RESUMEN
The myophage possesses a contractile tail that penetrates its host cell envelope. Except for investigations on the bacteriophage T4 with a rather complicated structure, the assembly pattern and tail contraction mechanism of myophage remain largely unknown. Here, we present the fine structure of a freshwater Myoviridae cyanophage Pam3, which has an icosahedral capsid of ~680 Å in diameter, connected via a three-section neck to an 840-Å-long contractile tail, ending with a three-module baseplate composed of only six protein components. This simplified baseplate consists of a central hub-spike surrounded by six wedge heterotriplexes, to which twelve tail fibers are covalently attached via disulfide bonds in alternating upward and downward configurations. In vitro reduction assays revealed a putative redox-dependent mechanism of baseplate assembly and tail sheath contraction. These findings establish a minimal myophage that might become a user-friendly chassis phage in synthetic biology.
Asunto(s)
Myoviridae , Ensamble de Virus , Bacteriófago T4/química , Cápside , Proteínas de la Cápside/química , Microscopía por Crioelectrón , Myoviridae/químicaRESUMEN
The coordination of carbon and nitrogen metabolism is essential for bacteria to adapt to nutritional variations in the environment, but the underlying mechanism remains poorly understood. In autotrophic cyanobacteria, high CO2 levels favor the carboxylase activity of ribulose 1,5 bisphosphate carboxylase/oxygenase (RuBisCO) to produce 3-phosphoglycerate, whereas low CO2 levels promote the oxygenase activity of RuBisCO, leading to 2-phosphoglycolate (2-PG) production. Thus, the 2-PG level is reversely correlated with that of 2-oxoglutarate (2-OG), which accumulates under a high carbon/nitrogen ratio and acts as a nitrogen-starvation signal. The LysR-type transcriptional repressor NAD(P)H dehydrogenase regulator (NdhR) controls the expression of genes related to carbon metabolism. Based on genetic and biochemical studies, we report here that 2-PG is an inducer of NdhR, while 2-OG is a corepressor, as found previously. Furthermore, structural analyses indicate that binding of 2-OG at the interface between the two regulatory domains (RD) allows the NdhR tetramer to adopt a repressor conformation, whereas 2-PG binding to an intradomain cleft of each RD triggers drastic conformational changes leading to the dissociation of NdhR from its target DNA. We further confirmed the effect of 2-PG or 2-OG levels on the transcription of the NdhR regulon. Together with previous findings, we propose that NdhR can sense 2-OG from the Krebs cycle and 2-PG from photorespiration, two key metabolites that function together as indicators of intracellular carbon/nitrogen status, thus representing a fine sensor for the coordination of carbon and nitrogen metabolism in cyanobacteria.
Asunto(s)
Carbono/metabolismo , Cianobacterias/metabolismo , Genes Reguladores , NAD(P)H Deshidrogenasa (Quinona)/metabolismo , Nitrógeno/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Dióxido de Carbono/metabolismo , Cianobacterias/genética , Regulación Bacteriana de la Expresión Génica , Glicolatos/metabolismo , Ácidos Cetoglutáricos/metabolismo , NAD(P)H Deshidrogenasa (Quinona)/genética , Ribulosa-Bifosfato Carboxilasa/genética , Ribulosa-Bifosfato Carboxilasa/metabolismo , Transducción de SeñalRESUMEN
Cyanophages, widespread in aquatic systems, are a class of viruses that specifically infect cyanobacteria. Though they play important roles in modulating the homeostasis of cyanobacterial populations, little is known about the freshwater cyanophages, especially those hypothetical proteins of unknown function. Mic1 is a freshwater siphocyanophage isolated from the Lake Chaohu. It encodes three hypothetical proteins Gp65, Gp66, and Gp72, which share an identity of 61.6% to 83%. However, we find these three homologous proteins differ from each other in oligomeric state. Moreover, we solve the crystal structure of Gp72 at 2.3 Å, which represents a novel fold in the α + ß class. Structural analyses combined with redox assays enable us to propose a model of disulfide bond mediated oligomerization for Gp72. Altogether, these findings provide structural and biochemical basis for further investigations on the freshwater cyanophage Mic1.
