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

2021 ◽  
Vol 17 (2) ◽  
pp. e1009282
Author(s):  
Kim M. Bouwman ◽  
Ilhan Tomris ◽  
Hannah L. Turner ◽  
Roosmarijn van der Woude ◽  
Tatiana M. Shamorkina ◽  
...  
Keyword(s):  
Receptor Binding ◽  
Expression Profiles ◽  
Receptor Binding Domain ◽  
Binding Studies ◽  
Binding Properties ◽  
Experimental Animals ◽  
Trimeric Complex ◽  
High Mannose ◽  
Mutant Cells ◽  
Spike Proteins

Receptor binding studies on sarbecoviruses would benefit from an available toolkit of recombinant spike proteins, or domains thereof, that recapitulate receptor binding properties of native viruses. We hypothesized that trimeric Receptor Binding Domain (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, complex 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 complex glycosylated trimeric RBD proteins are attractive to analyze sarbecovirus receptor binding and explore ACE2 expression profiles in tissues.

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 Unraveling the stability landscape of mutations in the SARS-CoV-2 receptor-binding domain

2020 ◽  
Author(s):  
Mohamed Raef Smaoui ◽  
Hamdi Yahyaoui
Keyword(s):  
Receptor Binding ◽  
Single Point ◽  
Protein Structures ◽  
Point Mutations ◽  
Binding Domain ◽  
Spike Protein ◽  
Receptor Binding Domain ◽  
Point Mutant ◽  
The Stability ◽  
Spike Proteins

Abstract The interaction between the receptor-binding domain of the SARS-CoV-2 spike glycoprotein and the ACE2 enzyme is believed to be the entry point of the virus into various cells in the body, including the lungs, heart, liver, and kidneys. The current focus of several therapeutic design efforts explore attempts at affecting the binding interaction between the two proteins to limit the activity of the virus and disease progression. In this work, we analyze the stability of the spike protein under all possible single-point mutations in the receptor-binding domain and computationally explore mutations that can affect the binding with the ACE2 enzyme. We unravel the mutation landscape of the receptor region and assess the toxicity potential of single and multi-point mutations, generating insights for future vaccine efforts on potential mutations that might further stabilize the spike protein and increase its infectivity. We developed a tool, called SpikeMutator, to construct full atomic protein structures of the mutant spike proteins and shared a database of 3,800 single-point mutant structures. We analyzed the recent 65,000 reported spike sequences across the globe and observed the emergence of stable multi-point mutant structures. Using the landscape, we searched through 7.5 million possible 2-point mutation combinations and report that the (R355D K424E) mutation produces one of the strongest spike proteins that therapeutic efforts should investigate for the sake of developing an effective vaccine.

scholarly journals Decreased neutralization of SARS-CoV-2 global variants by therapeutic anti-spike protein monoclonal antibodies

2021 ◽  
Author(s):  
Takuya Tada ◽  
Belinda M. Dcosta ◽  
Hao Zhou ◽  
Ada Vaill ◽  
Wes Kazmierski ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Receptor Binding ◽  
Binding Domain ◽  
Spike Protein ◽  
Receptor Binding Domain ◽  
Neutralization Activity ◽  
Protein Mutations ◽  
Spike Proteins

AbstractMonoclonal antibodies against the SARS-CoV-2 spike protein, notably, those developed by Regeneron Pharmaceuticals and Eli Lilly and Company have proven to provide protection against severe COVID-19. The emergence of SARS-CoV-2 variants with heavily mutated spike proteins raises the concern that the therapy could become less effective if any of the mutations disrupt epitopes engaged by the antibodies. In this study, we tested monoclonal antibodies REGN10933 and REGN10987 that are used in combination, for their ability to neutralize SARS-CoV-2 variants B.1.1.7, B.1.351, mink cluster 5 and COH.20G/677H. We report that REGN10987 maintains most of its neutralization activity against viruses with B.1.1.7, B.1.351 and mink cluster 5 spike proteins but that REGN10933 has lost activity against B.1.351 and mink cluster 5. The failure of REGN10933 to neutralize B.1.351 is caused by the K417N and E484K mutations in the receptor binding domain; the failure to neutralize the mink cluster 5 spike protein is caused by the Y453F mutation. The REGN10933 and REGN10987 combination was 9.1-fold less potent on B.1.351 and 16.2-fold less potent on mink cluster 5, raising concerns of reduced efficacy in the treatment of patients infected with variant viruses. The results suggest that there is a need to develop additional monoclonal antibodies that are not affected by the current spike protein mutations.

