Molecular mimicry in multisystem inflammatory syndrome in children.

Multisystem inflammatory syndrome in children (MIS-C) is a severe, post-infectious sequela of SARS-CoV-2 infection1,2, yet the pathophysiological mechanism connecting the infection to the broad inflammatory syndrome remains unknown. Here we leveraged a large set of samples from patients with MIS-C to identify a distinct set of host proteins targeted by patient autoantibodies including a particular autoreactive epitope within SNX8, a protein involved in regulating an antiviral pathway associated with MIS-C pathogenesis. In parallel, we also probed antibody responses from patients with MIS-C to the complete SARS-CoV-2 proteome and found enriched reactivity against a distinct domain of the SARS-CoV-2 nucleocapsid protein. The immunogenic regions of the viral nucleocapsid and host SNX8 proteins bear remarkable sequence similarity. Consequently, we found that many children with anti-SNX8 autoantibodies also have cross-reactive T cells engaging both the SNX8 and the SARS-CoV-2 nucleocapsid protein epitopes. Together, these findings suggest that patients with MIS-C develop a characteristic immune response to the SARS-CoV-2 nucleocapsid protein that is associated with cross-reactivity to the self-protein SNX8, demonstrating a mechanistic link between the infection and the inflammatory syndrome, with implications for better understanding a range of post-infectious autoinflammatory diseases.

Enhanced native chemical ligation by peptide conjugation in trifluoroacetic acid.

Chemical ligation of peptides is increasingly used to generate proteins not readily accessible by recombinant approaches. However, a robust method to ligate "difficult" peptides remains to be developed. Here, we report an enhanced native chemical ligation strategy mediated by peptide conjugation in trifluoroacetic acid (TFA). The conjugation between a carboxyl-terminal peptide thiosalicylaldehyde thioester and a 1,3-dithiol-containing peptide in TFA proceeds rapidly to form a thioacetal-linked intermediate, which is readily converted into the desired native amide bond product through simple postligation treatment. The effectiveness and practicality of the method was demonstrated by the successful synthesis of several challenging proteins, including the SARS-CoV-2 transmembrane Envelope (E) protein and nanobodies. Because of the ability of TFA to dissolve virtually all peptides and prevent the formation of unreactive peptide structures, the method is expected to open new opportunities for synthesizing all families of proteins, particularly those with aggregable or colloidal peptide segments.

Adsorption-Driven Deformation and Footprints of the RBD Proteins in SARS-CoV-2 Variants on Biological and Inanimate Surfaces.

Respiratory viruses, carried through airborne microdroplets, frequently adhere to surfaces, including plastics and metals. However, our understanding of the interactions between viruses and materials remains limited, particularly in scenarios involving polarizable surfaces. Here, we investigate the role of the receptor-binding domain (RBD) of the spike protein mutations on the adsorption of SARS-CoV-2 to hydrophobic and hydrophilic surfaces employing molecular simulations. To contextualize our findings, we contrast the interactions on inanimate surfaces with those on native biological interfaces, specifically the angiotensin-converting enzyme 2. Notably, we identify a 2-fold increase in structural deformations for the protein's receptor binding motif (RBM) onto inanimate surfaces, indicative of enhanced shock-absorbing mechanisms. Furthermore, the distribution of adsorbed amino acids (landing footprints) on the inanimate surface reveals a distinct regional asymmetry relative to the biological interface, with roughly half of the adsorbed amino acids arranged in opposite sites. In spite of the H-bonds formed at the hydrophilic substrate, the simulations consistently show a higher number of contacts and interfacial area with the hydrophobic surface, where the wild-type RBD adsorbs more strongly than the Delta or Omicron RBDs. In contrast, the adsorption of Delta and Omicron to hydrophilic surfaces was characterized by a distinctive hopping-pattern. The novel shock-absorbing mechanisms identified in the virus adsorption on inanimate surfaces show the embedded high-deformation capacity of the RBD without losing its secondary structure, which could lead to current experimental strategies in the design of virucidal surfaces.

Assessing pH-Dependent Conformational Changes in the Fusion Peptide Proximal Region of the SARS-CoV-2 Spike Glycoprotein.

