RESUMEN
Coronavirus spike proteins mediate receptor binding and membrane fusion, making them prime targets for neutralizing antibodies. In the cases of severe acute respiratory syndrome coronavirus, severe acute respiratory syndrome coronavirus 2 and Middle East respiratory syndrome coronavirus, spike proteins transition freely between open and closed conformations to balance host cell attachment and immune evasion1-5. Spike opening exposes domain S1B, allowing it to bind to proteinaceous receptors6,7, and is also thought to enable protein refolding during membrane fusion4,5. However, with a single exception, the pre-fusion spike proteins of all other coronaviruses studied so far have been observed exclusively in the closed state. This raises the possibility of regulation, with spike proteins more commonly transitioning to open states in response to specific cues, rather than spontaneously. Here, using cryogenic electron microscopy and molecular dynamics simulations, we show that the spike protein of the common cold human coronavirus HKU1 undergoes local and long-range conformational changes after binding a sialoglycan-based primary receptor to domain S1A. This binding triggers the transition of S1B domains to the open state through allosteric interdomain crosstalk. Our findings provide detailed insight into coronavirus attachment, with possibilities of dual receptor usage and priming of entry as a means of immune escape.
Asunto(s)
Betacoronavirus , Polisacáridos , Ácidos Siálicos , Glicoproteína de la Espiga del Coronavirus , Humanos , Regulación Alostérica , Betacoronavirus/química , Betacoronavirus/ultraestructura , Resfriado Común/virología , Microscopía por Crioelectrón , Simulación de Dinámica Molecular , Polisacáridos/química , Polisacáridos/metabolismo , Unión Proteica , Conformación Proteica , Ácidos Siálicos/química , Ácidos Siálicos/metabolismo , Glicoproteína de la Espiga del Coronavirus/química , Glicoproteína de la Espiga del Coronavirus/metabolismo , Glicoproteína de la Espiga del Coronavirus/ultraestructura , Evasión InmuneRESUMEN
The ongoing COVID-19 pandemic has had great societal and health consequences. Despite the availability of vaccines, infection rates remain high due to immune evasive Omicron sublineages. Broad-spectrum antivirals are needed to safeguard against emerging variants and future pandemics. We used messenger RNA (mRNA) display under a reprogrammed genetic code to find a spike-targeting macrocyclic peptide that inhibits SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) Wuhan strain infection and pseudoviruses containing spike proteins of SARS-CoV-2 variants or related sarbecoviruses. Structural and bioinformatic analyses reveal a conserved binding pocket between the receptor-binding domain, N-terminal domain, and S2 region, distal to the angiotensin-converting enzyme 2 receptor-interaction site. Our data reveal a hitherto unexplored site of vulnerability in sarbecoviruses that peptides and potentially other drug-like molecules can target.
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COVID-19 , SARS-CoV-2 , Humanos , SARS-CoV-2/metabolismo , Glicoproteína de la Espiga del Coronavirus/metabolismo , Pandemias/prevención & control , Péptidos/farmacologíaRESUMEN
Activated FXII (FXIIa) is the principal initiator of the plasma contact system and can activate both procoagulant and proinflammatory pathways. Its activity is important in the pathophysiology of hereditary angioedema (HAE). Here, we describe a high-resolution cryoelectron microscopy (cryo-EM) structure of the beta-chain from FXIIa (ßFXIIa) complexed with the Fab fragment of garadacimab. Garadacimab binds to ßFXIIa through an unusually long CDR-H3 that inserts into the S1 pocket in a non-canonical way. This structural mechanism is likely the primary contributor to the inhibition of activated FXIIa proteolytic activity in HAE. Garadacimab Fab-ßFXIIa structure also reveals critical determinants of high-affinity binding of garadacimab to activated FXIIa. Structural analysis with other bona fide FXIIa inhibitors, such as benzamidine and C1-INH, reveals a surprisingly similar mechanism of ßFXIIa inhibition by garadacimab. In summary, the garadacimab Fab-ßFXIIa structure provides crucial insights into its mechanism of action and delineates primary and auxiliary paratopes/epitopes.
