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1.
Proc Natl Acad Sci U S A ; 119(34): e2204256119, 2022 Aug 23.
Article in English | MEDLINE | ID: covidwho-1991767

ABSTRACT

Antibody therapeutics for the treatment of COVID-19 have been highly successful. However, the recent emergence of the Omicron variant has posed a challenge, as it evades detection by most existing SARS-CoV-2 neutralizing antibodies (nAbs). Here, we successfully generated a panel of SARS-CoV-2/SARS-CoV cross-neutralizing antibodies by sequential immunization of the two pseudoviruses. Of the potential candidates, we found that nAbs X01, X10, and X17 offer broad neutralizing potential against most variants of concern, with X17 further identified as a Class 5 nAb with undiminished neutralization against the Omicron variant. Cryo-electron microscopy structures of the three antibodies together in complex with each of the spike proteins of the prototypical SARS-CoV, SARS-CoV-2, and Delta and Omicron variants of SARS-CoV-2 defined three nonoverlapping conserved epitopes on the receptor-binding domain. The triple-antibody mixture exhibited enhanced resistance to viral evasion and effective protection against infection of the Beta variant in hamsters. Our findings will aid the development of antibody therapeutics and broad vaccines against SARS-CoV-2 and its emerging variants.


Subject(s)
COVID-19 , SARS Virus , Antibodies, Neutralizing , Antibodies, Viral , COVID-19 Vaccines , Cryoelectron Microscopy , Epitopes , Humans , Neutralization Tests , SARS-CoV-2/genetics , Spike Glycoprotein, Coronavirus/genetics
2.
Proc Natl Acad Sci U S A ; 119(33): e2208144119, 2022 Aug 16.
Article in English | MEDLINE | ID: covidwho-1984601

ABSTRACT

Pattern recognition molecules (PRMs) form an important part of innate immunity, where they facilitate the response to infections and damage by triggering processes such as inflammation. The pentraxin family of soluble PRMs comprises long and short pentraxins, with the former containing unique N-terminal regions unrelated to other proteins or each other. No complete high-resolution structural information exists about long pentraxins, unlike the short pentraxins, where there is an abundance of both X-ray and cryoelectron microscopy (cryo-EM)-derived structures. This study presents a high-resolution structure of the prototypical long pentraxin, PTX3. Cryo-EM yielded a 2.5-Å map of the C-terminal pentraxin domains that revealed a radically different quaternary structure compared to other pentraxins, comprising a glycosylated D4 symmetrical octameric complex stabilized by an extensive disulfide network. The cryo-EM map indicated α-helices that extended N terminal of the pentraxin domains that were not fully resolved. AlphaFold was used to predict the remaining N-terminal structure of the octameric PTX3 complex, revealing two long tetrameric coiled coils with two hinge regions, which was validated using classification of cryo-EM two-dimensional averages. The resulting hybrid cryo-EM/AlphaFold structure allowed mapping of ligand binding sites, such as C1q and fibroblast growth factor-2, as well as rationalization of previous biochemical data. Given the relevance of PTX3 in conditions ranging from COVID-19 prognosis, cancer progression, and female infertility, this structure could be used to inform the understanding and rational design of therapies for these disorders and processes.


Subject(s)
COVID-19 , Serum Amyloid P-Component , C-Reactive Protein/metabolism , Complement Activation , Cryoelectron Microscopy , Female , Humans , Immunity, Innate , Ligands , Serum Amyloid P-Component/chemistry , Serum Amyloid P-Component/metabolism
3.
Trends Biochem Sci ; 47(2): 173-186, 2022 02.
Article in English | MEDLINE | ID: covidwho-1400539

ABSTRACT

Viruses are macromolecular machineries that hijack cellular metabolism for replication. Enveloped viruses comprise a large variety of RNA and DNA viruses, many of which are notorious human or animal pathogens. Despite their importance, the presence of lipid bilayers in their assembly has made most enveloped viruses too pleomorphic to be reconstructed as a whole by traditional structural biology methods. Furthermore, structural biology of the viral lifecycle was hindered by the sample thickness. Here, I review the recent advances in the applications of cryo-electron tomography (cryo-ET) on enveloped viral structures and intracellular viral activities.