One of the entry mechanisms of the SARS-CoV-2 coronavirus into host cells involves endosomal acidification. It has been proposed that under acidic conditions, the fusion peptide proximal region (FPPR) of the SARS-CoV-2 spike glycoprotein acts as a pH-dependent switch, modulating immune response accessibility by influencing the positioning of the receptor binding domain (RBD). This would provide indirect coupling of RBD opening to the environmental pH. Here, we explored this possible pH-dependent conformational equilibrium of the FPPR within the SARS-CoV-2 spike glycoprotein. We analyzed hundreds of experimentally determined spike structures from the Protein Data Bank and carried out pH-replica exchange molecular dynamics to explore the extent to which the FPPR conformation depends on pH and the positioning of the RBD. A meta-analysis of experimental structures identified alternate conformations of the FPPR among structures in which this flexible regions was resolved. However, the results did not support a correlation between the FPPR conformation and either RBD position or the reported pH of the cryo-EM experiment. We calculated pKa values for titratable side chains in the FPPR region using PDB structures, but these pKa values showed large differences between alternate PDB structures that otherwise adopt the same FPPR conformation type. This hampers the comparison of pKa values in different FPPR conformations to rationalize a pH-dependent conformational change. We supplemented these PDB-based analyses with all-atom simulations and used constant-pH replica exchange molecular dynamics to estimate pKa values in the context of flexibility and explicit water. The resulting titration curves show good reproducibility between simulations, but they also suggest that the titration curves of the different FPPR conformations are the same within the error bars. In summary, we were unable to find evidence supporting the previously published hypothesis of an FPPR pH-dependent equilibrium: neither from existing experimental data nor from constant-pH MD simulations. The study underscores the complexity of the spike system and opens avenues for further exploration into the interplay between pH and SARS-CoV-2 viral entry mechanisms.

Cryo2RT: a high-throughput method for room-temperature macromolecular crystallography from cryo-cooled crystals.

Advances in structural biology have relied heavily on synchrotron cryo-crystallography and cryogenic electron microscopy to elucidate biological processes and for drug discovery. However, disparities between cryogenic and room-temperature (RT) crystal structures pose challenges. Here, Cryo2RT, a high-throughput RT data-collection method from cryo-cooled crystals that leverages the cryo-crystallography workflow, is introduced. Tested on endothiapepsin crystals with four soaked fragments, thaumatin and SARS-CoV-2 3CLpro, Cryo2RT reveals unique ligand-binding poses, offers a comparable throughput to cryo-crystallography and eases the exploration of structural dynamics at various temperatures.

Unnatural lipids for simultaneous mRNA delivery and metabolic cell labeling.

Lipids have demonstrated tremendous promise for mRNA delivery, as evidenced by the success of Covid-19 mRNA vaccines. However, existing lipids are mostly used as delivery vehicles and lack the ability to monitor and further modulate the target cells. Here, for the first time, we report a class of unnatural lipids (azido-DOTAP) that can efficiently deliver mRNAs into cells and meanwhile metabolically label cells with unique chemical tags (e.g., azido groups). The azido tags expressed on the cell membrane enable the monitoring of transfected cells, and can mediate subsequent conjugation of cargos via efficient click chemistry for further modulation of transfected cells. We further demonstrate that the dual-functional unnatural lipid is applicable to different types of cells including dendritic cells, the prominent type of antigen presenting cells, potentially opening a new avenue to developing enhanced mRNA vaccines.

Development of a high-performance liquid chromatography using rhodamine B hydrazide as the derivatization reagent for determination of ß propiolactone residues in inactivated COVID-19 vaccines.

