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1.
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
2.
Sci Rep ; 11(1): 3238, 2021 02 05.
Artículo en Inglés | MEDLINE | ID: mdl-33547334

RESUMEN

The rampant spread of COVID-19, an infectious disease caused by SARS-CoV-2, all over the world has led to over millions of deaths, and devastated the social, financial and political entities around the world. Without an existing effective medical therapy, vaccines are urgently needed to avoid the spread of this disease. In this study, we propose an in silico deep learning approach for prediction and design of a multi-epitope vaccine (DeepVacPred). By combining the in silico immunoinformatics and deep neural network strategies, the DeepVacPred computational framework directly predicts 26 potential vaccine subunits from the available SARS-CoV-2 spike protein sequence. We further use in silico methods to investigate the linear B-cell epitopes, Cytotoxic T Lymphocytes (CTL) epitopes, Helper T Lymphocytes (HTL) epitopes in the 26 subunit candidates and identify the best 11 of them to construct a multi-epitope vaccine for SARS-CoV-2 virus. The human population coverage, antigenicity, allergenicity, toxicity, physicochemical properties and secondary structure of the designed vaccine are evaluated via state-of-the-art bioinformatic approaches, showing good quality of the designed vaccine. The 3D structure of the designed vaccine is predicted, refined and validated by in silico tools. Finally, we optimize and insert the codon sequence into a plasmid to ensure the cloning and expression efficiency. In conclusion, this proposed artificial intelligence (AI) based vaccine discovery framework accelerates the vaccine design process and constructs a 694aa multi-epitope vaccine containing 16 B-cell epitopes, 82 CTL epitopes and 89 HTL epitopes, which is promising to fight the SARS-CoV-2 viral infection and can be further evaluated in clinical studies. Moreover, we trace the RNA mutations of the SARS-CoV-2 and ensure that the designed vaccine can tackle the recent RNA mutations of the virus.


Asunto(s)
Aprendizaje Profundo , Glicoproteína de la Espiga del Coronavirus/inmunología , Alérgenos , /efectos adversos , /inmunología , Uso de Codones , Biología Computacional , Diseño de Fármacos , Epítopos de Linfocito B/inmunología , Epítopos de Linfocito T/inmunología , Humanos , Inmunogenicidad Vacunal , Modelos Moleculares , Simulación del Acoplamiento Molecular , Simulación de Dinámica Molecular , Mutación , Conformación Proteica , ARN Viral , /genética , Solubilidad , Glicoproteína de la Espiga del Coronavirus/química , Glicoproteína de la Espiga del Coronavirus/genética , Linfocitos T Citotóxicos/inmunología , Linfocitos T Colaboradores-Inductores/inmunología , Vacunas de Subunidad/química , Vacunas de Subunidad/inmunología
3.
Nat Commun ; 12(1): 867, 2021 02 08.
Artículo en Inglés | MEDLINE | ID: mdl-33558520

RESUMEN

Statins are effective cholesterol-lowering drugs. Lovastatin, one of the precursors of statins, is formed from dihydromonacolin L (DML), which is synthesized by lovastatin nonaketide synthase (LovB), with the assistance of a separate trans-acting enoyl reductase (LovC). A full DML synthesis comprises 8 polyketide synthetic cycles with about 35 steps. The assembling of the LovB-LovC complex, and the structural basis for the iterative and yet permutative functions of the megasynthase have remained a mystery. Here, we present the cryo-EM structures of the LovB-LovC complex at 3.60 Å and the core LovB at 2.91 Å resolution. The domain organization of LovB is an X-shaped face-to-face dimer containing eight connected domains. The binding of LovC laterally to the malonyl-acetyl transferase domain allows the completion of a L-shaped catalytic chamber consisting of six active domains. This architecture and the structural details of the megasynthase provide the basis for the processing of the intermediates by the individual catalytic domains. The detailed architectural model provides structural insights that may enable the re-engineering of the megasynthase for the generation of new statins.


