Disulfide bonds play a critical role in the structure and function of the receptor-binding domain of the SARS-CoV-2 Spike antigen

The emergence of severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) and the subsequent COVID-19 pandemic has significantly impacted the world not just with disease and death but also economic turmoil. The rapid development and deployment of extremely effective vaccines against SARS-CoV-2 has made the end of the pandemic a reality within reach. However, as the virus spreads it has acquired mutations; and thus, variants of concern have emerged that are more infectious and reduce the efficacy of existing vaccines. While promising efforts are underway to combat these variants, the evolutionary pressures leading to these variants are poorly understood. To that end, here we have studied the effects of three amino-acid substitutions on the structure and function of the SARS-CoV-2 spike glycoprotein receptor-binding domain found in several variants of concern such as B.1.1.7, B.1.351 and P.1 that are now circulating. We found that these substitutions alter the RBD structure and stability, as well as its ability to bind to ACE2, which may have opposing and compensatory effects. These findings provide new insights into how these Variants of Concern (VOC) may have been selected to optimize infectivity while maintaining the structure and stability of the receptor binding domain.

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Structure and Function of Colicin S4, a Colicin with a Duplicated Receptor-binding Domain

Journal of Biological Chemistry ◽

10.1074/jbc.m808504200 ◽

2008 ◽

Vol 284 (10) ◽

pp. 6403-6413 ◽

Cited By ~ 29

Author(s):

Thomas Arnold ◽

Kornelius Zeth ◽

Dirk Linke

Keyword(s):

Receptor Binding ◽

Structure And Function ◽

Binding Domain ◽

Receptor Binding Domain ◽

And Function

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Structures and function of locked conformations of SARS-CoV-2 spike

10.1101/2021.03.10.434733 ◽

2021 ◽

Author(s):

Kun Qu ◽

Xiaoli Xiong ◽

Katarzyna A. Ciazynska ◽

Andrew P. Carter ◽

John A. G. Briggs

Keyword(s):

Receptor Binding ◽

Functional Characterization ◽

Protein S ◽

Binding Domain ◽

Receptor Binding Domain ◽

Neutral Ph ◽

And Function ◽

Cellular Compartments ◽

Domain Dynamics ◽

Spike Dynamics

AbstractThe spike protein (S) of SARS-CoV-2 has been observed in three distinct pre-fusion conformations: locked, closed and open. Of these, the locked conformation was not previously observed for SARS-CoV-1 S and its function remains poorly understood. Here we engineered a SARS-CoV-2 S protein construct “S-R/x3” to arrest SARS-CoV-2 spikes in the locked conformation by a disulfide bond. Using this construct we determined high-resolution structures revealing two distinct locked states, with or without the D614G substitution that has become fixed in the globally circulating SARS-CoV-2 strains. The D614G mutation induces a structural change in domain D from locked-1 to locked-2 conformation to alter spike dynamics, promoting transition into the closed conformation from which opening of the receptor binding domain is permitted. The transition from locked to closed conformations is additionally promoted by a change from low to neutral pH. We propose that the locked conformations of S are present in the acidic cellular compartments where virus is assembled and egresses. In this model, release of the virion into the neutral pH extracellular space would favour transition to the closed form which itself can stochastically transition into the open form. The S-R/x3 construct provides a tool for the further structural and functional characterization of the locked conformations of S, as well as how sequence changes might alter S assembly and regulation of receptor binding domain dynamics.

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SARS-CoV-2, an evolutionary perspective of interaction with human ACE2 reveals undiscovered amino acids necessary for complex stability

10.1101/2020.03.21.001933 ◽

2020 ◽

Cited By ~ 4

Author(s):

Vinicio Armijos-Jaramillo ◽

Justin Yeager ◽

Claire Muslin ◽

Yunierkis Perez-Castillo

Keyword(s):

