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

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.

scholarly journals Comparative Immunogenicity of the Recombinant Receptor-Binding Domain of Protein S SARS-CoV-2 Obtained in Prokaryotic and Mammalian Expression Systems

Vaccines ◽  
2022 ◽  
Vol 10 (1) ◽  
pp. 96
Author(s):  
Iuliia A. Merkuleva ◽  
Dmitry N. Shcherbakov ◽  
Mariya B. Borgoyakova ◽  
Daniil V. Shanshin ◽  
Andrey P. Rudometov ◽  
...  
Keyword(s):  
Receptor Binding ◽  
Neutralizing Antibodies ◽  
Expression System ◽  
Protein S ◽  
Binding Domain ◽  
Receptor Binding Domain ◽  
Expression Systems ◽  
Mice Model ◽  
Mammalian Expression ◽  
Recombinant Receptor

The receptor-binding domain (RBD) of the protein S SARS-CoV-2 is considered to be one of the appealing targets for developing a vaccine against COVID-19. The choice of an expression system is essential when developing subunit vaccines, as it ensures the effective synthesis of the correctly folded target protein, and maintains its antigenic and immunogenic properties. Here, we describe the production of a recombinant RBD protein using prokaryotic (pRBD) and mammalian (mRBD) expression systems, and compare the immunogenicity of prokaryotic and mammalian-expressed RBD using a BALB/c mice model. An analysis of the sera from mice immunized with both variants of the protein revealed that the mRBD expressed in CHO cells provides a significantly stronger humoral immune response compared with the RBD expressed in E.coli cells. A specific antibody titer of sera from mice immunized with mRBD was ten-fold higher than the sera from the mice that received pRBD in ELISA, and about 100-fold higher in a neutralization test. The data obtained suggests that mRBD is capable of inducing neutralizing antibodies against SARS-CoV-2.

scholarly journals Biochemical, Biophysical, and Functional Characterization of Bacterially Expressed and Refolded Receptor Binding Domain ofPlasmodium vivaxDuffy-binding Protein

2001 ◽  
Vol 276 (20) ◽  
pp. 17111-17116 ◽  
Author(s):  
Sanjay Singh ◽  
Kailash Pandey ◽  
Rana Chattopadhayay ◽  
Syed Shams Yazdani ◽  
Andrew Lynn ◽  
...  
Keyword(s):  
Receptor Binding ◽  
Binding Protein ◽  
Functional Characterization ◽  
Binding Domain ◽  
Receptor Binding Domain

scholarly journals An exploration of the SARS-CoV-2 spike receptor binding domain (RBD) – a complex palette of evolutionary and structural features

2020 ◽  
Author(s):  
Dwipanjan Sanyal ◽  
Sourav Chowdhury ◽  
Vladimir N. Uversky ◽  
Krishnananda Chattopadhyay
Keyword(s):  
Receptor Binding ◽  
Sequence Space ◽  
Structural Integrity ◽  
Protein S ◽  
Structural Features ◽  
Binding Domain ◽  
Evolutionary Analysis ◽  
Receptor Binding Domain ◽  
Comprehensive Picture ◽  
Fuzzy C Means Clustering

AbstractSARS-CoV-2 spike protein (S) is associated with the entry of virus inside the host cell by recruiting its loop dominant receptor binding domain (RBD) and interacting with the host ACE2 receptor. Our study deploying a two-tier approach encompassing evolutionary and structural analysis provides a comprehensive picture of the RBD, which could be of potential use for better understanding the RBD and address its druggability issues. Resorting to an ensemble of sequence space exploratory tools including co-evolutionary analysis and deep mutational scans we provide a quantitative insight into the evolutionarily constrained subspace of the RBD sequence space. Guided by structure network analysis and Monte Carlo simulation we highlight regions inside the RBD, which are critical for providing structural integrity and conformational flexibility of the binding cleft. We further deployed fuzzy C-means clustering by plugging the evolutionary and structural features of discrete structure blocks of RBD to understand which structure blocks share maximum overlap based on their evolutionary and structural features. Deploying this multi-tier interlinked approach, which essentially distilled the evolutionary and structural features of RBD, we highlight discrete region, which could be a potential druggable pocket thereby destabilizing the structure and addressing evolutionary routes.

scholarly journals SARS-CoV-2 Variant of Concern Substitutions Alter Spike Glycoprotein Receptor Binding Domain Structure and Stability

2021 ◽  
Author(s):  
Daniel L Moss ◽  
Jay Rappaport
Keyword(s):  
Receptor Binding ◽  
Rapid Development ◽  
Structure And Function ◽  
Binding Domain ◽  
Amino Acid Substitutions ◽  
Receptor Binding Domain ◽  
Spike Glycoprotein ◽  
Structure And Stability ◽  
The World ◽  
And Function