Asunto(s)
Lovastatina/biosíntesis , Lovastatina/química , Biocatálisis , Modelos Moleculares , Naftalenos/metabolismo , Oxidorreductasas actuantes sobre Donantes de Grupo CH-CH/química , Oxidorreductasas actuantes sobre Donantes de Grupo CH-CH/metabolismo , Sintasas Poliquetidas/química , Sintasas Poliquetidas/metabolismo , Sintasas Poliquetidas/ultraestructura , Dominios Proteicos , Especificidad por Sustrato
4.
Nat Commun ; 12(1): 856, 2021 02 08.
Artículo en Inglés | MEDLINE | ID: mdl-33558528

RESUMEN

Through the efforts of many groups, a wide range of fluorescent protein reporters and sensors based on green fluorescent protein and its relatives have been engineered in recent years. Here we explore the incorporation of sensing modalities into de novo designed fluorescence-activating proteins, called mini-fluorescence-activating proteins (mFAPs), that bind and stabilize the fluorescent cis-planar state of the fluorogenic compound DFHBI. We show through further design that the fluorescence intensity and specificity of mFAPs for different chromophores can be tuned, and the fluorescence made sensitive to pH and Ca2+ for real-time fluorescence reporting. Bipartite split mFAPs enable real-time monitoring of protein-protein association and (unlike widely used split GFP reporter systems) are fully reversible, allowing direct readout of association and dissociation events. The relative ease with which sensing modalities can be incorporated and advantages in smaller size and photostability make de novo designed fluorescence-activating proteins attractive candidates for optical sensor engineering.


Asunto(s)
Proteínas Luminiscentes/metabolismo , Acetilcolina/metabolismo , Animales , Células COS , Calcio/metabolismo , Chlorocebus aethiops , Fluorescencia , Colorantes Fluorescentes/metabolismo , Proteínas Fluorescentes Verdes/metabolismo , Células HEK293 , Humanos , Concentración de Iones de Hidrógeno , Proteínas Luminiscentes/química , Modelos Moleculares
5.
Molecules ; 26(4)2021 Feb 10.
Artículo en Inglés | MEDLINE | ID: mdl-33578831

RESUMEN

Currently, SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) has infected people among all countries and is a pandemic as declared by the World Health Organization (WHO). SARS-CoVID-2 main protease is one of the therapeutic drug targets that has been shown to reduce virus replication, and its high-resolution 3D structures in complex with inhibitors have been solved. Previously, we had demonstrated the potential of natural compounds such as serine protease inhibitors eventually leading us to hypothesize that FDA-approved marine drugs have the potential to inhibit the biological activity of SARS-CoV-2 main protease. Initially, field-template and structure-activity atlas models were constructed to understand and explain the molecular features responsible for SARS-CoVID-2 main protease inhibitors, which revealed that Eribulin Mesylate, Plitidepsin, and Trabectedin possess similar characteristics related to SARS-CoVID-2 main protease inhibitors. Later, protein-ligand interactions are studied using ensemble molecular-docking simulations that revealed that marine drugs bind at the active site of the main protease. The three-dimensional reference interaction site model (3D-RISM) studies show that marine drugs displace water molecules at the active site, and interactions observed are favorable. These computational studies eventually paved an interest in further in vitro studies. Finally, these findings are new and indeed provide insights into the role of FDA-approved marine drugs, which are already in clinical use for cancer treatment as a potential alternative to prevent and treat infected people with SARS-CoV-2.


Asunto(s)
Péptido Hidrolasas/química , Péptido Hidrolasas/metabolismo , Inhibidores de Serina Proteinasa/farmacología , Dominio Catalítico , Depsipéptidos/química , Depsipéptidos/farmacología , Reposicionamiento de Medicamentos , Furanos/química , Furanos/farmacología , Humanos , Cetonas/química , Cetonas/farmacología , Modelos Moleculares , Simulación del Acoplamiento Molecular , Relación Estructura-Actividad Cuantitativa , Inhibidores de Serina Proteinasa/química , Trabectedina/química , Trabectedina/farmacología , Proteínas Virales/antagonistas & inhibidores , Replicación Viral/efectos de los fármacos
6.
Nat Commun ; 12(1): 883, 2021 02 09.
Artículo en Inglés | MEDLINE | ID: mdl-33563988

