RESUMEN
Betacoronaviruses are a genus within the Coronaviridae family of RNA viruses. They are capable of infecting vertebrates and causing epidemics as well as global pandemics in humans. Mitigating the threat posed by Betacoronaviruses requires an understanding of their molecular diversity. The development of novel antivirals hinges on understanding the key regulatory elements within the viral RNA genomes, in particular the 5'-proximal region, which is pivotal for viral protein synthesis. Using a combination of cryo-electron microscopy, atomic force microscopy, chemical probing, and computational modeling, we determined the structures of 5'-proximal regions in RNA genomes of Betacoronaviruses from four subgenera: OC43-CoV, SARS-CoV-2, MERS-CoV, and Rousettus bat-CoV. We obtained cryo-electron microscopy maps and determined atomic-resolution models for the stem-loop-5 (SL5) region at the translation start site and found that despite low sequence similarity and variable length of the helical elements it exhibits a remarkable structural conservation. Atomic force microscopy imaging revealed a common domain organization and a dynamic arrangement of structural elements connected with flexible linkers across all four Betacoronavirus subgenera. Together, these results reveal common features of a critical regulatory region shared between different Betacoronavirus RNA genomes, which may allow targeting of these RNAs by broad-spectrum antiviral therapeutics.
Asunto(s)
Betacoronavirus , ARN Viral , Betacoronavirus/genética , Microscopía por Crioelectrón , Genoma Viral/genética , ARN Viral/química , ARN Viral/genética , ARN Viral/ultraestructura , SARS-CoV-2/genéticaRESUMEN
Artificial protein cages have great potential in diverse fields including as vaccines and drug delivery vehicles. TRAP-cage is an artificial protein cage notable for the way in which the interface between its ring-shaped building blocks can be modified such that the conditions under which cages disassemble can be controlled. To date, TRAP-cages have been constructed from homo-11mer rings, i.e., hendecamers. This is interesting as convex polyhedra with identical regular faces cannot be formed from hendecamers. TRAP-cage overcomes this limitation due to intrinsic flexibility, allowing slight deformation to absorb any error. The resulting TRAP-cage made from 24 TRAP 11mer rings is very close to regular with only very small errors necessary to allow the cage to form. The question arises as to the limits of the error that can be absorbed by a protein structure in this way before the formation of an apparently regular convex polyhedral becomes impossible. Here we use a naturally occurring TRAP variant consisting of twelve identical monomers (i.e., a dodecamer) to probe these limits. We show that it is able to form an apparently regular protein cage consisting of twelve TRAP rings. Comparison of the cryo-EM structure of the new cage with theoretical models and related cages gives insight into the rules of cage formation and allows us to predict other cages that may be formed given TRAP-rings consisting of different numbers of monomers.
Asunto(s)
ProteínasRESUMEN
Artificial protein cages made from multiple copies of a single protein can be produced such that they only assemble upon addition of a metal ion. Consequently, the ability to remove the metal ion triggers protein-cage disassembly. Controlling assembly and disassembly has many potential uses including cargo loading/unloading and hence drug delivery. TRAP-cage is an example of such a protein cage which assembles due to linear coordination bond formation with Au(I) which acts to bridge constituent proteins. Here we describe the method for production and purification of TRAP-cage.
Asunto(s)
Sistemas de Liberación de Medicamentos , OroRESUMEN
Hypusination is a unique post-translational modification of the eukaryotic translation factor 5A (eIF5A) that is essential for overcoming ribosome stalling at polyproline sequence stretches. The initial step of hypusination, the formation of deoxyhypusine, is catalyzed by deoxyhypusine synthase (DHS), however, the molecular details of the DHS-mediated reaction remained elusive. Recently, patient-derived variants of DHS and eIF5A have been linked to rare neurodevelopmental disorders. Here, we present the cryo-EM structure of the human eIF5A-DHS complex at 2.8 Å resolution and a crystal structure of DHS trapped in the key reaction transition state. Furthermore, we show that disease-associated DHS variants influence the complex formation and hypusination efficiency. Hence, our work dissects the molecular details of the deoxyhypusine synthesis reaction and reveals how clinically-relevant mutations affect this crucial cellular process.
