scholarly journals Mutations in the B.1.1.7 SARS-CoV-2 Spike Protein Reduce Receptor-Binding Affinity and Induce a Flexible Link to the Fusion Peptide

Biomedicines ◽  
2021 ◽  
Vol 9 (5) ◽  
pp. 525
Author(s):  
Eileen Socher ◽  
Marcus Conrad ◽  
Lukas Heger ◽  
Friedrich Paulsen ◽  
Heinrich Sticht ◽  
...  
Keyword(s):  
Binding Affinity ◽  
Receptor Binding ◽  
Salt Bridge ◽  
Fusion Peptide ◽  
Spike Protein ◽  
Spatial Proximity ◽  
Wild Type Virus ◽  
Wild Type ◽  
Protein Variant ◽  
Linear Interaction

The B.1.1.7 variant of the SARS-CoV-2 virus shows enhanced infectiousness over the wild type virus, leading to increasing patient numbers in affected areas. Amino acid exchanges within the SARS-CoV-2 spike protein variant of B.1.1.7 affect inter-monomeric contact sites within the trimer (A570D and D614G) as well as the ACE2-receptor interface region (N501Y), which comprises the receptor-binding domain (RBD) of the spike protein. However, the molecular consequences of mutations within B.1.1.7 on spike protein dynamics and stability or ACE2 binding are largely unknown. Here, molecular dynamics simulations comparing SARS-CoV-2 wild type with the B.1.1.7 variant revealed inter-trimeric contact rearrangements, altering the structural flexibility within the spike protein trimer. Furthermore, we found increased flexibility in direct spatial proximity of the fusion peptide due to salt bridge rearrangements induced by the D614G mutation in B.1.1.7. This study also implies a reduced binding affinity for B.1.1.7 with ACE2, as the N501Y mutation restructures the RBD–ACE2 interface, significantly decreasing the linear interaction energy between the RBD and ACE2. Our results demonstrate how mutations found within B.1.1.7 enlarge the flexibility around the fusion peptide and change the RBD–ACE2 interface. We anticipate our findings to be starting points for in depth biochemical and cell biological analyses of B.1.1.7.

scholarly journals Mutations in the B.1.1.7 SARS-CoV-2 spike protein reduce receptor-binding affinity and induce a flexible link to the fusion peptide

2021 ◽  
Author(s):  
Eileen Socher ◽  
Marcus Conrad ◽  
Lukas Heger ◽  
Friedrich Paulsen ◽  
Heinrich Sticht ◽  
...  
Keyword(s):  
Amino Acid ◽  
Binding Affinity ◽  
Receptor Binding ◽  
Fusion Peptide ◽  
Cell Surface Receptor ◽  
Spike Protein ◽  
Single Amino Acid ◽  
Wild Type Virus ◽  
Wild Type ◽  
Linear Interaction

AbstractThe B.1.1.7 variant of the SARS-CoV-2 virus shows enhanced infectiousness over the wild type virus, leading to increasing patient numbers in affected areas. A number of single amino acid exchanges and deletions within the trimeric viral spike protein characterize this new SARS-CoV-2 variant. Crucial for viral entry into the host cell is the interaction of the spike protein with the cell surface receptor angiotensin-converting enzyme 2 (ACE2) as well as integration of the viral fusion peptide into the host membrane. Respective amino acid exchanges within the SARS-CoV-2 variant B.1.1.7 affect inter-monomeric contact sites within the spike protein (A570D and D614G) as well as the ACE2-receptor interface region (N501Y), which comprises the receptor-binding domain (RBD) of the viral spike protein. However, the molecular consequences of mutations within B.1.1.7 on spike protein dynamics and stability, the fusion peptide, and ACE2 binding are largely unknown. Here, molecular dynamics simulations comparing SARS-CoV-2 wild type with the B.1.1.7 variant revealed inter-trimeric contact rearrangements, altering the structural flexibility within the spike protein trimer. In addition to reduced flexibility in the N-terminal domain of the spike protein, we found increased flexibility in direct spatial proximity of the fusion peptide. This increase in flexibility is due to salt bridge rearrangements induced by the D614G mutation in B.1.1.7 found in pre- and post-cleavage state at the S2’ site. Our results also imply a reduced binding affinity for B.1.1.7 with ACE2, as the N501Y mutation restructures the RBD-ACE2 interface, significantly decreasing the linear interaction energy between the RBD and ACE2.Our results demonstrate how mutations found within B.1.1.7 enlarge the flexibility around the fusion peptide and change the RBD-ACE2 interface, which, in combination, might explain the higher infectivity of B.1.1.7. We anticipate our findings to be starting points for in depth biochemical and cell biological analyses of B.1.1.7, but also other highly contagious SARS-CoV-2 variants, as many of them likewise exhibit a combination of the D614G and N501Y mutation.