RESUMEN

Recent advances in computational methods have enabled the predictive design of self-assembling protein nanomaterials with atomic-level accuracy. These design strategies focus exclusively on a single target structure, without consideration of the mechanism or dynamics of assembly. However, understanding the assembly process, and in particular its robustness to perturbation, will be critical for translating this class of materials into useful technologies. Here we investigate the assembly of two computationally designed, 120-subunit icosahedral complexes in detail using several complementary biochemical methods. We found that assembly of each material from its two constituent protein building blocks was highly cooperative and yielded exclusively complete, 120-subunit complexes except in one non-stoichiometric regime for one of the materials. Our results suggest that in vitro assembly provides a robust and controllable route for the manufacture of designed protein nanomaterials and confirm that cooperative assembly can be an intrinsic, rather than evolved, feature of hierarchically structured protein complexes.


Asunto(s)
Química Computacional , Nanoestructuras/química , Proteínas/química , Modelos Moleculares , Conformación Proteica , Multimerización de Proteína , Estabilidad Proteica , Subunidades de Proteína/química
7.
Molecules ; 26(4)2021 Feb 04.
Artículo en Inglés | MEDLINE | ID: mdl-33557115

RESUMEN

The 3CL-Protease appears to be a very promising medicinal target to develop anti-SARS-CoV-2 agents. The availability of resolved structures allows structure-based computational approaches to be carried out even though the lack of known inhibitors prevents a proper validation of the performed simulations. The innovative idea of the study is to exploit known inhibitors of SARS-CoV 3CL-Pro as a training set to perform and validate multiple virtual screening campaigns. Docking simulations using four different programs (Fred, Glide, LiGen, and PLANTS) were performed investigating the role of both multiple binding modes (by binding space) and multiple isomers/states (by developing the corresponding isomeric space). The computed docking scores were used to develop consensus models, which allow an in-depth comparison of the resulting performances. On average, the reached performances revealed the different sensitivity to isomeric differences and multiple binding modes between the four docking engines. In detail, Glide and LiGen are the tools that best benefit from isomeric and binding space, respectively, while Fred is the most insensitive program. The obtained results emphasize the fruitful role of combining various docking tools to optimize the predictive performances. Taken together, the performed simulations allowed the rational development of highly performing virtual screening workflows, which could be further optimized by considering different 3CL-Pro structures and, more importantly, by including true SARS-CoV-2 3CL-Pro inhibitors (as learning set) when available.


Asunto(s)
/virología , /enzimología , Antivirales/química , Antivirales/farmacología , Sitios de Unión , /antagonistas & inhibidores , Diseño de Fármacos , Evaluación Preclínica de Medicamentos/métodos , Reposicionamiento de Medicamentos/métodos , Humanos , Modelos Moleculares , Simulación del Acoplamiento Molecular/métodos , Péptido Hidrolasas/metabolismo , Inhibidores de Proteasas/química , Inhibidores de Proteasas/farmacología , Conformación Proteica
8.
J Vis Exp ; (167)2021 01 16.
Artículo en Inglés | MEDLINE | ID: mdl-33522501

RESUMEN

Protein structure elucidation using X-ray crystallography requires both high quality diffracting crystals and computational solution of the diffraction phase problem. Novel structures that lack a suitable homology model are often derivatized with heavy atoms to provide experimental phase information. The presented protocol efficiently generates derivatized protein crystals by combining random microseeding matrix screening with derivatization with a heavy atom molecule I3C (5-amino-2,4,6-triiodoisophthalic acid). By incorporating I3C into the crystal lattice, the diffraction phase problem can be efficiently solved using single wavelength anomalous dispersion (SAD) phasing. The equilateral triangle arrangement of iodine atoms in I3C allows for rapid validation of a correct anomalous substructure. This protocol will be useful to structural biologists who solve macromolecular structures using crystallography-based techniques with interest in experimental phasing.


