ABSTRACT
The aim of this study was to evaluate the impact of the most frequent Asn501 polar uncharged amino acid mutations upon important structural properties of SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) Surface Glycoprotein RBD–hACE2 (human angiotensin-converting enzyme 2) heterodimer. Mutations N501Y, N501T and N501S were considered and their impact upon complex solubility, secondary motifs formation and intermolecular hydrogen bonding interface was analyzed. Results and findings are reported based on 50 ns run in Gromacs molecular dynamics simulation software. Special attention is paid on the biomechanical shifts in the receptor-binding domain (RBD) [499-505]: ProThrAsn(Tyr)GlyValGlyTyr, having substituted Asparagine to Tyrosine at position 501. The main findings indicate that the N501S mutation increases SARS-CoV-2 S-protein RBD–hACE2 solubility over N501T, N501 (wild type): (Formula presented.), (Formula presented.). The N501Y mutation shifts (Formula presented.) -helix S-protein RBD [366-370]: SerValLeuTyrAsn into π-helix for t > 38.5 ns. An S-protein RBD [503-505]: ValGlyTyr shift from (Formula presented.) -helix into a turn is observed due to the N501Y mutation in t > 33 ns. An empirical proof for the presence of a Y501-binding pocket, based on RBD [499-505]: PTYGVGY (Formula presented.) 's RMSF peak formation is presented. There is enhanced electrostatic interaction between Tyr505 (RBD) phenolic -OH group and Glu37 (hACE2) side chain oxygen atoms due to the N501Y mutation. The N501Y mutation shifts the (Formula presented.) hydrogen bond into permanent polar contact;(Formula presented.);(Formula presented.). © 2023 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.
ABSTRACT
An effect of receptor-binding domain (RBD) of SARS-CoV-2 S-protein on structural parameters of model lipid membranes presented by dimyristoylphosphatidylcholine (DMPC) systems with cholesterol and melatonin impurities is studied by small angle neutron scattering (SANS). It is shown that an increase in melatonin concentration in the lipid membrane leads to a decrease in the thickness of the lipid bilayer, while an increase in the concentration of cholesterol leads to an increase in its thickness. It is suggested that increasing the concentration of melatonin in a membrane prevents the interaction of coronaviral S-protein with a lipid membrane of a cell. In the presence of cholesterol in the system, the interaction of a lipid membrane with an active part of S-protein occurs depending on a phase state of the lipid: in the case of a gel phase, there is no changes in structural parameters, but at higher temperatures in the case of a liquid crystal phase, an addition of RBD SARS-CoV-2 to the system causes a reduce in the membrane thickness. © 2023 Wiley-VCH GmbH.
ABSTRACT
The coronavirus disease 2019 (COVID-19) pandemic caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has serious consequences on global public health and social development. The binding of receptor binding domain (RBD) of spike protein to angiotensin converting enzyme 2 (ACE2) on the surface of SARS-CoV-2 host cell initiates the infection progress. Spike and ACE2 are both glycoproteins, the impact of glycosylation on protein structures and protein-protein interactions remains largely elusive. Characterizing the structural and dynamics of protein-protein binding progress will improve mechanism understanding of viral infection and facilitate targeted drug design. Structural mass spectrometry (MS) method is widely used in protein structural studies, providing complementary information to conventional biophysical methods, such as X-ray crystallography, nuclear magnetic resonance (NMR) spectroscopy and cryo-electron microscopy (cryo-EM). Native mass spectrometry (native MS) is an emerging technology that enables the study of intact protein, non-covalent protein-protein, and protein-ligand complexes in their biological state, which can provide structural stability, binding stoichiometry, and spatial arrangement information. Here, native MS was used to examine the interaction between RBD and ACE2 as well as the impact of deglycosylation on the interaction stability of the RBD-ACE2 complex. The results revealed that both RBD and ACE2 are highly glycosylated, ACE2 presents as a dimer while RBD as a monomer, and they form a (RBD-ACE2)2 complex. The conditions of using PNGasc F to remove the N-glycan were optimized. At least two Oglycans including NcuAc(2) and GalNAcC 1) Gal( 1) NcuAc(2) or GlcNAcd ) Gal(l) NeuAc(2) were observed for the N-glycan removed RBD. Furthermore, the stability of the complexes formed by glycosylated and deglycosylated RBD with ACE2 was compared, and the results showed that the removal of N-glycan significantly drops the interaction stability of the RBD-ACE2 complex. Therefore, we recommend that glycosyla-tion should not be removed for structural and functional studies. Additional glycosyla-tion, structural and dynamics studies on Spike (including separated RBD) and ACE2 complexes would help us to understand the process of viral infection, advance drug design and vaccine developments. Nowadays, a comprehensive MS-based toolbox has been developed for the analysis of protein structure, function, and dynamics, including hydrogen-deuterium exchange MS (HDX-MS), native top-down (nTD) MS, cross-linking MS (XL-MS), and covalent labelling MS (CL-MS), etc. Through integrating structural MS methods, more detailed and comprehensive structural information about glycoproteins and their complexes will be uncovered. © 2022 Chinese Society for Mass Spectrometry. All rights reserved.