ß-propiolactone (BPL) is an alkylating agent used for inactivation of biological samples such as vaccines. Due to its known carcinogenic properties, complete hydrolysis of BPL is essential, and the detection of trace amounts is crucial. In this study a novel High-Performance Liquid Chromatography-Ultraviolet (HPLC-UV) method was developed. Rhodamine B hydrazide (RBH) was synthesized and utilized as a derivatizing reagent to react with BPL. The reaction was optimized in a weak acidic solution, resulting in a high yield. The separation of the RBH-derivatized BPL was achieved on a C8 column and detected by a UV detector at a wavelength of 560 nm. The method's validation demonstrated a high linearity (r2 > 0.99) over a concentration range of 0.5-50 µg/mL, with detection and quantification limits of 0.17 µg/mL and 0.5 µg/mL, respectively. The average recovery of samples was 85.20 % with a relative standard deviation (RSD) of 1.75 %. This method was successfully applied for BPL residue analysis in inactivated COVID-19 vaccines. This novel derivatization method offers a promising solution for monitoring BPL residues in the vaccine production process for quality control purposes and compliance with regulatory standards.

Complete combinatorial mutational enumeration of a protein functional site enables sequence-landscape mapping and identifies highly-mutated variants that retain activity.

Understanding how proteins evolve under selective pressure is a longstanding challenge. The immensity of the search space has limited efforts to systematically evaluate the impact of multiple simultaneous mutations, so mutations have typically been assessed individually. However, epistasis, or the way in which mutations interact, prevents accurate prediction of combinatorial mutations based on measurements of individual mutations. Here, we use artificial intelligence to define the entire functional sequence landscape of a protein binding site in silico, and we call this approach Complete Combinatorial Mutational Enumeration (CCME). By leveraging CCME, we are able to construct a comprehensive map of the evolutionary connectivity within this functional sequence landscape. As a proof of concept, we applied CCME to the ACE2 binding site of the SARS-CoV-2 spike protein receptor binding domain. We selected representative variants from across the functional sequence landscape for testing in the laboratory. We identified variants that retained functionality to bind ACE2 despite changing over 40% of evaluated residue positions, and the variants now escape binding and neutralization by monoclonal antibodies. This work represents a crucial initial stride toward achieving precise predictions of pathogen evolution, opening avenues for proactive mitigation.

Light-Induced Transformation of Virus-Like Particles on TiO2.

Titanium dioxide (TiO2) shows significant potential as a self-cleaning material to inactivate severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and prevent virus transmission. This study provides insights into the impact of UV-A light on the photocatalytic inactivation of adsorbed SARS-CoV-2 virus-like particles (VLPs) on a TiO2 surface at the molecular and atomic levels. X-ray photoelectron spectroscopy, combined with density functional theory calculations, reveals that spike proteins can adsorb on TiO2 predominantly via their amine and amide functional groups in their amino acids blocks. We employ atomic force microscopy and grazing-incidence small-angle X-ray scattering (GISAXS) to investigate the molecular-scale morphological changes during the inactivation of VLPs on TiO2 under light irradiation. Notably, in situ measurements reveal photoinduced morphological changes of VLPs, resulting in increased particle diameters. These results suggest that the denaturation of structural proteins induced by UV irradiation and oxidation of the virus structure through photocatalytic reactions can take place on the TiO2 surface. The in situ GISAXS measurements under an N2 atmosphere reveal that the virus morphology remains intact under UV light. This provides evidence that the presence of both oxygen and UV light is necessary to initiate photocatalytic reactions on the surface and subsequently inactivate the adsorbed viruses. The chemical insights into the virus inactivation process obtained in this study contribute significantly to the development of solid materials for the inactivation of enveloped viruses.

Binding of SARS-CoV-2 Nonstructural Protein 1 to 40S Ribosome Inhibits mRNA Translation.

Experimental evidence has established that SARS-CoV-2 NSP1 acts as a factor that restricts cellular gene expression and impedes mRNA translation within the ribosome's 40S subunit. However, the precise molecular mechanisms underlying this phenomenon have remained elusive. To elucidate this issue, we employed a combination of all-atom steered molecular dynamics and coarse-grained alchemical simulations to explore the binding affinity of mRNA to the 40S ribosome, both in the presence and absence of SARS-CoV-2 NSP1. Our investigations revealed that the binding of SARS-CoV-2 NSP1 to the 40S ribosome leads to a significant enhancement in the binding affinity of mRNA. This observation, which aligns with experimental findings, strongly suggests that SARS-CoV-2 NSP1 has the capability to inhibit mRNA translation. Furthermore, we identified electrostatic interactions between mRNA and the 40S ribosome as the primary driving force behind mRNA translation. Notably, water molecules were found to play a pivotal role in stabilizing the mRNA-40S ribosome complex, underscoring their significance in this process. We successfully pinpointed the specific SARS-CoV-2 NSP1 residues that play a critical role in triggering the translation arrest.