scholarly journals Bioinformatics analysis of SARS-CoV-2 RBD mutant variants and insights into antibody and ACE2 receptor binding

2021 ◽  
Author(s):  
Prashant Ranjan ◽  
Neha   ◽  
Chandra Devi ◽  
Parimal Das
Keyword(s):  
Binding Affinity ◽  
Receptor Binding ◽  
Double Mutant ◽  
Structural Changes ◽  
Protective Efficacy ◽  
Spike Protein ◽  
Wild Type ◽  
Current Vaccine ◽  
New Variant ◽  
The Impact

Prevailing COVID-19 vaccines are based on the spike protein of earlier SARS-CoV-2 strain that emerged in Wuhan, China. Continuously evolving nature of SARS-CoV-2 resulting emergence of new variant/s raise the risk of immune absconds. Several RBD (receptor-binding domain) variants have been reported to affect the vaccine efficacy considerably. In the present study, we performed in silico structural analysis of spike protein of double mutant (L452R & E484Q), a new variant of SARS-CoV-2 recently reported in India along with K417G variants and earlier reported RBD variants and found structural changes in RBD region after comparing with the wild type. Comparison of the binding affinity of the double mutant and earlier reported RBD variant for ACE2 (angiotensin 2 altered enzymes) receptor and CR3022 antibody with the wild-type strain revealed the lowest binding affinity of the double mutant for CR3022 among all other variants. These findings suggest that the newly emerged double mutant could significantly reduce the impact of the current vaccine which threatens the protective efficacy of current vaccine therapy.

scholarly journals Structural Analysis And Molecular Characteristics of SARSCoV-2 Spike Protein Following S494P Point Mutation: Molecular Dynamics Study

2021 ◽  
Author(s):  
Mehr Ali Mahmood Janlou ◽  
Hassan sahebjamee ◽  
Shademan Shokravi
Keyword(s):  
Structural Analysis ◽  
Binding Affinity ◽  
Receptor Binding ◽  
Conformational Changes ◽  
Spike Protein ◽  
Molecular Characteristics ◽  
Wild Type ◽  
Natural Mutation ◽  
The Stability ◽  
Different Strains

Abstract The emergence of some mutations in the SARS-CoV-2 receptor binding domain (RBD) can increase the spread and pathogenicity due to the conformational changes and increase the stability of Spike protein. Due to the formation of different strains of SARS-CoV-2 by mutations, and their catastrophic effect on public health, the study of the effect of mutations by scientists and researchers around the world is inevitable. According to available evidence, the S494P variant is observed in several SARS-CoV-2 strains from Michigan, USA. To investigate how the S494P natural mutation alters receptor binding affinity in RBD, we performed structural analysis of wild-type and mutant spike proteins using some bioinformatics and computational tools. The results show that S494P mutation increases the spike protein stability. Also, applying docking by HADDOCK displayed higher binding affinity to hACE2 for mutant spike than wild type possibly due to the increased β-strand and Turn secondary structures which increases surface accessibly surface area (SASA) and chance of interaction. The analysis of S494P as a critical RBD mutation may provide the continuing surveillance of spike mutations to aid in the development of COVID-19 drugs and vaccines.

Structural-bioinformatics analysis of SARS-CoV-2 variants reveals higher hACE2 receptor binding affinity for Omicron B.1.1.529 spike RBD compared to wild-type reference.