Subject(s)
Electron Microscope Tomography , Viruses , Animals , Cryoelectron Microscopy/methods , Electron Microscope Tomography/methods , Lipid Bilayers , Viruses/chemistry , Viruses/metabolism
4.
Nat Commun ; 13(1): 4399, 2022 Aug 05.
Article in English | MEDLINE | ID: covidwho-1977993

ABSTRACT

The coronavirus membrane protein (M) is the most abundant viral structural protein and plays a central role in virus assembly and morphogenesis. However, the process of M protein-driven virus assembly are largely unknown. Here, we report the cryo-electron microscopy structure of the SARS-CoV-2 M protein in two different conformations. M protein forms a mushroom-shaped dimer, composed of two transmembrane domain-swapped three-helix bundles and two intravirion domains. M protein further assembles into higher-order oligomers. A highly conserved hinge region is key for conformational changes. The M protein dimer is unexpectedly similar to SARS-CoV-2 ORF3a, a viral ion channel. Moreover, the interaction analyses of M protein with nucleocapsid protein (N) and RNA suggest that the M protein mediates the concerted recruitment of these components through the positively charged intravirion domain. Our data shed light on the M protein-driven virus assembly mechanism and provide a structural basis for therapeutic intervention targeting M protein.


Subject(s)
COVID-19 , SARS-CoV-2 , Cryoelectron Microscopy , Humans , Membrane Proteins , Virus Assembly
5.
J Vis Exp ; (185)2022 07 20.
Article in English | MEDLINE | ID: covidwho-1974967

ABSTRACT

Interest in liquid-electron microscopy (liquid-EM) has skyrocketed in recent years as scientists can now observe real-time processes at the nanoscale. It is extremely desirable to pair high-resolution cryo-EM information with dynamic observations as many events occur at rapid timescales - in the millisecond range or faster. Improved knowledge of flexible structures can also assist in the design of novel reagents to combat emerging pathogens, such as SARS-CoV-2. More importantly, viewing biological materials in a fluid environment provides a unique glimpse of their performance in the human body. Presented here are newly developed methods to investigate the nanoscale properties of virus assemblies in liquid and vitreous ice. To accomplish this goal, well-defined samples were used as model systems. Side-by-side comparisons of sample preparation methods and representative structural information are presented. Sub-nanometer features are shown for structures resolved in the range of ~3.5-Å-10 Å. Other recent results that support this complementary framework include dynamic insights of vaccine candidates and antibody-based therapies imaged in liquid. Overall, these correlative applications advance our ability to visualize molecular dynamics, providing a unique context for their use in human health and disease.


Subject(s)
COVID-19 , Ice , Cryoelectron Microscopy/methods , Humans , SARS-CoV-2 , Specimen Handling
6.
J Vis Exp ; (185)2022 07 12.
Article in English | MEDLINE | ID: covidwho-1964147

ABSTRACT

Cryo-electron tomography (cryo-ET) has been gaining momentum in recent years, especially since the introduction of direct electron detectors, improved automated acquisition strategies, preparative techniques that expand the possibilities of what the electron microscope can image at high-resolution using cryo-ET and new subtomogram averaging software. Additionally, data acquisition has become increasingly streamlined, making it more accessible to many users. The SARS-CoV-2 pandemic has further accelerated remote cryo-electron microscopy (cryo-EM) data collection, especially for single-particle cryo-EM, in many facilities globally, providing uninterrupted user access to state-of-the-art instruments during the pandemic. With the recent advances in Tomo5 (software for 3D electron tomography), remote cryo-ET data collection has become robust and easy to handle from anywhere in the world. This article aims to provide a detailed walk-through, starting from the data collection setup in the tomography software for the process of a (remote) cryo-ET data collection session with detailed troubleshooting. The (remote) data collection protocol is further complemented with the workflow for structure determination at near-atomic resolution by subtomogram averaging with emClarity, using apoferritin as an example.


Subject(s)
COVID-19 , Electron Microscope Tomography , Cryoelectron Microscopy/methods , Data Collection , Electron Microscope Tomography/methods , Humans , Image Processing, Computer-Assisted/methods , SARS-CoV-2
7.
PLoS Pathog ; 18(7): e1010733, 2022 07.
Article in English | MEDLINE | ID: covidwho-1951572