Neuropilin-1 Protein May Serve as a Receptor for SARS-CoV-2 Infection: Evidence from Molecular Dynamics Simulations.

The binding of the virus to host cells is the first step in viral infection. Human cell angiotensin converting enzyme 2 (ACE2) is the most popular receptor for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), while other receptors have recently been observed in experiments. Neuropilin-1 protein (NRP1) is one of them, but the mechanism of its binding to the wild type (WT) and different variants of the virus remain unclear at the atomic level. In this work, all-atom umbrella sampling simulations were performed to clarify the binding mechanism of NRP1 to the spike protein fragments 679-685 of the WT, Delta, and Omicron BA.1 variants. We found that the Delta variant binds most strongly to NRP1, while the affinity for Omicron BA.1 slightly decreases for NRP1 compared to that of WT, and the van der Waals interaction plays a key role in stabilizing the studied complexes. The change in the protonation state of the His amino acid results in different binding free energies between variants. Consistent with the experiment, decreasing the pH was shown to increase the binding affinity of the virus to NRP1. Our results indicate that Delta and Omicron mutations not only affect fusogenicity but also affect NRP1 binding. In addition, we argue that viral evolution does not further improve NRP1 binding affinity which remains in the µM range but may increase immune evasion.

Structural basis for mouse receptor recognition by bat SARS2-like coronaviruses.

The animal origin of SARS-CoV-2 remains elusive, lacking a plausible evolutionary narrative that may account for its emergence. Its spike protein resembles certain segments of BANAL-236 and RaTG13, two bat coronaviruses considered possible progenitors of SARS-CoV-2. Additionally, its spike contains a furin motif, a common feature of rodent coronaviruses. To explore the possible involvement of rodents in the emergence of SARS-CoV-2 spike, we examined the crystal structures of the spike receptor-binding domains (RBDs) of BANAL-236 and RaTG13 each complexed with mouse receptor ACE2. Both RBDs have residues at positions 493 and 498 that align well with two virus-binding hotspots on mouse ACE2. Our biochemical evidence supports that both BANAL-236 and RaTG13 spikes can use mouse ACE2 as their entry receptor. These findings point to a scenario in which these bat coronaviruses may have coinfected rodents, leading to a recombination of their spike genes and a subsequent acquisition of a furin motif in rodents, culminating in the emergence of SARS-CoV-2.

Competitive Interaction of the SGFRKMAF Peptide with 3CLpro Dimerization Intermediates: A Brownian Dynamics Investigation.

The SGFRKMAF peptide is known to inhibit the dimerization of 3CLpro monomers, which is essential for SARS-CoV-2 replication. The mechanism behind this, however, is largely unknown. In this work, we used Brownian dynamics simulations to compare and contrast 3CLpro monomer-monomer interactions and 3CLpro monomer-SGFRKMAF peptide interactions. We found that formation of the 3CLpro wild-type dimer could potentially involve formation of three intermediates that are primarily stabilized by G11-G124, S1-S301, and T118-G278 interactions. Analysis of 3CLpro monomer interaction with the SGFRKMAF peptide, however, revealed the presence of eight basins of interactions where the peptide assumes the highest local densities at the 3CLPro monomer surface. The second highest-density basin was found to coincide with the interface region of the wild-type 3CLpro dimer, thereby directly blocking the 3CLpro dimer-dimer interactions. The other basins, however, were found to lie far from the interface region. Notably, we found that only 6% of the BD trajectories end up directly into the basin at the interface region and ∼39% of the trajectories end up into those basins lying away from the interface region, indicating a greater role for peptide binding at sites away from the dimer interface region. Importantly, the locations of the basins lying away from the interface were found to coincide with the 3CLpro residues involved in stabilization of the 3CLpro monomer-monomer intermediates. Given that the rate constant of the peptide reaching the monomer surface was found to be almost an order of magnitude higher than the rate constant of monomer-monomer association, the SGFRKMAF peptide has the potential to inhibit dimerization of 3CLpro monomers not only through blocking the interface region but also through blocking the formation of the intermediates involved in the dimerization process. This could potentially open new avenues for 3CLpro dimerization inhibitors that transcend traditional X-ray-based discovery approaches.