2021 ◽  
Author(s):  
Vedat Durmaz ◽  
Katharina Köchl ◽  
Amit Singh ◽  
Michael Hetmann ◽  
Lena Parigger ◽  
...  
Keyword(s):  
Binding Affinity ◽  
Receptor Binding ◽  
Bioinformatics Analysis ◽  
Structural Bioinformatics ◽  
Binding Energies ◽  
Large Set ◽  
Wild Type ◽  
Linear Interaction ◽  
Binding Interface ◽  
The Impact

Abstract To date, more than 263 million people have been infected with SARS-CoV-2 during the COVID-19 pandemic. In many countries, the global spread came in several pandemic waves characterized by the emergence of new SARS-CoV-2 variants. Here, we report on a sequence- and structural-bioinformatics analysis by which we estimate the impact of amino acid exchanges on the affinity of the SARS-CoV-2 spike receptor-binding domain (RBD) to the human receptor hACE2. This is carried out by qualitative electrostatics and hydrophobicity analysis as well as through molecular dynamics simulations used for the development of a highly accurate linear interaction energy (LIE) binding affinity model that was calibrated on a large set of experimental binding energies. For the newest variant of concern (VOC), B.1.1.529 Omicron, our Halo difference point cloud studies reveal the largest impact on the RBD binding interface compared to any other VOC. Moreover, according to our LIE model, Omicron achieved a substantially higher ACE2 binding affinity than the wild-type and in particular the highest among all VOCs except for Alpha and therefore requires special attention and monitoring. Using this prediction model we provide early structural insight and binding properties before experimentally determined complex structures and binding affinity data become available in the upcoming months.

scholarly journals Ultrapotent bispecific antibodies neutralize emerging SARS-CoV-2 variants

2021 ◽  
Author(s):  
Hyeseon Cho ◽  
Kristina Kay Gonzales-Wartz ◽  
Deli Huang ◽  
Meng Yuan ◽  
Mary Peterson ◽  
...  
Keyword(s):  
Bispecific Antibody ◽  
Complete Loss ◽  
Memory B Cells ◽  
Spike Protein ◽  
Bispecific Antibodies ◽  
Wild Type Virus ◽  
Type Virus ◽  
Wild Type ◽  
Hamster Model

The emergence of SARS-CoV-2 variants that threaten the efficacy of existing vaccines and therapeutic antibodies underscores the urgent need for new antibody-based tools that potently neutralize variants by targeting multiple sites of the spike protein. We isolated 216 monoclonal antibodies targeting SARS-CoV-2 from plasmablasts and memory B cells of COVID-19 patients. The three most potent antibodies targeted distinct regions of the RBD, and all three neutralized the SARS-CoV-2 variants B.1.1.7 and B.1.351. The crystal structure of the most potent antibody, CV503, revealed that it binds to the ridge region of SARS-CoV-2 RBD, competes with the ACE2 receptor, and has limited contact with key variant residues K417, E484 and N501. We designed bispecific antibodies by combining non-overlapping specificities and identified five ultrapotent bispecific antibodies that inhibit authentic SARS-CoV-2 infection at concentrations of <1 ng/mL. Through a novel mode of action three bispecific antibodies cross-linked adjacent spike proteins using dual NTD/RBD specificities. One bispecific antibody was >100-fold more potent than a cocktail of its parent monoclonals in vitro and prevented clinical disease in a hamster model at a 2.5 mg/kg dose. Notably, six of nine bispecific antibodies neutralized B.1.1.7, B.1.351 and the wild-type virus with comparable potency, despite partial or complete loss of activity of at least one parent monoclonal antibody against B.1.351. Furthermore, a bispecific antibody that neutralized B.1.351 protected against SARS-CoV-2 expressing the crucial E484K mutation in the hamster model. Thus, bispecific antibodies represent a promising next-generation countermeasure against SARS-CoV-2 variants of concern.

scholarly journals Multimerization- and glycosylation-dependent receptor binding of SARS-CoV-2 spike proteins

2020 ◽  
Author(s):  
Kim M. Bouwman ◽  
Ilhan Tomris ◽  
Hannah L. Turner ◽  
Roosmarijn van der Woude ◽  
Gerlof P. Bosman ◽  
...  
Keyword(s):  
Binding Affinity ◽  
Cell Lines ◽  
Receptor Binding ◽  
Expression Profiles ◽  
Spike Protein ◽  
Binding Studies ◽  
Binding Properties ◽  
Experimental Animals ◽  
High Mannose ◽  
Mutant Cells

