Angiotensin-converting Enzyme 2 Binding Research Articles

Single-molecule imaging reveals allosteric stimulation of SARS-CoV-2 spike receptor binding domain by host sialic acid.

Conformational dynamics of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike glycoprotein (S) mediate exposure of the binding site for the cellular receptor, angiotensin-converting enzyme 2 (ACE2). The N-terminal domain (NTD) of S binds terminal sialic acid (SA) moieties on the cell surface, but the functional role of this interaction in virus entry is unknown. Here, we report that NTD-SA interaction enhances both S-mediated virus attachment and ACE2 binding. Through single-molecule Förster resonance energy transfer imaging of individual S trimers, we demonstrate that SA binding to the NTD allosterically shifts the S conformational equilibrium, favoring enhanced exposure of the ACE2-binding site. Antibodies that target the NTD block SA binding, which contributes to their mechanism of neutralization. These findings inform on mechanisms of S activation at the cell surface.

Temperature-dependent Spike-ACE2 interaction of Omicron subvariants is associated with viral transmission.

The continued evolution of severe acute respiratory syndrome 2 (SARS-CoV-2) requires persistent monitoring of its subvariants. Omicron subvariants are responsible for the vast majority of SARS-CoV-2 infections worldwide, with XBB and BA.2.86 sublineages representing more than 90% of circulating strains as of January 2024. To better understand parameters involved in viral transmission, we characterized the functional properties of Spike glycoproteins from BA.2.75, CH.1.1, DV.7.1, BA.4/5, BQ.1.1, XBB, XBB.1, XBB.1.16, XBB.1.5, FD.1.1, EG.5.1, HK.3, BA.2.86 and JN.1. We tested their capacity to evade plasma-mediated recognition and neutralization, binding to angiotensin-converting enzyme 2 (ACE2), their susceptibility to cold inactivation, Spike processing, as well as the impact of temperature on Spike-ACE2 interaction. We found that compared to the early wild-type (D614G) strain, most Omicron subvariants' Spike glycoproteins evolved to escape recognition and neutralization by plasma from individuals who received a fifth dose of bivalent (BA.1 or BA.4/5) mRNA vaccine and improve ACE2 binding, particularly at low temperatures. Moreover, BA.2.86 had the best affinity for ACE2 at all temperatures tested. We found that Omicron subvariants' Spike processing is associated with their susceptibility to cold inactivation. Intriguingly, we found that Spike-ACE2 binding at low temperature was significantly associated with growth rates of Omicron subvariants in humans. Overall, we report that Spikes from newly emerged Omicron subvariants are relatively more stable and resistant to plasma-mediated neutralization, present improved affinity for ACE2 which is associated, particularly at low temperatures, with their growth rates.IMPORTANCEThe persistent evolution of SARS-CoV-2 gave rise to a wide range of variants harboring new mutations in their Spike glycoproteins. Several factors have been associated with viral transmission and fitness such as plasma-neutralization escape and ACE2 interaction. To better understand whether additional factors could be of importance in SARS-CoV-2 variants' transmission, we characterize the functional properties of Spike glycoproteins from several Omicron subvariants. We found that the Spike glycoprotein of Omicron subvariants presents an improved escape from plasma-mediated recognition and neutralization, Spike processing, and ACE2 binding which was further improved at low temperature. Intriguingly, Spike-ACE2 interaction at low temperature is strongly associated with viral growth rate, as such, low temperatures could represent another parameter affecting viral transmission.

Sites and Zones of Maximum Reactivity of the most Stable Structure of the Receptor-binding Domain of Wild-type SARS-CoV-2 Spike Protein: A Quantum Density Functional Theory Study

