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1.
Phys Rev Lett ; 126(4): 048101, 2021 Jan 29.
Artículo en Inglés | MEDLINE | ID: mdl-33576647

RESUMEN

Recent advances in microscopy techniques make it possible to study the growth, dynamics, and response of complex biophysical systems at single-cell resolution, from bacterial communities to tissues and organoids. In contrast to ordered crystals, it is less obvious how one can reliably distinguish two amorphous yet structurally different cellular materials. Here, we introduce a topological earth mover's (TEM) distance between disordered structures that compares local graph neighborhoods of the microscopic cell-centroid networks. Leveraging structural information contained in the neighborhood motif distributions, the TEM metric allows an interpretable reconstruction of equilibrium and nonequilibrium phase spaces and embedded pathways from static system snapshots alone. Applied to cell-resolution imaging data, the framework recovers time ordering without prior knowledge about the underlying dynamics, revealing that fly wing development solves a topological optimal transport problem. Extending our topological analysis to bacterial swarms, we find a universal neighborhood size distribution consistent with a Tracy-Widom law.


Asunto(s)
Modelos Teóricos , Reconocimiento de Normas Patrones Automatizadas/métodos , Algoritmos , Animales , Fenómenos Biofísicos , Coloides/química , Microscopía por Crioelectrón , Drosophila , Entropía , Células Epiteliales/citología , Interpretación de Imagen Asistida por Computador/métodos , Modelos Biológicos , Modelos Químicos , ARN/química
2.
Nat Commun ; 12(1): 1100, 2021 02 17.
Artículo en Inglés | MEDLINE | ID: mdl-33597543

RESUMEN

Photosystem I (PSI) and II (PSII) balance their light energy distribution absorbed by their light-harvesting complexes (LHCs) through state transition to maintain the maximum photosynthetic performance and to avoid photodamage. In state 2, a part of LHCII moves to PSI, forming a PSI-LHCI-LHCII supercomplex. The green alga Chlamydomonas reinhardtii exhibits state transition to a far larger extent than higher plants. Here we report the cryo-electron microscopy structure of a PSI-LHCI-LHCII supercomplex in state 2 from C. reinhardtii at 3.42 Å resolution. The result reveals that the PSI-LHCI-LHCII of C. reinhardtii binds two LHCII trimers in addition to ten LHCI subunits. The PSI core subunits PsaO and PsaH, which were missed or not well-resolved in previous Cr-PSI-LHCI structures, are observed. The present results reveal the organization and assembly of PSI core subunits, LHCI and LHCII, pigment arrangement, and possible pathways of energy transfer from peripheral antennae to the PSI core.


Asunto(s)
Proteínas Algáceas/metabolismo , Chlamydomonas reinhardtii/metabolismo , Complejos de Proteína Captadores de Luz/metabolismo , Complejo de Proteína del Fotosistema I/metabolismo , Proteínas Algáceas/química , Proteínas Algáceas/ultraestructura , Clorofila/metabolismo , Microscopía por Crioelectrón , Transferencia de Energía , Complejos de Proteína Captadores de Luz/química , Complejos de Proteína Captadores de Luz/ultraestructura , Modelos Moleculares , Fotosíntesis , Complejo de Proteína del Fotosistema I/química , Complejo de Proteína del Fotosistema I/ultraestructura , Complejo de Proteína del Fotosistema II/química , Complejo de Proteína del Fotosistema II/metabolismo , Complejo de Proteína del Fotosistema II/ultraestructura , Unión Proteica , Conformación Proteica , Multimerización de Proteína , Tilacoides/metabolismo , Tilacoides/ultraestructura
3.
Nat Commun ; 12(1): 739, 2021 02 02.
Artículo en Inglés | MEDLINE | ID: mdl-33531497

RESUMEN

The proteasome activator PA28αß affects MHC class I antigen presentation by associating with immunoproteasome core particles (iCPs). However, due to the lack of a mammalian PA28αß-iCP structure, how PA28αß regulates proteasome remains elusive. Here we present the complete architectures of the mammalian PA28αß-iCP immunoproteasome and free iCP at near atomic-resolution by cryo-EM, and determine the spatial arrangement between PA28αß and iCP through XL-MS. Our structures reveal a slight leaning of PA28αß towards the α3-α4 side of iCP, disturbing the allosteric network of the gatekeeper α2/3/4 subunits, resulting in a partial open iCP gate. We find that the binding and activation mechanism of iCP by PA28αß is distinct from those of constitutive CP by the homoheptameric TbPA26 or PfPA28. Our study sheds lights on the mechanism of enzymatic activity stimulation of immunoproteasome and suggests that PA28αß-iCP has experienced profound remodeling during evolution to achieve its current level of function in immune response.


