RESUMEN
Plants adapt to fluctuating environmental conditions by adjusting their metabolism and gene expression to maintain fitness1. In legumes, nitrogen homeostasis is maintained by balancing nitrogen acquired from soil resources with nitrogen fixation by symbiotic bacteria in root nodules2-8. Here we show that zinc, an essential plant micronutrient, acts as an intracellular second messenger that connects environmental changes to transcription factor control of metabolic activity in root nodules. We identify a transcriptional regulator, FIXATION UNDER NITRATE (FUN), which acts as a sensor, with zinc controlling the transition between an inactive filamentous megastructure and an active transcriptional regulator. Lower zinc concentrations in the nodule, which we show occur in response to higher levels of soil nitrate, dissociates the filament and activates FUN. FUN then directly targets multiple pathways to initiate breakdown of the nodule. The zinc-dependent filamentation mechanism thus establishes a concentration readout to adapt nodule function to the environmental nitrogen conditions. In a wider perspective, these results have implications for understanding the roles of metal ions in integration of environmental signals with plant development and optimizing delivery of fixed nitrogen in legume crops.
Asunto(s)
Regulación de la Expresión Génica de las Plantas , Nitratos , Fijación del Nitrógeno , Nódulos de las Raíces de las Plantas , Factores de Transcripción , Zinc , Zinc/metabolismo , Factores de Transcripción/metabolismo , Nitratos/metabolismo , Nódulos de las Raíces de las Plantas/metabolismo , Nitrógeno/metabolismo , Medicago truncatula/metabolismo , Medicago truncatula/genética , Simbiosis , Proteínas de Plantas/metabolismo , Proteínas de Plantas/genéticaRESUMEN
Biofilm-protected pathogenic Staphylococcus aureus causes chronic infections that are difficult to treat. An essential building block of these biofilms are functional amyloid fibrils that assemble from phenol-soluble modulins (PSMs). PSMα1 cross-seeds other PSMs into cross-ß amyloid folds and is therefore a key element in initiating biofilm formation. However, the paucity of high-resolution structures hinders efforts to prevent amyloid assembly and biofilm formation. Here, we present a 3.5 Å resolution density map of the major PSMα1 fibril form revealing a left-handed cross-ß fibril composed of two C2-symmetric U-shaped protofilaments whose subunits are unusually tilted out-of-plane. Monomeric α-helical PSMα1 is extremely cytotoxic to cells, despite the moderate toxicity of the cross-ß fibril. We suggest mechanistic insights into the PSM functional amyloid formation and conformation transformation on the path from monomer-to-fibril formation. Details of PSMα1 assembly and fibril polymorphism suggest how S. aureus utilizes functional amyloids to form biofilms and establish a framework for developing therapeutics against infection and antimicrobial resistance.
Asunto(s)
Amiloide , Biopelículas , Staphylococcus aureus , Staphylococcus aureus/metabolismo , Staphylococcus aureus/fisiología , Biopelículas/crecimiento & desarrollo , Amiloide/metabolismo , Amiloide/química , Toxinas Bacterianas/metabolismo , Toxinas Bacterianas/química , Conformación Proteica , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/química , Modelos MolecularesRESUMEN
Melanocortin receptors have emerged as important targets with a very unusual versatility, as their widespread distribution on multiple tissues (e.g. skin, adrenal glands, brain, immune cells, exocrine glands) together with the variety of physiological processes they control (pigmentation, cortisol release, satiety mechanism, inflammation, secretions), place this family of receptors as genuine therapeutic targets for many disorders. This review focuses in the journey of the development of melanocortin receptors as therapeutic targets from the discovery of their existence in the early 1990 s to the approval of the first few drugs of this class. Two major areas of development characterise the current state of melanocortin drug development: their role in obesity, recently culminated with the approval of setmelanotide, and their potential for the treatment of chronic inflammatory and autoimmune diseases like rheumatoid arthritis, multiple sclerosis or fibrosis. The pro-resolving nature of these drugs offers the advantage of acting by mimicking the way our body naturally resolves inflammation, expecting fewer side effects and a more balanced (i.e. non-immunosuppressive) response from them. Here we also review the approaches followed for the design and development of novel compounds, the importance of the GPCR nature of these receptors in the process of drug development, therapeutic value, current challenges and successes, and the potential for the implementation of precision medicine approaches through the incorporation of genetics advances.