Today, it is well known that Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has four types of proteins within its structure, between them the spike protein (S). The infection mechanism is carried out by the entry of the virus into the human host cell through the S protein, which strongly interacts with the human cell receptor angiotensin-converting enzyme 2 (ACE2). In this work, we propose an atomic model of the Receptor Binding Domain (RBD) of the S spike protein of the wild-type SARS-CoV-2 virus. The molecular structure of the model was composed of 50 amino acids that were chemically bonded, starting with Leucine and ending with one amino acid Tyrosine. The novelty of our work lies in the importance of knowing the sites and zones of maximum reactivity of the RBD from the fundamental levels of quantum mechanics considering the atomic structure of matter. For this, the local and global reactivity indices of the RBD were calculated, such as frontier orbitals, Highest Occupied Molecular Orbital (HOMO) and Lowest Unoccupied Molecular Orbital (LUMO), Fukui indices, chemical potential, chemical hardness, electrophilicity index; with this, it will be possible to know what type of molecules are more likely to interact with the RBD structure, and in this way, new knowledge will be generated at the quantum, atomic and molecular level to inhibit the virulent effects of wild-type SARS-CoV-2. Finally, in order to identify the functional groups within the most stable structure and thereby verify the future reactions that can be carried out between the RBD structure and biomolecules, the Infrared (IR) absorption spectrum was calculated. For this work, we used Material Studio v6.0 which uses the density functional theory (DFT) implemented in its DMol3 computational code. The IR spectrum was obtained using the Spartan ‘94 computer code. One novelty would be that we found nine amino acids more that could make the RBD and ACE2 binding further the already known. Thus, the Mulliken charge distribution indicates that the highest concentrations of positive and negative charge are found in the zones 477S, 478T, 484E, and 501N amino acids letting ionic or Van der Waals possible interactions with other structures.

Application of a Biomimetic Nanoparticle-Based Mock Virus to Determine SARS-CoV-2 Neutralizing Antibody Levels in Blood Samples Using a Lateral Flow Assay.

The presence of neutralizing antibodies against SARS-CoV-2 in blood, acquired through previous infection or vaccination, is known to prevent the (re)occurrence of outbreaks unless the virus mutates. Therefore, the measurement of neutralizing antibodies constitutes an indispensable tool in assessing an individual's and a population's immunity against SARS-CoV-2. For this reason, we have developed an innovative lateral flow assay (LFA) capable of detecting blood-derived neutralizing antibodies using a biomimetic SARS-CoV-2 mock virus system. Here, functionalized gold nanoparticles (AuNPs) featuring the trimeric spike (S) protein at its surface imitate the virus's structure and are applied to monitor the presence and efficacy of neutralizing antibodies in blood samples. The detection principle relies on the interaction between mock virus and the immobilized angiotensin-converting enzyme 2 (ACE2) receptor, which is inhibited when neutralizing antibodies are present. To further enhance the sensitivity of our competitive assay and identify low titers of neutralizing antibodies, an additional mixing pad is embedded into the device to increase the interaction time between mock virus and neutralizing antibodies. The developed LFA is benchmarked against the WHO International Standard (21/338) and demonstrated reliable quantification of neutralizing antibodies that inhibit ACE2 binding events down to a detection limit of an antibody titer of 59 IU/mL. Additional validation using whole blood and plasma samples showed reproducible results and good comparability to a laboratory-based reference test, thus highlighting its applicability for point-of-care testing.

Structural basis and analysis of hamster ACE2 binding to different SARS-CoV-2 spike RBDs.

Pet golden hamsters were first identified being infected with the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) delta variant of concern (VOC) and transmitted the virus back to humans in Hong Kong in January 2022. Here, we studied the binding of two hamster (golden hamster and Chinese hamster) angiotensin-converting enzyme 2 (ACE2) proteins to the spike protein receptor-binding domains (RBDs) of SARS-CoV-2 prototype and eight variants, including alpha, beta, gamma, delta, and four omicron sub-variants (BA.1, BA.2, BA.3, and BA.4/BA.5). We found that the two hamster ACE2s present slightly lower affinity for the RBDs of all nine SARS-CoV-2 viruses tested than human ACE2 (hACE2). Furthermore, the similar infectivity to host cells expressing hamster ACE2s and hACE2 was confirmed with the nine pseudotyped SARS-CoV-2 viruses. Additionally, we determined two cryo-electron microscopy (EM) complex structures of golden hamster ACE2 (ghACE2)/delta RBD and ghACE2/omicron BA.3 RBD. The residues Q34 and N82, which exist in many rodent ACE2s, are responsible for the lower binding affinity of ghACE2 compared to hACE2. These findings suggest that all SARS-CoV-2 VOCs may infect hamsters, highlighting the necessity of further surveillance of SARS-CoV-2 in these animals.IMPORTANCESARS-CoV-2 can infect many domestic animals, including hamsters. There is an urgent need to understand the binding mechanism of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants to hamster receptors. Herein, we showed that two hamster angiotensin-converting enzyme 2s (ACE2s) (golden hamster ACE2 and Chinese hamster ACE2) can bind to the spike protein receptor-binding domains (RBDs) of SARS-CoV-2 prototype and eight variants and that pseudotyped SARS-CoV-2 viruses can infect hamster ACE2-expressing cells. The binding pattern of golden hamster ACE2 to SARS-CoV-2 RBDs is similar to that of Chinese hamster ACE2. The two hamster ACE2s present slightly lower affinity for the RBDs of all nine SARS-CoV-2 viruses tested than human ACE2. We solved the cryo-electron microscopy (EM) structures of golden hamster ACE2 in complex with delta RBD and omicron BA.3 RBD and found that residues Q34 and N82 are responsible for the lower binding affinity of ghACE2 compared to hACE2. Our work provides valuable information for understanding the cross-species transmission mechanism of SARS-CoV-2.