Asunto(s)
Cristalografía por Rayos X , Proteínas/química , Ácidos Triyodobenzoicos/química , Animales , Pollos , Análisis de Datos , Difusión , Imagenología Tridimensional , Litio/química , Modelos Moleculares , Muramidasa/química
9.
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
10.
Nat Commun ; 12(1): 786, 2021 02 04.
Artículo en Inglés | MEDLINE | ID: mdl-33542228

RESUMEN

The anion channel TMEM16A is activated by intracellular Ca2+ in a highly cooperative process. By combining electrophysiology and autocorrelation analysis, we investigated the mechanism of channel activation and the concurrent rearrangement of the gate in the narrow part of the pore. Features in the fluctuation characteristics of steady-state current indicate the sampling of intermediate conformations that are successively occupied during gating. The initial step is related to conformational changes induced by Ca2+ binding, which is ensued by rearrangements that open the pore. Mutations in the gate shift the equilibrium of transitions in a manner consistent with a progressive destabilization of this region during pore opening. We come up with a mechanism of channel activation where the binding of Ca2+ induces conformational changes in the protein that, in a sequential manner, propagate from the binding site and couple to the gate in the narrow pore to allow ion permeation.


Asunto(s)
Anoctamina-1/metabolismo , Calcio/metabolismo , Activación del Canal Iónico , Modelos Moleculares , Proteínas de Neoplasias/metabolismo , Regulación Alostérica , Anoctamina-1/genética , Anoctamina-1/ultraestructura , Sitios de Unión/genética , Cationes Bivalentes/metabolismo , Cloruros/metabolismo , Células HEK293 , Humanos , Cinética , Método de Montecarlo , Mutación , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/ultraestructura , Técnicas de Placa-Clamp , Distribución de Poisson , Unión Proteica/genética , Conformación Proteica en Hélice alfa
11.
Commun Biol ; 4(1): 193, 2021 02 09.
Artículo en Inglés | MEDLINE | ID: mdl-33564093

RESUMEN

SARS-CoV-2 Nsp15 is a uridine-specific endoribonuclease with C-terminal catalytic domain belonging to the EndoU family that is highly conserved in coronaviruses. As endoribonuclease activity seems to be responsible for the interference with the innate immune response, Nsp15 emerges as an attractive target for therapeutic intervention. Here we report the first structures with bound nucleotides and show how the enzyme specifically recognizes uridine moiety. In addition to a uridine site we present evidence for a second base binding site that can accommodate any base. The structure with a transition state analog, uridine vanadate, confirms interactions key to catalytic mechanisms. In the presence of manganese ions, the enzyme cleaves unpaired RNAs. This acquired knowledge was instrumental in identifying Tipiracil, an FDA approved drug that is used in the treatment of colorectal cancer, as a potential anti-COVID-19 drug. Using crystallography, biochemical, and whole-cell assays, we demonstrate that Tipiracil inhibits SARS-CoV-2 Nsp15 by interacting with the uridine binding pocket in the enzyme's active site. Our findings provide new insights for the development of uracil scaffold-based drugs.


Asunto(s)
Antivirales/farmacología , /virología , Endorribonucleasas/antagonistas & inhibidores , Inhibidores Enzimáticos/farmacología , Pirrolidinas/farmacología , /enzimología , Timina/farmacología , Proteínas no Estructurales Virales/antagonistas & inhibidores , Células A549 , Antivirales/química , Antivirales/farmacocinética , Dominio Catalítico , Cristalografía por Rayos X , Endorribonucleasas/química , Endorribonucleasas/metabolismo , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacocinética , Humanos , Ligandos , Modelos Moleculares , Conformación Proteica , Pirrolidinas/química , Pirrolidinas/farmacocinética , Timina/química , Timina/farmacocinética , Uridina/metabolismo , Proteínas no Estructurales Virales/química , Proteínas no Estructurales Virales/metabolismo
12.
PLoS One ; 16(2): e0246731, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33571241

