRACT

Matrix proteins are encoded by many enveloped viruses, including influenza viruses, herpes viruses, and coronaviruses. Underneath the viral envelope of influenza virus, matrix protein 1 (M1) forms an oligomeric layer critical for particle stability and pH-dependent RNA genome release. However, high-resolution structures of full-length monomeric M1 and the matrix layer have not been available, impeding antiviral targeting and understanding of the pH-dependent transitions involved in cell entry. Here, purification and extensive mutagenesis revealed protein-protein interfaces required for the formation of multilayered helical M1 oligomers similar to those observed in virions exposed to the low pH of cell entry. However, single-layered helical oligomers with biochemical and ultrastructural similarity to those found in infectious virions before cell entry were observed upon mutation of a single amino acid. The highly ordered structure of the single-layered oligomers and their likeness to the matrix layer of intact virions prompted structural analysis by cryo-electron microscopy (cryo-EM). The resulting 3.4-Å-resolution structure revealed the molecular details of M1 folding and its organization within the single-shelled matrix. The solution of the full-length M1 structure, the identification of critical assembly interfaces, and the development of M1 assembly assays with purified proteins are crucial advances for antiviral targeting of influenza viruses.


Subject(s)
Imaging, Three-Dimensional , Viral Matrix Proteins/chemistry , Amino Acid Sequence , Cross-Linking Reagents/chemistry , Hydrogen-Ion Concentration , Models, Molecular , Mutation/genetics , Protein Multimerization , Protein Structure, Secondary , Protein Subunits/chemistry , Recombination, Genetic/genetics , Viral Matrix Proteins/genetics , Virion/ultrastructure
19.
Sci Adv ; 6(42)2020 10.
Article in English | MEDLINE | ID: covidwho-781066

ABSTRACT

To combat severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) and any unknown emerging pathogens in the future, the development of a rapid and effective method to generate high-affinity antibodies or antibody-like proteins is of critical importance. We here report high-speed in vitro selection of multiple high-affinity antibody-like proteins against various targets including the SARS-CoV-2 spike protein. The sequences of monobodies against the SARS-CoV-2 spike protein were successfully procured within only 4 days. Furthermore, the obtained monobody efficiently captured SARS-CoV-2 particles from the nasal swab samples of patients and exhibited a high neutralizing activity against SARS-CoV-2 infection (half-maximal inhibitory concentration, 0.5 nanomolar). High-speed in vitro selection of antibody-like proteins is a promising method for rapid development of a detection method for, and of a neutralizing protein against, a virus responsible for an ongoing, and possibly a future, pandemic.


Subject(s)
Betacoronavirus/immunology , Peptidyl-Dipeptidase A/immunology , Single-Domain Antibodies/immunology , Spike Glycoprotein, Coronavirus/immunology , Amino Acid Sequence , Angiotensin-Converting Enzyme 2 , Antibodies, Immobilized/chemistry , Antibodies, Immobilized/immunology , Antibodies, Neutralizing/chemistry , Antibodies, Neutralizing/immunology , Antibodies, Neutralizing/metabolism , Betacoronavirus/genetics , Betacoronavirus/isolation & purification , COVID-19 , Cell Surface Display Techniques/methods , Coronavirus Infections/pathology , Coronavirus Infections/virology , Dimerization , Humans , Kinetics , Pandemics , Peptides/chemistry , Peptides/immunology , Peptidyl-Dipeptidase A/chemistry , Peptidyl-Dipeptidase A/metabolism , Pneumonia, Viral/pathology , Pneumonia, Viral/virology , Protein Domains/immunology , Protein Subunits/chemistry , Protein Subunits/immunology , Protein Subunits/metabolism , RNA, Viral/metabolism , SARS-CoV-2 , Single-Domain Antibodies/chemistry , Single-Domain Antibodies/metabolism , Spike Glycoprotein, Coronavirus/chemistry
20.
Nature ; 588(7837): 327-330, 2020 12.
Article in English | MEDLINE | ID: covidwho-780015

ABSTRACT

Infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is initiated by virus binding to the ACE2 cell-surface receptors1-4, followed by fusion of the virus and cell membranes to release the virus genome into the cell. Both receptor binding and membrane fusion activities are mediated by the virus spike glycoprotein5-7. As with other class-I membrane-fusion proteins, the spike protein is post-translationally cleaved, in this case by furin, into the S1 and S2 components that remain associated after cleavage8-10. Fusion activation after receptor binding is proposed to involve the exposure of a second proteolytic site (S2'), cleavage of which is required for the release of the fusion peptide11,12. Here we analyse the binding of ACE2 to the furin-cleaved form of the SARS-CoV-2 spike protein using cryo-electron microscopy. We classify ten different molecular species, including the unbound, closed spike trimer, the fully open ACE2-bound trimer and dissociated monomeric S1 bound to ACE2. The ten structures describe ACE2-binding events that destabilize the spike trimer, progressively opening up, and out, the individual S1 components. The opening process reduces S1 contacts and unshields the trimeric S2 core, priming the protein for fusion activation and dissociation of ACE2-bound S1 monomers. The structures also reveal refolding of an S1 subdomain after ACE2 binding that disrupts interactions with S2, which involves Asp61413-15 and leads to the destabilization of the structure of S2 proximal to the secondary (S2') cleavage site.


Subject(s)
Angiotensin-Converting Enzyme 2/chemistry , Angiotensin-Converting Enzyme 2/metabolism , Membrane Fusion/physiology , Protein Binding , Receptors, Coronavirus/metabolism , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/metabolism , Angiotensin-Converting Enzyme 2/ultrastructure , Cryoelectron Microscopy , Furin/metabolism , Humans , Models, Molecular , Protein Folding , Protein Subunits/chemistry , Protein Subunits/metabolism , Proteolysis , Receptors, Coronavirus/chemistry , Receptors, Coronavirus/ultrastructure , Spike Glycoprotein, Coronavirus/ultrastructure
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