Asunto(s)
Artritis Reumatoide , Melanocortinas , Humanos , Proteínas Portadoras , Inflamación/tratamiento farmacológico , Inflamación/metabolismo , Melanocortinas/metabolismo , Receptores de Melanocortina/genética , Receptores de Melanocortina/metabolismoRESUMEN
Type 4 P-type ATPases (P4-ATPases) are lipid flippases that drive the active transport of phospholipids from exoplasmic or luminal leaflets to cytosolic leaflets of eukaryotic membranes. The molecular architecture of P4-ATPases and the mechanism through which they recognize and transport lipids have remained unknown. Here we describe the cryo-electron microscopy structure of the P4-ATPase Drs2p-Cdc50p, a Saccharomyces cerevisiae lipid flippase that is specific to phosphatidylserine and phosphatidylethanolamine. Drs2p-Cdc50p is autoinhibited by the C-terminal tail of Drs2p, and activated by the lipid phosphatidylinositol-4-phosphate (PtdIns4P or PI4P). We present three structures that represent the complex in an autoinhibited, an intermediate and a fully activated state. The analysis highlights specific features of P4-ATPases and reveals sites of autoinhibition and PI4P-dependent activation. We also observe a putative lipid translocation pathway in this flippase that involves a conserved PISL motif in transmembrane segment 4 and polar residues of transmembrane segments 2 and 5, in particular Lys1018, in the centre of the lipid bilayer.
Asunto(s)
ATPasas Transportadoras de Calcio/química , ATPasas Transportadoras de Calcio/metabolismo , Microscopía por Crioelectrón , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimología , Sitios de Unión , Transporte Biológico , ATPasas Transportadoras de Calcio/antagonistas & inhibidores , ATPasas Transportadoras de Calcio/ultraestructura , Activación Enzimática , Membrana Dobles de Lípidos/metabolismo , Modelos Biológicos , Modelos Moleculares , Fosfatidiletanolaminas/metabolismo , Fosfatos de Fosfatidilinositol/química , Fosfatos de Fosfatidilinositol/metabolismo , Fosfatidilserinas/metabolismo , Dominios Proteicos , Proteínas de Saccharomyces cerevisiae/antagonistas & inhibidores , Proteínas de Saccharomyces cerevisiae/ultraestructuraRESUMEN
Clostridioides difficile infections have emerged as the leading cause of healthcare-associated infectious diarrhea. Disease symptoms are mainly caused by the virulence factors, TcdA and TcdB, which are large homologous multidomain proteins. Here, we report a 2.8 Å resolution cryo-EM structure of native TcdA, unveiling its conformation at neutral pH. The structure uncovers the dynamic movement of the CROPs domain which is induced in response to environmental acidification. Furthermore, the structure reveals detailed information about the interaction area between the CROPs domain and the tip of the delivery and receptor-binding domain, which likely serves to shield the C-terminal part of the hydrophobic pore-forming region from solvent exposure. Similarly, extensive interactions between the globular subdomain and the N-terminal part of the pore-forming region suggest that the globular subdomain shields the upper part of the pore-forming region from exposure to the surrounding solvent. Hence, the TcdA structure provides insights into the mechanism of preventing premature unfolding of the pore-forming region at neutral pH, as well as the pH-induced inter-domain dynamics.
Asunto(s)
Toxinas Bacterianas , Clostridioides difficile , Proteínas Bacterianas/metabolismo , Toxinas Bacterianas/metabolismo , Clostridioides , Enterotoxinas/química , Enterotoxinas/metabolismoRESUMEN
Cable bacteria are multicellular, filamentous bacteria that use internal conductive fibers to transfer electrons over centimeter distances from donors within anoxic sediment layers to oxygen at the surface. We extracted the fibers and used them as free-standing bio-based electrodes to investigate their electrocatalytic behavior. The fibers catalyzed the reversible interconversion of oxygen and water, and an electric current was running through the fibers even when the potential difference was generated solely by a gradient of oxygen concentration. Oxygen reduction as well as oxygen evolution were confirmed by optical measurements. Within living cable bacteria, oxygen reduction by direct electrocatalysis on the fibers and not by membrane-bound proteins readily explains exceptionally high cell-specific oxygen consumption rates observed in the oxic zone, while electrocatalytic water oxidation may provide oxygen to cells in the anoxic zone.
