Cryo-EM structure of the SARS-CoV-2 Omicron spike.

The recently reported B.1.1.529 Omicron variant of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) includes 34 mutations in the spike protein relative to the Wuhan strain, including 15 mutations in the receptor-binding domain (RBD). Functional studies have shown Omicron to substantially escape the activity of many SARS-CoV-2-neutralizing antibodies. Here, we report a 3.1 Å-resolution cryoelectron microscopy (cryo-EM) structure of the Omicron spike protein ectodomain. The structure depicts a spike that is exclusively in the 1-RBD-up conformation with high mobility of RBD. Many mutations cause steric clashes and/or altered interactions at antibody-binding surfaces, whereas others mediate changes of the spike structure in local regions to interfere with antibody recognition. Overall, the structure of the Omicron spike reveals how mutations alter its conformation and explains its extraordinary ability to evade neutralizing antibodies.

Structural and functional characterizations of infectivity and immune evasion of SARS-CoV-2 Omicron.

The SARS-CoV-2 Omicron variant with increased fitness is spreading rapidly worldwide. Analysis of cryo-EM structures of the spike (S) from Omicron reveals amino acid substitutions forging interactions that stably maintain an active conformation for receptor recognition. The relatively more compact domain organization confers improved stability and enhances attachment but compromises the efficiency of the viral fusion step. Alterations in local conformation, charge, and hydrophobic microenvironments underpin the modulation of the epitopes such that they are not recognized by most NTD- and RBD-antibodies, facilitating viral immune escape. Structure of the Omicron S bound with human ACE2, together with the analysis of sequence conservation in ACE2 binding region of 25 sarbecovirus members, as well as heatmaps of the immunogenic sites and their corresponding mutational frequencies, sheds light on conserved and structurally restrained regions that can be used for the development of broad-spectrum vaccines and therapeutics.

Structural mechanism of SARS-CoV-2 neutralization by two murine antibodies targeting the RBD.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has necessitated the rapid development of antibody-based therapies and vaccines as countermeasures. Here, we use cryoelectron microscopy (cryo-EM) to characterize two protective anti-SARS-CoV-2 murine monoclonal antibodies (mAbs) in complex with the spike protein, revealing similarities between epitopes targeted by human and murine B cells. The more neutralizing mAb, 2B04, binds the receptor-binding motif (RBM) of the receptor-binding domain (RBD) and competes with angiotensin-converting enzyme 2 (ACE2). By contrast, 2H04 binds adjacent to the RBM and does not compete for ACE2 binding. Naturally occurring sequence variants of SARS-CoV-2 and corresponding neutralization escape variants selected in vitro map to our structurally defined epitopes, suggesting that SARS-CoV-2 might evade therapeutic antibodies with a limited set of mutations, underscoring the importance of combination mAb therapeutics. Finally, we show that 2B04 neutralizes SARS-CoV-2 infection by preventing ACE2 engagement, whereas 2H04 reduces host cell attachment without directly disrupting ACE2-RBM interactions, providing distinct inhibitory mechanisms used by RBD-specific mAbs.

D614G mutation in the SARS-CoV-2 spike protein enhances viral fitness by desensitizing it to temperature-dependent denaturation.

The D614G mutation in the spike protein of SARS-CoV-2 alters the fitness of the virus, leading to the dominant form observed in the COVID-19 pandemic. However, the molecular basis of the mechanism by which this mutation enhances fitness is not clear. Here we demonstrated by cryo-electron microscopy that the D614G mutation resulted in increased propensity of multiple receptor-binding domains (RBDs) in an upward conformation poised for host receptor binding. Multiple substates within the one RBD-up or two RBD-up conformational space were determined. According to negative staining electron microscopy, differential scanning calorimetry, and differential scanning fluorimetry, the most significant impact of the mutation lies in its ability to eliminate the unusual cold-induced unfolding characteristics and to significantly increase the thermal stability under physiological pH. The D614G spike variant also exhibited exceptional long-term stability when stored at 37 °C for up to 2 months. Our findings shed light on how the D614G mutation enhances the infectivity of SARS-CoV-2 through a stabilizing mutation and suggest an approach for better design of spike protein-based conjugates for vaccine development.