Asunto(s)
Microscopía por Crioelectrón/métodos , Complejo de la Endopetidasa Proteasomal/inmunología , Complejo de la Endopetidasa Proteasomal/ultraestructura , Presentación de Antígeno/inmunología , Antígenos de Histocompatibilidad Clase I/inmunología , Humanos , Complejo de la Endopetidasa Proteasomal/metabolismo
4.
Nat Commun ; 12(1): 1074, 2021 02 16.
Artículo en Inglés | MEDLINE | ID: mdl-33594077

RESUMEN

Pentameric ligand-gated ion channels (pLGICs) of the Cys-loop receptor family are key players in fast signal transduction throughout the nervous system. They have been shown to be modulated by the lipid environment, however the underlying mechanism is not well understood. We report three structures of the Cys-loop 5-HT3A serotonin receptor (5HT3R) reconstituted into saposin-based lipid bilayer discs: a symmetric and an asymmetric apo state, and an asymmetric agonist-bound state. In comparison to previously published 5HT3R conformations in detergent, the lipid bilayer stabilises the receptor in a more tightly packed, 'coupled' state, involving a cluster of highly conserved residues. In consequence, the agonist-bound receptor conformation adopts a wide-open pore capable of conducting sodium ions in unbiased molecular dynamics (MD) simulations. Taken together, we provide a structural basis for the modulation of 5HT3R by the membrane environment, and a model for asymmetric activation of the receptor.


Asunto(s)
Membrana Dobles de Lípidos/metabolismo , Multimerización de Proteína , Receptores de Serotonina 5-HT3/química , Receptores de Serotonina 5-HT3/metabolismo , Animales , Apoproteínas/química , Apoproteínas/metabolismo , Línea Celular , Microscopía por Crioelectrón , Lípidos/química , Ratones , Modelos Biológicos , Modelos Moleculares , Conformación Proteica , Subunidades de Proteína/química , Subunidades de Proteína/metabolismo , Receptores de Serotonina 5-HT3/ultraestructura , Serotonina/farmacología
5.
Nat Commun ; 12(1): 758, 2021 02 03.
Artículo en Inglés | MEDLINE | ID: mdl-33536435

RESUMEN

RNA polymerase (Pol) I transcribes the ribosomal RNA precursor in all eukaryotes. The mechanisms 'activation by cleft contraction' and 'hibernation by dimerization' are unique to the regulation of this enzyme, but structure-function analysis is limited to baker's yeast. To understand whether regulation by such strategies is specific to this model organism or conserved among species, we solve three cryo-EM structures of Pol I from Schizosaccharomyces pombe in different functional states. Comparative analysis of structural models derived from high-resolution reconstructions shows that activation is accomplished by a conserved contraction of the active center cleft. In contrast to current beliefs, we find that dimerization of the S. pombe polymerase is also possible. This dimerization is achieved independent of the 'connector' domain but relies on two previously undescribed interfaces. Our analyses highlight the divergent nature of Pol I transcription systems from their counterparts and suggest conservation of regulatory mechanisms among organisms.


Asunto(s)
ARN Polimerasa I/química , Proteínas de Schizosaccharomyces pombe/química , Schizosaccharomyces/enzimología , Transcripción Genética , Secuencia de Aminoácidos , Secuencia de Bases , Microscopía por Crioelectrón , Modelos Moleculares , Conformación Proteica , Multimerización de Proteína , Subunidades de Proteína/química , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismo , ARN Polimerasa I/genética , ARN Polimerasa I/metabolismo , ARN Ribosómico/genética , Saccharomyces cerevisiae/enzimología , Saccharomyces cerevisiae/genética , Schizosaccharomyces/genética , Proteínas de Schizosaccharomyces pombe/genética , Proteínas de Schizosaccharomyces pombe/metabolismo , Homología de Secuencia de Aminoácido
6.
Nat Commun ; 12(1): 837, 2021 02 05.
Artículo en Inglés | MEDLINE | ID: mdl-33547281