Asunto(s)
Sedimentos Geológicos , Sulfuros , Transporte de Electrón , Sedimentos Geológicos/microbiología , Sulfuros/metabolismo , Oxidación-Reducción , Bacterias/metabolismo , Oxígeno/metabolismo , Agua/metabolismo , ElectrodosRESUMEN
The human peptide transporter hPEPT1 (SLC15A1) is responsible for uptake of dietary di- and tripeptides and a number of drugs from the small intestine by utilizing the proton electrochemical gradient, and hence an important target for peptide-like drug design and drug delivery. hPEPT1 belongs to the ubiquitous major facilitator superfamily that all contain a 12TM core structure, with global conformational changes occurring during the transport cycle. Several bacterial homologues of these transporters have been characterized, providing valuable insight into the transport mechanism of this family. Here we report the overexpression and purification of recombinant hPEPT1 in a detergent-solubilized state. Thermostability profiling of hPEPT1 at different pH values revealed that hPEPT1 is more stable at pH 6 as compared to pH 7 and 8. Micro-scale thermophoresis (MST) confirmed that the purified hPEPT1 was able to bind di- and tripeptides respectively. To assess the in-solution oligomeric state of hPEPT1, negative stain electron microscopy was performed, demonstrating a predominantly monomeric state.
Asunto(s)
Expresión Génica , Transportador de Péptidos 1 , Calor , Humanos , Concentración de Iones de Hidrógeno , Transportador de Péptidos 1/biosíntesis , Transportador de Péptidos 1/química , Transportador de Péptidos 1/genética , Transportador de Péptidos 1/aislamiento & purificación , Estabilidad Proteica , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificaciónRESUMEN
Ice active bacteria can catalyze water freezing at high subzero temperatures using ice nucleating proteins (INPs) located at their outer cell walls. INPs are the most effective ice nucleators known and are of significant interest for agriculture, climate research, and freeze/antifreeze technologies. The aggregation of INPs into large ice nucleation sites is a key step for effective ice nucleation. It has been proposed that ice active bacteria can drive the aggregation of INPs and thereby trigger ice nucleation. However, the mechanism of INP aggregate assembly and the molecular processes behind the activation are still unclear. Both biochemical pathways and activation through electrostatics have been proposed based on experiments with lysed ice active bacteria. For a more direct view on the assembly of INPs, we follow the structure and water interactions of a synthetic model INP of the well-studied ice bacterium Pseudomonas syringae at the air-water interface as a function of the subphase pH. By combining sum frequency generation spectroscopy with two-dimensional infrared spectra, we conclude that self-assembly and electrostatic interactions drive the formation of ordered INP structures capable of aligning interfacial water.
Asunto(s)
Proteínas de la Membrana Bacteriana Externa , Hielo , Bacterias/metabolismo , Proteínas de la Membrana Bacteriana Externa/química , Proteínas de la Membrana Bacteriana Externa/metabolismo , Congelación , Electricidad Estática , Agua/químicaRESUMEN
Herein, we characterize the cellular uptake of a DNA structure generated by rolling circle DNA amplification. The structure, termed nanoflower, was fluorescently labeled by incorporation of ATTO488-dUTP allowing the intracellular localization to be followed. The nanoflower had a hydrodynamic diameter of approximately 300 nanometer and was non-toxic for all mammalian cell lines tested. It was internalized specifically by mammalian macrophages by phagocytosis within a few hours resulting in specific compartmentalization in phagolysosomes. Maximum uptake was observed after eight hours and the nanoflower remained stable in the phagolysosomes with a half-life of 12 h. Interestingly, the nanoflower co-localized with both Mycobacterium tuberculosis and Leishmania infantum within infected macrophages although these pathogens escape lysosomal degradation by affecting the phagocytotic pathway in very different manners. These results suggest an intriguing and overlooked potential application of DNA structures in targeted treatment of infectious diseases such as tuberculosis and leishmaniasis that are caused by pathogens that escape the human immune system by modifying macrophage biology.