RESUMEN

SARS-CoV-2 antibodies develop within two weeks of infection, but wane relatively rapidly post-infection, raising concerns about whether antibody responses will provide protection upon re-exposure. Here we revisit T-B cooperation as a prerequisite for effective and durable neutralizing antibody responses centered on a mutationally constrained RBM B cell epitope. T-B cooperation requires co-processing of B and T cell epitopes by the same B cell and is subject to MHC-II restriction. We evaluated MHC-II constraints relevant to the neutralizing antibody response to a mutationally-constrained B cell epitope in the receptor binding motif (RBM) of the spike protein. Examining common MHC-II alleles, we found that peptides surrounding this key B cell epitope are predicted to bind poorly, suggesting a lack MHC-II support in T-B cooperation, impacting generation of high-potency neutralizing antibodies in the general population. Additionally, we found that multiple microbial peptides had potential for RBM cross-reactivity, supporting previous exposures as a possible source of T cell memory.


Asunto(s)
Anticuerpos Neutralizantes/inmunología , Epítopos de Linfocito B/inmunología , Antígenos de Histocompatibilidad Clase II/inmunología , Glicoproteína de la Espiga del Coronavirus/inmunología , Secuencias de Aminoácidos , Anticuerpos Antivirales/inmunología , Formación de Anticuerpos , Linfocitos B/inmunología , Simulación por Computador , Epítopos de Linfocito B/química , Humanos , Modelos Moleculares , Péptidos/química , Péptidos/inmunología , Glicoproteína de la Espiga del Coronavirus/química , Linfocitos T/inmunología
13.
Nat Commun ; 12(1): 1028, 2021 02 15.
Artículo en Inglés | MEDLINE | ID: mdl-33589610

RESUMEN

Upon binding to DNA breaks, poly(ADP-ribose) polymerase 1 (PARP1) ADP-ribosylates itself and other factors to initiate DNA repair. Serine is the major residue for ADP-ribosylation upon DNA damage, which strictly depends on HPF1. Here, we report the crystal structures of human HPF1/PARP1-CAT ΔHD complex at 1.98 Å resolution, and mouse and human HPF1 at 1.71 Å and 1.57 Å resolution, respectively. Our structures and mutagenesis data confirm that the structural insights obtained in a recent HPF1/PARP2 study by Suskiewicz et al. apply to PARP1. Moreover, we quantitatively characterize the key residues necessary for HPF1/PARP1 binding. Our data show that through salt-bridging to Glu284/Asp286, Arg239 positions Glu284 to catalyze serine ADP-ribosylation, maintains the local conformation of HPF1 to limit PARP1 automodification, and facilitates HPF1/PARP1 binding by neutralizing the negative charge of Glu284. These findings, along with the high-resolution structural data, may facilitate drug discovery targeting PARP1.


Asunto(s)
Proteínas Portadoras/química , ADN/química , Histonas/química , Proteínas Nucleares/química , Poli(ADP-Ribosa) Polimerasa-1/química , Serina/metabolismo , ADP-Ribosilación , Secuencia de Aminoácidos , Animales , Sitios de Unión , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Clonación Molecular , Cristalografía por Rayos X , ADN/genética , ADN/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Expresión Génica , Vectores Genéticos/química , Vectores Genéticos/metabolismo , Glutamina/metabolismo , Histonas/genética , Histonas/metabolismo , Humanos , Ratones , Modelos Moleculares , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Poli(ADP-Ribosa) Polimerasa-1/genética , Poli(ADP-Ribosa) Polimerasa-1/metabolismo , 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 , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Electricidad Estática
14.
Commun Biol ; 4(1): 228, 2021 02 15.
Artículo en Inglés | MEDLINE | ID: mdl-33589648

RESUMEN

SARS-CoV-2 has been mutating since it was first sequenced in early January 2020. Here, we analyze 45,494 complete SARS-CoV-2 geneome sequences in the world to understand their mutations. Among them, 12,754 sequences are from the United States. Our analysis suggests the presence of four substrains and eleven top mutations in the United States. These eleven top mutations belong to 3 disconnected groups. The first and second groups consisting of 5 and 8 concurrent mutations are prevailing, while the other group with three concurrent mutations gradually fades out. Moreover, we reveal that female immune systems are more active than those of males in responding to SARS-CoV-2 infections. One of the top mutations, 27964C > T-(S24L) on ORF8, has an unusually strong gender dependence. Based on the analysis of all mutations on the spike protein, we uncover that two of four SASR-CoV-2 substrains in the United States become potentially more infectious.