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Data acquisition and processing for cryo-electron tomography can be a significant bottleneck for users. To simplify and streamline the cryo-ET workflow, Tomo Live, an on-the-fly solution that automates the alignment and reconstruction of tilt-series data, enabling real-time data-quality assessment, has been developed. Through the integration of Tomo Live into the data-acquisition workflow for cryo-ET, motion correction is performed directly after each of the acquired tilt angles. Immediately after the tilt-series acquisition has completed, an unattended tilt-series alignment and reconstruction into a 3D volume is performed. The results are displayed in real time in a dedicated remote web platform that runs on the microscope hardware. Through this web platform, users can review the acquired data (aligned stack and 3D volume) and several quality metrics that are obtained during the alignment and reconstruction process. These quality metrics can be used for fast feedback for subsequent acquisitions to save time. Parameters such as Alignment Accuracy, Deleted Tilts and Tilt Axis Correction Angle are visualized as graphs and can be used as filters to export only the best tomograms (raw data, reconstruction and intermediate data) for further processing. Here, the Tomo Live algorithms and workflow are described and representative results on several biological samples are presented. The Tomo Live workflow is accessible to both expert and non-expert users, making it a valuable tool for the continued advancement of structural biology, cell biology and histology.
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Tomografía con Microscopio Electrónico , Procesamiento de Imagen Asistido por Computador , Tomografía con Microscopio Electrónico/métodos , Microscopía por Crioelectrón/métodos , Procesamiento de Imagen Asistido por Computador/métodos , Exactitud de los Datos , Flujo de TrabajoRESUMEN
Porcine deltacoronavirus (PDCoV) is an emerging enteric pathogen that has recently been detected in humans. Despite this zoonotic concern, the antigenic structure of PDCoV remains unknown. The virus relies on its spike (S) protein for cell entry, making it a prime target for neutralizing antibodies. Here, we generate and characterize a set of neutralizing antibodies targeting the S protein, shedding light on PDCoV S interdomain crosstalk and its vulnerable sites. Among the four identified antibodies, one targets the S1A domain, causing local and long-range conformational changes, resulting in partial exposure of the S1B domain. The other antibodies bind the S1B domain, disrupting binding to aminopeptidase N (APN), the entry receptor for PDCoV. Notably, the epitopes of these S1B-targeting antibodies are concealed in the prefusion S trimer conformation, highlighting the necessity for conformational changes for effective antibody binding. The binding footprint of one S1B binder entirely overlaps with APN-interacting residues and thus targets a highly conserved epitope. These findings provide structural insights into the humoral immune response against the PDCoV S protein, potentially guiding vaccine and therapeutic development for this zoonotic pathogen.
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Anticuerpos Neutralizantes , Anticuerpos Antivirales , Deltacoronavirus , Epítopos , Glicoproteína de la Espiga del Coronavirus , Glicoproteína de la Espiga del Coronavirus/inmunología , Glicoproteína de la Espiga del Coronavirus/metabolismo , Glicoproteína de la Espiga del Coronavirus/química , Animales , Anticuerpos Neutralizantes/inmunología , Porcinos , Anticuerpos Antivirales/inmunología , Epítopos/inmunología , Humanos , Deltacoronavirus/inmunología , Deltacoronavirus/metabolismo , Antígenos CD13/metabolismo , Antígenos CD13/inmunología , Infecciones por Coronavirus/inmunología , Infecciones por Coronavirus/virología , Dominios Proteicos , Unión Proteica , Enfermedades de los Porcinos/virología , Enfermedades de los Porcinos/inmunología , Células HEK293RESUMEN
PURPOSE: Estrogen receptor (ER) alpha signaling is a known driver of ER-positive (ER+)/human epidermal growth factor receptor 2 negative (HER2-) breast cancer. Combining endocrine therapy (ET) such as fulvestrant with CDK4/6, mTOR, or PI3K inhibitors has become a central strategy in the treatment of ER+ advanced breast cancer. However, suboptimal ER inhibition and resistance resulting from the ESR1 mutation dictates that new therapies are needed. EXPERIMENTAL DESIGN: A medicinal chemistry campaign identified vepdegestrant (ARV-471), a selective, orally bioavailable, and potent small molecule PROteolysis-TArgeting Chimera (PROTAC) degrader of ER. We used biochemical and intracellular target engagement assays to demonstrate the mechanism of action of vepdegestrant, and ESR1 wild-type (WT) and mutant ER+ preclinical breast cancer models to demonstrate ER degradation-mediated tumor growth inhibition (TGI). RESULTS: Vepdegestrant induced ≥90% degradation of wild-type and mutant ER, inhibited ER-dependent breast cancer cell line proliferation in vitro, and achieved substantial TGI (87%-123%) in MCF7 orthotopic xenograft models, better than those of the ET agent fulvestrant (31%-80% TGI). In the hormone independent (HI) mutant ER Y537S patient-derived xenograft (PDX) breast cancer model ST941/HI, vepdegestrant achieved tumor regression and was similarly efficacious in the ST941/HI/PBR palbociclib-resistant model (102% TGI). Vepdegestrant-induced robust tumor regressions in combination with each of the CDK4/6 inhibitors palbociclib, abemaciclib, and ribociclib; the mTOR inhibitor everolimus; and the PI3K inhibitors alpelisib and inavolisib. CONCLUSIONS: Vepdegestrant achieved greater ER degradation in vivo compared with fulvestrant, which correlated with improved TGI, suggesting vepdegestrant could be a more effective backbone ET for patients with ER+/HER2- breast cancer.
