RESUMEN

Tailed bacteriophages are one of the most widespread biological entities on Earth. Their singular structures, such as spikes or fibers are of special interest given their potential use in a wide range of biotechnological applications. In particular, the long fibers present at the termini of the T4 phage tail have been studied in detail and are important for host recognition and adsorption. Although significant progress has been made in elucidating structural mechanisms of model phages, the high-resolution structural description of the vast population of marine phages is still unexplored. In this context, we present here the crystal structure of C24, a putative receptor-binding tip-like protein from Bizionia argentinensis JUB59, a psychrotolerant bacterium isolated from the marine surface waters of Potter Cove, Antarctica. The structure resembles the receptor-binding tip from the bacteriophage T4 long tail fiber yet showing marked differences in its domain organization, size, sequence identity and metal binding nature. We confirmed the viral origin of C24 by induction experiments using mitomycin C. Our results reveal the presence of a novel uncharacterized prophage in the genome of B. argentinensis JUB59, whose morphology is compatible with the order Caudovirales and that carries the nucleotide sequence of C24 in its genome. This work provides valuable information to expand our current knowledge on the viral machinery prevalent in the oceans.

Asunto(s)

Bacteriófagos/genética , Flavobacteriaceae/virología , Regiones Antárticas , Genoma Bacteriano/genética , Genoma Viral/genética , Unión Proteica/genética

RESUMEN

Survival and infectivity of trypanosomatids rely on cell-surface and secreted glycoconjugates, many of which contain a variable number of galactose residues. Incorporation of galactose to proteins and lipids occurs along the secretory pathway from UDP-galactose (UDP-Gal). Before being used in glycosylation reactions, however, this activated sugar donor must first be transported across the endoplasmic reticulum and Golgi membranes by a specific nucleotide sugar transporter (NST). In this study, we identified an UDP-Gal transporter (named TcNST2 and encoded by the TcCLB.504085.60 gene) from Trypanosoma cruzi, the etiological agent of Chagas disease. TcNST2 was identified by heterologous expression of selected putative nucleotide sugar transporters in a mutant Chinese Hamster Ovary cell line. TcNST2 mRNA levels were detected in all T. cruzi life-cycle forms, with an increase in expression in axenic amastigotes. Confocal microscope analysis indicated that the transporter is specifically localized to the Golgi apparatus. A three-dimensional model of TcNST2 suggested an overall structural conservation as compared with members of the metabolite transporter superfamily and also suggested specific features that could be related to its activity. The identification of this transporter is an important step toward a better understanding of glycoconjugate biosynthesis and the role NSTs play in this process in trypanosomatids.

Asunto(s)

Aparato de Golgi/metabolismo , Proteínas de Transporte de Monosacáridos/metabolismo , Proteínas Protozoarias/genética , Trypanosoma cruzi/genética , Animales , Células CHO , Cricetulus , Proteínas Protozoarias/química , Proteínas Protozoarias/metabolismo , Análisis de Secuencia de Proteína , Trypanosoma cruzi/metabolismo

RESUMEN

The histidine kinase (HK) domain belonging to the light-oxygen-voltage histidine kinase (LOV-HK) from Brucella abortus is a member of the HWE family, for which no structural information is available, and has low sequence identity (20%) to the closest HK present in the PDB. The `off-edge' S-SAD method in macromolecular X-ray crystallography was used to solve the structure of the HK domain from LOV-HK at low resolution from crystals in a low-symmetry space group (P21) and with four copies in the asymmetric unit (∼108 kDa). Data were collected both from multiple crystals (diffraction limit varying from 2.90 to 3.25 Å) and from multiple orientations of the same crystal, using the κ-geometry goniostat on SOLEIL beamline PROXIMA 1, to obtain `true redundancy'. Data from three different crystals were combined for structure determination. An optimized HK construct bearing a shorter cloning artifact yielded crystals that diffracted X-rays to 2.51 Šresolution and that were used for final refinement of the model. Moreover, a thorough a posteriori analysis using several different combinations of data sets allowed us to investigate the impact of the data-collection strategy on the success of the structure determination.