Asunto(s)
Bacteriófagos/química , Cianobacterias/virología , Disulfuros/química , Proteínas Virales/química , Secuencia de Aminoácidos , Bacteriófagos/genética , Bacteriófagos/metabolismo , Sitios de Unión , Clonación Molecular , Cristalografía por Rayos X , Disulfuros/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Agua Dulce/microbiología , Agua Dulce/virología , Expresión Génica , Vectores Genéticos/química , Vectores Genéticos/metabolismo , Modelos Moleculares , Oxidación-Reducción , Unión Proteica , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Pliegue de Proteína , Dominios y Motivos de Interacción de Proteínas , Multimerización de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Proteínas Virales/genética , Proteínas Virales/metabolismoRESUMEN
Despite over 3300 protein-DNA complex structures have been reported in the past decades, there remain some unknown recognition patterns between protein and target DNA. The silkgland-specific transcription factor FMBP-1 from the silkworm Bombyx mori contains a unique DNA-binding domain of four tandem STPRs, namely the score and three amino acid peptide repeats. Here we report three structures of this STPR domain (termed BmSTPR) in complex with DNA of various lengths. In the presence of target DNA, BmSTPR adopts a zig-zag structure of three or four tandem α-helices that run along the major groove of DNA. Structural analyses combined with binding assays indicate BmSTPR prefers the AT-rich sequences, with each α-helix covering a DNA sequence of 4 bp. The successive AT-rich DNAs adopt a wider major groove, which is in complementary in shape and size to the tandem α-helices of BmSTPR. Substitutions of DNA sequences and affinity comparison further prove that BmSTPR recognizes the major groove mainly via shape readout. Multiple-sequence alignment suggests this unique DNA-binding pattern should be highly conserved for the STPR domain containing proteins which are widespread in animals. Together, our findings provide structural insights into the specific interactions between a novel DNA-binding protein and a unique deformed B-DNA.
Asunto(s)
Proteínas de Unión al ADN/química , ADN/química , Proteínas de Insectos/química , Factores de Transcripción/química , Animales , Sitios de Unión , Bombyx , ADN/metabolismo , Proteínas de Unión al ADN/metabolismo , Proteínas de Insectos/metabolismo , Modelos Moleculares , Conformación de Ácido Nucleico , Unión Proteica , Conformación Proteica en Hélice alfa , Dominios Proteicos , Secuencias Repetitivas de Ácidos Nucleicos , Factores de Transcripción/metabolismoRESUMEN
Invertases catalyze the hydrolysis of sucrose to glucose and fructose, thereby playing a key role in primary metabolism and plant development. According to the optimum pH, invertases are classified into acid invertases (Ac-Invs) and alkaline/neutral invertases (A/N-Invs), which share no sequence homology. Compared with Ac-Invs that have been extensively studied, the structure and catalytic mechanism of A/N-Invs remain unknown. Here we report the crystal structures of Anabaena alkaline invertase InvA, which was proposed to be the ancestor of modern plant A/N-Invs. These structures are the first in the GH100 family. InvA exists as a hexamer in both crystal and solution. Each subunit consists of an (α/α)6 barrel core structure in addition to an insertion of three helices. A couple of structures in complex with the substrate or products enabled us to assign the subsites -1 and +1 specifically binding glucose and fructose, respectively. Structural comparison combined with enzymatic assays indicated that Asp-188 and Glu-414 are putative catalytic residues. Further analysis of the substrate binding pocket demonstrated that InvA possesses a stringent substrate specificity toward the α1,2-glycosidic bond of sucrose. Together, we suggest that InvA and homologs represent a novel family of glucosidases.