Asunto(s)
/virología , Mutación/genética , /genética , Regiones no Traducidas 5'/genética , Secuencia de Aminoácidos , /metabolismo , Evolución Molecular , Femenino , Humanos , Masculino , Modelos Moleculares , Nucleocápside/metabolismo , Sistemas de Lectura Abierta/genética , Polimorfismo de Nucleótido Simple/genética , Unión Proteica , Dominios Proteicos , Pliegue de Proteína , Glicoproteína de la Espiga del Coronavirus/química , Glicoproteína de la Espiga del Coronavirus/genética , Termodinámica , Estados Unidos
15.
Nat Commun ; 12(1): 1063, 2021 02 16.
Artículo en Inglés | MEDLINE | ID: mdl-33594061

RESUMEN

The most advanced P. falciparum circumsporozoite protein-based malaria vaccine, RTS,S/AS01 (RTS,S), confers partial protection but with antibody titers that wane relatively rapidly, highlighting the need to elicit more potent and durable antibody responses. Here, we elucidate crystal structures, binding affinities and kinetics, and in vivo protection of eight anti-NANP antibodies derived from an RTS,S phase 2a trial and encoded by three different heavy-chain germline genes. The structures reinforce the importance of homotypic Fab-Fab interactions in protective antibodies and the overwhelmingly dominant preference for a germline-encoded aromatic residue for recognition of the NANP motif. In this study, antibody apparent affinity correlates best with protection in an in vivo mouse model, with the more potent antibodies also recognizing epitopes with repeating secondary structural motifs of type I ß- and Asn pseudo 310 turns; such insights can be incorporated into design of more effective immunogens and antibodies for passive immunization.


Asunto(s)
Anticuerpos Antiprotozoarios/inmunología , Malaria Falciparum/inmunología , Malaria Falciparum/prevención & control , Plasmodium falciparum/inmunología , Secuencias Repetitivas de Aminoácido , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Animales , Afinidad de Anticuerpos/inmunología , Cristalografía por Rayos X , Epítopos/química , Epítopos/inmunología , Fragmentos Fab de Inmunoglobulinas/química , Fragmentos Fab de Inmunoglobulinas/inmunología , Cinética , Ratones Endogámicos C57BL , Modelos Moleculares , Parásitos/inmunología , Péptidos/química , Péptidos/metabolismo , Unión Proteica
16.
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
17.
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
18.
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
19.
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
20.
Nat Commun ; 12(1): 848, 2021 02 08.
Artículo en Inglés | MEDLINE | ID: mdl-33558493

RESUMEN

The causative agent of the COVID-19 pandemic, SARS-CoV-2, is steadily mutating during continuous transmission among humans. Such mutations can occur in the spike (S) protein that binds to the ACE2 receptor and is cleaved by TMPRSS2. However, whether S mutations affect SARS-CoV-2 cell entry remains unknown. Here, we show that naturally occurring S mutations can reduce or enhance cell entry via ACE2 and TMPRSS2. A SARS-CoV-2 S-pseudotyped lentivirus exhibits substantially lower entry than that of SARS-CoV S. Among S variants, the D614G mutant shows the highest cell entry, as supported by structural and binding analyses. Nevertheless, the D614G mutation does not affect neutralization by antisera against prototypic viruses. Taken together, we conclude that the D614G mutation increases cell entry by acquiring higher affinity to ACE2 while maintaining neutralization susceptibility. Based on these findings, further worldwide surveillance is required to understand SARS-CoV-2 transmissibility among humans.


Asunto(s)
/metabolismo , Mutación , Glicoproteína de la Espiga del Coronavirus/genética , Internalización del Virus , Unión Competitiva , /virología , Humanos , Modelos Moleculares , Pandemias , Unión Proteica , Dominios Proteicos , Receptores Virales/metabolismo , Serina Endopeptidasas/metabolismo , Glicoproteína de la Espiga del Coronavirus/química , Glicoproteína de la Espiga del Coronavirus/metabolismo
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