ABSTRACT

Emerging SARS-CoV-2 variants are creating major challenges in the ongoing COVID-19 pandemic. Being able to predict mutations that could arise in SARS-CoV-2 leading to increased transmissibility or immune evasion would be extremely valuable in development of broad-acting therapeutics and vaccines, and prioritising viral monitoring and containment. Here we use in vitro evolution to seek mutations in SARS-CoV-2 receptor binding domain (RBD) that would substantially increase binding to ACE2. We find a double mutation, S477N and Q498H, that increases affinity of RBD for ACE2 by 6.5-fold. This affinity gain is largely driven by the Q498H mutation. We determine the structure of the mutant-RBD:ACE2 complex by cryo-electron microscopy to reveal the mechanism for increased affinity. Addition of Q498H to SARS-CoV-2 RBD variants is found to boost binding affinity of the variants for human ACE2 and confer a new ability to bind rat ACE2 with high affinity. Surprisingly however, in the presence of the common N501Y mutation, Q498H inhibits binding, due to a clash between H498 and Y501 side chains. To achieve an intermolecular bonding network, affinity gain and cross-species binding similar to Q498H alone, RBD variants with the N501Y mutation must acquire instead the related Q498R mutation. Thus, SARS-CoV-2 RBD can access large affinity gains and cross-species binding via two alternative mutational routes involving Q498, with route selection determined by whether a variant already has the N501Y mutation. These mutations are now appearing in emerging SARS-CoV-2 variants where they have the potential to influence human-to-human and cross-species transmission.


Subject(s)
COVID-19 , SARS-CoV-2 , Angiotensin-Converting Enzyme 2/genetics , Animals , COVID-19/genetics , Cryoelectron Microscopy , Humans , Mutation , Pandemics , Peptidyl-Dipeptidase A/metabolism , Protein Binding , Rats , Receptors, Virus/metabolism , SARS-CoV-2/genetics , Spike Glycoprotein, Coronavirus/metabolism
8.
Proc Natl Acad Sci U S A ; 119(31): e2205412119, 2022 Aug 02.
Article in English | MEDLINE | ID: covidwho-1947766

ABSTRACT

Camelid single-domain antibodies, also known as nanobodies, can be readily isolated from naïve libraries for specific targets but often bind too weakly to their targets to be immediately useful. Laboratory-based genetic engineering methods to enhance their affinity, termed maturation, can deliver useful reagents for different areas of biology and potentially medicine. Using the receptor binding domain (RBD) of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein and a naïve library, we generated closely related nanobodies with micromolar to nanomolar binding affinities. By analyzing the structure-activity relationship using X-ray crystallography, cryoelectron microscopy, and biophysical methods, we observed that higher conformational entropy losses in the formation of the spike protein-nanobody complex are associated with tighter binding. To investigate this, we generated structural ensembles of the different complexes from electron microscopy maps and correlated the conformational fluctuations with binding affinity. This insight guided the engineering of a nanobody with improved affinity for the spike protein.


Subject(s)
Antibodies, Neutralizing , Antibodies, Viral , Antibody Affinity , SARS-CoV-2 , Single-Domain Antibodies , Spike Glycoprotein, Coronavirus , Antibodies, Neutralizing/chemistry , Antibodies, Neutralizing/genetics , Antibodies, Viral/chemistry , Antibodies, Viral/genetics , Antibody Affinity/genetics , Cryoelectron Microscopy , Entropy , Genetic Engineering , Humans , Protein Binding , Protein Domains , SARS-CoV-2/immunology , Single-Domain Antibodies/chemistry , Single-Domain Antibodies/genetics , Spike Glycoprotein, Coronavirus/immunology
9.
Structure ; 30(8): 1062-1074.e4, 2022 Aug 04.
Article in English | MEDLINE | ID: covidwho-1946637

ABSTRACT

The COVID-19 pandemic has prompted a rapid response in vaccine and drug development. Herein, we modeled a complete membrane-embedded SARS-CoV-2 spike glycoprotein and used molecular dynamics simulations with benzene probes designed to enhance discovery of cryptic pockets. This approach recapitulated lipid and host metabolite binding sites previously characterized by cryo-electron microscopy, revealing likely ligand entry routes, and uncovered a novel cryptic pocket with promising druggable properties located underneath the 617-628 loop. A full representation of glycan moieties was essential to accurately describe pocket dynamics. A multi-conformational behavior of the 617-628 loop in simulations was validated using hydrogen-deuterium exchange mass spectrometry experiments, supportive of opening and closing dynamics. The pocket is the site of multiple mutations associated with increased transmissibility found in SARS-CoV-2 variants of concern including Omicron. Collectively, this work highlights the utility of the benzene mapping approach in uncovering potential druggable sites on the surface of SARS-CoV-2 targets.