Asunto(s)
Anabaena/enzimología , Proteínas Bacterianas/química , beta-Fructofuranosidasa/química , Anabaena/genética , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Cristalografía por Rayos X , Fructosa/química , Fructosa/metabolismo , Glucosa/química , Glucosa/metabolismo , Dominios Proteicos , Sacarosa/química , Sacarosa/metabolismo , beta-Fructofuranosidasa/genética , beta-Fructofuranosidasa/metabolismoRESUMEN
BACKGROUND: Renal ischemia/reperfusion (I/R)-induced acute kidney injury remains to be a troublesome condition in clinical practice. Although the exact molecular mechanisms underlying renal I/R injury are incompletely understood, the deleterious progress of renal I/R injury involves inflammation, apoptosis, and oxidative stress. Diosmetin is a member of the flavonoid glycosides family, which suppresses the inflammatory response and cellular apoptosis and enhances antioxidant activity. The purpose of this study was to investigate the protective effect of diosmetin on I/R-induced renal injury in mice. METHODS: Thirty BALB/c mice were randomly divided into five groups. Four groups of mice received diosmetin (0.25, 0.5, and 1 mg/kg) or vehicle (I/R group) before ischemia. Another group received vehicle without ischemia to serve as a negative control (sham-operated group). Twenty-four hours after reperfusion, serum and renal tissues were harvested to evaluate renal function and histopathologic features. In addition, the expression of inflammation-related proteins, apoptotic molecules, and antioxidant enzymes was analyzed. RESULTS: Compared with sham mice, the I/R group significantly exacerbated renal function and renal tube architecture and increased the inflammatory response and renal tubule apoptosis. Nevertheless, pretreatment with diosmetin reversed these changes. In addition, diosmetin treatment resulted in a marked increase in antioxidant protein expression compared with I/R mice. CONCLUSIONS: The renoprotective effects of diosmetin involved suppression of the nuclear factor-κB and mitochondrial apoptosis pathways, as well as activation of the nuclear factor erythroid 2-related factor 2/heme oxygenase-1 pathway. Diosmetin has significant potential as a therapeutic intervention to ameliorate renal injury after renal I/R.
Asunto(s)
Lesión Renal Aguda/prevención & control , Flavonoides/uso terapéutico , Sustancias Protectoras/uso terapéutico , Daño por Reperfusión/prevención & control , Lesión Renal Aguda/etiología , Lesión Renal Aguda/metabolismo , Animales , Biomarcadores/metabolismo , Western Blotting , Esquema de Medicación , Inmunohistoquímica , Etiquetado Corte-Fin in Situ , Masculino , Ratones , Ratones Endogámicos BALB C , Distribución Aleatoria , Reacción en Cadena en Tiempo Real de la Polimerasa , Daño por Reperfusión/metabolismo , Resultado del TratamientoRESUMEN
The soilborne fungus Verticillium dahliae is the major pathogen that causes the verticillium wilt disease of plants, which leads to huge economic loss worldwide. At the early stage of infection, growth of the pathogen is subject to the nutrition stress of limited nitrogen. To investigate the secreted pathogenic proteins that play indispensable roles during invasion at this stage, we compared the profiles of secreted proteins of V. dahliae under nitrogen starvation and normal conditions by using in-gel and in-solution digestion combined with liquid chromatography-nano-electrospray ionization tandem mass spectrometry (LC-nanoESI-MS). In total, we identified 212 proteins from the supernatant of liquid medium, including 109 putative secreted proteins. Comparative analysis indicated that the expression of 76 proteins was induced, whereas that of 9 proteins was suppressed under nitrogen starvation. Notably, 24 proteins are constitutively expressed. Further bioinformatic exploration enabled us to classify the stress-induced proteins into seven functional groups: cell wall degradation (10.5%), reactive oxygen species (ROS) scavenging and stress response (11.8%), lipid effectors (5.3%), protein metabolism (21.1%), carbohydrate metabolism (15.8%), electron-proton transport and energy metabolism (14.5%), and other (21.0%). In addition, most stress-suppressed proteins are involved in the cell-wall remodeling. Taken together, our analyses provide insights into the pathogenesis of V. dahliae and might give hints for the development of novel strategy against the verticillium wilt disease.