An atomistic model of the coronavirus replication-transcription complex as a hexamer assembled around nsp15.

The SARS-CoV-2 replication-transcription complex is an assembly of nonstructural viral proteins that collectively act to reproduce the viral genome and generate mRNA transcripts. While the structures of the individual proteins involved are known, how they assemble into a functioning superstructure is not. Applying molecular modeling tools, including protein-protein docking, to the available structures of nsp7-nsp16 and the nucleocapsid, we have constructed an atomistic model of how these proteins associate. Our principal finding is that the complex is hexameric, centered on nsp15. The nsp15 hexamer is capped on two faces by trimers of nsp14/nsp16/(nsp10)2, which then recruit six nsp12/nsp7/(nsp8)2 polymerase subunits to the complex. To this, six subunits of nsp13 are arranged around the superstructure, but not evenly distributed. Polymerase subunits that coordinate dimers of nsp13 are capable of binding the nucleocapsid, which positions the 5'-UTR TRS-L RNA over the polymerase active site, a state distinguishing transcription from replication. Analysis of the viral RNA path through the complex indicates the dsRNA that exits the polymerase passes over the nsp14 exonuclease and nsp15 endonuclease sites before being unwound by a convergence of zinc fingers from nsp10 and nsp14. The template strand is then directed away from the complex, while the nascent strand is directed to the sites responsible for mRNA capping. The model presents a cohesive picture of the multiple functions of the coronavirus replication-transcription complex and addresses fundamental questions related to proofreading, template switching, mRNA capping, and the role of the endonuclease.

ZoomQA: residue-level protein model accuracy estimation with machine learning on sequential and 3D structural features.

MOTIVATION: The Estimation of Model Accuracy problem is a cornerstone problem in the field of Bioinformatics. As of CASP14, there are 79 global QA methods, and a minority of 39 residue-level QA methods with very few of them working on protein complexes. Here, we introduce ZoomQA, a novel, single-model method for assessing the accuracy of a tertiary protein structure/complex prediction at residue level, which have many applications such as drug discovery. ZoomQA differs from others by considering the change in chemical and physical features of a fragment structure (a portion of a protein within a radius $r$ of the target amino acid) as the radius of contact increases. Fourteen physical and chemical properties of amino acids are used to build a comprehensive representation of every residue within a protein and grade their placement within the protein as a whole. Moreover, we have shown the potential of ZoomQA to identify problematic regions of the SARS-CoV-2 protein complex. RESULTS: We benchmark ZoomQA on CASP14, and it outperforms other state-of-the-art local QA methods and rivals state of the art QA methods in global prediction metrics. Our experiment shows the efficacy of these new features and shows that our method is able to match the performance of other state-of-the-art methods without the use of homology searching against databases or PSSM matrices. AVAILABILITY: http://zoomQA.renzhitech.com.

Structures of signaling complexes of lipid receptors S1PR1 and S1PR5 reveal mechanisms of activation and drug recognition.

Sphingosine-1-phosphate (S1P) is an important bioactive lipid molecule in cell membrane metabolism and binds to G protein-coupled S1P receptors (S1PRs) to regulate embryonic development, physiological homeostasis, and pathogenic processes in various organs. S1PRs are lipid-sensing receptors and are therapeutic targets for drug development, including potential treatment of COVID-19. Herein, we present five cryo-electron microscopy structures of S1PRs bound to diverse drug agonists and the heterotrimeric Gi protein. Our structural and functional assays demonstrate the different binding modes of chemically distinct agonists of S1PRs, reveal the mechanical switch that activates these receptors, and provide a framework for understanding ligand selectivity and G protein coupling.

Cryo-EM analysis of the HCoV-229E spike glycoprotein reveals dynamic prefusion conformational changes.