scholarly journals Design, expression, purification and characterization of a YFP-tagged 2019-nCoV spike receptor-binding domain construct

2020 ◽  
Author(s):  
Tobias Bierig ◽  
Gabriella Collu ◽  
Alain Blanc ◽  
Emiliya Poghosyan ◽  
Roger. M. Benoit
Keyword(s):  
Fusion Protein ◽  
Receptor Binding ◽  
Fluorescent Protein ◽  
Yellow Fluorescent Protein ◽  
Fluorescent Labeling ◽  
Binding Domain ◽  
Size Exclusion ◽  
Antibody Detection ◽  
Receptor Binding Domain ◽  
Binding Studies

Abstract2019-nCoV is the causative agent of the serious, still ongoing, worldwide COVID-19 pandemic. High quality recombinant virus proteins are required for research related to the development of vaccines and improved assays, and to the general understanding of virus action. The receptor-binding domain (RBD) of the 2019-nCoV spike (S) protein contains disulfide bonds and N-linked glycosylations, therefore, it is typically produced by secretion. Here, we describe a construct and protocol for the expression and purification of yellow fluorescent protein (YFP) labeled 2019-nCoV spike RBD. The fusion protein, in the vector pcDNA 4/TO, comprises an N-terminal interferon alpha 2 (IFNα2) signal peptide, an eYFP, a FLAG-tag, a human rhinovirus 3C protease cleavage site, the RBD of the 2019-nCoV spike protein and a C-terminal 8x His-tag. We stably transfected HEK 293 cells. Following expansion of the cells, the fusion protein was secreted from adherent cells into serum-free medium. Ni-NTA IMAC purification resulted in very high protein purity, based on analysis by SDS-PAGE. The fusion protein was soluble and monodisperse, as confirmed by size-exclusion chromatography (SEC) and negative staining electron microscopy. Deglycosylation experiments confirmed the presence of N-linked glycosylations in the secreted protein. Complex formation with the peptidase domain of human angiotensin-converting enzyme 2 (ACE2), the receptor for the 2019-nCoV spike RBD, was confirmed by SEC, both for the YFP-fused spike RBD and for spike RBD alone, after removal of YFP by proteolytic cleavage. Possible applications for the fusion protein include binding studies on cells or in vitro, fluorescent labeling of potential virus-binding sites on cells, the use as an antigen for immunization studies or as a tool for the development of novel virus- or antibody-detection assays.

scholarly journals Structural Analysis of Receptor Binding Domain Mutations in SARS-CoV-2 Variants of Concern that Modulate ACE2 and Antibody Binding

2021 ◽  
Author(s):  
Dhiraj Mannar ◽  
James W Saville ◽  
Xing Zhu ◽  
Shanti S. Srivastava ◽  
Alison M. Berezuk ◽  
...  
Keyword(s):  
Structural Analysis ◽  
South African ◽  
Receptor Binding ◽  
Antibody Binding ◽  
Binding Domain ◽  
Spike Protein ◽  
Receptor Binding Domain ◽  
Biochemical Assays ◽  
The Uk ◽  
Spike Proteins