Asunto(s)
Proteínas Fúngicas/análisis , Proteínas Fúngicas/metabolismo , Nitrógeno/deficiencia , Verticillium/metabolismo , Secuencia de Aminoácidos , Pared Celular/metabolismo , Cromatografía Liquida , Electroforesis en Gel de Poliacrilamida , Depuradores de Radicales Libres/metabolismo , Espectrometría de Masas , Datos de Secuencia Molecular , Nitrógeno/metabolismo , Enfermedades de las Plantas/microbiología , Proteoma/análisis , Proteoma/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Estrés Fisiológico , Verticillium/crecimiento & desarrollo , Verticillium/patogenicidadRESUMEN
Saccharomyces cerevisiae Gre2 (EC1.1.1.283) serves as a versatile enzyme that catalyzes the stereoselective reduction of a broad range of substrates including aliphatic and aromatic ketones, diketones, as well as aldehydes, using NADPH as the cofactor. Here we present the crystal structures of Gre2 from S. cerevisiae in an apo-form at 2.00Å and NADPH-complexed form at 2.40Å resolution. Gre2 forms a homodimer, each subunit of which contains an N-terminal Rossmann-fold domain and a variable C-terminal domain, which participates in substrate recognition. The induced fit upon binding to the cofactor NADPH makes the two domains shift toward each other, producing an interdomain cleft that better fits the substrate. Computational simulation combined with site-directed mutagenesis and enzymatic activity analysis enabled us to define a potential substrate-binding pocket that determines the stringent substrate stereoselectivity for catalysis.
Asunto(s)
Apoenzimas/química , Coenzimas/química , NADP/química , Oxidorreductasas/química , Subunidades de Proteína/química , Proteínas de Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/química , Secuencia de Aminoácidos , Apoenzimas/genética , Apoenzimas/metabolismo , Coenzimas/metabolismo , Cristalografía por Rayos X , Cinética , Simulación del Acoplamiento Molecular , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , NADP/metabolismo , Oxidorreductasas/genética , Oxidorreductasas/metabolismo , Unión Proteica , Multimerización de Proteína , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/enzimología , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Alineación de Secuencia , Especificidad por Sustrato , TermodinámicaRESUMEN
The cyanosiphophage Mic1 specifically infects the bloom-forming Microcystis aeruginosa FACHB 1339 from Lake Chaohu, China. Previous genomic analysis showed that its 92,627 bp double-stranded DNA genome consists of 98 putative open reading frames, 63% of which are of unknown function. Here, we investigated the transcriptome dynamics of Mic1 and its host using RNA sequencing. In the early, middle, and late phases of the 10 h lytic cycle, the Mic1 genes are sequentially expressed and could be further temporally grouped into two distinct clusters in each phase. Notably, six early genes, including gp49 that encodes a TnpB-like transposase, immediately reach the highest transcriptional level in half an hour, representing a pioneer cluster that rapidly regulates and redirects host metabolism toward the phage. An in-depth analysis of the host transcriptomic profile in response to Mic1 infection revealed significant upregulation of a polyketide synthase pathway and a type III-B CRISPR system, accompanied by moderate downregulation of the photosynthesis and key metabolism pathways. The constant increase of phage transcripts and relatively low replacement rate over the host transcripts indicated that Mic1 utilizes a unique strategy to gradually take over a small portion of host metabolism pathways after infection. In addition, genomic analysis of a less-infective Mic1 and a Mic1-resistant host strain further confirmed their dynamic interplay and coevolution via the frequent horizontal gene transfer. These findings provide insights into the mutual benefit and symbiosis of the highly polymorphic cyanobacteria M. aeruginosa and cyanophages. IMPORTANCE: The highly polymorphic Microcystis aeruginosa is one of the predominant bloom-forming cyanobacteria in eutrophic freshwater bodies and is infected by diverse and abundant cyanophages. The presence of a large number of defense systems in M. aeruginosa genome suggests a dynamic interplay and coevolution with the cyanophages. In this study, we investigated the temporal gene expression pattern of Mic1 after infection and the corresponding transcriptional responses of its host. Moreover, the identification of a less-infective Mic1 and a Mic1-resistant host strain provided the evolved genes in the phage-host coevolution during the multiple-generation cultivation in the laboratory. Our findings enrich the knowledge on the interplay and coevolution of M. aeruginosa and its cyanophages and lay the foundation for the future application of cyanophage as a potential eco-friendly and bio-safe agent in controlling the succession of harmful cyanobacterial blooms.
