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1.
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
2.
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
3.
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
4.
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
5.
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
6.
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
7.
Nat Commun ; 12(1): 833, 2021 02 05.
Artículo en Inglés | MEDLINE | ID: mdl-33547280

RESUMEN

The structure of proline prevents it from adopting an optimal position for rapid protein synthesis. Poly-proline-tract (PPT) associated ribosomal stalling is resolved by highly conserved eIF5A, the only protein to contain the amino acid hypusine. We show that de novo heterozygous EIF5A variants cause a disorder characterized by variable combinations of developmental delay, microcephaly, micrognathia and dysmorphism. Yeast growth assays, polysome profiling, total/hypusinated eIF5A levels and PPT-reporters studies reveal that the variants impair eIF5A function, reduce eIF5A-ribosome interactions and impair the synthesis of PPT-containing proteins. Supplementation with 1 mM spermidine partially corrects the yeast growth defects, improves the polysome profiles and restores expression of PPT reporters. In zebrafish, knockdown eif5a partly recapitulates the human phenotype that can be rescued with 1 µM spermidine supplementation. In summary, we uncover the role of eIF5A in human development and disease, demonstrate the mechanistic complexity of EIF5A-related disorder and raise possibilities for its treatment.


Asunto(s)
Discapacidades del Desarrollo/genética , Regulación del Desarrollo de la Expresión Génica , Microcefalia/genética , Micrognatismo/genética , Factores de Iniciación de Péptidos/genética , Proteínas de Unión al ARN/genética , Adolescente , Secuencia de Aminoácidos , Animales , Niño , Discapacidades del Desarrollo/metabolismo , Discapacidades del Desarrollo/patología , Embrión no Mamífero , Femenino , Humanos , Lisina/análogos & derivados , Lisina/genética , Lisina/metabolismo , Masculino , Microcefalia/metabolismo , Microcefalia/patología , Micrognatismo/metabolismo , Micrognatismo/patología , Factores de Iniciación de Péptidos/deficiencia , Péptidos/genética , Péptidos/metabolismo , Biosíntesis de Proteínas , Conformación Proteica , Isoformas de Proteínas/deficiencia , Isoformas de Proteínas/genética , Ribosomas/genética , Ribosomas/metabolismo , Saccharomyces cerevisiae/efectos de los fármacos , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crecimiento & desarrollo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Espermidina/farmacología , Pez Cebra , Proteínas de Pez Cebra/genética , Proteínas de Pez Cebra/metabolismo
8.
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
9.
Sci Rep ; 11(1): 1156, 2021 01 13.
Artículo en Inglés | MEDLINE | ID: mdl-33441985

RESUMEN

Several viruses of the corona family interact, via their spike (S) proteins, with human cellular receptors. Spike proteins of SARS-CoV-1 and SARS-CoV-2 virions, being structurally related but not identical, mediate attachment to the human angiotensin-converting enzyme 2 (hACE2) receptor in similar but non-identical ways. Molecular-level understanding of interactions between spike proteins and hACE2 can aid strategies for blocking attachment of SARS-CoV-1, a potentially reemerging health threat, to human cells. We have identified dominant molecular-level interactions, some attractive and some repulsive, between the receptor binding domain of SARS-CoV-1 spike proteins (S-RBD) and hACE2. We performed fragment-based quantum-biochemical calculations which directly relate biomolecular structure to the hACE2...S-RBD interaction energy. Consistent with X-ray crystallography and cryo-EM, the interaction energy between hACE2 and S-RBD ([Formula: see text]26 kcal/mol) corresponds to a net intermolecular attraction which is significantly enhanced by inclusion of dispersion van der Waals forces. Protein fragments at the hACE2...S-RBD interface, that dominate host-virus attraction, have been identified together with their constituent amino acid residues. Two hACE2 fragments which include residues (GLU37, ASP38, TYR41, GLN42) and (GLU329, LYS353, GLY354), respectively, as well as three S-RBD fragments which include residues (TYR436), (ARG426) and (THR487, GLY488, TYR491), respectively, have been identified as primary attractors at the hACE2...S-RBD interface.