A highly specific aptamer for the SARS-CoV-2 spike protein from the authentic strain.

DNA aptamers are oligonucleotides that specifically bind to target molecules, similar to how antibodies bind to antigens. We identified an aptamer named MEZ that is highly specific to the receptor-binding domain, RBD, of the SARS-CoV-2 spike protein from the Wuhan-Hu-1 strain. The SELEX procedure was utilized to enrich the initial 31-mer oligonucleotide library with the target aptamer. The aptamer identification was performed using the novel protocol based on nanopore sequencing developed in this study. The MEZ aptamer was chemically synthesized and tested for binding with the SARS-CoV-2 RBD of the spike protein from different strains. The Kd is 6.5 nM for the complex with the RBD from the Wuhan-Hu-1 strain, which is comparable with known aptamers; the advantage is that the MEZ aptamer is smaller than known analogs. The proposed aptamer is highly selective for the RBD protein from the Wuhan-Hu-1 strain and does not form complexes with the RBD from Beta, Delta and Omicron strains. Experimental and theoretical studies together revealed the molecular mechanism of aptamer binding. The aptamer occupies the same binding site as ACE2 when bound to the RBD. The 3'-end of the MEZ aptamer is important for complex formation and is responsible for the discrimination of the RBD protein from a specific strain. The 5'-end is responsible for the formation of a loop in the 3D structure of the aptamer, which is important for proper binding.

Sensitivity-improved SERS detection of SARS-CoV-2 spike protein by Au NPs/COFs integrated with catalytic-hairpin-assembly amplification technology.

BACKGROUND: The COVID-19 pandemic, caused by the novel coronavirus, has had a profound impact on global health and economies worldwide. This unprecedented crisis has affected individuals, communities, and nations in diverse manners. Developing simple and accurate diagnostic methods is an imperative task for frequent testing to mitigate the spread of the virus. Among these methods, SARS-CoV-2 antigen tests in clinical specimens have emerged as a promising diagnostic method for COVID-19 due to their sensitive and accurate detection of spike (S) protein, which plays a crucial role in viral infection initiation. RESULTS: In this work, a dual-signal amplification surface enhanced Raman scattering (SERS)-based S protein biosensor was constructed based on Au NPs/COFs and enzyme-free catalytic hairpin assembly (CHA) amplification method. The approach relies on a released free DNA sequence (T), which is generated from the competition reaction between Aptamer/T and Aptamer/S protein, to trigger a CHA reaction. Due to the high binding affinity and selectivity between the S protein and its aptamer, CHA process was triggered with the maximum SERS tags (H2-conjugated Au@4-mercaptobenzonitrile@Ag) anchored onto Au NPs/COFs substrate surface. This SERS platform could detect the S protein at concentrations with high sensitivity (limit of detection = 3.0 × 10-16 g/mL), wide detection range (1 × 10-16 to 1 × 10-11 g/mL), acceptable reproducibility (relative standard deviation = 7.01 %) and excellent specificity. The biosensor was also employed to detect S protein in artificial human salivas. SIGNIFICANCE: Thus, this study not only developed a novel Au NPs/COFs substrate exhibiting strong SERS enhancement ability and high reproducibility, but also proposed a promising dual-signal amplification SERS-based diagnostic method for COVID-19, holding immense potential for the detection of a wide range of antigens and infectious diseases in future applications.

SARS-CoV-2 Omicron XBB lineage spike structures, conformations, antigenicity, and receptor recognition.