Subject(s)
SARS-CoV-2 , Spike Glycoprotein, Coronavirus , Benzene , Cryoelectron Microscopy , Molecular Dynamics Simulation , Protein Binding , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/genetics
10.
Cell Rep ; 39(13): 111009, 2022 Jun 28.
Article in English | MEDLINE | ID: covidwho-1944463

ABSTRACT

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Omicron BA.2 sub-lineage has gained in proportion relative to BA.1. Because spike (S) protein variations may underlie differences in their pathobiology, here we determine cryoelectron microscopy (cryo-EM) structures of the BA.2 S ectodomain and compare these with previously determined BA.1 S structures. BA.2 receptor-binding domain (RBD) mutations induce remodeling of the RBD structure, resulting in tighter packing and improved thermostability. Interprotomer RBD interactions are enhanced in the closed (or 3-RBD-down) BA.2 S, while the fusion peptide is less accessible to antibodies than in BA.1. Binding and pseudovirus neutralization assays reveal extensive immune evasion while defining epitopes of two outer RBD face-binding antibodies, DH1044 and DH1193, that neutralize both BA.1 and BA.2. Taken together, our results indicate that stabilization of the closed state through interprotomer RBD-RBD packing is a hallmark of the Omicron variant and show differences in key functional regions in the BA.1 and BA.2 S proteins.


Subject(s)
COVID-19 , SARS-CoV-2 , Antibodies, Viral , Cryoelectron Microscopy , Humans , Receptors, Virus/metabolism , Spike Glycoprotein, Coronavirus
11.
J Phys Chem B ; 126(28): 5194-5206, 2022 07 21.
Article in English | MEDLINE | ID: covidwho-1931300

ABSTRACT

Since the introduction of the novel SARS-CoV-2 virus (COVID-19) in late 2019, various new variants have appeared with mutations that confer resistance to the vaccines and monoclonal antibodies that were developed in response to the wild-type virus. As we continue through the pandemic, an accurate and efficient methodology is needed to help predict the effects certain mutations will have on both our currently produced therapeutics and those that are in development. Using published cryo-electron microscopy and X-ray crystallography structures of the spike receptor binding domain region with currently known antibodies, in the present study, we created and cross-validated an intermolecular interaction modeling-based multi-layer perceptron machine learning approach that can accurately predict the mutation-caused shifts in the binding affinity between the spike protein (wild-type or mutant) and various antibodies. This validated artificial intelligence (AI) model was used to predict the binding affinity (Kd) of reported SARS-CoV-2 antibodies with various variants of concern, including the most recently identified "Deltamicron" (or "Deltacron") variant. This AI model may be employed in the future to predict the Kd of developed novel antibody therapeutics to overcome the challenging antibody resistance issue and develop structural bases for the effects of both current and new mutants of the spike protein. In addition, the similar AI strategy and approach based on modeling of the intermolecular interactions may be useful in development of machine learning models predicting binding affinities for other protein-protein binding systems, including other antibodies binding with their antigens.


Subject(s)
COVID-19 , Spike Glycoprotein, Coronavirus , Angiotensin-Converting Enzyme 2 , Artificial Intelligence , Cryoelectron Microscopy , Humans , Machine Learning , Mutation , Protein Binding , SARS-CoV-2/genetics , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/metabolism
12.
Front Immunol ; 13: 863831, 2022.
Article in English | MEDLINE | ID: covidwho-1924097

ABSTRACT

The emergence of SARS-CoV-2 variants that escape from immune neutralization are challenging vaccines and antibodies developed to stop the COVID-19 pandemic. Thus, it is important to establish therapeutics directed toward multiple or specific SARS-CoV-2 variants. The envelope spike (S) glycoprotein of SARS-CoV-2 is the key target of neutralizing antibodies (Abs). We selected a panel of nine nanobodies (Nbs) from dromedary camels immunized with the receptor-binding domain (RBD) of the S, and engineered Nb fusions as humanized heavy chain Abs (hcAbs). Nbs and derived hcAbs bound with subnanomolar or picomolar affinities to the S and its RBD, and S-binding cross-competition clustered them in two different groups. Most of the hcAbs hindered RBD binding to its human ACE2 (hACE2) receptor, blocked cell entry of viruses pseudotyped with the S protein and neutralized SARS-CoV-2 infection in cell cultures. Four potent neutralizing hcAbs prevented the progression to lethal SARS-CoV-2 infection in hACE2-transgenic mice, demonstrating their therapeutic potential. Cryo-electron microscopy identified Nb binding epitopes in and out the receptor binding motif (RBM), and showed different ways to prevent virus binding to its cell entry receptor. The Nb binding modes were consistent with its recognition of SARS-CoV-2 RBD variants; mono and bispecific hcAbs efficiently bound all variants of concern except omicron, which emphasized the immune escape capacity of this latest variant.