Asunto(s)
/química , Glicoproteína de la Espiga del Coronavirus/química , Glicoproteína de la Espiga del Coronavirus/metabolismo , Humanos , Modelos Moleculares , Unión Proteica , Conformación Proteica , Termodinámica
10.
BMC Bioinformatics ; 22(1): 8, 2021 Jan 06.
Artículo en Inglés | MEDLINE | ID: mdl-33407077

RESUMEN

BACKGROUND: Protein inter-residue contact and distance prediction are two key intermediate steps essential to accurate protein structure prediction. Distance prediction comes in two forms: real-valued distances and 'binned' distograms, which are a more finely grained variant of the binary contact prediction problem. The latter has been introduced as a new challenge in the 14th Critical Assessment of Techniques for Protein Structure Prediction (CASP14) 2020 experiment. Despite the recent proliferation of methods for predicting distances, few methods exist for evaluating these predictions. Currently only numerical metrics, which evaluate the entire prediction at once, are used. These give no insight into the structural details of a prediction. For this reason, new methods and tools are needed. RESULTS: We have developed a web server for evaluating predicted inter-residue distances. Our server, DISTEVAL, accepts predicted contacts, distances, and a true structure as optional inputs to generate informative heatmaps, chord diagrams, and 3D models. All of these outputs facilitate visual and qualitative assessment. The server also evaluates predictions using other metrics such as mean absolute error, root mean squared error, and contact precision. CONCLUSIONS: The visualizations generated by DISTEVAL complement each other and collectively serve as a powerful tool for both quantitative and qualitative assessments of predicted contacts and distances, even in the absence of a true 3D structure.


Asunto(s)
Biología Computacional/métodos , Internet , Modelos Moleculares , Proteínas , Aminoácidos/química , Aminoácidos/metabolismo , Conformación Proteica , Proteínas/química , Proteínas/metabolismo
11.
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
12.
Nat Commun ; 12(1): 250, 2021 01 11.
Artículo en Inglés | MEDLINE | ID: mdl-33431856

RESUMEN

Understanding the mechanism for antibody neutralization of SARS-CoV-2 is critical for the development of effective therapeutics and vaccines. We recently isolated a large number of monoclonal antibodies from SARS-CoV-2 infected individuals. Here we select the top three most potent yet variable neutralizing antibodies for in-depth structural and functional analyses. Crystal structural comparisons reveal differences in the angles of approach to the receptor binding domain (RBD), the size of the buried surface areas, and the key binding residues on the RBD of the viral spike glycoprotein. One antibody, P2C-1F11, most closely mimics binding of receptor ACE2, displays the most potent neutralizing activity in vitro and conferred strong protection against SARS-CoV-2 infection in Ad5-hACE2-sensitized mice. It also occupies the largest binding surface and demonstrates the highest binding affinity to RBD. More interestingly, P2C-1F11 triggers rapid and extensive shedding of S1 from the cell-surface expressed spike glycoprotein, with only minimal such effect by the remaining two antibodies. These results offer a structural and functional basis for potent neutralization via disruption of the very first and critical steps for SARS-CoV-2 cell entry.


Asunto(s)
/química , Anticuerpos Monoclonales/química , Anticuerpos Neutralizantes/inmunología , /inmunología , Animales , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/uso terapéutico , Anticuerpos Antivirales/inmunología , Sitios de Unión , /virología , Modelos Animales de Enfermedad , Epítopos , Células HEK293 , Humanos , Ratones , Ratones Endogámicos BALB C , Modelos Moleculares , Unión Proteica , Conformación Proteica , Receptores Virales/inmunología , Receptores Virales/metabolismo , Glicoproteína de la Espiga del Coronavirus/química , Glicoproteína de la Espiga del Coronavirus/metabolismo , Internalización del Virus
13.
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
14.
Nat Commun ; 12(1): 288, 2021 01 12.
Artículo en Inglés | MEDLINE | ID: mdl-33436577

RESUMEN

Vaccines and therapeutics are urgently needed for the pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Here, we screen human monoclonal antibodies (mAb) targeting the receptor binding domain (RBD) of the viral spike protein via antibody library constructed from peripheral blood mononuclear cells of a convalescent patient. The CT-P59 mAb potently neutralizes SARS-CoV-2 isolates including the D614G variant without antibody-dependent enhancement effect. Complex crystal structure of CT-P59 Fab/RBD shows that CT-P59 blocks interaction regions of RBD for angiotensin converting enzyme 2 (ACE2) receptor with an orientation that is notably different from previously reported RBD-targeting mAbs. Furthermore, therapeutic effects of CT-P59 are evaluated in three animal models (ferret, hamster, and rhesus monkey), demonstrating a substantial reduction in viral titer along with alleviation of clinical symptoms. Therefore, CT-P59 may be a promising therapeutic candidate for COVID-19.