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Enfermedades Neurodegenerativas , Trastornos del Neurodesarrollo , Oxidorreductasas actuantes sobre Donantes de Grupo CH-NH , Factores de Iniciación de Péptidos , Humanos , Microscopía por Crioelectrón , Factores de Iniciación de Péptidos/química , Procesamiento Proteico-Postraduccional , Oxidorreductasas actuantes sobre Donantes de Grupo CH-NH/química , Factor 5A Eucariótico de Iniciación de TraducciónRESUMEN
Artificial protein cages are constructed from multiple protein subunits. The interaction between the subunits, notably the angle formed between them, controls the geometry of the resulting cage. Here, using the artificial protein cage, "TRAP-cage", we show that a simple alteration in the position of a single amino acid responsible for Au(I)-mediated subunit-subunit interactions in the constituent ring-shaped building blocks results in a more acute dihedral angle between them. In turn, this causes a dramatic shift in the structure from a 24-ring cage with an octahedral symmetry to a 20-ring cage with a C2 symmetry. This symmetry change is accompanied by a decrease in the number of Au(I)-mediated bonds between cysteines and a concomitant change in biophysical properties of the cage.
RESUMEN
Artificial protein cages have great potential in a number of areas including cargo capture and delivery and as artificial vaccines. Here, we investigate an artificial protein cage whose assembly is triggered by gold nanoparticles. Using biochemical and biophysical methods we were able to determine both the mechanical properties and the gross compositional features of the cage which, combined with mathematical models and biophysical data, allowed the structure of the cage to be predicted. The accuracy of the overall geometrical prediction was confirmed by the cryo-EM structure determined to sub-5 Å resolution. This showed the cage to be nonregular but similar to a dodecahedron, being constructed from 12 11-membered rings. Surprisingly, the structure revealed that the cage also contained a single, small gold nanoparticle at each 3-fold axis meaning that each cage acts as a synthetic framework for regular arrangement of 20 gold nanoparticles in a three-dimensional lattice.
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Nanopartículas del Metal , Nanopartículas , Oro/química , Nanopartículas del Metal/química , Proteínas/químicaRESUMEN
Virus-like particles (VLPs) have significant potential as artificial vaccines and drug delivery systems. The ability to control their size has wide ranging utility but achieving such controlled polymorphism using a single protein subunit is challenging as it requires altering VLP geometry. Here we achieve size control of MS2 bacteriophage VLPs via insertion of amino acid sequences in an external loop to shift morphology to significantly larger forms. The resulting VLP size and geometry is controlled by altering the length and type of the insert. Cryo electron microscopy structures of the new VLPs, in combination with a kinetic model of their assembly, show that the abundance of wild type (T = 3), T = 4, D3 and D5 symmetrical VLPs can be biased in this way. We propose a mechanism whereby the insert leads to a change in the dynamic behavior of the capsid protein dimer, affecting the interconversion between the symmetric and asymmetric conformers and thus determining VLP size and morphology.
RESUMEN
Engineered protein cages are promising tools that can be customized for applications in medicine and nanotechnology. A major challenge is developing a straightforward strategy for endowing cages with bespoke, inducible disassembly. Such cages would allow release of encapsulated cargoes at desired timing and location. Here, we achieve such programmable disassembly using protein cages, in which the subunits are held together by different molecular cross-linkers. This modular system enables cage disassembly to be controlled in a condition-dependent manner. Structural details of the resulting cages were determined using cryoelectron microscopy, which allowed observation of bridging cross-linkers at intended positions. Triggered disassembly was demonstrated by high-speed atomic force microscopy and subsequent cargo release using an encapsulated Förster resonance energy transfer pair whose signal depends on the quaternary structure of the cage.