RESUMEN

Coronaviruses of bats and pangolins have been implicated in the origin and evolution of the pandemic SARS-CoV-2. We show that spikes from Guangdong Pangolin-CoVs, closely related to SARS-CoV-2, bind strongly to human and pangolin ACE2 receptors. We also report the cryo-EM structure of a Pangolin-CoV spike protein and show it adopts a fully-closed conformation and that, aside from the Receptor-Binding Domain, it resembles the spike of a bat coronavirus RaTG13 more than that of SARS-CoV-2.


Asunto(s)
/prevención & control , Evolución Molecular , Glicoproteína de la Espiga del Coronavirus/genética , /metabolismo , Animales , Unión Competitiva , /virología , Microscopía por Crioelectrón , Humanos , Modelos Moleculares , Pandemias , Unión Proteica , Dominios Proteicos , /fisiología , Glicoproteína de la Espiga del Coronavirus/química , Glicoproteína de la Espiga del Coronavirus/metabolismo
7.
Proc Natl Acad Sci U S A ; 118(7)2021 02 16.
Artículo en Inglés | MEDLINE | ID: mdl-33526596

RESUMEN

The RNA polymerase inhibitor favipiravir is currently in clinical trials as a treatment for infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), despite limited information about the molecular basis for its activity. Here we report the structure of favipiravir ribonucleoside triphosphate (favipiravir-RTP) in complex with the SARS-CoV-2 RNA-dependent RNA polymerase (RdRp) bound to a template:primer RNA duplex, determined by electron cryomicroscopy (cryoEM) to a resolution of 2.5 Å. The structure shows clear evidence for the inhibitor at the catalytic site of the enzyme, and resolves the conformation of key side chains and ions surrounding the binding pocket. Polymerase activity assays indicate that the inhibitor is weakly incorporated into the RNA primer strand, and suppresses RNA replication in the presence of natural nucleotides. The structure reveals an unusual, nonproductive binding mode of favipiravir-RTP at the catalytic site of SARS-CoV-2 RdRp, which explains its low rate of incorporation into the RNA primer strand. Together, these findings inform current and future efforts to develop polymerase inhibitors for SARS coronaviruses.


Asunto(s)
Amidas/farmacología , Inhibidores Enzimáticos/farmacología , Pirazinas/farmacología , /ultraestructura , Amidas/química , /química , Microscopía por Crioelectrón/métodos , Inhibidores Enzimáticos/química , Pirazinas/química , Ribonucleótidos/química , /enzimología , Imagen Individual de Molécula/métodos
8.
Nat Commun ; 12(1): 875, 2021 02 08.
Artículo en Inglés | MEDLINE | ID: mdl-33558536

RESUMEN

Systemic AL amyloidosis is a debilitating and potentially fatal disease that arises from the misfolding and fibrillation of immunoglobulin light chains (LCs). The disease is patient-specific with essentially each patient possessing a unique LC sequence. In this study, we present two ex vivo fibril structures of a λ3 LC. The fibrils were extracted from the explanted heart of a patient (FOR005) and consist of 115-residue fibril proteins, mainly from the LC variable domain. The fibril structures imply that a 180° rotation around the disulfide bond and a major unfolding step are necessary for fibrils to form. The two fibril structures show highly similar fibril protein folds, differing in only a 12-residue segment. Remarkably, the two structures do not represent separate fibril morphologies, as they can co-exist at different z-axial positions within the same fibril. Our data imply the presence of structural breaks at the interface of the two structural forms.