Receptor Binding ◽

Evolutionary Dynamics ◽

Hot Spot ◽

Critical Role ◽

Human Cells ◽

Binding Domain ◽

Host Cells ◽

Spike Protein ◽

Receptor Binding Domain ◽

Salt Bridges

AbstractThe emergence of SARS-CoV-2 has resulted in more than 200,000 infections and nearly 9,000 deaths globally so far. This novel virus is thought to have originated from an animal reservoir, and acquired the ability to infect human cells using the SARS-CoV cell receptor hACE2. In the wake of a global pandemic it is essential to improve our understanding of the evolutionary dynamics surrounding the origin and spread of a novel infectious disease. One way theory predicts selection pressures should shape viral evolution is to enhance binding with host cells. We first assessed evolutionary dynamics in select betacoronavirus spike protein genes to predict where these genomic regions are under directional or purifying selection between divergent viral lineages at various scales of relatedness. With this analysis, we determine a region inside the receptor-binding domain with putative sites under positive selection interspersed among highly conserved sites, which are implicated in structural stability of the viral spike protein and its union with human receptor hACE2. Next, to gain further insights into factors associated with coronaviruses recognition of the human host receptor, we performed modeling studies of five different coronaviruses and their potential binding to hACE2. Modeling results indicate that interfering with the salt bridges at hot spot 353 could be an effective strategy for inhibiting binding, and hence for the prevention of coronavirus infections. We also propose that a glycine residue at the receptor binding domain of the spike glycoprotein can have a critical role in permitting bat variants of the coronaviruses to infect human cells.

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A Structural Landscape of Neutralizing Antibodies Against SARS-CoV-2 Receptor Binding Domain

Frontiers in Immunology ◽

10.3389/fimmu.2021.647934 ◽

2021 ◽

Vol 12 ◽

Author(s):

Ling Niu ◽

Kathryn N. Wittrock ◽

Gage C. Clabaugh ◽

Vikram Srivastava ◽

Michael W. Cho

Keyword(s):

Receptor Binding ◽

Neutralizing Antibodies ◽

Critical Role ◽

Binding Domain ◽

The Novel ◽

Receptor Binding Domain ◽

Correlates Of Protection ◽

Immune Correlates ◽

Virus Life Cycle ◽

Novel Coronavirus

SARS-CoV-2, the novel coronavirus responsible for the ongoing COVID-19 pandemic, has been spreading rampantly. The global scientific community has responded rapidly to understand immune correlates of protection to develop vaccines and immunotherapeutics against the virus. The major goal of this mini review is to summarize current understanding of the structural landscape of neutralizing antibodies (nAbs) that target the receptor binding domain (RBD) of viral spike (S) glycoprotein. The RBD plays a critical role in the very first step of the virus life cycle. Better understanding of where and how nAbs bind the RBD should enable identification of sites of vulnerability and facilitate better vaccine design and formulation of immunotherapeutics. Towards this goal, we compiled 38 RBD-binding nAbs with known structures. Review of these nAb structures showed that (1) nAbs can be divided into five general clusters, (2) there are distinct non-neutralizing faces on the RBD, and (3) maximum of potentially four nAbs could bind the RBD simultaneously. Since most of these nAbs were isolated from virus-infected patients, additional analyses of vaccine-induced nAbs could facilitate development of improved vaccines.

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Bacterial expression and purification of functional recombinant SARS-CoV-2 spike receptor binding domain

10.1101/2021.02.03.429601 ◽

2021 ◽

Author(s):

Janani Prahlad ◽

Lucas R. Struble ◽

William E. Lutz ◽

Savanna A. Wallin ◽

Surender Khurana ◽

...

Keyword(s):

Receptor Binding ◽

Disulfide Bonds ◽

Rapid Diagnosis ◽

Binding Domain ◽

Spike Protein ◽

Receptor Binding Domain ◽

Expression And Purification ◽

Protein Receptor ◽

The World ◽

Active Antigen

AbstractThe COVID-19 pandemic caused by SARS-CoV-2 has applied significant pressure on overtaxed healthcare around the world, underscoring the urgent need for rapid diagnosis and treatment. We have developed a bacterial strategy for the expression and purification of the SARS-CoV-2 spike protein receptor binding domain using the CyDisCo system to create and maintain the correct disulfide bonds for protein integrity and functionality. We show that it is possible to quickly and inexpensively produce functional, active antigen in bacteria capable of recognizing and binding to the ACE2 (angiotensin-converting enzyme) receptor as well as antibodies in COVID-19 patient sera.