Coronaviruses spike (S) glycoproteins mediate viral entry into host cells by binding to host receptors. However, how the S1 subunit undergoes conformational changes for receptor recognition has not been elucidated in Alphacoronavirus. Here, we report the cryo-EM structures of the HCoV-229E S trimer in prefusion state with two conformations. The activated conformation may pose the potential exposure of the S1-RBDs by decreasing of the interaction area between the S1-RBDs and the surrounding S1-NTDs and S1-RBDs compared to the closed conformation. Furthermore, structural comparison of our structures with the previously reported HCoV-229E S structure showed that the S trimers trended to open the S2 subunit from the closed conformation to open conformation, which could promote the transition from pre- to postfusion. Our results provide insights into the mechanisms involved in S glycoprotein-mediated Alphacoronavirus entry and have implications for vaccine and therapeutic antibody design.

A Trimeric Hydrophobic Zipper Mediates the Intramembrane Assembly of SARS-CoV-2 Spike.

The S protein of SARS-CoV-2 is a type I membrane protein that mediates membrane fusion and viral entry. A vast amount of structural information is available for the ectodomain of S, a primary target by the host immune system, but much less is known regarding its transmembrane domain (TMD) and its membrane-proximal regions. Here, we determined the NMR structure of the S protein TMD in bicelles that closely mimic a lipid bilayer. The TMD structure is a transmembrane α-helix (TMH) trimer that assembles spontaneously in a membrane. The trimer structure shows an extensive hydrophobic core along the 3-fold axis that resembles that of a trimeric leucine/isoleucine zipper, but with tetrad, not heptad, repeats. The trimeric core is strong in bicelles, resisting hydrogen-deuterium exchange for weeks. Although highly stable, structural guided mutagenesis identified single mutations that can completely dissociate the TMD trimer. Multiple studies have shown that the membrane anchors of viral fusion proteins can form highly specific oligomers, but the exact function of these oligomers remains unclear. Our findings should guide future experiments to address the above question for SARS coronaviruses.

Molecular Simulations suggest Vitamins, Retinoids and Steroids as Ligands of the Free Fatty Acid Pocket of the SARS-CoV-2 Spike Protein*.

We investigate binding of linoleate and other potential ligands to the recently discovered fatty acid binding site in the SARS-CoV-2 spike protein, using docking and molecular dynamics simulations. Simulations suggest that linoleate and dexamethasone stabilize the locked spike conformation, thus reducing the opportunity for ACE2 interaction. In contrast, cholesterol may expose the receptor-binding domain by destabilizing the closed structure, preferentially binding to a different site in the hinge region of the open structure. We docked a library of FDA-approved drugs to the fatty acid site using an approach that reproduces the structure of the linoleate complex. Docking identifies steroids (including dexamethasone and vitamin D); retinoids (some known to be active in vitro, and vitamin A); and vitamin K as potential ligands that may stabilize the closed conformation. The SARS-CoV-2 spike fatty acid site may bind a diverse array of ligands, including dietary components, and therefore provides a promising target for therapeutics or prophylaxis.

Impact of temperature on the affinity of SARS-CoV-2 Spike glycoprotein for host ACE2.

The seasonal nature of outbreaks of respiratory viral infections with increased transmission during low temperatures has been well established. Accordingly, temperature has been suggested to play a role on the viability and transmissibility of SARS-CoV-2, the virus responsible for the COVID-19 pandemic. The receptor-binding domain (RBD) of the Spike glycoprotein is known to bind to its host receptor angiotensin-converting enzyme 2 (ACE2) to initiate viral fusion. Using biochemical, biophysical, and functional assays to dissect the effect of temperature on the receptor-Spike interaction, we observed a significant and stepwise increase in RBD-ACE2 affinity at low temperatures, resulting in slower dissociation kinetics. This translated into enhanced interaction of the full Spike glycoprotein with the ACE2 receptor and higher viral attachment at low temperatures. Interestingly, the RBD N501Y mutation, present in emerging variants of concern (VOCs) that are fueling the pandemic worldwide (including the B.1.1.7 (α) lineage), bypassed this requirement. This data suggests that the acquisition of N501Y reflects an adaptation to warmer climates, a hypothesis that remains to be tested.