RESUMEN
Cage forming proteins have numerous potential applications in biomedicine and biotechnology, where the iron storage ferritin is a widely used example. However, controlling ferritin cage assembly/disassembly remains challenging, typically requiring extreme conditions incompatible with many desirable cargoes, particularly for more fragile biopharmaceuticals. Recently, a ferritin from the hyperthermophile bacterium Thermotoga maritima (TmFtn) has been shown to have reversible assembly under mild conditions, offering greater potential biocompatibility in terms of cargo access and encapsulation. Like Archeoglobus fulgidus ferritin (AfFtn), TmFtn forms 24mer cages mediated by metal ions (Mg2+). We have solved the crystal structure of the wild type TmFtn and several mutants displaying different assembly/disassembly properties. These data combined with other biophysical studies allow us to suggest candidate interfacial amino acids crucial in controlling assembly. This work deepens our understanding of how these ferritin complexes assemble and is a useful step towards production of triggerable ferritins in which these properties can be finely designed and controlled.
Asunto(s)
Ferritinas , Hierro , Ferritinas/genética , Hierro/metabolismo , Thermotoga maritimaRESUMEN
DNA glycosylases are emerging as relevant pharmacological targets in inflammation, cancer and neurodegenerative diseases. Consequently, the search for inhibitors of these enzymes has become a very active research field. As a continuation of previous work that showed that 2-thioxanthine (2TX) is an irreversible inhibitor of zinc finger (ZnF)-containing Fpg/Nei DNA glycosylases, we designed and synthesized a mini-library of 2TX-derivatives (TXn) and evaluated their ability to inhibit Fpg/Nei enzymes. Among forty compounds, four TXn were better inhibitors than 2TX for Fpg. Unexpectedly, but very interestingly, two dithiolated derivatives more selectively and efficiently inhibit the zincless finger (ZnLF)-containing enzymes (human and mimivirus Neil1 DNA glycosylases hNeil1 and MvNei1, respectively). By combining chemistry, biochemistry, mass spectrometry, blind and flexible docking and X-ray structure analysis, we localized new TXn binding sites on Fpg/Nei enzymes. This endeavor allowed us to decipher at the atomic level the mode of action for the best TXn inhibitors on the ZnF-containing enzymes. We discovered an original inhibition mechanism for the ZnLF-containing Fpg/Nei DNA glycosylases by disulfide cyclic trimeric forms of dithiopurines. This work paves the way for the design and synthesis of a new structural class of inhibitors for selective pharmacological targeting of hNeil1 in cancer and neurodegenerative diseases.
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ADN Glicosilasas/antagonistas & inhibidores , ADN Glicosilasas/química , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Purinas/química , Purinas/farmacología , Compuestos de Sulfhidrilo/química , Compuestos de Sulfhidrilo/farmacología , Bacterias/enzimología , Sitios de Unión , Cristalografía por Rayos X , Reparación del ADN , Relación Dosis-Respuesta a Droga , Activación Enzimática/efectos de los fármacos , Humanos , Modelos Moleculares , Conformación Molecular , Estructura Molecular , Unión Proteica , Relación Estructura-Actividad , Tioxantenos/química , Tioxantenos/farmacologíaRESUMEN
Symmetrical protein cages have evolved to fulfil diverse roles in nature, including compartmentalization and cargo delivery1, and have inspired synthetic biologists to create novel protein assemblies via the precise manipulation of protein-protein interfaces. Despite the impressive array of protein cages produced in the laboratory, the design of inducible assemblies remains challenging2,3. Here we demonstrate an ultra-stable artificial protein cage, the assembly and disassembly of which can be controlled by metal coordination at the protein-protein interfaces. The addition of a gold (I)-triphenylphosphine compound to a cysteine-substituted, 11-mer protein ring triggers supramolecular self-assembly, which generates monodisperse cage structures with masses greater than 2 MDa. The geometry of these structures is based on the Archimedean snub cube and is, to our knowledge, unprecedented. Cryo-electron microscopy confirms that the assemblies are held together by 120 S-Aui-S staples between the protein oligomers, and exist in two chiral forms. The cage shows extreme chemical and thermal stability, yet it readily disassembles upon exposure to reducing agents. As well as gold, mercury(II) is also found to enable formation of the protein cage. This work establishes an approach for linking protein components into robust, higher-order structures, and expands the design space available for supramolecular assemblies to include previously unexplored geometries.