Two Receptor Binding Strategy of SARS-CoV-2 Is Mediated by Both the N-Terminal and Receptor-Binding Spike Domain.

It is not well understood why severe acute respiratory syndrome (SARS)-CoV-2 spreads much faster than other β-coronaviruses such as SARS-CoV and Middle East respiratory syndrome (MERS)-CoV. In a previous publication, we predicted the binding of the N-terminal domain (NTD) of SARS-CoV-2 spike to sialic acids (SAs). Here, we experimentally validate this interaction and present simulations that reveal a second possible interaction between SAs and the spike protein via a binding site located in the receptor-binding domain (RBD). The predictions from molecular-dynamics simulations and the previously-published 2D-Zernike binding-site recognition approach were validated through flow-induced dispersion analysis (FIDA)─which reveals the capability of the SARS-CoV-2 spike to bind to SA-containing (glyco)lipid vesicles, and flow-cytometry measurements─which show that spike binding is strongly decreased upon inhibition of SA expression on the membranes of angiotensin converting enzyme-2 (ACE2)-expressing HEK cells. Our analyses reveal that the SA binding of the NTD and RBD strongly enhances the infection-inducing ACE2 binding. Altogether, our work provides in silico, in vitro, and cellular evidence that the SARS-CoV-2 virus utilizes a two-receptor (SA and ACE2) strategy. This allows the SARS-CoV-2 spike to use SA moieties on the cell membrane as a binding anchor, which increases the residence time of the virus on the cell surface and aids in the binding of the main receptor, ACE2, via 2D diffusion.

Studying SARS-CoV-2 interactions using phage-displayed receptor binding domain as a model protein

SARS-CoV-2 receptor binding domain (RBD) mediates viral entry into human cells through its interaction with angiotensin converting enzyme 2 (ACE2). Most neutralizing antibodies elicited by infection or vaccination target this domain. Such a functional relevance, together with large RBD sequence variability arising during viral spreading, point to the need of exploring the complex landscape of interactions between RBD-derived variants, ACE2 and antibodies. The current work was aimed at developing a simple platform to do so. Biologically active and antigenic Wuhan-Hu-1 RBD, as well as mutated RBD variants found in nature, were successfully displayed on filamentous phages. Mutational scanning confirmed the global plasticity of the receptor binding motif within RBD, highlighted residues playing a critical role in receptor binding, and identified mutations strengthening the interaction. The ability of vaccine-induced antibodies to inhibit ACE2 binding of many mutated RBD variants, albeit at different extents, was shown. Amino acid replacements which could compromise such inhibitory potential were underscored. The expansion of our approach could be the starting point for a large-scale phage-based exploration of diversity within RBD of SARS-CoV-2 and related coronaviruses, useful to understand structure–function relationships, to engineer RBD proteins, and to anticipate changes to watch during viral evolution.

ACE2 Receptor-Targeted Inhaled Nanoemulsions Inhibit SARS-CoV-2 and Attenuate Inflammatory Responses.