Asunto(s)
Anticuerpos Neutralizantes/farmacología , Unión Proteica/efectos de los fármacos , Glicoproteína de la Espiga del Coronavirus/efectos de los fármacos , /química , Animales , Anticuerpos Monoclonales/inmunología , Anticuerpos Neutralizantes/química , Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/inmunología , Chlorocebus aethiops , Modelos Animales de Enfermedad , Femenino , Hurones , Humanos , Leucocitos Mononucleares , Macaca mulatta , Masculino , Mesocricetus , Modelos Moleculares , Conformación Proteica , Glicoproteína de la Espiga del Coronavirus/química , Células Vero
15.
Nat Commun ; 12(1): 44, 2021 01 04.
Artículo en Inglés | MEDLINE | ID: mdl-33398001

RESUMEN

In Bacteroidetes, one of the dominant phyla of the mammalian gut, active uptake of large nutrients across the outer membrane is mediated by SusCD protein complexes via a "pedal bin" transport mechanism. However, many features of SusCD function in glycan uptake remain unclear, including ligand binding, the role of the SusD lid and the size limit for substrate transport. Here we characterise the ß2,6 fructo-oligosaccharide (FOS) importing SusCD from Bacteroides thetaiotaomicron (Bt1762-Bt1763) to shed light on SusCD function. Co-crystal structures reveal residues involved in glycan recognition and suggest that the large binding cavity can accommodate several substrate molecules, each up to ~2.5 kDa in size, a finding supported by native mass spectrometry and isothermal titration calorimetry. Mutational studies in vivo provide functional insights into the key structural features of the SusCD apparatus and cryo-EM of the intact dimeric SusCD complex reveals several distinct states of the transporter, directly visualising the dynamics of the pedal bin transport mechanism.


Asunto(s)
Proteínas Bacterianas/metabolismo , Microbioma Gastrointestinal , Polisacáridos/metabolismo , Simbiosis , Proteínas Bacterianas/química , Microscopía por Crioelectrón , Ligandos , Espectroscopía de Resonancia Magnética , Modelos Moleculares , Oligosacáridos/química , Polisacáridos/química , Conformación Proteica , Relación Estructura-Actividad
16.
Nat Commun ; 12(1): 128, 2021 01 04.
Artículo en Inglés | MEDLINE | ID: mdl-33397917

RESUMEN

Voltage-gated sodium (NaV) channels initiate action potentials in excitable cells, and their function is altered by potent gating-modifier toxins. The α-toxin LqhIII from the deathstalker scorpion inhibits fast inactivation of cardiac NaV1.5 channels with IC50 = 11.4 nM. Here we reveal the structure of LqhIII bound to NaV1.5 at 3.3 Å resolution by cryo-EM. LqhIII anchors on top of voltage-sensing domain IV, wedged between the S1-S2 and S3-S4 linkers, which traps the gating charges of the S4 segment in a unique intermediate-activated state stabilized by four ion-pairs. This conformational change is propagated inward to weaken binding of the fast inactivation gate and favor opening the activation gate. However, these changes do not permit Na+ permeation, revealing why LqhIII slows inactivation of NaV channels but does not open them. Our results provide important insights into the structural basis for gating-modifier toxin binding, voltage-sensor trapping, and fast inactivation of NaV channels.


Asunto(s)
Miocardio/metabolismo , Canal de Sodio Activado por Voltaje NAV1.5/química , Canal de Sodio Activado por Voltaje NAV1.5/metabolismo , Venenos de Escorpión/toxicidad , Animales , Sitios de Unión , Microscopía por Crioelectrón , Células HEK293 , Humanos , Activación del Canal Iónico/efectos de los fármacos , Simulación de Dinámica Molecular , Canal de Sodio Activado por Voltaje NAV1.5/ultraestructura , Conformación Proteica , Ratas , Venenos de Escorpión/química , Sodio/metabolismo
17.
Nat Commun ; 12(1): 121, 2021 01 05.
Artículo en Inglés | MEDLINE | ID: mdl-33402676