Asunto(s)
Amiloide/ultraestructura , Microscopía por Crioelectrón , Amiloidosis de Cadenas Ligeras de las Inmunoglobulinas/metabolismo , Secuencia de Aminoácidos , Femenino , Humanos , Cadenas Ligeras de Inmunoglobulina/metabolismo , Persona de Mediana Edad , Mutación/genética , Agregado de Proteínas , Conformación Proteica
9.
Nat Commun ; 12(1): 815, 2021 02 05.
Artículo en Inglés | MEDLINE | ID: mdl-33547286

RESUMEN

Narcolepsy type 1 (NT1) is a chronic neurological disorder that impairs the brain's ability to control sleep-wake cycles. Current therapies are limited to the management of symptoms with modest effectiveness and substantial adverse effects. Agonists of the orexin receptor 2 (OX2R) have shown promise as novel therapeutics that directly target the pathophysiology of the disease. However, identification of drug-like OX2R agonists has proven difficult. Here we report cryo-electron microscopy structures of active-state OX2R bound to an endogenous peptide agonist and a small-molecule agonist. The extended carboxy-terminal segment of the peptide reaches into the core of OX2R to stabilize an active conformation, while the small-molecule agonist binds deep inside the orthosteric pocket, making similar key interactions. Comparison with antagonist-bound OX2R suggests a molecular mechanism that rationalizes both receptor activation and inhibition. Our results enable structure-based discovery of therapeutic orexin agonists for the treatment of NT1 and other hypersomnia disorders.


Asunto(s)
Aminopiridinas/química , Azepinas/química , Antagonistas de los Receptores de Orexina/química , Receptores de Orexina/química , Péptidos/química , Fármacos Inductores del Sueño/química , Sulfonamidas/química , Triazoles/química , Aminopiridinas/metabolismo , Azepinas/metabolismo , Sitios de Unión , Clonación Molecular , Microscopía por Crioelectrón , Escherichia coli/genética , Escherichia coli/metabolismo , Expresión Génica , Vectores Genéticos/química , Vectores Genéticos/metabolismo , Células HEK293 , Humanos , Simulación de Dinámica Molecular , Antagonistas de los Receptores de Orexina/metabolismo , Receptores de Orexina/agonistas , Receptores de Orexina/metabolismo , Péptidos/metabolismo , Unión Proteica , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Dominios y Motivos de Interacción de Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Fármacos Inductores del Sueño/metabolismo , Sulfonamidas/metabolismo , Triazoles/metabolismo
10.
Nat Commun ; 12(1): 819, 2021 02 05.
Artículo en Inglés | MEDLINE | ID: mdl-33547302

RESUMEN

Regulated cell death is essential in development and cellular homeostasis. Multi-protein platforms, including the Death-Inducing Signaling Complex (DISC), co-ordinate cell fate via a core FADD:Caspase-8 complex and its regulatory partners, such as the cell death inhibitor c-FLIP. Here, using electron microscopy, we visualize full-length procaspase-8 in complex with FADD. Our structural analysis now reveals how the FADD-nucleated tandem death effector domain (tDED) helical filament is required to orientate the procaspase-8 catalytic domains, enabling their activation via anti-parallel dimerization. Strikingly, recruitment of c-FLIPS into this complex inhibits Caspase-8 activity by altering tDED triple helix architecture, resulting in steric hindrance of the canonical tDED Type I binding site. This prevents both Caspase-8 catalytic domain assembly and tDED helical filament elongation. Our findings reveal how the plasticity, composition and architecture of the core FADD:Caspase-8 complex critically defines life/death decisions not only via the DISC, but across multiple key signaling platforms including TNF complex II, the ripoptosome, and RIPK1/RIPK3 necrosome.


Asunto(s)
Proteína Reguladora de Apoptosis Similar a CASP8 y FADD/química , Caspasa 8/química , Proteína de Dominio de Muerte Asociada a Fas/química , Proteína Serina-Treonina Quinasas de Interacción con Receptores/química , Proteína Reguladora de Apoptosis Similar a CASP8 y FADD/genética , Proteína Reguladora de Apoptosis Similar a CASP8 y FADD/metabolismo , Caspasa 8/genética , Caspasa 8/metabolismo , Dominio Catalítico , Clonación Molecular , Microscopía por Crioelectrón , Proteínas Adaptadoras de Señalización del Receptor del Dominio de Muerte/química , Proteínas Adaptadoras de Señalización del Receptor del Dominio de Muerte/genética , Proteínas Adaptadoras de Señalización del Receptor del Dominio de Muerte/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Proteína de Dominio de Muerte Asociada a Fas/genética , Proteína de Dominio de Muerte Asociada a Fas/metabolismo , Expresión Génica , Vectores Genéticos/química , Vectores Genéticos/metabolismo , Células HEK293 , Humanos , Modelos Moleculares , Unión Proteica , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Dominios y Motivos de Interacción de Proteínas , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Multimerización de Proteína , Proteína Serina-Treonina Quinasas de Interacción con Receptores/genética , Proteína Serina-Treonina Quinasas de Interacción con Receptores/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Muerte Celular Regulada/genética , Factor de Necrosis Tumoral alfa/química , Factor de Necrosis Tumoral alfa/genética , Factor de Necrosis Tumoral alfa/metabolismo
11.
Nat Commun ; 12(1): 807, 2021 02 05.
Artículo en Inglés | MEDLINE | ID: mdl-33547325