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Monoclonal Antibody 667 Recognizes the Variable Region A Motif of the Ecotropic Retrovirus CasBrE Envelope Glycoprotein and Inhibits Env Binding to the Viral Receptor

Journal of Virology ◽

10.1128/jvi.77.20.10984-10993.2003 ◽

2003 ◽

Vol 77 (20) ◽

pp. 10984-10993 ◽

Cited By ~ 8

Author(s):

Hanna Dreja ◽

Laurent Gros ◽

Sylvie Villard ◽

Estanislao Bachrach ◽

Anna Oates ◽

...

Keyword(s):

Amino Acids ◽

Monoclonal Antibody ◽

Receptor Binding ◽

Envelope Glycoprotein ◽

Critical Role ◽

Variable Region ◽

Binding Domain ◽

Target Cells ◽

Kinetic Properties ◽

Receptor Binding Domain

ABSTRACT Monoclonal antibody (MAb) 667 is a neutralizing mouse monoclonal antibody recognizing the envelope glycoprotein (Env) of the ecotropic neurotropic murine retrovirus CasBrE but not that of other murine retroviruses. Since 667 can be used for preclinical studies of antiviral gene therapy as well as for studying the early events of retroviral infection, we have cloned its cDNAs and molecularly characterized it in detail. Spot technique-based experiments showed that 667 recognizes a linear epitope of 12 amino acids located in the variable region A of the receptor binding domain. Alanine scanning experiments showed that six amino acids within the epitope are critical for MAb binding. One of them, D57, is not present in any other murine retroviral Env, which suggests a critical role for this residue in the selectivity of 667. MAb 667 heavy- and light-chain cDNAs were functionally characterized by transient transfection into Cos-7 cells. Enzyme-linked immunosorbent assays and Biacore studies showed that the specificities as well as the antigen-binding thermodynamic and kinetic properties of the recombinant 667 MAb (r667) produced by Cos-7 cells and those of the parental hybridoma-produced MAb (h667) were similar. However, h667 was shown to contain contaminating retroviral and/or retrovirus-like particles which interfere with both viral binding and neutralization experiments. These contaminants could successfully be removed by a stringent purification protocol. Importantly, this purified 667 could completely prevent retrovirus binding to target cells and was as efficient as the r667 MAb produced by transfected Cos-7 cells in neutralization assays. In conclusion, this study shows that the primary mechanism of virus neutralization by MAb 667 is the blocking of the retroviral receptor binding domain of CasBrE Env. In addition, the findings of this study constitute a warning against the direct use of hybridoma cell culture supernatants for studying the initial events of retroviral cell infection as well as for carrying out in vivo neutralization experiments and suggest that either recombinant antibodies or highly purified antibodies are preferable for these purposes.

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Peptide inhibitors of the interaction of the SARS-CoV-2 receptor-binding domain with the ACE2 cell receptor

Biomeditsinskaya Khimiya ◽

10.18097/pbmc20216703244 ◽

2021 ◽

Vol 67 (3) ◽

pp. 244-250

Author(s):

R.Sh. Bibilashvili ◽

M.V. Sidorova ◽

U.S. Dudkina ◽

M.E. Palkeeva ◽

A.S. Molokoedov ◽

...

Keyword(s):