RESUMEN

p97, also known as valosin-containing protein (VCP) or Cdc48, plays a central role in cellular protein homeostasis. Human p97 mutations are associated with several neurodegenerative diseases. Targeting p97 and its cofactors is a strategy for cancer drug development. Despite significant structural insights into the fungal homolog Cdc48, little is known about how human p97 interacts with its cofactors. Recently, the anti-alcohol abuse drug disulfiram was found to target cancer through Npl4, a cofactor of p97, but the molecular mechanism remains elusive. Here, using single-particle cryo-electron microscopy (cryo-EM), we uncovered three Npl4 conformational states in complex with human p97 before ATP hydrolysis. The motion of Npl4 results from its zinc finger motifs interacting with the N domain of p97, which is essential for the unfolding activity of p97. In vitro and cell-based assays showed that the disulfiram derivative bis-(diethyldithiocarbamate)-copper (CuET) can bypass the copper transporter system and inhibit the function of p97 in the cytoplasm by releasing cupric ions under oxidative conditions, which disrupt the zinc finger motifs of Npl4, locking the essential conformational switch of the complex.


Asunto(s)
Coenzimas/química , Ditiocarba/análogos & derivados , Péptidos y Proteínas de Señalización Intracelular/química , Proteínas Nucleares/química , Compuestos Organometálicos/química , Ubiquitina/química , Proteína que Contiene Valosina/química , Adenosina Trifosfato/análogos & derivados , Adenosina Trifosfato/química , Adenosina Trifosfato/metabolismo , Sitios de Unión , Clonación Molecular , Coenzimas/genética , Coenzimas/metabolismo , Microscopía por Crioelectrón , Disulfiram/química , Disulfiram/metabolismo , Ditiocarba/química , Ditiocarba/metabolismo , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Expresión Génica , Vectores Genéticos/química , Vectores Genéticos/metabolismo , Humanos , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Modelos Moleculares , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Compuestos Organometálicos/metabolismo , Unión Proteica , Conformación Proteica , Dominios y Motivos de Interacción de Proteínas , Desplegamiento Proteico , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Especificidad por Sustrato , Ubiquitina/genética , Ubiquitina/metabolismo , Proteína que Contiene Valosina/antagonistas & inhibidores , Proteína que Contiene Valosina/genética , Proteína que Contiene Valosina/metabolismo , Dedos de Zinc
18.
J Phys Chem B ; 125(3): 850-873, 2021 01 28.
Artículo en Inglés | MEDLINE | ID: mdl-33448856

RESUMEN

The rapidly growing body of structural and biochemical studies of the SARS-CoV-2 spike glycoprotein has revealed a variety of distinct functional states with radically different arrangements of the receptor-binding domain, highlighting a remarkable function-driven conformational plasticity and adaptability of the spike proteins. In this study, we examined molecular mechanisms underlying conformational and dynamic changes in the SARS-CoV-2 spike mutant trimers through the lens of dynamic analysis of allosteric interaction networks and atomistic modeling of signal transmission. Using an integrated approach that combined coarse-grained molecular simulations, protein stability analysis, and perturbation-based modeling of residue interaction networks, we examined how mutations in the regulatory regions of the SARS-CoV-2 spike protein can differentially affect dynamics and allosteric signaling in distinct functional states. The results of this study revealed key functional regions and regulatory centers that govern collective dynamics, allosteric interactions, and control signal transmission in the SARS-CoV-2 spike proteins. We found that the experimentally confirmed regulatory hotspots that dictate dynamic switching between conformational states of the SARS-CoV-2 spike protein correspond to the key hinge sites and global mediating centers of the allosteric interaction networks. The results of this study provide a novel insight into allosteric regulatory mechanisms of SARS-CoV-2 spike proteins showing that mutations at the key regulatory positions can differentially modulate distribution of states and determine topography of signal communication pathways operating through state-specific cascades of control switch points. This analysis provides a plausible strategy for allosteric probing of the conformational equilibrium and therapeutic intervention by targeting specific hotspots of allosteric interactions and communications in the SARS-CoV-2 spike proteins.