Asunto(s)

Brucella abortus/enzimología , Proteínas Quinasas/química , Brucella abortus/química , Cristalización , Cristalografía por Rayos X/métodos , Histidina Quinasa , Modelos Moleculares , Conformación Proteica , Estructura Terciaria de Proteína

RESUMEN

Phytochromes give rise to the largest photosensor family known to date. However, they are underrepresented in the Protein Data Bank. Plant, cyanobacterial, fungal and bacterial phytochromes share a canonical architecture consisting of an N-terminal photosensory module (PAS2-GAF-PHY domains) and a C-terminal variable output module. The bacterium Xanthomonas campestris pv. campestris, a worldwide agricultural pathogen, codes for a single bacteriophytochrome (XccBphP) that has this canonical architecture, bearing a C-terminal PAS9 domain as the output module. Full-length XccBphP was cloned, expressed and purified to homogeneity by nickel-NTA affinity and size-exclusion chromatography and was then crystallized at room temperature bound to its cofactor biliverdin. A complete native X-ray diffraction data set was collected to a maximum resolution of 3.25 Å. The crystals belonged to space group P43212, with unit-cell parameters a = b = 103.94, c = 344.57 Šand a dimer in the asymmetric unit. Refinement is underway after solving the structure by molecular replacement.

Asunto(s)

Fitocromo/química , Xanthomonas campestris/química , Secuencia de Aminoácidos , Cristalización , Cristalografía por Rayos X , Datos de Secuencia Molecular

RESUMEN

Riboflavin synthase (RS) catalyzes the last step of riboflavin biosynthesis in microorganisms and plants, which corresponds to the dismutation of two molecules of 6,7-dimethyl-8-ribityllumazine to yield one molecule of riboflavin and one molecule of 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione. Owing to the absence of this enzyme in animals and the fact that most pathogenic bacteria show a strict dependence on riboflavin biosynthesis, RS has been proposed as a potential target for antimicrobial drug development. Eubacterial, fungal and plant RSs assemble as homotrimers lacking C3 symmetry. Each monomer can bind two substrate molecules, yet there is only one active site for the whole enzyme, which is located at the interface between two neighbouring chains. This work reports the crystallographic structure of RS from the pathogenic bacterium Brucella abortus (the aetiological agent of the disease brucellosis) in its apo form, in complex with riboflavin and in complex with two different product analogues, being the first time that the structure of an intact RS trimer with bound ligands has been solved. These crystal models support the hypothesis of enhanced flexibility in the particle and also highlight the role of the ligands in assembling the unique active site. Kinetic and binding studies were also performed to complement these findings. The structural and biochemical information generated may be useful for the rational design of novel RS inhibitors with antimicrobial activity.

Asunto(s)

Brucella abortus/enzimología , Riboflavina Sintasa/química , Riboflavina Sintasa/metabolismo , Secuencia de Aminoácidos , Sitios de Unión , Dominio Catalítico , Cristalografía por Rayos X , Cinética , Modelos Moleculares , Datos de Secuencia Molecular , Conformación Proteica , Riboflavina/química , Riboflavina Sintasa/genética , Homología de Secuencia de Aminoácido