Asunto(s)
ADN/química , ADN/metabolismo , Leishmania infantum/metabolismo , Macrófagos/microbiología , Macrófagos/parasitología , Mycobacterium tuberculosis/metabolismo , Fagosomas/metabolismo , ADN/análisis , Replicación del ADN , Fluorescencia , Semivida , Humanos , Leishmaniasis/terapia , Macrófagos/citología , Macrófagos/inmunología , Nanoestructuras/análisis , Nanoestructuras/química , Técnicas de Amplificación de Ácido Nucleico , Fagocitosis , Fagosomas/química , Fagosomas/microbiología , Fagosomas/parasitología , Tuberculosis/terapiaRESUMEN
Cable bacteria of the family Desulfobulbaceae form centimeter-long filaments comprising thousands of cells. They occur worldwide in the surface of aquatic sediments, where they connect sulfide oxidation with oxygen or nitrate reduction via long-distance electron transport. In the absence of pure cultures, we used single-filament genomics and metagenomics to retrieve draft genomes of 3 marine Candidatus Electrothrix and 1 freshwater Ca. Electronema species. These genomes contain >50% unknown genes but still share their core genomic makeup with sulfate-reducing and sulfur-disproportionating Desulfobulbaceae, with few core genes lost and 212 unique genes (from 197 gene families) conserved among cable bacteria. Last common ancestor analysis indicates gene divergence and lateral gene transfer as equally important origins of these unique genes. With support from metaproteomics of a Ca. Electronema enrichment, the genomes suggest that cable bacteria oxidize sulfide by reversing the canonical sulfate reduction pathway and fix CO2 using the Wood-Ljungdahl pathway. Cable bacteria show limited organotrophic potential, may assimilate smaller organic acids and alcohols, fix N2, and synthesize polyphosphates and polyglucose as storage compounds; several of these traits were confirmed by cell-level experimental analyses. We propose a model for electron flow from sulfide to oxygen that involves periplasmic cytochromes, yet-unidentified conductive periplasmic fibers, and periplasmic oxygen reduction. This model proposes that an active cable bacterium gains energy in the anodic, sulfide-oxidizing cells, whereas cells in the oxic zone flare off electrons through intense cathodic oxygen respiration without energy conservation; this peculiar form of multicellularity seems unparalleled in the microbial world.
Asunto(s)
Proteínas Bacterianas/metabolismo , Evolución Biológica , Deltaproteobacteria/genética , Deltaproteobacteria/fisiología , Genoma Bacteriano , Proteoma/análisis , Secuencia de Aminoácidos , Proteínas Bacterianas/genética , Ciclo del Carbono , Movimiento Celular , Quimiotaxis , Citocromos/metabolismo , Deltaproteobacteria/clasificación , Transporte de Electrón , Sedimentos Geológicos/microbiología , Nitratos/metabolismo , Oxidación-Reducción , Oxígeno/metabolismo , Filogenia , Homología de Secuencia , Sulfuros/metabolismoRESUMEN
Polymer-lipid hybrid vesicles are an emerging type of nano-assemblies that show potential as artificial organelles among others. Phospholipids and poly(cholesteryl methacrylate)-block-poly(methionine methacryloyloxyethyl ester (METMA)-random-2-carboxyethyl acrylate (CEA)) labeled with a Förster resonance energy transfer (FRET) reporter pair are used for the assembly of small and giant hybrid vesicles with homogenous distribution of both building blocks in the membrane as confirmed by the FRET effect. These hybrid vesicles have no inherent cytotoxicity when incubated with HepG2 cells up to 1.1 × 1011 hybrid vesicles per mL, and they are internalized by the cells. In contrast to the fluorescent signal originating from the block copolymer, the fluorescent signal coming from the lipids is barely detectable in cells incubated with hybrid vesicles for 6 h followed by 24 h in cell media, suggesting that the two building blocks have a different intracellular fate. These findings provide important insight into the design criteria of artificial organelles with potential structural integrity.