RESUMEN

Ryanodine Receptors (RyRs) are massive channels that release Ca2+ from the endoplasmic and sarcoplasmic reticulum. Hundreds of mutations are linked to malignant hyperthermia (MH), myopathies, and arrhythmias. Here, we explore the first MH mutation identified in humans by providing cryo-EM snapshots of the pig homolog, R615C, showing that it affects an interface between three solenoid regions. We also show the impact of apo-calmodulin (apoCaM) and how it can induce opening by bending of the bridging solenoid, mediated by its N-terminal lobe. For R615C RyR1, apoCaM binding abolishes a pathological 'intermediate' conformation, distributing the population to a mixture of open and closed channels, both different from the structure without apoCaM. Comparisons show that the mutation primarily affects the closed state, inducing partial movements linked to channel activation. This shows that disease mutations can cause distinct pathological conformations of the RyR and facilitate channel opening by disrupting interactions between different solenoid regions.


Asunto(s)
Apoproteínas/química , Calcio/química , Calmodulina/química , Hipertermia Maligna/metabolismo , Canal Liberador de Calcio Receptor de Rianodina/química , Sustitución de Aminoácidos , Animales , Apoproteínas/genética , Apoproteínas/metabolismo , Arginina/química , Arginina/metabolismo , Calcio/metabolismo , Calmodulina/genética , Calmodulina/metabolismo , Microscopía por Crioelectrón , Cisteína/química , Cisteína/metabolismo , Expresión Génica , Humanos , Transporte Iónico , Hipertermia Maligna/genética , Hipertermia Maligna/patología , Modelos Moleculares , Músculo Esquelético/química , Músculo Esquelético/metabolismo , Mutación , Unión Proteica , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Dominios y Motivos de Interacción de Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Canal Liberador de Calcio Receptor de Rianodina/genética , Canal Liberador de Calcio Receptor de Rianodina/metabolismo , Retículo Sarcoplasmático/química , Retículo Sarcoplasmático/metabolismo , Homología de Secuencia de Aminoácido , Especificidad por Sustrato , Porcinos
12.
BMC Bioinformatics ; 22(1): 55, 2021 Feb 08.
Artículo en Inglés | MEDLINE | ID: mdl-33557750

RESUMEN

BACKGROUND: Identification and selection of protein particles in cryo-electron micrographs is an important step in single particle analysis. In this study, we developed a deep learning-based particle picking network to automatically detect particle centers from cryoEM micrographs. This is a challenging task due to the nature of cryoEM data, having low signal-to-noise ratios with variable particle sizes, shapes, distributions, grayscale variations as well as other undesirable artifacts. RESULTS: We propose a double convolutional neural network (CNN) cascade for automated detection of particles in cryo-electron micrographs. This approach, entitled Deep Regression Picker Network or "DRPnet", is simple but very effective in recognizing different particle sizes, shapes, distributions and grayscale patterns corresponding to 2D views of 3D particles. Particles are detected by the first network, a fully convolutional regression network (FCRN), which maps the particle image to a continuous distance map that acts like a probability density function of particle centers. Particles identified by FCRN are further refined to reduce false particle detections by the second classification CNN. DRPnet's first CNN pretrained with only a single cryoEM dataset can be used to detect particles from different datasets without retraining. Compared to RELION template-based autopicking, DRPnet results in better particle picking performance with drastically reduced user interactions and processing time. DRPnet also outperforms the state-of-the-art particle picking networks in terms of the supervised detection evaluation metrics recall, precision, and F-measure. To further highlight quality of the picked particle sets, we compute and present additional performance metrics assessing the resulting 3D reconstructions such as number of 2D class averages, efficiency/angular coverage, Rosenthal-Henderson plots and local/global 3D reconstruction resolution. CONCLUSION: DRPnet shows greatly improved time-savings to generate an initial particle dataset compared to manual picking, followed by template-based autopicking. Compared to other networks, DRPnet has equivalent or better performance. DRPnet excels on cryoEM datasets that have low contrast or clumped particles. Evaluating other performance metrics, DRPnet is useful for higher resolution 3D reconstructions with decreased particle numbers or unknown symmetry, detecting particles with better angular orientation coverage.