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Oro/química , Proteínas/química , Microscopía por Crioelectrón , Cisteína/química , Mercurio/química , Modelos Moleculares , Proteínas/ultraestructuraRESUMEN
In oncology, the "Warburg effect" describes the elevated production of energy by glycolysis in cancer cells. The ubiquitous and hypoxia-induced 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (PFKFB3) plays a noteworthy role in the regulation of glycolysis by producing fructose-2,6-biphosphate (F-2,6-BP), a potent activator of the glycolysis rate-limiting phosphofructokinase PFK-1. Series of amides and sulfonamides derivatives based on a N-aryl 6-aminoquinoxaline scaffold were synthesized and tested for their inhibition of PFKFB3 in vitro in a biochemical assay as well as in HCT116 cells. The carboxamide series displayed satisfactory kinetic solubility and metabolic stability, and within this class, potent lead compounds with low nanomolar activity have been identified with a suitable profile for further in vivo evaluation.
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Amidas/química , Fosfofructoquinasa-2/antagonistas & inhibidores , Quinoxalinas/química , Quinoxalinas/farmacología , Sulfonamidas/química , Células HCT116 , Humanos , Cinética , SolubilidadRESUMEN
Many plant-derived compounds possess antimicrobial, antioxidant and even anticancer activities. Therefore, they are considered as substances that can be used instead of synthetic compounds in various applications. In this work, the essential oil from hop cones was extracted and analyzed, and then its effects on model bacteria membranes were studied to verify whether the hop essential oils could be used as ecological pesticides. The experiments involved surface pressure-area measurements, penetration studies and Brewster angle microscopy (BAM) imaging of lipid monolayers as well as hydrodynamic diameter, zeta potential, steady-state fluorescence anisotropy and Cryo-Transmission Electron Microscopy (cryo-TEM) measurements of liposomes. Finally the bactericidal tests on plant pathogen bacteria Pseudomonas syringae pv. lachrymans PCM 1410 were performed. The obtained results showed that the components of the essential oils from hop cones incorporate into lipid monolayers and bilayers and alter their fluidity. However, the observed effect is determined by the system composition, its condensation and the oil concentration. Interestingly, at a given dose, the effect of the essential oil on membranes was found to stabilize. Moreover, BAM images proved that hop oil prevents the formation of a large fraction of a condensed phase at the interface. Both the studies on model membranes as well as the in vitro tests allow one to conclude that the hop essential oil could likely be considered as the candidate to be used in agriculture as a natural pesticide.
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Antibacterianos/farmacología , Humulus/química , Membrana Dobles de Lípidos/química , Aceites Volátiles/farmacología , Liposomas Unilamelares/química , Antibacterianos/química , Antibacterianos/aislamiento & purificación , Cardiolipinas/química , Fluidez de la Membrana/efectos de los fármacos , Pruebas de Sensibilidad Microbiana , Aceites Volátiles/química , Aceites Volátiles/aislamiento & purificación , Fosfatidiletanolaminas/química , Fosfatidilgliceroles/química , Pseudomonas syringae/química , Pseudomonas syringae/efectos de los fármacos , Pseudomonas syringae/crecimiento & desarrolloRESUMEN
A number of studies were devoted to understanding an immunological effect of pressure-treated ß-lactoglobulin. In our previous work we have proved that high pressure significantly modifies ß-lactoglobulin conformation and consequently its physicochemical properties. Here, structure of ß-lactoglobulin complex with myristic acid determined at the highest accepted by the crystal pressure value of 550â¯MPa is reported. Our results structurally prove that pressure noticeably modifies positions of the major ß-lactoglobulin epitopes. Considering the biological impact of observed changes in epitope regions, high pressure ß-lactoglobulin structure presents a step forward in understanding the pressure modification of food protein allergenicity. The conformational changes of pressurized ß-lactoglobulin did not support the hypothesis that proteolytic digestion facilitated by pressure is caused by an exposure of the digestive sites. Our findings demonstrate that high pressure protein crystallography can potentially identify the most pressure-sensitive fragments in allergens, and can therefore support development of hypoallergenic food products.