Asunto(s)
Bacteriófagos , Microcystis , Microcystis/virología , Microcystis/genética , Microcystis/metabolismo , Bacteriófagos/genética , Bacteriófagos/fisiología , China , Transcriptoma , Lagos/microbiología , Lagos/virología , Genoma Viral/genética , Evolución MolecularRESUMEN
The primary role of yeast Ara1, previously mis-annotated as a D-arabinose dehydrogenase, is to catalyze the reduction of a variety of toxic α,ß-dicarbonyl compounds using NADPH as a cofactor at physiological pH levels. Here, crystal structures of Ara1 in apo and NADPH-complexed forms are presented at 2.10 and 2.00â Å resolution, respectively. Ara1 exists as a homodimer, each subunit of which adopts an (α/ß)8-barrel structure and has a highly conserved cofactor-binding pocket. Structural comparison revealed that induced fit upon NADPH binding yielded an intact active-site pocket that recognizes the substrate. Moreover, the crystal structures combined with computational simulation defined an open substrate-binding site to accommodate various substrates that possess a dicarbonyl group.
Asunto(s)
NADP/metabolismo , Oxidorreductasas Actuantes sobre Donantes de Grupos Aldehído u Oxo/química , Oxidorreductasas Actuantes sobre Donantes de Grupos Aldehído u Oxo/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimología , Deshidrogenasas del Alcohol de Azúcar/química , Deshidrogenasas del Alcohol de Azúcar/metabolismo , Secuencia de Aminoácidos , Sitios de Unión , Biocatálisis , Cristalografía por Rayos X , Modelos Moleculares , Datos de Secuencia Molecular , Multimerización de Proteína , Alineación de Secuencia , Especificidad por SustratoRESUMEN
In order to explore the characteristics of exhaust gas emissions, environmental impact, and human health risks in the pesticide manufacturing industry, two typical pesticide manufacturing enterprises were selected as the research objects, and samples were collected and analyzed for all exhaust pipes of each enterprise. The following results were noted:there were certain differences in the pollutants produced by different enterprises due to different products and production links. The main pollutants in enterprise A were ammonia and VOCs. The concentration of ammonia in enterprise A ranged from 0 to 847.83 mg·m-3, and the concentration of VOCs ranged from 4.21 to 91.68 mg·m-3. The main pollutants in enterprise B were VOCs, and the concentration of VOCs ranged from 3.37 to 197.30 mg·m-3. The ozone formation potential (OFP) ranged from 1.96 to 107.24 mg·m-3. Substances that required further attention in terms of ozone formation potential:enterprise A mainly included ethanol, methanol, toluene, xylene, and other substances; enterprise B mainly included 1, 1-dichloroethylene, 1, 2-dichloroethane, toluene, methylal, and other substances. The secondary organic aerosol formation potential (SOAFP) ranged from 0.94 to 74.72 mg·m-3. The main contributors to the secondary organic aerosol formation potential were aromatic hydrocarbons and oxygen-containing organics. In addition, ammonia also required additional attention. The odorous substances in pesticide enterprises were more complex, and there were differences in the exhaust pipes of different enterprises and different production links of the same enterprise. There were certain health risks in the gas pollutants of pesticide enterprises. The main carcinogens were 1, 2-dichloroethane, trichloroethylene, tetrachloroethylene, methyl chloride, and benzene. In addition, pyridine and hexachloroethane had certain non-carcinogenic risks in pesticide production enterprises.