SummaryThe recently emerged SARS-CoV-2 South African (B. 1.351) and Brazil/Japan (P.1) variants of concern (VoCs) include a key mutation (N501Y) found in the UK variant that enhances affinity of the spike protein for its receptor, ACE2. Additional mutations are found in these variants at residues 417 and 484 that appear to promote antibody evasion. In contrast, the Californian VoCs (B.1.427/429) lack the N501Y mutation, yet exhibit antibody evasion. We engineered spike proteins to express these RBD VoC mutations either in isolation, or in different combinations, and analyzed the effects using biochemical assays and cryo-EM structural analyses. Overall, our findings suggest that the emergence of new SARS-CoV-2 variant spikes can be rationalized as the result of mutations that confer either increased ACE2 affinity, increased antibody evasion, or both, providing a framework to dissect the molecular factors that drive VoC evolution.

scholarly journals SARS-CoV-2 Prion-Like Domains in Spike Proteins Enables Higher Affinity to ACE2

2020 ◽  
Author(s):  
George Tetz ◽  
Victor Tetz
Keyword(s):  
Receptor Binding ◽  
Cell Receptor ◽  
Binding Domain ◽  
Spike Protein ◽  
Striking Difference ◽  
Receptor Binding Domain ◽  
Viral Receptor ◽  
Functional Roles ◽  
Binding Domains ◽  
Spike Proteins

Currently, the world is struggling with the coronavirus disease 2019 (COVID-19) pandemic, caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Prion-like domains are critical for virulence and the development of therapeutic targets; however, the prion-like domains in the SARS-CoV-2 proteome have not been analyzed. In this in silico study, using the PLAAC algorithm, we identified the presence of prion-like domains in SARS-CoV-2 spike protein. Compared with other viruses, a striking difference was observed in the distribution of prion-like domains in the spike, since SARS-CoV-2 was the only coronavirus with a prion-like domain found in the receptor-binding domain of the S1 region of the spike protein. The presence and unique distribution of prion-like domains in the SARS-CoV-2 receptor-binding domains of spike proteins is particularly interesting, since although SARS-CoV-2 and SARS-CoV S share the same host cell receptor, angiotensin-converting enzyme 2 (ACE2), SARS-CoV-2 demonstrates a 10- to 20-fold higher affinity for ACE2. Finally, we identified prion-like domains in the α1 helix of the ACE2 receptor that interacts with the viral receptor-binding domain of SARS-CoV-2. Taken together, the present findings indicate that the identified PrDs in the SARS-CoV-2 receptor-binding domain (RBD) and ACE2 region that interacts with RBD have important functional roles in viral adhesion and entry.

scholarly journals Unraveling the stability landscape of mutations in the SARS-CoV-2 receptor-binding domain

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Mohamed Raef Smaoui ◽  
Hamdi Yahyaoui
Keyword(s):  
Receptor Binding ◽  
Single Point ◽  
Protein Structures ◽  
Point Mutations ◽  
Binding Domain ◽  
Spike Protein ◽  
Receptor Binding Domain ◽  
Point Mutant ◽  
The Stability ◽  
Spike Proteins

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

scholarly journals Structure and binding properties of Pangolin-CoV spike glycoprotein inform the evolution of SARS-CoV-2

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Antoni G. Wrobel ◽  
Donald J. Benton ◽  
Pengqi Xu ◽  
Lesley J. Calder ◽  
Annabel Borg ◽  
...  
Keyword(s):  
Receptor Binding ◽  
Binding Domain ◽  
Spike Protein ◽  
Receptor Binding Domain ◽  
Spike Glycoprotein ◽  
Binding Properties ◽  
Origin And Evolution ◽  
Closed Conformation ◽  
Bat Coronavirus

AbstractCoronaviruses of bats and pangolins have been implicated in the origin and evolution of the pandemic SARS-CoV-2. We show that spikes from Guangdong Pangolin-CoVs, closely related to SARS-CoV-2, bind strongly to human and pangolin ACE2 receptors. We also report the cryo-EM structure of a Pangolin-CoV spike protein and show it adopts a fully-closed conformation and that, aside from the Receptor-Binding Domain, it resembles the spike of a bat coronavirus RaTG13 more than that of SARS-CoV-2.

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