Three kinds of coronaviruses are highly pathogenic to humans, and two of them mainly infect humans through Angiotensin-converting enzyme 2 （ACE2）receptors. Therefore, specifically blocking ACE2 binding at the interface with the receptor-binding domain is promising to achieve both preventive and therapeutic effects of coronaviruses. Alternatively, drug-targeted delivery based on ACE2 receptors can further improve the efficacy and safety of inhalation drugs. Here, these two approaches are innovatively combined by designing a nanoemulsion (NE) drug delivery system (termed NE-AYQ) for inhalation that targets binding to ACE2 receptors. This inhalation-delivered remdesivir nanoemulsion (termed RDSV-NE-AYQ) effectively inhibits the infection of target cells by both wild-type and mutant viruses. The RDSV-NE-AYQ strongly inhibits Severe acute respiratory syndrome coronavirus 2 at two dimensions: they not only block the binding of the virus to host cells at the cell surface but also restrict virus replication intracellularly. Furthermore, in the mouse model of acute lung injury, the inhaled drug delivery system loaded with anti-inflammatory drugs (TPCA-1-NE-AYQ) can significantly alleviate the lung tissue injury of mice. This smart combination provides a new choice for dealing with possible emergencies in the future and for the rapid development of inhaled drugs for the treatment of respiratory diseases.

Enhancement of SARS-CoV-2 Infection via Crosslinking of Adjacent Spike Proteins by N-Terminal Domain-Targeting Antibodies.

The entry of SARS-CoV-2 into host cells is mediated by the interaction between the spike receptor-binding domain (RBD) and host angiotensin-converting enzyme 2 (ACE2). Certain human antibodies, which target the spike N-terminal domain (NTD) at a distant epitope from the host cell binding surface, have been found to augment ACE2 binding and enhance SARS-CoV-2 infection. Notably, these antibodies exert their effect independently of the antibody fragment crystallizable (Fc) region, distinguishing their mode of action from previously described antibody-dependent infection-enhancing (ADE) mechanisms. Building upon previous hypotheses and experimental evidence, we propose that these NTD-targeting infection-enhancing antibodies (NIEAs) achieve their effect through the crosslinking of neighboring spike proteins. In this study, we present refined structural models of NIEA fragment antigen-binding region (Fab)-NTD complexes, supported by molecular dynamics simulations and hydrogen-deuterium exchange mass spectrometry (HDX-MS). Furthermore, we provide direct evidence confirming the crosslinking of spike NTDs by NIEAs. Collectively, our findings advance our understanding of the molecular mechanisms underlying NIEAs and their impact on SARS-CoV-2 infection.

Heparin-Induced Allosteric Changes in SARS-CoV-2 Spike Protein Facilitate ACE2 Binding and Viral Entry.

Understanding the entry of severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) into host cells is crucial in the battle against COVID-19. Using atomic force microscopy (AFM), we probed the interaction between the virus's spike protein and heparan sulfate (HS) as a potential attachment factor. Our AFM studies revealed a moderate-affinity interaction between the spike protein and HS on both model surfaces and living cells, highlighting HS's role in early viral attachment. Remarkably, we observed an interplay between HS and the host cell receptor angiotensin-converting enzyme 2 (ACE2), with HS engagement resulting in enhanced ACE2 binding and subsequent viral entry. Our research furthers our understanding of SARS-CoV-2 infection mechanisms and reveals potential interventions targeting viral entry. These insights are valuable as we navigate the evolving landscape of viral threats and seek effective strategies to combat emerging infectious diseases.

Low-Entropy Hydration Shells at the Spike RBD's Binding Site May Reveal the Contagiousness of SARS-CoV-2 Variants.

The infectivity of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is primarily determined by the binding affinity between the receptor-binding domain (RBD) of the spike protein and the angiotensin-converting enzyme 2 (ACE2) receptor. Here, through screening off pseudo hydrophilic groups on protein surfaces, the distribution of low-entropy regions on hydration shells of the ACE2 receptor and the RBDs of multiple SARS-CoV-2 variants was demonstrated. Shape matching between the low-entropy hydration shells of multiple SARS-CoV-2 variants and the ACE2 receptor has been identified as a mechanism that drives hydrophobic attraction between the RBDs and the ACE2 receptor, which estimates the binding affinity. Low-entropy regions of the hydration shells, which play important roles in determining the binding of other viruses and their receptors, are demonstrated. The RBD-ACE2 binding is thus found to be guided by hydrophobic collapse between the shape-matched low-entropy regions of the hydration shells of the proteins. A measure of the low-entropy status of the hydration shells can be estimated by calculating genuine hydrophilic groups within the binding sites. An important indicator of the contagiousness of SARS-CoV-2 variants is the low-entropy level of its hydration shells at the spike protein binding site.

A potent and broad-spectrum neutralizing nanobody for SARS-CoV-2 viruses, including all major Omicron strains.