RESUMEN
BACKGROUND: Along with the fast development and urbanization in developing countries, the waterbodies aside the growing cities become heavily polluted and highly eutrophic, thus leading to the seasonal outbreak of cyanobacterial bloom. Systematic isolation and characterization of freshwater cyanophages might provide a biological solution to control the awful blooms. However, genomic sequences and related investigations on the freshwater cyanophages remain very limited to date. RESULTS: Following our recently reported five cyanophages Pam1~Pam5 from Lake Chaohu in China, here we isolated another five cyanophages, termed Pan1~Pan5, which infect the cyanobacterium Pseudanabaena sp. Chao 1811. Whole-genome sequencing showed that they all contain a double-stranded DNA genome of 37.2 to 72.0 kb in length, with less than half of the putative open reading frames annotated with known functions. Remarkably, the siphophage Pan1 encodes an auxiliary metabolic gene phoH and constitutes, together with the host, a complete queuosine modification pathway. Proteomic analyses revealed that although Pan1~Pan5 are distinct from each other in evolution, Pan1 and Pan3 are somewhat similar to our previously identified cyanophages Pam3 and Pam1 at the genomic level, respectively. Moreover, phylogenetic analyses suggested that Pan1 resembles the α-proteobacterial phage vB_DshS-R5C, revealing direct evidence for phage-mediated horizontal gene transfer between cyanobacteria and α-proteobacteria. CONCLUSION: In addition to the previous reports of Pam1~Pam5, the present findings on Pan1~Pan5 largely enrich the library of reference freshwater cyanophages. The abundant genomic information provides a pool to identify novel genes and proteins of unknown function. Moreover, we found for the first time the evolutionary traces in the cyanophage that horizontal gene transfer might occur at the level of not only inter-species, but even inter-phylum. It indicates that the bacteriophage or cyanophage could be developed as a powerful tool for gene manipulation among various species or phyla.
RESUMEN
BACKGROUND: As important producers using photosynthesis on Earth, cyanobacteria contribute to the oxygenation of atmosphere and the primary production of biosphere. However, due to the eutrophication of urban waterbodies and global warming, uncontrollable growth of cyanobacteria usually leads to the seasonal outbreak of cyanobacterial blooms. Cyanophages, a group of viruses that specifically infect and lyse cyanobacteria, are considered as potential environment-friendly agents to control the harmful blooms. Compared to the marine counterparts, only a few freshwater cyanophages have been isolated and genome sequenced to date, largely limiting their characterizations and applications. RESULTS: Here, we isolated five freshwater cyanophages varying in tail morphology, termed Pam1~Pam5, all of which infect the cyanobacterium Pseudanabaena mucicola Chao 1806 that was isolated from the bloom-suffering Lake Chaohu in Anhui, China. The whole-genome sequencing showed that cyanophages Pam1~Pam5 all contain a dsDNA genome, varying in size from 36 to 142 Kb. Phylogenetic analyses suggested that Pam1~Pam5 possess different DNA packaging mechanisms and are evolutionarily distinct from each other. Notably, Pam1 and Pam5 have lysogeny-associated gene clusters, whereas Pam2 possesses 9 punctuated DNA segments identical to the CRISPR spacers in the host genome. Metagenomic data-based calculation of the relative abundance of Pam1~Pam5 at the Nanfei estuary towards the Lake Chaohu revealed that the short-tailed Pam1 and Pam5 account for the majority of the five cyanophages. Moreover, comparative analyses of the reference genomes of Pam1~Pam5 and previously reported cyanophages enabled us to identify three circular and seven linear contigs of virtual freshwater cyanophages from the metagenomic data of the Lake Chaohu. CONCLUSIONS: We propose a high-throughput strategy to systematically identify cyanophages based on the currently available metagenomic data and the very limited reference genomes of experimentally isolated cyanophages. This strategy could be applied to mine the complete or partial genomes of unculturable bacteriophages and viruses. Transformation of the synthesized whole genomes of these virtual phages/viruses to proper hosts will enable the rescue of bona fide viral particles and eventually enrich the library of microorganisms that exist on Earth. Video abstract.