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Neoplasias de la Mama , Quinasa 4 Dependiente de la Ciclina , Quinasa 6 Dependiente de la Ciclina , Transducción de Señal , Serina-Treonina Quinasas TOR , Ensayos Antitumor por Modelo de Xenoinjerto , Humanos , Femenino , Animales , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/patología , Neoplasias de la Mama/metabolismo , Quinasa 6 Dependiente de la Ciclina/antagonistas & inhibidores , Quinasa 4 Dependiente de la Ciclina/antagonistas & inhibidores , Ratones , Serina-Treonina Quinasas TOR/metabolismo , Serina-Treonina Quinasas TOR/antagonistas & inhibidores , Línea Celular Tumoral , Transducción de Señal/efectos de los fármacos , Receptor alfa de Estrógeno/metabolismo , Receptor alfa de Estrógeno/genética , Receptor alfa de Estrógeno/antagonistas & inhibidores , Piperazinas/farmacología , Piperazinas/administración & dosificación , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Inhibidores de las Quinasa Fosfoinosítidos-3/farmacología , Inhibidores de las Quinasa Fosfoinosítidos-3/administración & dosificación , Receptores de Estrógenos/metabolismo , Piridinas/administración & dosificación , Piridinas/farmacología , Inhibidores de Proteínas Quinasas/farmacología , Proliferación Celular/efectos de los fármacosRESUMEN
Membrane proteins are the largest group of therapeutic targets in a variety of disease areas and yet, they remain particularly difficult to investigate. We have developed a novel one-step approach for the incorporation of membrane proteins directly from cells into lipid Salipro nanoparticles. Here, with the pannexin1 channel as a case study, we demonstrate the applicability of this method for structure-function analysis using SPR and cryo-EM.
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Proteínas de la Membrana , Proteínas de la Membrana/metabolismo , Microscopía por Crioelectrón/métodos , Membrana Celular/metabolismoRESUMEN
The ongoing evolution of SARS-CoV-2 has resulted in the emergence of Omicron, which displays notable immune escape potential through mutations at key antigenic sites on the spike protein. Many of these mutations localize to the spike protein ACE2 receptor binding domain, annulling the neutralizing activity of therapeutic antibodies that were effective against other variants of concern (VOCs) earlier in the pandemic. Here, we identified a receptor-blocking human monoclonal antibody, 87G7, that retained potent in vitro neutralizing activity against SARS-CoV-2 variants including the Alpha, Beta, Gamma, Delta, and Omicron (BA.1/BA.2) VOCs. Using cryo-electron microscopy and site-directed mutagenesis experiments, we showed that 87G7 targets a patch of hydrophobic residues in the ACE2-binding site that are highly conserved in SARS-CoV-2 variants, explaining its broad neutralization capacity. 87G7 protected mice and hamsters prophylactically against challenge with all current SARS-CoV-2 VOCs and showed therapeutic activity against SARS-CoV-2 challenge in both animal models. Our findings demonstrate that 87G7 holds promise as a prophylactic or therapeutic agent for COVID-19 that is more resilient to SARS-CoV-2 antigenic diversity.