Asunto(s)
Modelos Biológicos , Mutación , Glicoproteína de la Espiga del Coronavirus/química , Regulación Alostérica , Sitios de Unión , Cisteína/genética , Simulación de Dinámica Molecular , Unión Proteica , Conformación Proteica , Estabilidad Proteica , Subunidades de Proteína , Transducción de Señal/genética , Glicoproteína de la Espiga del Coronavirus/genética
19.
Nucleic Acids Res ; 49(2): 601-620, 2021 01 25.
Artículo en Inglés | MEDLINE | ID: mdl-33406242

RESUMEN

It is a well-known and intensively studied phenomenon that the levels of many miRNAs are differentiated in cancer. miRNA biogenesis and functional expression are complex processes orchestrated by many proteins cumulatively called miRNA biogenesis proteins. To characterize cancer somatic mutations in the miRNA biogenesis genes and investigate their potential impact on the levels of miRNAs, we analyzed whole-exome sequencing datasets of over 10 000 cancer/normal sample pairs deposited within the TCGA repository. We identified and characterized over 3600 somatic mutations in 29 miRNA biogenesis genes and showed that some of the genes are overmutated in specific cancers and/or have recurrent hotspot mutations (e.g. SMAD4 in PAAD, COAD and READ; DICER1 in UCEC; PRKRA in OV and LIN28B in SKCM). We identified a list of miRNAs whose level is affected by particular types of mutations in either SMAD4, SMAD2 or DICER1 and showed that hotspot mutations in the RNase domains in DICER1 not only decrease the level of 5p-miRNAs but also increase the level of 3p-miRNAs, including many well-known cancer-related miRNAs. We also showed an association of the mutations with patient survival. Eventually, we created an atlas/compendium of miRNA biogenesis alterations providing a useful resource for different aspects of biomedical research.


Asunto(s)
ARN Helicasas DEAD-box/genética , MicroARNs/biosíntesis , Mutación , Proteínas de Neoplasias/genética , Neoplasias/genética , ARN Neoplásico/biosíntesis , Ribonucleasa III/genética , Proteína Smad2/genética , Proteína Smad4/genética , ARN Helicasas DEAD-box/metabolismo , Conjuntos de Datos como Asunto , Regulación Neoplásica de la Expresión Génica , Genes Relacionados con las Neoplasias , Estudio de Asociación del Genoma Completo , Humanos , MicroARNs/genética , Modelos Moleculares , Mutación Missense , Proteínas de Neoplasias/metabolismo , Neoplasias/metabolismo , Neoplasias/mortalidad , Conformación Proteica , ARN Neoplásico/genética , Ribonucleasa III/metabolismo , Proteína Smad2/química , Proteína Smad2/metabolismo , Proteína Smad4/química , Proteína Smad4/metabolismo
20.
Nucleic Acids Res ; 49(2): 700-712, 2021 01 25.
Artículo en Inglés | MEDLINE | ID: mdl-33410883

RESUMEN

Aptamers are single-stranded oligonucleotides that bind to a specific target with high affinity, and are widely applied in biomedical diagnostics and drug development. However, the use of aptamers has largely been limited to simple binders or inhibitors that interfere with the function of a target protein. Here, we show that an aptamer can also act as a positive allosteric modulator that enhances the activation of a receptor by stabilizing the binding of a ligand to that receptor. We developed an aptamer, named IR-A43, which binds to the insulin receptor, and confirmed that IR-A43 and insulin bind to the insulin receptor with mutual positive cooperativity. IR-A43 alone is inactive, but, in the presence of insulin, it potentiates autophosphorylation and downstream signaling of the insulin receptor. By using the species-specific activity of IR-A43 at the human insulin receptor, we demonstrate that residue Q272 in the cysteine-rich domain is directly involved in the insulin-enhancing activity of IR-A43. Therefore, we propose that the region containing residue Q272 is a hotspot that can be used to enhance insulin receptor activation. Moreover, our study implies that aptamers are promising reagents for the development of allosteric modulators that discriminate a specific conformation of a target receptor.


Asunto(s)
Antígenos CD/efectos de los fármacos , Aptámeros de Nucleótidos/farmacología , Receptor de Insulina/efectos de los fármacos , Regulación Alostérica , Animales , Antígenos CD/química , Antígenos CD/metabolismo , Células Cultivadas , Cricetinae , Glutamina/química , Humanos , Insulina/metabolismo , Ratones , Fosforilación , Unión Proteica , Conformación Proteica , Dominios Proteicos , Procesamiento Proteico-Postraduccional , Ratas , Receptor IGF Tipo 1/química , Receptor IGF Tipo 1/efectos de los fármacos , Receptor IGF Tipo 1/metabolismo , Receptor de Insulina/química , Receptor de Insulina/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/efectos de los fármacos , Proteínas Recombinantes/metabolismo , Técnica SELEX de Producción de Aptámeros , Estimulación Química
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