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Alérgenos/inmunología , Hipersensibilidad a los Alimentos/etiología , Lactoglobulinas/química , Presión , Epítopos , HumanosRESUMEN
Energy and biomass production in cancer cells are largely supported by aerobic glycolysis in what is called the Warburg effect. The process is regulated by key enzymes, among which phosphofructokinase PFK-2 plays a significant role by producing fructose-2,6-biphosphate; the most potent activator of the glycolysis rate-limiting step performed by phosphofructokinase PFK-1. Herein, the synthesis, biological evaluation and structure-activity relationship of novel inhibitors of 6-phosphofructo-2-kinase/fructose-2,6-biphosphataseâ 3 (PFKFB3), which is the ubiquitous and hypoxia-induced isoform of PFK-2, are reported. X-ray crystallography and docking were instrumental in the design and optimisation of a series of N-aryl 6-aminoquinoxalines. The most potent representative, N-(4-methanesulfonylpyridin-3-yl)-8-(3-methyl-1-benzothiophen-5-yl)quinoxalin-6-amine, displayed an IC50 of 14â nm for the target and an IC50 of 0.49â µm for fructose-2,6-biphosphate production in human colon carcinoma HCT116 cells. This work provides a new entry in the field of PFKFB3 inhibitors with potential for development in oncology.
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Descubrimiento de Drogas , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Fosfofructoquinasa-2/antagonistas & inhibidores , Quinoxalinas/química , Quinoxalinas/farmacología , Supervivencia Celular/efectos de los fármacos , Cristalografía por Rayos X , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/síntesis química , Células HCT116 , Humanos , Ácido Láctico/antagonistas & inhibidores , Ácido Láctico/biosíntesis , Modelos Moleculares , Estructura Molecular , Fosfofructoquinasa-2/metabolismo , Quinoxalinas/síntesis química , Relación Estructura-ActividadRESUMEN
DNA glycosylases from the Fpg/Nei structural superfamily are base excision repair enzymes involved in the removal of a wide variety of mutagen and potentially lethal oxidized purines and pyrimidines. Although involved in genome stability, the recent discovery of synthetic lethal relationships between DNA glycosylases and other pathways highlights the potential of DNA glycosylase inhibitors for future medicinal chemistry development in cancer therapy. By combining biochemical and structural approaches, the physical target of 2-thioxanthine (2TX), an uncompetitive inhibitor of Fpg, was identified. 2TX interacts with the zinc finger (ZnF) DNA binding domain of the enzyme. This explains why the zincless hNEIL1 enzyme is resistant to 2TX. Crystal structures of the enzyme bound to DNA in the presence of 2TX demonstrate that the inhibitor chemically reacts with cysteine thiolates of ZnF and induces the loss of zinc. The molecular mechanism by which 2TX inhibits Fpg may be generalized to all prokaryote and eukaryote ZnF-containing Fpg/Nei-DNA glycosylases. Cell experiments show that 2TX can operate in cellulo on the human Fpg/Nei DNA glycosylases. The atomic elucidation of the determinants for the interaction of 2TX to Fpg provides the foundation for the future design and synthesis of new inhibitors with high efficiency and selectivity.