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Enzima Convertidora de Angiotensina 2 , Anticuerpos Neutralizantes , Tratamiento Farmacológico de COVID-19 , SARS-CoV-2 , Enzima Convertidora de Angiotensina 2/antagonistas & inhibidores , Animales , Anticuerpos Neutralizantes/farmacología , Microscopía por Crioelectrón , Humanos , Glicoproteínas de Membrana , Ratones , Pruebas de Neutralización , SARS-CoV-2/genética , Glicoproteína de la Espiga del Coronavirus/genética , Proteínas del Envoltorio ViralRESUMEN
The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants with potential resistance to existing drugs emphasizes the need for new therapeutic modalities with broad variant activity. Here we show that ensovibep, a trispecific DARPin (designed ankyrin repeat protein) clinical candidate, can engage the three units of the spike protein trimer of SARS-CoV-2 and inhibit ACE2 binding with high potency, as revealed by cryo-electron microscopy analysis. The cooperative binding together with the complementarity of the three DARPin modules enable ensovibep to inhibit frequent SARS-CoV-2 variants, including Omicron sublineages BA.1 and BA.2. In Roborovski dwarf hamsters infected with SARS-CoV-2, ensovibep reduced fatality similarly to a standard-of-care monoclonal antibody (mAb) cocktail. When used as a single agent in viral passaging experiments in vitro, ensovibep reduced the emergence of escape mutations in a similar fashion to the same mAb cocktail. These results support further clinical evaluation of ensovibep as a broad variant alternative to existing targeted therapies for Coronavirus Disease 2019 (COVID-19).
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COVID-19 , SARS-CoV-2 , Animales , Cricetinae , Humanos , SARS-CoV-2/genética , Proteínas de Repetición de Anquirina Diseñadas , Microscopía por Crioelectrón , Anticuerpos Monoclonales/uso terapéutico , Terapéutica Combinada de Anticuerpos , Anticuerpos NeutralizantesRESUMEN
G protein-coupled receptors (GPCRs) are the largest class of cell surface drug targets. Advances in stabilization of GPCR:transducer complexes, together with improvements in cryoelectron microscopy (cryo-EM) have recently been applied to structure-assisted drug design for GPCR agonists. Nonetheless, limitations in the commercial application of these approaches, including the use of nanobody 35 (Nb35) to aid complex stabilization and the high cost of 300 kV imaging, have restricted broad application of cryo-EM in drug discovery. Here, using the PF 06882961-bound GLP-1R as exemplar, we validated the formation of stable complexes with a modified Gs protein in the absence of Nb35. In parallel, we compare 200 versus 300 kV image acquisition using a Falcon 4 or K3 direct electron detector. Moreover, the 200 kV Glacios-Falcon 4 yielded a 3.2 Å map with clear density for bound drug and multiple structurally ordered waters. Our work paves the way for broader commercial application of cryo-EM for GPCR drug discovery.
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Microscopía por Crioelectrón/métodos , Descubrimiento de Drogas/métodos , Receptores Acoplados a Proteínas G/química , Animales , Células CHO , Cricetinae , Cricetulus , Humanos , Unión Proteica , Receptores Acoplados a Proteínas G/agonistas , Receptores Acoplados a Proteínas G/metabolismo , Células Sf9 , Anticuerpos de Dominio Único/química , Anticuerpos de Dominio Único/farmacología , Bibliotecas de Moléculas Pequeñas/química , Bibliotecas de Moléculas Pequeñas/farmacología , SpodopteraRESUMEN
The emergence of SARS-CoV-2 antibody escape mutations highlights the urgent need for broadly neutralizing therapeutics. We previously identified a human monoclonal antibody, 47D11, capable of cross-neutralizing SARS-CoV-2 and SARS-CoV and protecting against the associated respiratory disease in an animal model. Here, we report cryo-EM structures of both trimeric spike ectodomains in complex with the 47D11 Fab. 47D11 binds to the closed receptor-binding domain, distal to the ACE2 binding site. The CDRL3 stabilizes the N343 glycan in an upright conformation, exposing a mutationally constrained hydrophobic pocket, into which the CDRH3 loop inserts two aromatic residues. 47D11 stabilizes a partially open conformation of the SARS-CoV-2 spike, suggesting that it could be used effectively in combination with other antibodies targeting the exposed receptor-binding motif. Together, these results reveal a cross-protective epitope on the SARS-CoV-2 spike and provide a structural roadmap for the development of 47D11 as a prophylactic or postexposure therapy for COVID-19.