SARS-CoV-2 viruses are highly transmissible and immune evasive. It is critical to develop broad-spectrum prophylactic and therapeutic antibodies for potential future pandemics. Here, we used the phage display method to discover nanobodies (Nbs) for neutralizing SARS-CoV-2 viruses especially Omicron strains. The leading nanobody (Nb), namely, Nb4, with excellent physicochemical properties, can neutralize Delta and Omicron subtypes, including BA.1, BA.1.1 (BA.1+R346K), BA.2, BA.5, BQ.1, and XBB.1. The crystal structure of Nb4 in complex with the receptor-binding domain (RBD) of BA.1 Spike protein reveals that Nb4 interacts with an epitope on the RBD overlapping with the receptor-binding motif, and thus competes with angiotensin-converting enzyme 2 (ACE2) binding. Nb4 is expected to be effective for neutralizing most recent Omicron variants, since the epitopes are evolutionarily conserved among them. Indeed, trivalent Nb4 interacts with the XBB1.5 Spike protein with low nM affinity and competes for ACE2 binding. Prophylactic and therapeutic experiments in mice indicated that Nb4 could reduce the Omicron virus loads in the lung. In particular, in prophylactic experiments, intranasal administration of multivalent Nb4 completely protected mice from Omicron infection. Taken together, these results demonstrated that Nb4 could serve as a potent and broad-spectrum prophylactic and therapeutic Nb for COVID-19.

Molecular dynamic simulation reveals spider antimicrobial peptide Latarcin-1 and human eosinophil cationic protein as peptide inhibitors of SARS-CoV-2 variants

COVID-19 has rapidly proliferated around 180 countries, and new cases are reported frequently. No peptide medication has been developed that can reliably block SARS-CoV-2 infection. The investigation focuses on the crucial host receptors angiotensin-converting enzyme 2 (ACE2) , which can bind receptor-binding domain (RBD) on the SARS-CoV-2 spike protein (S). To investigate the inhibitory effects of human Eosinophil Cationic Protein (hECP) and Latarcin-1 (L1)on SARS-CoV-2 infection, we have selected them as research subjects. Further, we ran extensive molecular dynamics simulations to bring the docked peptide-ACE2 complex into its equilibrium state. The outcomes were then evaluated with g_MMPBSA and interaction analysis. We have also considered the Delta and Omicron variants to examine these peptides’ inhibitory effects. The experimental findings revealed an enhanced capability of L1 and hECP as SARS-CoV-2 inhibitors, occupying hot spots and numerous key residues in ACE2. These include ASP30, ASP38, GLU35 and GLU75, which significantly inhibit the binding of RBD and ACE2 and are effective against two common variants in a similar manner. In addition, this study can serve as a springboard for future research on SARS-CoV-2 inhibitors. Communicated by Ramaswamy H. Sarma

Losartan Has a Comparable Effect to Human Recombinant ACE2 in Reducing Interleukin-6 (IL-6) Levels on Human Adipocytes Exposed to SARS-CoV-2 Spike Protein

BACKGROUND: High angiotensin-converting enzyme 2 (ACE2) expression in adipocyte cells facilitates the initiation of SARS-CoV-2 infection and triggers a cytokine storm. This finding suggests that obesity is an independent risk factor for the severity of the symptoms caused by COVID-19. The use of cardiovascular medications that focus on ACE2, such as angiotensin II receptor blockers, remains controversial, and their effects on inflammatory cytokine production and ACE2 expression in cells, especially adipocytes, remain inconsistent.METHODS: The human adipocytes were isolated from obese donor subcutaneous adipose tissue and infected with the subunit S1 spike protein from SARS-Cov-2. The adipocytes were later treated with either hrsACE2 or losartan. The levels of ACE2 and inflammatory cytokines interleukin (IL)-6, IL-1β, and tumor necrosis factor (TNF)-α were measured using enzyme linked immunosorbent assay (ELISA). ACE2 and S1 spike protein binding assays were also performed. RESULTS: ACE2, IL-6, and TNF-α levels were significantly increased in human adipocyte cells infected with SARS-Cov-2 but not IL-1β. There was a statistically significant positive correlation between ACE2 and IL-6 (r=0.878, p<0.001). Administration of losartan and hrsACE2 was shown to reduce ACE2 levels and its binding to the SARS-CoV-2 S1 spike protein, and IL-6 levels were statistically significant, but had no significant effect on IL-1β or TNF-α levels.CONCLUSION: This study shows that the administration of losartan in COVID-19 may not be harmful, but instead has a protective effect similar to that of hrsACE2 in preventing a cytokine storm, especially IL-6.KEYWORDS: obesity, SARS-CoV-2, losartan, IL-6, ACE2