ABSTRACT
The coronavirus disease 2019 (COVID-19) pandemic caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has serious consequences on global public health and social development. The binding of receptor binding domain (RBD) of spike protein to angiotensin converting enzyme 2 (ACE2) on the surface of SARS-CoV-2 host cell initiates the infection progress. Spike and ACE2 are both glycoproteins, the impact of glycosylation on protein structures and protein-protein interactions remains largely elusive. Characterizing the structural and dynamics of protein-protein binding progress will improve mechanism understanding of viral infection and facilitate targeted drug design. Structural mass spectrometry (MS) method is widely used in protein structural studies, providing complementary information to conventional biophysical methods, such as X-ray crystallography, nuclear magnetic resonance (NMR) spectroscopy and cryo-electron microscopy (cryo-EM). Native mass spectrometry (native MS) is an emerging technology that enables the study of intact protein, non-covalent protein-protein, and protein-ligand complexes in their biological state, which can provide structural stability, binding stoichiometry, and spatial arrangement information. Here, native MS was used to examine the interaction between RBD and ACE2 as well as the impact of deglycosylation on the interaction stability of the RBD-ACE2 complex. The results revealed that both RBD and ACE2 are highly glycosylated, ACE2 presents as a dimer while RBD as a monomer, and they form a (RBD-ACE2)2 complex. The conditions of using PNGasc F to remove the N-glycan were optimized. At least two Oglycans including NcuAc(2) and GalNAcC 1) Gal( 1) NcuAc(2) or GlcNAcd ) Gal(l) NeuAc(2) were observed for the N-glycan removed RBD. Furthermore, the stability of the complexes formed by glycosylated and deglycosylated RBD with ACE2 was compared, and the results showed that the removal of N-glycan significantly drops the interaction stability of the RBD-ACE2 complex. Therefore, we recommend that glycosyla-tion should not be removed for structural and functional studies. Additional glycosyla-tion, structural and dynamics studies on Spike (including separated RBD) and ACE2 complexes would help us to understand the process of viral infection, advance drug design and vaccine developments. Nowadays, a comprehensive MS-based toolbox has been developed for the analysis of protein structure, function, and dynamics, including hydrogen-deuterium exchange MS (HDX-MS), native top-down (nTD) MS, cross-linking MS (XL-MS), and covalent labelling MS (CL-MS), etc. Through integrating structural MS methods, more detailed and comprehensive structural information about glycoproteins and their complexes will be uncovered. © 2022 Chinese Society for Mass Spectrometry. All rights reserved.
ABSTRACT
Covid-19 has become a world pandemic for years. With the appearance of mutations, immune escape has become a problem, reducing the effectiveness of vaccines and antibodies. To reveal the mechanism of immune escape, we analyze the geometrical properties of the receptor-binding domain in the SARS-CoV-2 spike protein, which plays a vital role in the immune reaction. Several important variants are taken as examples, and the wild type model is prepared as a reference. The computational method is applied to simulate the behaviors of the models, and alpha shape algorithm is employed to extract geometrical data of the protein surface. Average moving distance of the surface atoms is used to quantify their activity. Our results show that the mutations changed the properties of the protein. The variants have different distributions of active sites, which may change the specific antigenicity and influence the binding abilities of drugs and antibodies. This study explains the mechanism of immune escape of SARS-CoV-2, and provides a geometrical method to find potential new target sites for the design of drugs and vaccines. © 2022 IEEE.
ABSTRACT
Adhesion of the SARS-CoV-2 virus spike protein was studied by vibrational spectroscopy using terahertz metamaterials. Specific features of metastructure absorption by histidine, albumin, and receptor-binding domain of spike protein films were investigated. An original method for quantitative estimation of the efficiency of virus adhesion on the surface of metamaterials has been proposed and experimentally tested. © 2022 IEEE.
ABSTRACT
The need to develop high-throughput diagnostic platforms for infectious diseases has never been more evident than with the emergence of SARS-CoV-2 and the ensued COVID-19 pandemic. Microfluidics, in tandem with its multiplexing capabilities, high sensitivity, and potential for automation, provides a unique advantage towards the development of high-throughput serological diagnostic platforms. Here, we present a microfluidic device that detects IgG or IgM raised against four SARS-CoV-2 antigens (spike, S;S1 subunit, S1;the receptor-binding domain, RBD;and nucleocapsid, N) from 50 serum samples in parallel. We validated the platform with a cross-sectional cohort of 66 samples from confirmed COVID-19 patients and a pre-pandemic control of 34 serum samples collected in 2018. The analysis of both antibodies against all four viral antigens provided a sensitivity of 90.4% and a specificity of 94.1%, with both parameters increasing to 100% in late-stage samples (21-30 days after symptoms onset). We expect our device to open the door to massive serological testing, impacting diagnostics, vaccine development, and epidemiological understanding of COVID-19. © 2021 MicroTAS 2021 - 25th International Conference on Miniaturized Systems for Chemistry and Life Sciences. All rights reserved.