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Anticuerpos Monoclonales , Anticuerpos Neutralizantes , Anticuerpos Antivirales , SARS-CoV-2 , Coronavirus Relacionado al Síndrome Respiratorio Agudo Severo , Anticuerpos Monoclonales/química , Anticuerpos Monoclonales/inmunología , Anticuerpos Neutralizantes/química , Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/química , Anticuerpos Antivirales/inmunología , Humanos , Coronavirus Relacionado al Síndrome Respiratorio Agudo Severo/química , Coronavirus Relacionado al Síndrome Respiratorio Agudo Severo/inmunología , SARS-CoV-2/química , SARS-CoV-2/inmunología , Relación Estructura-ActividadRESUMEN
The human betacoronaviruses HKU1 and OC43 (subgenus Embecovirus) arose from separate zoonotic introductions, OC43 relatively recently and HKU1 apparently much longer ago. Embecovirus particles contain two surface projections called spike (S) and haemagglutinin-esterase (HE), with S mediating receptor binding and membrane fusion, and HE acting as a receptor-destroying enzyme. Together, they promote dynamic virion attachment to glycan-based receptors, specifically 9-O-acetylated sialic acid. Here we present the cryo-EM structure of the ~80 kDa, heavily glycosylated HKU1 HE at 3.4 Å resolution. Comparison with existing HE structures reveals a drastically truncated lectin domain, incompatible with sialic acid binding, but with the structure and function of the esterase domain left intact. Cryo-EM and mass spectrometry analysis reveals a putative glycan shield on the now redundant lectin domain. The findings further our insight into the evolution and host adaptation of human embecoviruses, and demonstrate the utility of cryo-EM for studying small, heavily glycosylated proteins.
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Betacoronavirus/química , Betacoronavirus/fisiología , Infecciones por Coronavirus/virología , Hemaglutininas Virales/química , Proteínas Virales de Fusión/química , Betacoronavirus/clasificación , Sitios de Unión , Dominio Catalítico , Microscopía por Crioelectrón , Glicosilación , Células HEK293 , Hemaglutininas Virales/metabolismo , Hemaglutininas Virales/ultraestructura , Humanos , Lectinas/química , Lectinas/metabolismo , Espectrometría de Masas , Modelos Moleculares , Ácido N-Acetilneuramínico/metabolismo , Polisacáridos/química , Dominios Proteicos , Proteínas Virales de Fusión/metabolismo , Proteínas Virales de Fusión/ultraestructuraRESUMEN
Indanomycin is biosynthesized by a hybrid nonribosomal peptide synthase/polyketide synthase (NRPS/PKS) followed by a number of 'tailoring' steps to form the two ring systems that are present in the mature product. It had previously been hypothesized that the indane ring of indanomycin was formed by the action of IdmH using a Diels-Alder reaction. Here, the crystal structure of a selenomethionine-labelled truncated form of IdmH (IdmH-Δ99-107) was solved using single-wavelength anomalous dispersion (SAD) phasing. This truncated variant allows consistent and easy crystallization, but importantly the structure was used as a search model in molecular replacement, allowing the full-length IdmH structure to be determined to 2.7â Å resolution. IdmH is a homodimer, with the individual protomers consisting of an α+ß barrel. Each protomer contains a deep hydrophobic pocket which is proposed to constitute the active site of the enzyme. To investigate the reaction catalysed by IdmH, 88% of the backbone NMR resonances were assigned, and using chemical shift perturbation of [15N]-labelled IdmH it was demonstrated that indanomycin binds in the active-site pocket. Finally, combined quantum mechanical/molecular mechanical (QM/MM) modelling of the IdmH reaction shows that the active site of the enzyme provides an appropriate environment to promote indane-ring formation, supporting the assignment of IdmH as the key Diels-Alderase catalysing the final step in the biosynthesis of indanomycin through a similar mechanism to other recently characterized Diels-Alderases involved in polyketide-tailoring reactions. An animated Interactive 3D Complement (I3DC) is available in Proteopedia at https://proteopedia.org/w/Journal:IUCrJ:S2052252519012399.
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Cryo-electron microscopy (cryo-EM) can now be used to determine high-resolution structural information on a diverse range of biological specimens. Recent advances have been driven primarily by developments in microscopes and detectors, and through advances in image-processing software. However, for many single-particle cryo-EM projects, major bottlenecks currently remain at the sample-preparation stage; obtaining cryo-EM grids of sufficient quality for high-resolution single-particle analysis can require the careful optimization of many variables. Common hurdles to overcome include problems associated with the sample itself (buffer components, labile complexes), sample distribution (obtaining the correct concentration, affinity for the support film), preferred orientation, and poor reproducibility of the grid-making process within and between batches. This review outlines a number of methodologies used within the electron-microscopy community to address these challenges, providing a range of approaches which may aid in obtaining optimal grids for high-resolution data collection.