A recombinant lineage of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Omicron variant, named XBB, appeared in late 2022 and evolved descendants that successively swept local and global populations. XBB lineage members were noted for their improved immune evasion and transmissibility. Here, we determine cryoelectron microscopy (cryo-EM) structures of XBB.1.5, XBB.1.16, EG.5, and EG.5.1 spike (S) ectodomains to reveal reinforced 3-receptor binding domain (RBD)-down receptor-inaccessible closed states mediated by interprotomer RBD interactions previously observed in BA.1 and BA.2. Improved XBB.1.5 and XBB.1.16 RBD stability compensated for stability loss caused by early Omicron mutations, while the F456L substitution reduced EG.5 RBD stability. S1 subunit mutations had long-range impacts on conformation and epitope presentation in the S2 subunit. Our results reveal continued S protein evolution via simultaneous optimization of multiple parameters, including stability, receptor binding, and immune evasion, and the dramatic effects of relatively few residue substitutions in altering the S protein conformational landscape.

High-confidence placement of low-occupancy fragments into electron density using the anomalous signal of sulfur and halogen atoms.

Fragment-based drug design using X-ray crystallography is a powerful technique to enable the development of new lead compounds, or probe molecules, against biological targets. This study addresses the need to determine fragment binding orientations for low-occupancy fragments with incomplete electron density, an essential step before further development of the molecule. Halogen atoms play multiple roles in drug discovery due to their unique combination of electronegativity, steric effects and hydrophobic properties. Fragments incorporating halogen atoms serve as promising starting points in hit-to-lead development as they often establish halogen bonds with target proteins, potentially enhancing binding affinity and selectivity, as well as counteracting drug resistance. Here, the aim was to unambiguously identify the binding orientations of fragment hits for SARS-CoV-2 nonstructural protein 1 (nsp1) which contain a combination of sulfur and/or chlorine, bromine and iodine substituents. The binding orientations of carefully selected nsp1 analogue hits were focused on by employing their anomalous scattering combined with Pan-Dataset Density Analysis (PanDDA). Anomalous difference Fourier maps derived from the diffraction data collected at both standard and long-wavelength X-rays were compared. The discrepancies observed in the maps of iodine-containing fragments collected at different energies were attributed to site-specific radiation-damage stemming from the strong X-ray absorption of I atoms, which is likely to cause cleavage of the C-I bond. A reliable and effective data-collection strategy to unambiguously determine the binding orientations of low-occupancy fragments containing sulfur and/or halogen atoms while mitigating radiation damage is presented.

Development of Robust Freeze-Drying Process for Long-Term Stability of rVSV-SARS-CoV-2 Vaccine.

The thermostability of vaccines, particularly enveloped viral vectored vaccines, remains a challenge to their delivery wherever needed. The freeze-drying of viral vectored vaccines is a promising approach but remains challenging due to the water removal process from the outer and inner parts of the virus. In the case of enveloped viruses, freeze-drying induces increased stress on the envelope, which often leads to the inactivation of the virus. In this study, we designed a method to freeze-dry a recombinant vesicular stomatitis virus (VSV) expressing the SARS-CoV-2 spike glycoprotein. Since the envelope of VSV is composed of 50% lipids and 50% protein, the formulation study focused on both the protein and lipid portions of the vector. Formulations were prepared primarily using sucrose, trehalose, and sorbitol as cryoprotectants; mannitol as a lyoprotectant; and histidine as a buffer. Initially, the infectivity of rVSV-SARS-CoV-2 and the cake stability were investigated at different final moisture content levels. High recovery of the infectious viral titer (~0.5 to 1 log loss) was found at 3-6% moisture content, with no deterioration in the freeze-dried cakes. To further minimize infectious viral titer loss, the composition and concentration of the excipients were studied. An increase from 5 to 10% in both the cryoprotectants and lyoprotectant, together with the addition of 0.5% gelatin, resulted in the improved recovery of the infectious virus titer and stable cake formation. Moreover, the secondary drying temperature of the freeze-drying process showed a significant impact on the infectivity of rVSV-SARS-CoV-2. The infectivity of the vector declined drastically when the temperature was raised above 20 °C. Throughout a long-term stability study, formulations containing 10% sugar (sucrose/trehalose), 10% mannitol, 0.5% gelatin, and 10 mM histidine showed satisfactory stability for six months at 2-8 °C. The development of this freeze-drying process and the optimized formulation minimize the need for a costly cold chain distribution system.