Receptor binding studies using recombinant SARS-CoV proteins have been hampered due to challenges in approaches creating spike protein or domains thereof, that recapitulate receptor binding properties of native viruses. We hypothesized that trimeric RBD proteins would be suitable candidates to study receptor binding properties of SARS-CoV-1 and -2. Here we created monomeric and trimeric fluorescent RBD proteins, derived from adherent HEK293T, as well as in GnTI mutant cells, to analyze the effect of complex vs high mannose glycosylation on receptor binding. The results demonstrate that trimeric fully glycosylated proteins are superior in receptor binding compared to monomeric and immaturely glycosylated variants. Although differences in binding to commonly used cell lines were minimal between the different RBD preparations, substantial differences were observed when respiratory tissues of experimental animals were stained. The RBD trimers demonstrated distinct ACE2 expression profiles in bronchiolar ducts and confirmed the higher binding affinity of SARS-CoV-2 over SARS-CoV-1. Our results show that fully glycosylated trimeric RBD proteins are attractive to analyze receptor binding and explore ACE2 expression profiles in tissues.

scholarly journals A Spike protein-based subunit SARS-CoV-2 vaccine for pets: safety, immunogenicity, and protective efficacy in juvenile cats

2021 ◽  
Author(s):  
Kairat Tabynov ◽  
Madiana Orynbassar ◽  
Leila Yelchibayeva ◽  
Nurkeldi Turebekov ◽  
Toktassyn Yerubayev ◽  
...  
Keyword(s):  
Respiratory Tract ◽  
Neutralizing Antibodies ◽  
Large Scale ◽  
Protective Efficacy ◽  
Spike Protein ◽  
Wild Type Virus ◽  
Upper Respiratory Tract ◽  
Wild Type ◽  
Serum Samples ◽  
Virus Shedding

Abstract Whereas multiple vaccine types have been developed to curb the spread of Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) among humans, there are very few vaccines being developed for animals including pets. To combat the threat of human-to-animal, animal-to-animal and animal-to-human transmission and the generation of new virus variants, we developed a subunit SARS-CoV-2 vaccine which is based on recombinant spike protein extracellular domain expressed in insect cells then formulated with appropriate adjuvants. Sixteen 8-12-week-old outbred female and male kittens (n=4/group) were randomly assigned into four treatment groups: Group 1, Antigen alone; Group 2, Sepivac SWE™ adjuvant; Group 3, aluminum hydroxide adjuvant; Group 4, PBS administered control animals. All animals were vaccinated twice at day 0 and 14, intramuscularly in a volume of 0.5 mL (Groups 1-3: 5 µg of Spike protein). On days 0 and 28 serum samples were collected to evaluate anti-spike IgG, inhibition of spike binding to angiotensin-converting enzyme 2 (ACE-2), neutralizing antibodies to Wuhan-01 SARS-CoV-2 D614G (wild-type) and Delta variant viruses, and whole blood for hematology studies. At day 28, all groups were challenged with SARS-CoV-2 wild-type virus 106 TCID50 intranasally. On day 31, tissue samples (lung, heart, and nasal turbinates) were collected for histology, viral RNA detection and virus titration. Parameters evaluated in this study included safety, immunogenicity, and protection from infection with wild-type SARS-CoV-2 virus. After two immunizations, both vaccines induced high titers of serum anti-spike IgG, ACE-2 binding inhibitory and neutralizing antibodies against both wild-type and Delta variant virus in the juvenile cats. Both subunit vaccines provided protection of juvenile cats against virus shedding from the upper respiratory tract, and against viral replication in the lower respiratory tract and hearts. These promising data warrant ongoing evaluation of the vaccine’s ability to protect cats against SARS-CoV-2 Delta variant and in particular to prevent transmission of the infection to naïve cats, before proceeding with large-scale field trials.

scholarly journals Age-Dependent Reduction in Neutralization against Alpha and Beta Variants of BNT162b2 SARS-CoV-2 Vaccine-Induced Immunity

2021 ◽  
Author(s):  
Hitoshi Kawasuji ◽  
Yoshitomo Morinaga ◽  
Hideki Tani ◽  
Yumiko Saga ◽  
Makito Kaneda ◽  
...  
Keyword(s):  
Age Groups ◽  
Older Age ◽  
Spike Protein ◽  
Wild Type Virus ◽  
Type Virus ◽  
Wild Type ◽  
Neutralizing Activity ◽  
Age Dependent ◽  
Induced Immunity