Subject(s)
COVID-19 , Single-Domain Antibodies , Animals , Cryoelectron Microscopy , Epitopes/chemistry , Humans , Mice , Pandemics , SARS-CoV-2 , Spike Glycoprotein, Coronavirus
13.
Acta Crystallogr D Struct Biol ; 78(Pt 7): 806-816, 2022 Jul 01.
Article in English | MEDLINE | ID: covidwho-1922451

ABSTRACT

The availability of new artificial intelligence-based protein-structure-prediction tools has radically changed the way that cryo-EM maps are interpreted, but it has not eliminated the challenges of map interpretation faced by a microscopist. Models will continue to be locally rebuilt and refined using interactive tools. This inevitably results in occasional errors, among which register shifts remain one of the most difficult to identify and correct. Here, checkMySequence, a fast, fully automated and parameter-free method for detecting register shifts in protein models built into cryo-EM maps, is introduced. It is shown that the method can assist model building in cases where poorer map resolution hinders visual interpretation. It is also shown that checkMySequence could have helped to avoid a widely discussed sequence-register error in a model of SARS-CoV-2 RNA-dependent RNA polymerase that was originally detected thanks to a visual residue-by-residue inspection by members of the structural biology community. The software is freely available at https://gitlab.com/gchojnowski/checkmysequence.


Subject(s)
Artificial Intelligence , COVID-19 , Cryoelectron Microscopy/methods , Humans , Models, Molecular , Proteins/chemistry , RNA, Viral , SARS-CoV-2
14.
J Vis Exp ; (184)2022 06 08.
Article in English | MEDLINE | ID: covidwho-1911781

ABSTRACT

Lysophospholipids (LPLs) are bioactive lipids that include sphingosine 1-phosphate (S1P), lysophosphatidic acid, etc. S1P, a metabolic product of sphingolipids in the cell membrane, is one of the best-characterized LPLs that regulates a variety of cellular physiological responses via signaling pathways mediated by sphingosine 1-phosphate receptors (S1PRs). This implicated that the S1P-S1PRs signaling system is a remarkable potential therapeutic target for disorders, including multiple sclerosis (MS), autoimmune disorders, cancer, inflammation, and even COVID-19. S1PRs, a small subset of the class A G-protein coupled receptor (GPCR) family, are composed of five subtypes: S1PR1, S1PR2, S1PR3, S1PR4, and S1PR5. The lack of detailed structural information, however, impedes the drug discovery targeting S1PRs. Here, we applied the cryo-electron microscopy method to solve the structure of the S1P-S1PRs complex, and elucidated the mechanism of activation, selective drug recognition, and G-protein coupling by using cell-based functional assays. Other lysophospholipid receptors (LPLRs) and GPCRs can also be studied using this strategy.


Subject(s)
COVID-19 , Receptors, Lysosphingolipid , Cryoelectron Microscopy , Humans , Lysophospholipids , Receptors, Lysosphingolipid/metabolism , Signal Transduction , Sphingosine/analogs & derivatives , Sphingosine-1-Phosphate Receptors
15.
Virol J ; 19(1): 112, 2022 06 27.
Article in English | MEDLINE | ID: covidwho-1905664

ABSTRACT

BACKGROUND: Middle East respiratory syndrome coronavirus (MERS-CoV) causes severe respiratory disease in humans, with a case fatality rate of approximately 35%, thus posing a considerable threat to public health. The lack of approved vaccines or antivirals currently constitutes a barrier in controlling disease outbreaks and spread. METHODS: In this study, using a mammalian expression system, which is advantageous for maintaining correct protein glycosylation patterns, we constructed chimeric MERS-CoV virus-like particles (VLPs) and determined their immunogenicity and protective efficacy in mice. RESULTS: Western blot and cryo-electron microscopy analyses demonstrated that MERS-CoV VLPs were efficiently produced in cells co-transfected with MERS-CoV spike (S), envelope, membrane and murine hepatitis virus nucleocapsid genes. We examined their ability as a vaccine in a human dipeptidyl peptidase 4 knock-in C57BL/6 congenic mouse model. Mice immunized with MERS VLPs produced S-specific antibodies with virus neutralization activity. Furthermore, MERS-CoV VLP immunization provided complete protection against a lethal challenge with mouse-adapted MERS-CoV and improved virus clearance in the lung. CONCLUSIONS: Overall, these data demonstrate that MERS-CoV VLPs have excellent immunogenicity and represent a promising vaccine candidate.