Asunto(s)
Bacteriófagos , Genoma Viral , Minería de Datos , Agua Dulce/microbiología , Genoma Viral/genética , Metagenómica , Oligopéptidos , Filogenia , Receptor Toll-Like 2/agonistas , Receptor Toll-Like 9/agonistasRESUMEN
Despite previous structural analyses of bacteriophages, quite little is known about the structures and assembly patterns of cyanophages. Using cryo-EM combined with crystallography, we solve the near-atomic-resolution structure of a freshwater short-tailed cyanophage, Pam1, which comprises a 400-Å-long tail and an icosahedral capsid of 650 Å in diameter. The outer capsid surface is reinforced by trimeric cement proteins with a ß-sandwich fold, which structurally resemble the distal motif of Pam1's tailspike, suggesting its potential role in host recognition. At the portal vertex, the dodecameric portal and connected adaptor, followed by a hexameric needle head, form a DNA ejection channel, which is sealed by a trimeric needle. Moreover, we identify a right-handed rifling pattern that might help DNA to revolve along the wall of the ejection channel. Our study reveals the precise assembly pattern of a cyanophage and lays the foundation to support its practical biotechnological and environmental applications.
Asunto(s)
Bacteriófagos/química , Cápside/química , Cianobacterias/virología , Secuenciación Completa del Genoma/métodos , Microscopía por Crioelectrón , Cristalografía por Rayos X , Tamaño del Genoma , Genoma Viral , Modelos Moleculares , Conformación Molecular , Ensamble de VirusRESUMEN
The hemolymph of the fifth instar larvae of the silkworm Bombyx mori contains a group of homologous proteins with a molecular weight of approximately 30 kDa, termed B. mori low molecular weight lipoproteins (Bmlps), which account for about 5% of the total plasma proteins. These so-called "30 K proteins" have been reported to be involved in the innate immune response and transportation of lipid and/or sugar. To elucidate their molecular functions, we determined the crystal structure of a 30 K protein, Bmlp7, at 1.91Å. It has two distinct domains: an all-α N-terminal domain (NTD) and an all-ß C-terminal domain (CTD) of the ß-trefoil fold. Comparative structural analysis indicates that Bmlp7 represents a new family, adding to the 14 families currently identified, of the ß-trefoil superfamily. Structural comparison and simulation suggest that the NTD has a putative lipid-binding cavity, whereas the CTD has a potential sugar-binding site. However, we were unable to detect the binding of either lipid or sugar. Therefore, further investigations are needed to characterize the molecular function of this protein.
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Bombyx/química , Proteínas de Insectos/química , Secuencia de Aminoácidos , Animales , Secuencia Conservada , Cristalografía por Rayos X , Hemolinfa/química , Datos de Secuencia Molecular , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Alineación de SecuenciaRESUMEN
Yeast glutaredoxins Grx1 and Grx2 catalyze the reduction of both inter- and intra-molecular disulfide bonds using glutathione (GSH) as the electron donor. Although sharing the same dithiolic CPYC active site and a sequence identity of 64%, they have been proved to play different roles during oxidative stress and to possess different glutathione-disulfide reductase activities. To address the structural basis of these differences, we solved the crystal structures of Grx2 in oxidized and reduced forms, at 2.10 A and 1.50 A, respectively. With the Grx1 structures we previously reported, comparative structural analyses revealed that Grx1 and Grx2 share a similar GSH binding site, except for a single residue substitution from Asp89 in Grx1 to Ser123 in Grx2. Site-directed mutagenesis in combination with activity assays further proved this single residue variation is critical for the different activities of yeast Grx1 and Grx2.