Asunto(s)
Fosfolípidos , Polímeros , Supervivencia Celular , Transferencia Resonante de Energía de Fluorescencia , Células Hep G2 , Humanos , Fosfolípidos/química , Fosfolípidos/toxicidad , Polímeros/química , Polímeros/toxicidad , Vesículas Transportadoras/químicaRESUMEN
Type IV pili are important virulence factors on the surface of many pathogenic bacteria and have been implicated in a wide range of diverse functions, including attachment, twitching motility, biofilm formation, and horizontal gene transfer. The respiratory pathogen Streptococcus pneumoniae deploys type IV pili to take up DNA during transformation. These "competence pili" are composed of the major pilin protein ComGC and exclusively assembled during bacterial competence, but their biogenesis remains unclear. Here, we report the high resolution NMR structure of N-terminal truncated ComGC revealing a highly flexible and structurally divergent type IV pilin. It consists of only three α-helical segments forming a well-defined electronegative cavity and confined electronegative and hydrophobic patches. The structure is particularly flexible between the first and second α-helix with the first helical part exhibiting slightly slower dynamics than the rest of the pilin, suggesting that the first helix is involved in forming the pilus structure core and that parts of helices two and three are primarily surface-exposed. Taken together, our results provide the first structure of a type IV pilin protein involved in the formation of competence-induced pili in Gram-positive bacteria and corroborate the remarkable structural diversity among type IV pilin proteins.
Asunto(s)
Proteínas Fimbrias/química , Fimbrias Bacterianas/ultraestructura , Modelos Moleculares , Streptococcus pneumoniae/fisiología , Factores de Virulencia/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Microscopía por Crioelectrón , Dimerización , Proteínas Fimbrias/genética , Proteínas Fimbrias/metabolismo , Fimbrias Bacterianas/metabolismo , Eliminación de Gen , Interacciones Hidrofóbicas e Hidrofílicas , Cinética , Membrana Dobles de Lípidos/química , Membrana Dobles de Lípidos/metabolismo , Microscopía Electrónica de Transmisión , Resonancia Magnética Nuclear Biomolecular , Operón , Fragmentos de Péptidos/química , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/metabolismo , Conformación Proteica , Conformación Proteica en Hélice alfa , Proteínas Recombinantes de Fusión , Solubilidad , Streptococcus pneumoniae/ultraestructura , Transactivadores/genética , Transactivadores/metabolismo , Factores de Virulencia/genética , Factores de Virulencia/metabolismoRESUMEN
The human proton coupled folic acid transporter PCFT is the major import route for dietary folates. Mutations in the gene encoding PCFT cause hereditary folic acid malabsorption, which manifests itself by compromised folate absorption from the intestine and also in impaired folate transport into the central nervous system. Since its recent discovery, PCFT has been the subject of numerous biochemical studies aiming at understanding its structure and mechanism. One major focus has been its oligomeric state, with some reports supporting oligomers and others a monomer. Here, we report the overexpression and purification of recombinant PCFT. Following detergent screening, n-Dodecyl ß-D-maltoside (DDM) and lauryl maltose neopentyl glycol (LMNG) were chosen for further work as they exhibited the most optimal solubilization. We found that purified detergent solubilized PCFT was able to bind folic acid, thus indicating a functionally active protein. Size exclusion chromatography showed that PCFT in DDM was polydisperse; the LMNG preparation was clearly monodisperse but with shorter retention time than the major DDM peak. To assess the oligomeric state negative stain electron microscopy was performed which showed a particle with the size of a PCFT dimer.
Asunto(s)
Transportador de Folato Acoplado a Protón/química , Animales , Detergentes , Ácido Fólico/metabolismo , Glucósidos , Glicoles , Humanos , Ligandos , Microscopía Electrónica , Modelos Moleculares , Multimerización de Proteína , Estructura Cuaternaria de Proteína , Transportador de Folato Acoplado a Protón/metabolismo , Transportador de Folato Acoplado a Protón/ultraestructura , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/ultraestructura , Células Sf9 , Solubilidad , SpodopteraRESUMEN
The repressive Nucleosome Remodeling and histone Deacetylation (NuRD) complex remodels the chromatin structure by coupling ATP-dependent remodeling activity with histone deacetylase function and plays important roles in regulating gene transcription, DNA damage repair and chromatin assembly. The complex is composed of six subunits: Metastasis Associated proteins MTA1/2/3 initially recruit histone chaperones RBBP4/7 followed by the histone deacetylases HDAC1/2 forming a core complex. Further association of the CpG-binding protein MBD2/3, p66α/ß and the ATP-dependent helicase CDH3/4 constitutes the NuRD complex. Recent structural studies on truncated human proteins or orthologous have revealed that the stoichiometry of the MTA1-RBBP4 complex is 2:4. This study reports expression and purification of the intact human MTA2-RBBP7 complex using HEK293F cells as expression system. In analogy with findings on the Drosophila NuRD complex, we find that also the human MTA-RBBP can be isolated in vitro. Taken together with previous findings this suggests, that MTA-RBBP is a stable complex, with a central role in the initial assembly of the human NuRD complex. Refined 3D volumes of the complex generated from negative stain electron microscopy (EM) data reveals an elongated architecture that is capable of hinge like motion around the center of the particle.