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.

scholarly journals Stabilization of the SARS-CoV-2 Spike receptor-binding domain using deep mutational scanning and structure-based design

2021 ◽  
Author(s):  
Daniel Ellis ◽  
Natalie Brunette ◽  
Katherine H. D. Crawford ◽  
Alexandra C. Walls ◽  
Minh N. Pham ◽  
...  
Keyword(s):  
Receptor Binding ◽  
Large Scale ◽  
Protein S ◽  
Binding Pocket ◽  
Binding Domain ◽  
Effective Vaccine ◽  
Receptor Binding Domain ◽  
Wild Type ◽  
Vaccine Candidates ◽  
Generation Vaccine

The unprecedented global demand for SARS-CoV-2 vaccines has demonstrated the need for highly effective vaccine candidates that are thermostable and amenable to large-scale manufacturing. Nanoparticle immunogens presenting the receptor-binding domain (RBD) of the SARS-CoV-2 Spike protein (S) in repetitive arrays are being advanced as second-generation vaccine candidates, as they feature robust manufacturing characteristics and have shown promising immunogenicity in preclinical models. Here, we used previously reported deep mutational scanning (DMS) data to guide the design of stabilized variants of the RBD. The selected mutations fill a cavity in the RBD that has been identified as a linoleic acid binding pocket. Screening of several designs led to the selection of two lead candidates that expressed at higher yields than the wild-type RBD. These stabilized RBDs possess enhanced thermal stability and resistance to aggregation, particularly when incorporated into an icosahedral nanoparticle immunogen that maintained its integrity and antigenicity for 28 days at 35-40°C, while corresponding immunogens displaying the wild-type RBD experienced aggregation and loss of antigenicity. The stabilized immunogens preserved the potent immunogenicity of the original nanoparticle immunogen, which is currently being evaluated in a Phase I/II clinical trial. Our findings may improve the scalability and stability of RBD-based coronavirus vaccines in any format and more generally highlight the utility of comprehensive DMS data in guiding vaccine design.

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

2021 ◽  
pp. 167357
Author(s):  
Andrey M. Grishin ◽  
Nataliya V. Dolgova ◽  
Shelby Landreth ◽  
Olivier Fisette ◽  
Ingrid J. Pickering ◽  
...  
Keyword(s):  
Receptor Binding ◽  
Disulfide Bonds ◽  
Critical Role ◽  
Structure And Function ◽  
Binding Domain ◽  
Receptor Binding Domain ◽  
And Function

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

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.

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

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.

scholarly journals Stabilization of the SARS-CoV-2 Spike Receptor-Binding Domain Using Deep Mutational Scanning and Structure-Based Design

2021 ◽  
Vol 12 ◽  
Author(s):  
Daniel Ellis ◽  
Natalie Brunette ◽  
Katharine H. D. Crawford ◽  
Alexandra C. Walls ◽  
Minh N. Pham ◽  
...  
Keyword(s):  
Receptor Binding ◽  
Large Scale ◽  
Protein S ◽  
Binding Pocket ◽  
Binding Domain ◽  
Effective Vaccine ◽  
Receptor Binding Domain ◽  
Wild Type ◽  
Vaccine Candidates ◽  
Generation Vaccine

The unprecedented global demand for SARS-CoV-2 vaccines has demonstrated the need for highly effective vaccine candidates that are thermostable and amenable to large-scale manufacturing. Nanoparticle immunogens presenting the receptor-binding domain (RBD) of the SARS-CoV-2 Spike protein (S) in repetitive arrays are being advanced as second-generation vaccine candidates, as they feature robust manufacturing characteristics and have shown promising immunogenicity in preclinical models. Here, we used previously reported deep mutational scanning (DMS) data to guide the design of stabilized variants of the RBD. The selected mutations fill a cavity in the RBD that has been identified as a linoleic acid binding pocket. Screening of several designs led to the selection of two lead candidates that expressed at higher yields than the wild-type RBD. These stabilized RBDs possess enhanced thermal stability and resistance to aggregation, particularly when incorporated into an icosahedral nanoparticle immunogen that maintained its integrity and antigenicity for 28 days at 35-40°C, while corresponding immunogens displaying the wild-type RBD experienced aggregation and loss of antigenicity. The stabilized immunogens preserved the potent immunogenicity of the original nanoparticle immunogen, which is currently being evaluated in a Phase I/II clinical trial. Our findings may improve the scalability and stability of RBD-based coronavirus vaccines in any format and more generally highlight the utility of comprehensive DMS data in guiding vaccine design.

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