Since mRNA vaccines utilize wild-type SARS-CoV-2 spike protein as an antigen, there are potential concerns about acquiring immunity to variants of this virus. The neutralizing activity in BNT162b2-vaccinated individuals was higher against the wild-type virus than against its variants; this effect was more apparent in older age groups.

scholarly journals Characterisation of SARS-CoV-2 Lentiviral Pseudotypes and Correlation between Pseudotype-Based Neutralisation Assays and Live Virus-Based Micro Neutralisation Assays

Viruses ◽  
2020 ◽  
Vol 12 (9) ◽  
pp. 1011 ◽  
Author(s):  
Inesa Hyseni ◽  
Eleonora Molesti ◽  
Linda Benincasa ◽  
Pietro Piu ◽  
Elisa Casa ◽  
...  
Keyword(s):  
Wild Type Strain ◽  
Spike Protein ◽  
Wild Type Virus ◽  
Wild Type ◽  
Serum Samples ◽  
Live Virus ◽  
Rapid Spread ◽  
Recent Outbreak ◽  
Human Sera ◽  
Novel Coronavirus

The recent outbreak of a novel Coronavirus (SARS-CoV-2) and its rapid spread across the continents has generated an urgent need for assays to detect the neutralising activity of human sera or human monoclonal antibodies against SARS-CoV-2 spike protein and to evaluate the serological immunity in humans. Since the accessibility of live virus microneutralisation (MN) assays with SARS-CoV-2 is limited and requires enhanced bio-containment, the approach based on “pseudotyping” can be considered a useful complement to other serological assays. After fully characterising lentiviral pseudotypes bearing the SARS-CoV-2 spike protein, we employed them in pseudotype-based neutralisation assays in order to profile the neutralising activity of human serum samples from an Italian sero-epidemiological study. The results obtained with pseudotype-based neutralisation assays mirrored those obtained when the same panel of sera was tested against the wild type virus, showing an evident convergence of the pseudotype-based neutralisation and MN results. The overall results lead to the conclusion that the pseudotype-based neutralisation assay is a valid alternative to using the wild-type strain, and although this system needs to be optimised and standardised, it can not only complement the classical serological methods, but also allows serological assessments to be made when other methods cannot be employed, especially in a human pandemic context.

scholarly journals Cleavage of K-FGF produces a truncated molecule with increased biological activity and receptor binding affinity.

1993 ◽  
Vol 121 (3) ◽  
pp. 705-713 ◽  
Author(s):  
P Bellosta ◽  
D Talarico ◽  
D Rogers ◽  
C Basilico
Keyword(s):  
Amino Acids ◽  
Biological Activity ◽  
Growth Factor ◽  
Binding Affinity ◽  
Receptor Binding ◽  
Biologically Active ◽  
Heparin Binding ◽  
Wild Type ◽  
Mammalian Expression

The K-FGF/HST (FGF-4) growth factor is a member of the FGF family which is efficiently secreted and contains a single N-linked glycosylation signal. To study the role of glycosylation in the secretion of K-FGF, we mutated the human K-fgf cDNA to eliminate the glycosylation signal and the mutated cDNA was cloned into a mammalian expression vector. Studies of immunoprecipitation from the conditioned medium of cells expressing this plasmid revealed that the lack of glycosylation did not impair secretion, however the unglycosylated protein was immediately cleaved into two NH2-terminally truncated peptides of 13 and 15 kD, which appeared to be more biologically active than the wild-type protein. These two proteins also showed higher heparin binding affinity than that of wt K-FGF. We have expressed in bacteria the larger of these two proteins (K140), in which the NH2-terminal 36 amino acids present in the mature form of K-FGF have been deleted. Mitogenicity assays on several cell lines showed that purified recombinant K140 had approximately five times higher biological activity than wild-type recombinant K-FGF. Studies of receptor binding showed that K140 had higher affinity than wt K-FGF for two of the four members of FGF receptor's family, specifically for FGFR-1 (flg) and FGFR-2 (bek). K140 also had increased heparin binding ability, but this property does not appear to be responsible for the increased affinity for FGF receptors. Thus removal of the NH2-terminal 36 amino acids from the mature K-FGF produces growth factor molecules with an altered conformation, resulting in higher heparin affinity, and more efficient binding to FGF receptors. Although it is not clear whether cleavage of K-FGF to generate K140 occurs in vivo, this could represent a novel mechanism of modulation of growth factor activity.

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