Asunto(s)
Microscopía por Crioelectrón , Electrones , Procesamiento de Imagen Asistido por Computador , Análisis de Regresión , Imagenología Tridimensional , Redes Neurales de la Computación , Proteínas , Relación Señal-Ruido
13.
Nat Commun ; 12(1): 785, 2021 02 04.
Artículo en Inglés | MEDLINE | ID: mdl-33542223

RESUMEN

The binding of cytoplasmic Ca2+ to the anion-selective channel TMEM16A triggers a conformational change around its binding site that is coupled to the release of a gate at the constricted neck of an hourglass-shaped pore. By combining mutagenesis, electrophysiology, and cryo-electron microscopy, we identified three hydrophobic residues at the intracellular entrance of the neck as constituents of this gate. Mutation of each of these residues increases the potency of Ca2+ and results in pronounced basal activity. The structure of an activating mutant shows a conformational change of an α-helix that contributes to Ca2+ binding as a likely cause for the basal activity. Although not in physical contact, the three residues are functionally coupled to collectively contribute to the stabilization of the gate in the closed conformation of the pore, thus explaining the low open probability of the channel in the absence of Ca2+.


Asunto(s)
Anoctamina-1/metabolismo , Calcio/metabolismo , Activación del Canal Iónico , Proteínas de Neoplasias/metabolismo , Anoctamina-1/genética , Anoctamina-1/ultraestructura , Sitios de Unión/genética , Cationes Bivalentes/metabolismo , Cloruros/metabolismo , Microscopía por Crioelectrón , Células HEK293 , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Modelos Moleculares , Mutagénesis , Mutación , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/ultraestructura , Unión Proteica , Conformación Proteica en Hélice alfa
14.
Nat Struct Mol Biol ; 28(2): 128-131, 2021 02.
Artículo en Inglés | MEDLINE | ID: mdl-33402708

RESUMEN

The SARS-CoV-2 spike (S) protein, a primary target for COVID-19 vaccine development, presents its receptor binding domain in two conformations, the receptor-accessible 'up' or receptor-inaccessible 'down' states. Here we report that the commonly used stabilized S ectodomain construct '2P' is sensitive to cold temperatures, and this cold sensitivity is abrogated in a 'down' state-stabilized ectodomain. Our findings will impact structural, functional and vaccine studies that use the SARS-CoV-2 S ectodomain.


Asunto(s)
Glicoproteína de la Espiga del Coronavirus/química , Anticuerpos Antivirales/química , Frío , Microscopía por Crioelectrón , Ensayo de Inmunoadsorción Enzimática , Humanos , Desnaturalización Proteica , Dominios Proteicos , Estabilidad Proteica , Glicoproteína de la Espiga del Coronavirus/ultraestructura , Resonancia por Plasmón de Superficie
15.
Nat Struct Mol Biol ; 28(2): 202-209, 2021 02.
Artículo en Inglés | MEDLINE | ID: mdl-33432247

RESUMEN

Effective intervention strategies are urgently needed to control the COVID-19 pandemic. Human angiotensin-converting enzyme 2 (ACE2) is a membrane-bound carboxypeptidase that forms a dimer and serves as the cellular receptor for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). ACE2 is also a key negative regulator of the renin-angiotensin system that modulates vascular functions. We report here the properties of a trimeric ACE2 ectodomain variant, engineered using a structure-based approach. The trimeric ACE2 variant has a binding affinity of ~60 pM for the spike protein of SARS­CoV­2 (compared with 77 nM for monomeric ACE2 and 12-22 nM for dimeric ACE2 constructs), and its peptidase activity and the ability to block activation of angiotensin II receptor type 1 in the renin-angiotensin system are preserved. Moreover, the engineered ACE2 potently inhibits SARS­CoV­2 infection in cell culture. These results suggest that engineered, trimeric ACE2 may be a promising anti-SARS-CoV-2 agent for treating COVID-19.