Water-Triggered, Irreversible Conformational Change of SARS-CoV-2 Main Protease on Passing from the Solid State to Aqueous Solution.

The main protease from SARS-CoV-2 is a homodimer. Yet, a recent 0.1-ms-long molecular dynamics simulation performed by D. E. Shaw's research group shows that it readily undergoes a symmetry-breaking event on passing from the solid state to aqueous solution. As a result, the subunits present distinct conformations of the binding pocket. By analyzing this long simulation, we uncover a previously unrecognized role of water molecules in triggering the transition. Interestingly, each subunit presents a different collection of long-lived water molecules. Enhanced sampling simulations performed here, along with machine learning approaches, further establish that the transition to the asymmetric state is essentially irreversible.

The method utilized to purify the SARS-CoV-2 N protein can affect its molecular properties.

One of the main structural proteins of Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the nucleocapsid protein (N). The basic function of this protein is to bind genomic RNA and to form a protective nucleocapsid in the mature virion. The intrinsic ability of the N protein to interact with nucleic acids makes its purification very challenging. Therefore, typically employed purification methods appear to be insufficient for removing nucleic acid contamination. In this study, we present a novel purification protocol that enables the N protein to be prepared without any bound nucleic acids. We also performed comparative structural analysis of the N protein contaminated with nucleic acids and free of contamination and showed significant differences in the structural and phase separation properties of the protein. These results indicate that nucleic-acid contamination may severely affect molecular properties of the purified N protein. In addition, the notable ability of the N protein to form condensates whose morphology and behaviour suggest more ordered forms resembling gel-like or solid structures is described.

Reversible Lectin Binding to Glycan-Functionalized Graphene.

The monolayer character of two-dimensional materials predestines them for application as active layers of sensors. However, their inherent high sensitivity is always accompanied by a low selectivity. Chemical functionalization of two-dimensional materials has emerged as a promising way to overcome the selectivity issues. Here, we demonstrate efficient graphene functionalization with carbohydrate ligands-chitooligomers, which bind proteins of the lectin family with high selectivity. Successful grafting of a chitooligomer library was thoroughly characterized, and glycan binding to wheat germ agglutinin was studied by a series of methods. The results demonstrate that the protein quaternary structure remains intact after binding to the functionalized graphene, and that the lectin can be liberated from the surface by the addition of a binding competitor. The chemoenzymatic assay with a horseradish peroxidase conjugate also confirmed the intact catalytic properties of the enzyme. The present approach thus paves the way towards graphene-based sensors for carbohydrate-lectin binding.

Comparative protein structure network analysis on 3CLpro from SARS-CoV-1 and SARS-CoV-2.

The main protease Mpro , 3CLpro is an important target from coronaviruses. In spite of having 96% sequence identity among Mpros from SARS-CoV-1 and SARS-CoV-2; the inhibitors used to block the activity of SARS-CoV-1 Mpro so far, were found to have differential inhibitory effect on Mpro of SARS-CoV-2. The possible reason could be due to the difference of few amino acids among the peptidases. Since, overall 3-D crystallographic structure of Mpro from SARS-CoV-1 and SARS-CoV-2 is quite similar and mapping a subtle structural variation is seemingly impossible. Hence, we have attempted to study a structural comparison of SARS-CoV-1 and SARS-CoV-2 Mpro in apo and inhibitor bound states using protein structure network (PSN) based approach at contacts level. The comparative PSNs analysis of apo Mpros from SARS-CoV-1 and SARS-CoV-2 uncovers small but significant local changes occurring near the active site region and distributed throughout the structure. Additionally, we have shown how inhibitor binding perturbs the PSG and the communication pathways in Mpros . Moreover, we have also investigated the network connectivity on the quaternary structure of Mpro and identified critical residue pairs for complex formation using three centrality measurement parameters along with the modularity analysis. Taken together, these results on the comparative PSN provide an insight into conformational changes that may be used as an additional guidance towards specific drug development.