Asunto(s)
Ensamble y Desensamble de Cromatina/genética , Histona Desacetilasas/química , Complejo Desacetilasa y Remodelación del Nucleosoma Mi-2/química , Proteínas Represoras/química , Proteína 7 de Unión a Retinoblastoma/química , Secuencia de Aminoácidos/genética , Regulación de la Expresión Génica , Células HEK293 , Chaperonas de Histonas/química , Chaperonas de Histonas/aislamiento & purificación , Chaperonas de Histonas/metabolismo , Histona Desacetilasa 1/química , Histona Desacetilasa 1/genética , Histona Desacetilasa 1/metabolismo , Histona Desacetilasa 2/química , Histona Desacetilasa 2/genética , Histona Desacetilasa 2/metabolismo , Histona Desacetilasas/genética , Histona Desacetilasas/aislamiento & purificación , Humanos , Complejo Desacetilasa y Remodelación del Nucleosoma Mi-2/genética , Complejo Desacetilasa y Remodelación del Nucleosoma Mi-2/metabolismo , Proteínas Represoras/genética , Proteínas Represoras/aislamiento & purificación , Proteína 7 de Unión a Retinoblastoma/genética , Proteína 7 de Unión a Retinoblastoma/aislamiento & purificaciónRESUMEN
Exploiting the anomalous signal of the intrinsic S atoms to phase a protein structure is advantageous, as ideally only a single well diffracting native crystal is required. However, sulfur is a weak anomalous scatterer at the typical wavelengths used for X-ray diffraction experiments, and therefore sulfur SAD data sets need to be recorded with a high multiplicity. In this study, the structure of a small pilin protein was determined by sulfur SAD despite several obstacles such as a low anomalous signal (a theoretical Bijvoet ratio of 0.9% at a wavelength of 1.8 Å), radiation damage-induced reduction of the cysteines and a multiplicity of only 5.5. The anomalous signal was improved by merging three data sets from different volumes of a single crystal, yielding a multiplicity of 17.5, and a sodium ion was added to the substructure of anomalous scatterers. In general, all data sets were balanced around the threshold values for a successful phasing strategy. In addition, a collection of statistics on structures from the PDB that were solved by sulfur SAD are presented and compared with the data. Looking at the quality indicator R(anom)/R(p.i.m.), an inconsistency in the documentation of the anomalous R factor is noted and reported.
Asunto(s)
Cisteína/química , Proteínas Fimbrias/química , Azufre/química , Difracción de Rayos X/métodos , Cristalización , Cristalografía por Rayos X , Bases de Datos de Proteínas , Modelos Moleculares , Conformación ProteicaRESUMEN
BACKGROUND: Type IV pili are widely expressed among Gram-negative bacteria, where they are involved in biofilm formation, serve in the transfer of DNA, motility and in the bacterial attachment to various surfaces. Type IV pili in Shewanella oneidensis are also supposed to play an important role in extracellular electron transfer by the attachment to sediments containing electron acceptors and potentially forming conductive nanowires. RESULTS: The potential nanowire type IV pilin PilBac1 from S. oneidensis was characterized by a combination of complementary structural methods and the atomic structure was determined at a resolution of 1.67 Å by X-ray crystallography. PilBac1 consists of one long N-terminal α-helix packed against four antiparallel ß-strands, thus revealing the core fold of type IV pilins. In the crystal, PilBac1 forms a parallel dimer with a sodium ion bound to one of the monomers. Interestingly, our PilBac1 crystal structure reveals two unusual features compared to other type IVa pilins: an unusual position of the disulfide bridge and a straight α-helical section, which usually exhibits a pronounced kink. This straight helix leads to a distinct packing in a filament model of PilBac1 based on an EM model of a Neisseria pilus. CONCLUSIONS: In this study we have described the first structure of a pilin from Shewanella oneidensis. The structure possesses features of the common type IV pilin core, but also exhibits significant variations in the α-helical part and the D-region.