Asunto(s)
/química , Antivirales/química , /tratamiento farmacológico , /genética , Antivirales/uso terapéutico , Microscopía por Crioelectrón , Humanos , Modelos Moleculares , Ingeniería de Proteínas , Multimerización de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/uso terapéutico , /fisiología
16.
Nat Commun ; 12(1): 244, 2021 01 11.
Artículo en Inglés | MEDLINE | ID: mdl-33431842

RESUMEN

The trimeric spike (S) protein of SARS-CoV-2 is the primary focus of most vaccine design and development efforts. Due to intrinsic instability typical of class I fusion proteins, S tends to prematurely refold to the post-fusion conformation, compromising immunogenic properties and prefusion trimer yields. To support ongoing vaccine development efforts, we report the structure-based design of soluble S trimers with increased yields and stabilities, based on introduction of single point mutations and disulfide-bridges. We identify regions critical for stability: the heptad repeat region 1, the SD1 domain and position 614 in SD2. We combine a minimal selection of mostly interprotomeric mutations to create a stable S-closed variant with a 6.4-fold higher expression than the parental construct while no longer containing a heterologous trimerization domain. The cryo-EM structure reveals a correctly folded, predominantly closed pre-fusion conformation. Highly stable and well producing S protein and the increased understanding of S protein structure will support vaccine development and serological diagnostics.


Asunto(s)
Glicoproteína de la Espiga del Coronavirus/química , Glicoproteína de la Espiga del Coronavirus/metabolismo , /química , /virología , Microscopía por Crioelectrón , Humanos , Modelos Moleculares , Mutación , Conformación Proteica , Dominios Proteicos , Estabilidad Proteica , /genética , Glicoproteína de la Espiga del Coronavirus/genética
17.
Nat Commun ; 12(1): 230, 2021 01 11.
Artículo en Inglés | MEDLINE | ID: mdl-33431861

RESUMEN

Infection of the human stomach by Helicobacter pylori remains a worldwide problem and greatly contributes to peptic ulcer disease and gastric cancer. Without active intervention approximately 50% of the world population will continue to be infected with this gastric pathogen. Current eradication, called triple therapy, entails a proton-pump inhibitor and two broadband antibiotics, however resistance to either clarithromycin or metronidazole is greater than 25% and rising. Therefore, there is an urgent need for a targeted, high-specificity eradication drug. Gastric infection by H. pylori depends on the expression of a nickel-dependent urease in the cytoplasm of the bacteria. Here, we report the 2.0 Å resolution structure of the 1.1 MDa urease in complex with an inhibitor by cryo-electron microscopy and compare it to a ß-mercaptoethanol-inhibited structure at 2.5 Å resolution. The structural information is of sufficient detail to aid in the development of inhibitors with high specificity and affinity.


Asunto(s)
Microscopía por Crioelectrón , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Helicobacter pylori/enzimología , Ureasa/antagonistas & inhibidores , Ureasa/ultraestructura , Dominio Catalítico , Concentración de Iones de Hidrógeno , Modelos Moleculares
18.
Nat Commun ; 12(1): 264, 2021 01 11.
Artículo en Inglés | MEDLINE | ID: mdl-33431876

RESUMEN

The ongoing pandemic of coronavirus disease 2019 (COVID-19) is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Neutralizing antibodies against SARS-CoV-2 are an option for drug development for treating COVID-19. Here, we report the identification and characterization of two groups of mouse neutralizing monoclonal antibodies (MAbs) targeting the receptor-binding domain (RBD) on the SARS-CoV-2 spike (S) protein. MAbs 2H2 and 3C1, representing the two antibody groups, respectively, bind distinct epitopes and are compatible in formulating a noncompeting antibody cocktail. A humanized version of the 2H2/3C1 cocktail is found to potently neutralize authentic SARS-CoV-2 infection in vitro with half inhibitory concentration (IC50) of 12 ng/mL and effectively treat SARS-CoV-2-infected mice even when administered at as late as 24 h post-infection. We determine an ensemble of cryo-EM structures of 2H2 or 3C1 Fab in complex with the S trimer up to 3.8 Å resolution, revealing the conformational space of the antigen-antibody complexes and MAb-triggered stepwise allosteric rearrangements of the S trimer, delineating a previously uncharacterized dynamic process of coordinated binding of neutralizing antibodies to the trimeric S protein. Our findings provide important information for the development of MAb-based drugs for preventing and treating SARS-CoV-2 infections.