Exploring conformational landscapes and binding mechanisms of convergent evolution for the SARS-CoV-2 spike Omicron variant complexes with the ACE2 receptor using AlphaFold2-based structural ensembles and molecular dynamics simulations.

In this study, we combined AlphaFold-based approaches for atomistic modeling of multiple protein states and microsecond molecular simulations to accurately characterize conformational ensembles evolution and binding mechanisms of convergent evolution for the SARS-CoV-2 spike Omicron variants BA.1, BA.2, BA.2.75, BA.3, BA.4/BA.5 and BQ.1.1. We employed and validated several different adaptations of the AlphaFold methodology for modeling of conformational ensembles including the introduced randomized full sequence scanning for manipulation of sequence variations to systematically explore conformational dynamics of Omicron spike protein complexes with the ACE2 receptor. Microsecond atomistic molecular dynamics (MD) simulations provide a detailed characterization of the conformational landscapes and thermodynamic stability of the Omicron variant complexes. By integrating the predictions of conformational ensembles from different AlphaFold adaptations and applying statistical confidence metrics we can expand characterization of the conformational ensembles and identify functional protein conformations that determine the equilibrium dynamics for the Omicron spike complexes with the ACE2. Conformational ensembles of the Omicron RBD-ACE2 complexes obtained using AlphaFold-based approaches for modeling protein states and MD simulations are employed for accurate comparative prediction of the binding energetics revealing an excellent agreement with the experimental data. In particular, the results demonstrated that AlphaFold-generated extended conformational ensembles can produce accurate binding energies for the Omicron RBD-ACE2 complexes. The results of this study suggested complementarities and potential synergies between AlphaFold predictions of protein conformational ensembles and MD simulations showing that integrating information from both methods can potentially yield a more adequate characterization of the conformational landscapes for the Omicron RBD-ACE2 complexes. This study provides insights in the interplay between conformational dynamics and binding, showing that evolution of Omicron variants through acquisition of convergent mutational sites may leverage conformational adaptability and dynamic couplings between key binding energy hotspots to optimize ACE2 binding affinity and enable immune evasion.

Molecular interactions of the Omicron, Kappa, and Delta SARS-CoV-2 spike proteins with quantum dots of graphene oxide.

CONTEXT: The Omicron, Kappa, and Delta variants are different strains of the SARS-CoV-2 virus. Graphene oxide quantum dots (GOQDs) represent a burgeoning class of oxygen-enriched, zero-dimensional materials characterized by their sub-20-nm dimensions. Exhibiting pronounced quantum confinement and edge effects, GOQDs manifest exceptional physical-chemical attributes. This study delves into the potential of graphene oxide quantum dots, elucidating their inherent properties pertinent to the surface structures of SARS-CoV-2, employing an integrated computational approach for the repositioning of inhibitory agents. METHODS: Following rigorous adjustment tests, a spectrum of divergent bonding conformations emerged, with particular emphasis placed on identifying the conformation exhibiting optimal adjustment scores and interactions. The investigation employed molecular docking simulations integrating affinity energy evaluations, electrostatic potential clouds, molecular dynamics encompassing average square root calculations, and the computation of Gibbs-free energy. These values quantify the strength of interaction between GOQDs and SARS-CoV-2 spike protein variants. The receptor structures were optimized using the CHARM-GUI server employing force field AMBERFF14SB. The algorithm embedded in CHARMM offers an efficient interpolation scheme and automatic step size selection, enhancing the efficiency of the optimization process. The 3D structures of the ligands are constructed and optimized with density functional theory (DFT) method based on the most stable conformer of each binder. Autodock Vina Software (ADV) was utilized, where essential parameters were specified. Electrostatic potential maps (MEPs) provide a visual depiction of molecules' charge distributions and related properties. After this, molecular dynamics simulations employing the CHARM36 force field in Gromacs 2022.2 were conducted to investigate GOs' interactions with surface macromolecules of SARS-CoV-2 in an explicit aqueous environment. Furthermore, our investigation suggests that lower values indicate stronger binding. Notably, GO-E consistently showed the most negative values across interactions with different variants, suggesting a higher affinity compared to other GOQDs (GO-A to GO-D).