ABSTRACT
We present fiber optic surface plasmon resonance (FO-SPR) label-free (LF) bioassays for quantification and kinetic profiling of complete antibody isotypes against the receptor binding domain (RBD) of SARS-CoV-2 spike protein. This was accomplished not only in serum but also for the first time directly in whole blood of COVID-19 convalescent patients. The LF bioassay was correlated with the traditional FO-SPR sandwich bioassay, the latter also benchmarked with ELISA. Compared to other serological tests, our approach is superior in: (1) information about kinetics, (2) rapid insight into the amount of all antibody isotypes and (3) exceptional compatibility with whole blood samples. © 2021 MicroTAS 2021 - 25th International Conference on Miniaturized Systems for Chemistry and Life Sciences. All rights reserved.
ABSTRACT
A rapid, portable, and cost-effective method to detect the infection of SARS-CoV-2 is fundamental toward mitigating the current COVID-19 pandemic. A localized surface plasmon resonance (LSPR) sensor based on human angiotensin-converting enzyme 2 protein (ACE2) functionalized silver nanotriangle array is developed for rapid coronavirus detection. The sensor is validated by SARS-CoV-2 spike RBD protein and CoV NL63 virus with high sensitivity and specificity. A linear shift of the LSPR wavelength and transmission intensity at a fixed wavelength (750 nm) versus the logarithm of the concentration of the spike RBD protein and CoV NL63 is observed. The limits of detection for the spike RBD protein, CoV NL63 in untreated saliva are determined to be 0.38 pM, and 625 PFU/mL, respectively, while the detection time is found to be less than 20 min. Such a LSPR sensor could serve as a potential rapid point-of-care diagnostic platform for COVID-19. © 2022 SPIE
ABSTRACT
The recurrent zoonotic spillover of coronaviruses (CoVs) into the human population underscores the need for broadly active countermeasures. We employed a directed evolution approach to engineer three SARS-CoV-2 antibodies for enhanced neutralization breadth and potency. One of the affinity-matured variants, ADG-2, displays strong binding activity to a large panel of sarbecovirus receptor binding domains (RBDs) and neutralizes representative epidemic sarbecoviruses with high potency. Structural and biochemical studies demonstrate that ADG-2 employs a distinct angle of approach to recognize a highly conserved epitope overlapping the receptor binding site. In immunocompetent mouse models of SARS and COVID-19, prophylactic administration of ADG-2 provided complete protection against respiratory burden, viral replication in the lungs, and lung pathology. Altogether, ADG-2 represents a promising broad-spectrum therapeutic candidate against clade 1 sarbecoviruses.
ABSTRACT
SARS-CoV-2 is a novel virus that crossed over into humans in 2019 and declared a pandemic in early 2020. To understand how the virus infects a new host, we need to understand the mechanistic functions involved with the binding process. To address this need, we generate homology models of SARS-CoV-2 spikes as monomer and trimer to determine the feasibility of reduced computational requirements by using monomer structures. We further generate homology models of the conserved region of SARS-CoV-2 spike subunit s1 noted as the receptor binding domain (RBD) and the human angiotensin-converting enzyme 2 (ACE2). To determine functional breadth of spike monomer, trimer and RBD in relation with ACE2, we apply Coulombs Law to determine an electric force between combinations with ACE2 across the range of pH from 3.0 to 9.0 in 0.1 increments. The results indicate that spike trimer should be used to determine mechanistic binding function and these data indicate that variations of spike sequence influence breadth of function. Our results also indicate the RBD has a broader range of function across pH compared to spike trimer, but is influenced by the range of function presented by the spike trimer. © 2021 IEEE.
ABSTRACT
The end of 2019 marked the start of coronavirus disease (COVID-19) pandemic from China, which went on to envelope more than 190 countries and territories across the globe. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), from a group of betacoronaviruses, is responsible for COVID-19. The virulent factors include the presence of envelope and spike proteins having receptor bonding domains (RBD). Clinical manifestations can range from mild respiratory infections to fatal outcomes. The viability of virus ranges from 3 to 72 hours. Polymerase chain reaction (PCR) is the diagnostic test of choice in this pandemic situation. Due to the absence of specific antivirals and vaccine, adoption of preventive option can help to combat the specific life-threatening outcomes.