Subject(s)
Coronavirus Infections , Middle East Respiratory Syndrome Coronavirus , Vaccines, Virus-Like Particle , Viral Vaccines , Animals , Antibodies, Neutralizing , Antibodies, Viral , Cryoelectron Microscopy , Mammals , Mice , Mice, Inbred C57BL , Middle East Respiratory Syndrome Coronavirus/genetics , Spike Glycoprotein, Coronavirus/genetics , Vaccines, Virus-Like Particle/genetics , Viral Vaccines/genetics
16.
Science ; 377(6604): eabm3125, 2022 07 22.
Article in English | MEDLINE | ID: covidwho-1901907

ABSTRACT

Many pathogens exploit host cell-surface glycans. However, precise analyses of glycan ligands binding with heavily modified pathogen proteins can be confounded by overlapping sugar signals and/or compounded with known experimental constraints. Universal saturation transfer analysis (uSTA) builds on existing nuclear magnetic resonance spectroscopy to provide an automated workflow for quantitating protein-ligand interactions. uSTA reveals that early-pandemic, B-origin-lineage severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike trimer binds sialoside sugars in an "end-on" manner. uSTA-guided modeling and a high-resolution cryo-electron microscopy structure implicate the spike N-terminal domain (NTD) and confirm end-on binding. This finding rationalizes the effect of NTD mutations that abolish sugar binding in SARS-CoV-2 variants of concern. Together with genetic variance analyses in early pandemic patient cohorts, this binding implicates a sialylated polylactosamine motif found on tetraantennary N-linked glycoproteins deep in the human lung as potentially relevant to virulence and/or zoonosis.


Subject(s)
COVID-19 , Host-Pathogen Interactions , SARS-CoV-2 , Sialic Acids , Spike Glycoprotein, Coronavirus , COVID-19/transmission , Cryoelectron Microscopy , Genetic Variation , Humans , Nuclear Magnetic Resonance, Biomolecular , Polysaccharides/chemistry , Protein Binding , Protein Domains , SARS-CoV-2/chemistry , SARS-CoV-2/genetics , Sialic Acids/chemistry , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/genetics
17.
Biophys J ; 121(12): 2353-2370, 2022 06 21.
Article in English | MEDLINE | ID: covidwho-1899576

ABSTRACT

The newly developed finite element (FE) modeling at the atomic scale was used to predict the static and dynamic response of the α-helix (AH) and tropocollagen (TC) protein fragments, the main building blocks of the spike of the SARS-CoV-2. The geometry and morphology of the spike's stalk and its connection to the viral envelope were determined from the combination of most recent molecular dynamics (MD) simulation and images of cryoelectron microscopy. The stiffness parameters of the covalent bonds in the main chain of the helix were taken from the literature. The AH and TC were modeled using both beam elements (wire model) and shell elements (ribbon model) in FE analysis to predict their mechanical properties under tension. The asymptotic stiffening features of AH and TC under tensile loading were revealed and compared with a new analytical solution. The mechanical stiffnesses under other loading conditions, including compression, torsion, and bending, were also predicted numerically and correlated with the results of the existing MD simulations and tests. The mode shapes and natural frequencies of the spike were predicted using the built FE model. The frequencies were shown to be within the safe range of 1-20 MHz routinely used for medical imaging and diagnosis by means of ultrasound. These results provide a solid theoretical basis for using ultrasound to study damaging coronavirus through transient and resonant vibration at large deformations.