Amino Acid ◽

Receptor Binding ◽

Disulfide Bonds ◽

Protein S ◽

Cell Receptor ◽

Binding Domain ◽

Amino Acid Sequences ◽

Receptor Binding Domain ◽

Amino Acid Residues ◽

Chimeric Molecules

Computer simulation has been used to identify peptides that mimic the natural target of the SARS-CoV-2 coronavirus spike (S) protein, the angiotensin converting enzyme type 2 (ACE2) cell receptor. Based on the structure of the complex of the protein S receptor-binding domain (RBD) and ACE2, the design of chimeric molecules consisting of two 22-23-mer peptides linked to each other by disulfide bonds was carried out. The chimeric molecule X1 was a disulfide dimer, in which edge cysteine residues in the precursor molecules h1 and h2 were connected by the S-S bond. In the chimeric molecule X2, the disulfide bond was located in the middle of the molecule of each of the precursor peptides. The precursors h1 and h2 modelled amino acid sequences of α1- and α2-helices of the extracellular peptidase domain of ACE2, respectively, keeping intact most of the amino acid residues involved in the interaction with RBD. The aim of the work was to evaluate the binding efficiency of chimeric molecules and their RBD-peptides (particularly in dependence of the middle and edge methods of fixing the initial peptides h1 and h2). The proposed polypeptides and chimeric molecules were synthesized by chemical methods, purified (to 95-97% purity), and characterized by HPLC and MALDI-TOF mass spectrometry. The binding of the peptides to the SARS-CoV-2 RBD was evaluated by microthermophoresis with recombinant domains corresponding in sequence to the original Chinese (GenBank ID NC_045512.2) and the British (B. 1.1.7, GISAID EPI_ISL_683466) variants. Binding to the original RBD of the Chinese variant was detected in three synthesized peptides: linear h2 and both chimeric variants. Chimeric peptides were also bound to the RBD of the British variant with micromolar constants. The antiviral activity of the proposed peptides in Vero cell culture was also evaluated.

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N-Glycosylation of the SARS-CoV-2 Receptor Binding Domain Is Important for Functional Expression in Plants

Frontiers in Plant Science ◽

10.3389/fpls.2021.689104 ◽

2021 ◽

Vol 12 ◽

Author(s):

Yun-Ji Shin ◽

Julia König-Beihammer ◽

Ulrike Vavra ◽

Jennifer Schwestka ◽

Nikolaus F. Kienzl ◽

...

Keyword(s):

Protein Expression ◽

Recombinant Proteins ◽

Receptor Binding ◽

Posttranslational Modifications ◽

Functional Expression ◽

Binding Domain ◽

Site Directed Mutagenesis ◽

Receptor Binding Domain ◽

And Function ◽

The Impact

Nicotiana benthamiana is used worldwide as production host for recombinant proteins. Many recombinant proteins such as monoclonal antibodies, growth factors or viral antigens require posttranslational modifications like glycosylation for their function. Here, we transiently expressed different variants of the glycosylated receptor binding domain (RBD) from the SARS-CoV-2 spike protein in N. benthamiana. We characterized the impact of variations in RBD-length and posttranslational modifications on protein expression, yield and functionality. We found that a truncated RBD variant (RBD-215) consisting of amino acids Arg319-Leu533 can be efficiently expressed as a secreted soluble protein. Purified RBD-215 was mainly present as a monomer and showed binding to the conformation-dependent antibody CR3022, the cellular receptor angiotensin converting enzyme 2 (ACE2) and to antibodies present in convalescent sera. Expression of RBD-215 in glycoengineered ΔXT/FT plants resulted in the generation of complex N-glycans on both N-glycosylation sites. While site-directed mutagenesis showed that the N-glycans are important for proper RBD folding, differences in N-glycan processing had no effect on protein expression and function.

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Receptor-binding domain-specific human neutralizing monoclonal antibodies against SARS-CoV and SARS-CoV-2

Signal Transduction and Targeted Therapy ◽

10.1038/s41392-020-00318-0 ◽

2020 ◽

Vol 5 (1) ◽

Cited By ~ 1

Author(s):

Fei Yu ◽

Rong Xiang ◽

Xiaoqian Deng ◽

Lili Wang ◽

Zhengsen Yu ◽

...

Keyword(s):

Monoclonal Antibodies ◽

Receptor Binding ◽

Neutralizing Antibodies ◽

Binding Domain ◽

Receptor Binding Domain ◽

Global Public Health ◽

Domain Specific ◽

Treatment And Prevention ◽

T Cell Immune Responses ◽

And Function

Abstract The outbreaks of severe acute respiratory syndrome (SARS) and Coronavirus Disease 2019 (COVID-19) caused by SARS-CoV and SARS-CoV-2, respectively, have posed severe threats to global public health and the economy. Treatment and prevention of these viral diseases call for the research and development of human neutralizing monoclonal antibodies (NMAbs). Scientists have screened neutralizing antibodies using the virus receptor-binding domain (RBD) as an antigen, indicating that RBD contains multiple conformational neutralizing epitopes, which are the main structural domains for inducing neutralizing antibodies and T-cell immune responses. This review summarizes the structure and function of RBD and RBD-specific NMAbs against SARS-CoV and SARS-CoV-2 currently under development.

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