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Glutarredoxinas/química , Glutarredoxinas/fisiología , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/fisiología , Secuencia de Aminoácidos , Sitios de Unión , Dominio Catalítico , Cristalografía por Rayos X/métodos , Escherichia coli/metabolismo , Glutatión/química , Datos de Secuencia Molecular , Oxidación-Reducción , Estrés Oxidativo , Unión Proteica , Conformación Proteica , Homología de Secuencia de AminoácidoRESUMEN
The manganese-specific superoxide dismutase SOD2 from the yeast Saccharomyces cerevisiae is a protein that resides in the mitochondrion and protects it against attack by superoxide radicals. However, a high iron concentration in the mitochondria results in iron misincorporation at the active site, with subsequent inactivation of SOD2. Here, the crystal structures of SOD2 bound with the native metal manganese and with the `wrong' metal iron are presented at 2.05 and 1.79 Å resolution, respectively. Structural comparison of the two structures shows no significant conformational alteration in the overall structure or in the active site upon binding the non-native metal iron. Moreover, residues Asp163 and Lys80 are proposed to potentially be responsible for the metal specificity of the Mn-specific SOD. Additionally, the surface-potential distribution of SOD2 revealed a conserved positively charged electrostatic zone in the proximity of the active site that probably functions in the same way as in Cu/Zn-SODs by facilitating the diffusion of the superoxide anion to the metal ion.
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Hierro/química , Saccharomyces cerevisiae/enzimología , Superóxido Dismutasa/química , Secuencia de Aminoácidos , Biocatálisis , Cristalografía por Rayos X , Enlace de Hidrógeno , Modelos Moleculares , Datos de Secuencia Molecular , Estructura Terciaria de Proteína , Alineación de Secuencia , Electricidad Estática , Homología Estructural de ProteínaRESUMEN
Saccharomyces cerevisiae Hsp33/YOR391Cp is a member of the ThiI/DJ-1/PfpI superfamily. Hsp33 was overexpressed in Escherichia coli and its crystal structure was determined at 2.40â Å resolution. Structural comparison revealed that Hsp33 adopts an α/ß-hydrolase fold and possesses the putative Cys-His-Glu catalytic triad common to the Hsp31 family, suggesting that Hsp33 and Hsp31 share similar aminopeptidase activity, while structural deviations in helices α2-α3 of the core domain might be responsible for the access of different peptide substrates.
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Aminopeptidasas/química , Proteínas de Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/química , Secuencia de Aminoácidos , Dominio Catalítico , Cristalografía por Rayos X , Proteínas de Choque Térmico/química , Modelos Moleculares , Datos de Secuencia Molecular , Multimerización de Proteína , Estructura Secundaria de Proteína , Alineación de Secuencia , Homología Estructural de ProteínaRESUMEN
Autophagy-related protein Atg8 is ubiquitous in all eukaryotes. It is involved in the Atg8-PE ubiquitin-like conjugation system, which is essential for autophagosome formation. The structures of Atg8 from different species are very similar and share a ubiquitin-fold domain at the C-terminus. In the 2.40 A crystal structure of Atg8 from the silkworm Bombyx mori reported here, the ubiquitin fold at the C-terminus is preceded by two additional helices at the N-terminus.
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Autofagia , Bombyx/química , Proteínas de Insectos/química , Secuencia de Aminoácidos , Animales , Bombyx/citología , Cristalografía por Rayos X , Modelos Moleculares , Datos de Secuencia Molecular , Pliegue de Proteína , Estructura Cuaternaria de Proteína , Estructura Terciaria de Proteína , Alineación de Secuencia , Homología de Secuencia de AminoácidoRESUMEN
The folding and assembly of RuBisCO, the most abundant enzyme in nature, needs a series of chaperones, including the RuBisCO accumulation factor Raf1, which is highly conserved in cyanobacteria and plants. Here, we report the crystal structures of Raf1 from cyanobacteria Anabaena sp. PCC 7120 and its complex with RuBisCO large subunit RbcL. Structural analyses and biochemical assays reveal that each Raf1 dimer captures an RbcL dimer, with the C-terminal tail inserting into the catalytic pocket, and further mediates the assembly of RbcL dimers to form the octameric core of RuBisCO. Furthermore, the cryo-electron microscopy structures of the RbcL-Raf1-RbcS assembly intermediates enable us to see a dynamic assembly process from RbcL8Raf18 to the holoenzyme RbcL8RbcS8. In vitro assays also indicate that Raf1 can attenuate and reverse CcmM-mediated cyanobacterial RuBisCO condensation. Combined with previous findings, we propose a putative model for the assembly of cyanobacterial RuBisCO coordinated by the chaperone Raf1.