Effect of natural mutations of SARS-CoV-2 on spike structure, conformation, and antigenicity.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants with multiple spike mutations enable increased transmission and antibody resistance. We combined cryo-electron microscopy (cryo-EM), binding, and computational analyses to study variant spikes, including one that was involved in transmission between minks and humans, and others that originated and spread in human populations. All variants showed increased angiotensin-converting enzyme 2 (ACE2) receptor binding and increased propensity for receptor binding domain (RBD)-up states. While adaptation to mink resulted in spike destabilization, the B.1.1.7 (UK) spike balanced stabilizing and destabilizing mutations. A local destabilizing effect of the RBD E484K mutation was implicated in resistance of the B.1.1.28/P.1 (Brazil) and B.1.351 (South Africa) variants to neutralizing antibodies. Our studies revealed allosteric effects of mutations and mechanistic differences that drive either interspecies transmission or escape from antibody neutralization.

Structural insight into SARS-CoV-2 neutralizing antibodies and modulation of syncytia.

Infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is initiated by binding of the viral Spike protein to host receptor angiotensin-converting enzyme 2 (ACE2), followed by fusion of viral and host membranes. Although antibodies that block this interaction are in emergency use as early coronavirus disease 2019 (COVID-19) therapies, the precise determinants of neutralization potency remain unknown. We discovered a series of antibodies that potently block ACE2 binding but exhibit divergent neutralization efficacy against the live virus. Strikingly, these neutralizing antibodies can inhibit or enhance Spike-mediated membrane fusion and formation of syncytia, which are associated with chronic tissue damage in individuals with COVID-19. As revealed by cryoelectron microscopy, multiple structures of Spike-antibody complexes have distinct binding modes that not only block ACE2 binding but also alter the Spike protein conformational cycle triggered by ACE2 binding. We show that stabilization of different Spike conformations leads to modulation of Spike-mediated membrane fusion with profound implications for COVID-19 pathology and immunity.

Comparative Perturbation-Based Modeling of the SARS-CoV-2 Spike Protein Binding with Host Receptor and Neutralizing Antibodies: Structurally Adaptable Allosteric Communication Hotspots Define Spike Sites Targeted by Global Circulating Mutations.

In this study, we used an integrative computational approach to examine molecular mechanisms and determine functional signatures underlying the role of functional residues in the SARS-CoV-2 spike protein that are targeted by novel mutational variants and antibody-escaping mutations. Atomistic simulations and functional dynamics analysis are combined with alanine scanning and mutational sensitivity profiling of the SARS-CoV-2 spike protein complexes with the ACE2 host receptor and the REGN-COV2 antibody cocktail(REG10987+REG10933). Using alanine scanning and mutational sensitivity analysis, we have shown that K417, E484, and N501 residues correspond to key interacting centers with a significant degree of structural and energetic plasticity that allow mutants in these positions to afford the improved binding affinity with ACE2. Through perturbation-based network modeling and community analysis of the SARS-CoV-2 spike protein complexes with ACE2, we demonstrate that E406, N439, K417, and N501 residues serve as effector centers of allosteric interactions and anchor major intermolecular communities that mediate long-range communication in the complexes. The results provide support to a model according to which mutational variants and antibody-escaping mutations constrained by the requirements for host receptor binding and preservation of stability may preferentially select structurally plastic and energetically adaptable allosteric centers to differentially modulate collective motions and allosteric interactions in the complexes with the ACE2 enzyme and REGN-COV2 antibody combination. This study suggests that the SARS-CoV-2 spike protein may function as a versatile and functionally adaptable allosteric machine that exploits the plasticity of allosteric regulatory centers to fine-tune response to antibody binding without compromising the activity of the spike protein.