Asunto(s)
Anticuerpos Monoclonales/química , Anticuerpos Monoclonales/farmacología , Anticuerpos Antivirales/química , Anticuerpos Antivirales/farmacología , /efectos de los fármacos , Animales , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/uso terapéutico , Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/inmunología , Anticuerpos Antivirales/uso terapéutico , Microscopía por Crioelectrón , Mapeo Epitopo , Epítopos , Femenino , Ratones , Ratones Endogámicos BALB C , Modelos Moleculares , Unión Proteica/efectos de los fármacos , Conformación Proteica , Glicoproteína de la Espiga del Coronavirus/química , Glicoproteína de la Espiga del Coronavirus/inmunología
19.
Viruses ; 13(2)2021 Jan 20.
Artículo en Inglés | MEDLINE | ID: mdl-33498475

RESUMEN

The paper covers the history of the discovery and description of phiKZ, the first known giant bacteriophage active on Pseudomonas aeruginosa. It also describes its unique features, especially the characteristic manner of DNA packing in the head around a cylinder-shaped structure ("inner body"), which probably governs an ordered and tight packaging of the phage genome. Important properties of phiKZ-like phages include a wide range of lytic activity and the blue opalescence of their negative colonies, and provide a background for the search and discovery of new P. aeruginosa giant phages. The importance of the phiKZ species and of other giant phage species in practical phage therapy is noted given their broad use in commercial phage preparations.


Asunto(s)
Genoma Viral , Fagos Pseudomonas/genética , Fagos Pseudomonas/fisiología , Pseudomonas aeruginosa/virología , Microscopía por Crioelectrón , Terapia de Fagos , Filogenia , Fagos Pseudomonas/ultraestructura
20.
Science ; 371(6530)2021 02 12.
Artículo en Inglés | MEDLINE | ID: mdl-33436526

RESUMEN

The pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to spread, with devastating consequences. For passive immunization efforts, nanobodies have size and cost advantages over conventional antibodies. In this study, we generated four neutralizing nanobodies that target the receptor binding domain of the SARS-CoV-2 spike protein. We used x-ray crystallography and cryo-electron microscopy to define two distinct binding epitopes. On the basis of these structures, we engineered multivalent nanobodies with more than 100 times the neutralizing activity of monovalent nanobodies. Biparatopic nanobody fusions suppressed the emergence of escape mutants. Several nanobody constructs neutralized through receptor binding competition, whereas other monovalent and biparatopic nanobodies triggered aberrant activation of the spike fusion machinery. These premature conformational changes in the spike protein forestalled productive fusion and rendered the virions noninfectious.


Asunto(s)
Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/inmunología , /inmunología , Anticuerpos de Dominio Único/inmunología , Glicoproteína de la Espiga del Coronavirus/inmunología , Sustitución de Aminoácidos , Animales , Anticuerpos Neutralizantes/química , Anticuerpos Neutralizantes/metabolismo , Anticuerpos Antivirales/química , Anticuerpos Antivirales/metabolismo , Afinidad de Anticuerpos , Antígenos Virales/inmunología , Sitios de Unión de Anticuerpos , Línea Celular , Microscopía por Crioelectrón , Epítopos , Humanos , Fusión de Membrana , Mutación , Unión Proteica , Conformación Proteica , Dominios Proteicos , /genética , Anticuerpos de Dominio Único/química , Anticuerpos de Dominio Único/metabolismo , Glicoproteína de la Espiga del Coronavirus/química , Glicoproteína de la Espiga del Coronavirus/genética , Glicoproteína de la Espiga del Coronavirus/metabolismo , Replicación Viral
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