Asunto(s)
Proteínas Fimbrias/química , Fimbrias Bacterianas/química , Shewanella/genética , Cristalografía por Rayos X , Proteínas Fimbrias/genética , Fimbrias Bacterianas/genética , Modelos Moleculares , Estructura Secundaria de Proteína , Homología de Secuencia de Aminoácido , Shewanella/química , Shewanella/metabolismoRESUMEN
UNLABELLED: The members of the oligoadenylate synthetase (OAS) family of proteins are antiviral restriction factors that target a wide range of RNA and DNA viruses. They function as intracellular double-stranded RNA (dsRNA) sensors that, upon binding to dsRNA, undergo a conformational change and are activated to synthesize 2'-5'-linked oligoadenylates (2-5As). 2-5As of sufficient length act as second messengers to activate RNase L and thereby restrict viral replication. We expressed human OAS3 using the baculovirus system and purified it to homogeneity. We show that recombinant OAS3 is activated at a substantially lower concentration of dsRNA than OAS1, making it a potent in vivo sensor of dsRNA. Moreover, we find that OAS3 synthesizes considerably longer 2-5As than previously reported, and that OAS3 can activate RNase L intracellularly. The combined high affinity for dsRNA and the capability to produce 2-5As of sufficient length to activate RNase L suggests that OAS3 is a potent activator of RNase L. In addition, we provide experimental evidence to support one active site of OAS3 located in the C-terminal OAS domain and generate a low-resolution structure of OAS3 using SAXS. IMPORTANCE: We are the first to purify the OAS3 enzyme to homogeneity, which allowed us to characterize the mechanism utilized by OAS3 and identify the active site. We provide compelling evidence that OAS3 can produce 2'-5'-oligoadenylates of sufficient length to activate RNase L. This is contrary to what is described in the current literature but agrees with recent in vivo data showing that OAS3 harbors an antiviral activity requiring RNase L. Thus, our work redefines our understanding of the biological role of OAS3. Furthermore, we used a combination of mutagenesis and small-angle X-ray scattering to describe the active site and low-resolution structure of OAS3.
Asunto(s)
2',5'-Oligoadenilato Sintetasa/metabolismo , Nucleótidos de Adenina/metabolismo , Endorribonucleasas/metabolismo , Oligorribonucleótidos/metabolismo , Nucleótidos de Adenina/genética , Nucleótidos de Adenina/aislamiento & purificación , Secuencia de Aminoácidos , Baculoviridae/genética , Dominio Catalítico , Activación Enzimática , Expresión Génica , Vectores Genéticos , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Oligorribonucleótidos/genética , Oligorribonucleótidos/aislamiento & purificación , Conformación Proteica , ARN Bicatenario/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Dispersión del Ángulo Pequeño , Alineación de SecuenciaRESUMEN
High-throughput screening was used to find selective inhibitors of human 15-lipoxygenase-1 (15-LOX-1). One hit, a 1-benzoyl substituted pyrazole-3-carboxanilide (1a), was used as a starting point in a program to develop potent and selective 15-LOX-1 inhibitors.
Asunto(s)
Araquidonato 15-Lipooxigenasa/metabolismo , Inhibidores de la Lipooxigenasa/química , Inhibidores de la Lipooxigenasa/farmacología , Pirazoles/química , Pirazoles/farmacología , Amidas/química , Amidas/farmacología , HumanosRESUMEN
Investigation of 1N-substituted pyrazole-3-carboxanilides as 15-lipoxygenase-1 (15-LOX-1) inhibitors demonstrated that the 1N-substituent was not essential for activity or selectivity. Additional halogen substituents on the pyrazole ring, however, increased activity. Further development led to triazole-4-carboxanilides and 2-(3-pyrazolyl) benzoxazoles, which are potent and selective 15-LOX-1 inhibitors.