Subject(s)
COVID-19 , Tropocollagen , Cryoelectron Microscopy , Finite Element Analysis , Humans , Protein Conformation, alpha-Helical , SARS-CoV-2 , Tropocollagen/chemistry
18.
Science ; 376(6595): eabn6020, 2022 05 20.
Article in English | MEDLINE | ID: covidwho-1861569

ABSTRACT

The detyrosination-tyrosination cycle involves the removal and religation of the C-terminal tyrosine of α-tubulin and is implicated in cognitive, cardiac, and mitotic defects. The vasohibin-small vasohibin-binding protein (SVBP) complex underlies much, but not all, detyrosination. We used haploid genetic screens to identify an unannotated protein, microtubule associated tyrosine carboxypeptidase (MATCAP), as a remaining detyrosinating enzyme. X-ray crystallography and cryo-electron microscopy structures established MATCAP's cleaving mechanism, substrate specificity, and microtubule recognition. Paradoxically, whereas abrogation of tyrosine religation is lethal in mice, codeletion of MATCAP and SVBP is not. Although viable, defective detyrosination caused microcephaly, associated with proliferative defects during neurogenesis, and abnormal behavior. Thus, MATCAP is a missing component of the detyrosination-tyrosination cycle, revealing the importance of this modification in brain formation.


Subject(s)
Carboxypeptidases , Microtubule-Associated Proteins , Microtubules , Protein Processing, Post-Translational , Tubulin , Tyrosine , Animals , Carboxypeptidases/genetics , Cryoelectron Microscopy , Crystallography, X-Ray , Humans , Mice , Microtubule-Associated Proteins/chemistry , Microtubule-Associated Proteins/genetics , Microtubules/chemistry , Tubulin/chemistry , Tyrosine/chemistry
19.
Cell Rep ; 39(5): 110770, 2022 05 03.
Article in English | MEDLINE | ID: covidwho-1859379

ABSTRACT

The emergence of the SARS-CoV-2 Omicron variant is dominant in many countries worldwide. The high number of spike mutations is responsible for the broad immune evasion from existing vaccines and antibody drugs. To understand this, we first present the cryo-electron microscopy structure of ACE2-bound SARS-CoV-2 Omicron spike. Comparison to previous spike antibody structures explains how Omicron escapes these therapeutics. Secondly, we report structures of Omicron, Delta, and wild-type spikes bound to a patient-derived Fab antibody fragment (510A5), which provides direct evidence where antibody binding is greatly attenuated by the Omicron mutations, freeing spike to bind ACE2. Together with biochemical binding and 510A5 neutralization assays, our work establishes principles of binding required for neutralization and clearly illustrates how the mutations lead to antibody evasion yet retain strong ACE2 interactions. Structural information on spike with both bound and unbound antibodies collectively elucidates potential strategies for generation of therapeutic antibodies.


Subject(s)
COVID-19 , SARS-CoV-2 , Angiotensin-Converting Enzyme 2 , Antibodies, Neutralizing , Antibodies, Viral , Cryoelectron Microscopy , Humans , Immunoglobulin Fab Fragments , Spike Glycoprotein, Coronavirus
20.
Signal Transduct Target Ther ; 7(1): 139, 2022 04 27.
Article in English | MEDLINE | ID: covidwho-1815514

ABSTRACT

The SARS-CoV-2 Omicron variant shows substantial resistance to neutralization by infection- and vaccination-induced antibodies, highlighting the demands for research on the continuing discovery of broadly neutralizing antibodies (bnAbs). Here, we developed a panel of bnAbs against Omicron and other variants of concern (VOCs) elicited by vaccination of adenovirus-vectored COVID-19 vaccine (Ad5-nCoV). We also investigated the human longitudinal antibody responses following vaccination and demonstrated how the bnAbs evolved over time. A monoclonal antibody (mAb), named ZWD12, exhibited potent and broad neutralization against SARS-CoV-2 variants Alpha, Beta, Gamma, Kappa, Delta, and Omicron by blocking the spike protein binding to the angiotensin-converting enzyme 2 (ACE2) and provided complete protection in the challenged prophylactic and therapeutic K18-hACE2 transgenic mouse model. We defined the ZWD12 epitope by determining its structure in complex with the spike (S) protein via cryo-electron microscopy. This study affords the potential to develop broadly therapeutic mAb drugs and suggests that the RBD epitope bound by ZWD12 is a rational target for the design of a broad spectrum of vaccines.


Subject(s)
COVID-19 , SARS-CoV-2 , Animals , Antibodies, Monoclonal/genetics , Antibodies, Viral , Broadly Neutralizing Antibodies , COVID-19/prevention & control , COVID-19 Vaccines/genetics , Cryoelectron Microscopy , Epitopes , Humans , Mice , SARS-CoV-2/genetics , Spike Glycoprotein, Coronavirus/genetics , Vaccination , Viral Envelope Proteins
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