Design, structure and plasma binding of ancestral β-CoV scaffold antigens

We report the application of ancestral sequence reconstruction on coronavirus spike protein, resulting in stable and highly soluble ancestral scaffold antigens (AnSAs). The AnSAs interact with plasma of patients recovered from COVID-19 but do not bind to the human angiotensin-converting enzyme 2 (ACE2) receptor. Cryo-EM analysis of the AnSAs yield high resolution structures (2.6–2.8 Å) indicating a closed pre-fusion conformation in which all three receptor-binding domains (RBDs) are facing downwards. The structures reveal an intricate hydrogen-bonding network mediated by well-resolved loops, both within and across monomers, tethering the N-terminal domain and RBD together. We show that AnSA-5 can induce and boost a broad-spectrum immune response against the wild-type RBD as well as circulating variants of concern in an immune organoid model derived from tonsils. Finally, we highlight how AnSAs are potent scaffolds by replacing the ancestral RBD with the wild-type sequence, which restores ACE2 binding and increases the interaction with convalescent plasma.

Structural basis of the American mink ACE2 binding by Y453F trimeric spike glycoproteins of SARS-CoV-2.

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) enters the host cell by binding to angiotensin-converting enzyme 2 (ACE2). While evolutionarily conserved, ACE2 receptors differ across various species and differential interactions with Spike (S) glycoproteins of SARS-CoV-2 viruses impact species specificity. Reverse zoonoses led to SARS-CoV-2 outbreaks on multiple American mink (Mustela vison) farms during the pandemic and gave rise to mink-associated S substitutions known for transmissibility between mink and zoonotic transmission to humans. In this study, we used bio-layer interferometry (BLI) to discern the differences in binding affinity between multiple human and mink-derived S glycoproteins of SARS-CoV-2 and their respective ACE2 receptors. Further, we conducted a structural analysis of a mink variant S glycoprotein and American mink ACE2 (mvACE2) using cryo-electron microscopy (cryo-EM), revealing four distinct conformations. We discovered a novel intermediary conformation where the mvACE2 receptor is bound to the receptor-binding domain (RBD) of the S glycoprotein in a "down" position, approximately 34° lower than previously reported "up" RBD. Finally, we compared residue interactions in the S-ACE2 complex interface of S glycoprotein conformations with varying RBD orientations. These findings provide valuable insights into the molecular mechanisms of SARS-CoV-2 entry.

Effects of spike proteins on angiotensin converting enzyme 2 (ACE2)

The Coronavirus Disease 2019 (COVID-19) pandemic was caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), which enters host cells through interactions of its spike protein to Angiotensin-Converting Enzyme 2 (ACE2). ACE2 is a peptidase that cleaves Angiotensin II, a critical pathological mediator. This study investigated if the spike protein binding to ACE2 compromises its peptidase activity. Spike/ACE2 Binding Assays suggested that spike proteins of SARS-CoV-2, SARS-CoV and MERS-CoV, but not HKU1, bind to ACE2. S1 and receptor-binding domain (RBD), but not S2, extracellular domain (ECD) or CendR domain, bind to ACE2. While glycosylated spike proteins prepared in HEK293 cells bind to ACE2, non-glycosylated proteins produced in E. coli do not. Cysteine residues of the spike protein expressed in HEK293 cells are fully oxidized, while those of the protein expressed in E. coli are reduced. The deglycosylation of HEK cell-produced protein attenuates the ACE2 binding, while the oxidation of the E. coli protein does not promote the binding. The S1 protein of SARS-CoV-2 enhances the ACE2 peptidase activity, while SARS-CoV, MERS-CoV or HKU1 does not. The ACE2 activity is enhanced by RBD, but not ECD or CendR. In contrast to distinct ACE2 binding capacities of proteins expressed in HEK293 cells and in E. coli, spike proteins expressed in both systems enhance the ACE2 activity. Thus, the spike protein of SARS-CoV-2, but not other coronaviruses, enhances the ACE2 peptidase activity through its RBD in a glycosylation-independent manner.

Primary Exposure to SARS-CoV-2 via Infection or Vaccination Determines Mucosal Antibody-Dependent ACE2 Binding Inhibition.

Mucosal antibodies play a critical role in preventing SARS-CoV-2 infections or reinfections by blocking the interaction of the receptor-binding domain (RBD) with the angiotensin-converting enzyme 2 (ACE2) receptor on the cell surface. In this study, we investigated the difference between the mucosal antibody response after primary infection and vaccination. We assessed longitudinal changes in the quantity and capacity of nasal antibodies to neutralize the interaction of RBD with the ACE2 receptor using the spike protein and RBD from ancestral SARS-CoV-2 (Wuhan-Hu-1), as well as the RBD from the Delta and Omicron variants. Significantly higher mucosal IgA concentrations were detected postinfection vs postvaccination, while vaccination induced higher IgG concentrations. However, ACE2-inhibiting activity did not differ between the cohorts. Regarding whether IgA or IgG drove ACE2 inhibition, infection-induced binding inhibition was driven by both isotypes, while postvaccination binding inhibition was mainly driven by IgG. Our study provides new insights into the relationship between antibody isotypes and neutralization by using a sensitive and high-throughput ACE2 binding inhibition assay. Key differences are highlighted between vaccination and infection at the mucosal level, showing that despite differences in the response quantity, postinfection and postvaccination ACE2 binding inhibition capacity did not differ.

Structural basis of white-tailed deer, Odocoileus virginianus, ACE2 recognizing all the SARS-CoV-2 variants of concern with high affinity.

SARS-CoV-2 has been expanding its host range, among which the white-tailed deer (WTD), Odocoileus virginianus, became the first wildlife species infected on a large scale and might serve as a host reservoir for variants of concern (VOCs) in case no longer circulating in humans. In this study, we comprehensively assessed the binding of the WTD angiotensin-converting enzyme 2 (ACE2) receptor to the spike (S) receptor-binding domains (RBDs) from the SARS-CoV-2 prototype (PT) strain and multiple variants. We found that WTD ACE2 could be broadly recognized by all of the tested RBDs. We further determined the complex structures of WTD ACE2 with PT, Omicron BA.1, and BA.4/5 S trimer. Detailed structural comparison revealed the important roles of RBD residues on 486, 498, and 501 sites for WTD ACE2 binding. This study deepens our understanding of the interspecies transmission mechanisms of SARS-CoV-2 and further addresses the importance of constant monitoring on SARS-CoV-2 infections in wild animals. IMPORTANCE Even if we manage to eliminate the virus among humans, it will still circulate among wildlife and continuously be transmitted back to humans. A recent study indicated that WTD may serve as reservoir for nearly extinct SARS-CoV-2 strains. Therefore, it is critical to evaluate the binding abilities of SARS-CoV-2 variants to the WTD ACE2 receptor and elucidate the molecular mechanisms of binding of the RBDs to assess the risk of spillback events.

Aptamer-, heparin- or cocktail-based inhibition of S1-ACE2 protein complexes

The Spike protein (S1) from the Severe acute respiratory syndrome 2 virus binds to angiotensin converting enzyme 2 (ACE2) receptor to initiate infection. Hence, antiviral therapeutic targeting the S1-ACE2 interface is of interest. Herein, we compare the inhibition efficacy of an aptamer to heparin or their cocktail, against wild-type (WT), Omicron, Delta, and Lambda S1-ACE2 complexes. The aptamer-protein complexes had the dissociation constant KD values in the 2–13 nM range. The aptamer half-maximal inhibitory concentration against WT S1-ACE was 17 nM, with the % inhibition in the 12–35% range. Several aptamer-S1 protein complexes were also stable at low pH with 60% inhibition. Despite the similarity in S1 sequences, the extent of inhibition (2–27%) with heparin was highly dependent on the type of S1 protein. More importantly, heparin did not inhibit the WT S1-ACE2 complex but was effective with mutants. The aptamer-heparin cocktail was less effective compared to aptamer or heparin, individually. Modelling data show that either a direct or proximal binding to RBD sites by aptamer or heparin prevents the ACE2 binding. Overall, heparin was as an effective inhibitor as aptamer against certain variants, and represents the more cost-effective neutralizing agent against emerging coronaviruses.