RESUMEN

Winged-helix transcriptional factors play important roles in the control of gene expression in many organisms. In the plant pathogens Xylella fastidiosa and Agrobacterium tumefaciens, the winged-helix protein BigR, a member of the ArsR/SmtB family of metal sensors, regulates transcription of the bigR operon involved in bacterial biofilm growth. Previous studies showed that BigR represses transcription of its own operon through the occupation of the RNA polymerase-binding site; however, the signals that modulate its activity and the biological function of its operon are still poorly understood. Here we show that although BigR is a homodimer similar to metal sensors, it functions as a novel redox switch that derepresses transcription upon oxidation. Crystal structures of reduced and oxidized BigR reveal that formation of a disulfide bridge involving two critical cysteines induces conformational changes in the dimer that remarkably alter the topography of the winged-helix DNA-binding interface, precluding DNA binding. This structural mechanism of DNA association-dissociation is novel among winged-helix factors. Moreover, we demonstrate that the bigR operon is required for hydrogen sulfide detoxification through the action of a sulfur dioxygenase (Blh) and sulfite exporter. As hydrogen sulfide strongly inhibits cytochrome c oxidase, it must be eliminated to allow aerobic growth under low oxygen tension, an environmental condition found in bacterial biofilms, xylem vessels, and root tissues. Accordingly, we show that the bigR operon is critical to sustain bacterial growth under hypoxia. These results suggest that BigR integrates the transcriptional regulation of a sulfur oxidation pathway to an oxidative signal through a thiol-based redox switch.

Asunto(s)

Agrobacterium tumefaciens/metabolismo , Proteínas Bacterianas/metabolismo , Sulfuro de Hidrógeno/metabolismo , Oxígeno/metabolismo , Plantas/microbiología , Factores de Transcripción/metabolismo , Xylella/metabolismo , Agrobacterium tumefaciens/crecimiento & desarrollo , Agrobacterium tumefaciens/fisiología , Animales , Proteínas Bacterianas/química , Biopelículas/crecimiento & desarrollo , Secuencia Conservada , Cisteína , ADN Bacteriano/genética , ADN Bacteriano/metabolismo , Dioxigenasas/metabolismo , Disulfuros/química , Sulfuro de Hidrógeno/toxicidad , Ratones , Proteínas Mitocondriales/metabolismo , Modelos Moleculares , Operón/genética , Oxidación-Reducción , Estructura Secundaria de Proteína , Factores de Transcripción/química , Transcripción Genética , Xylella/crecimiento & desarrollo , Xylella/fisiología

RESUMEN

Leptospirosis is a world spread zoonosis caused by members of the genus Leptospira. Although leptospires were identified as the causal agent of leptospirosis almost 100 years ago, little is known about their biology, which hinders the development of new treatment and prevention strategies. One of the several aspects of the leptospiral biology not yet elucidated is the process by which outer membrane proteins (OMPs) traverse the periplasm and are inserted into the outer membrane. The crystal structure determination of the conserved hypothetical protein LIC12922 from Leptospira interrogans revealed a two domain protein homologous to the Escherichia coli periplasmic chaperone SurA. The LIC12922 NC-domain is structurally related to the chaperone modules of E. coli SurA and trigger factor, whereas the parvulin domain is devoid of peptidyl prolyl cis-trans isomerase activity. Phylogenetic analyses suggest a relationship between LIC12922 and the chaperones PrsA, PpiD and SurA. Based on our structural and evolutionary analyses, we postulate that LIC12922 is a periplasmic chaperone involved in OMPs biogenesis in Leptospira spp. Since LIC12922 homologs were identified in all spirochetal genomes sequenced to date, this assumption may have implications for the OMPs biogenesis studies not only in leptospires but in the entire Phylum Spirochaetes.

Asunto(s)

Proteínas de la Membrana Bacteriana Externa/química , Proteínas de la Membrana Bacteriana Externa/metabolismo , Leptospira/metabolismo , Periplasma/metabolismo , Secuencia de Aminoácidos , Modelos Moleculares , Datos de Secuencia Molecular , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Homología de Secuencia de Aminoácido

RESUMEN

Leptospirosis is a world spread zoonosis caused by members of the genus Leptospira. Although leptospireswere identified as the causal agent of leptospirosis almost 100 years ago, little is known about their biology,which hinders the development of new treatment and prevention strategies. One of the severalaspects of the leptospiral biology not yet elucidated is the process by which outer membrane proteins (OMPs) traverse the periplasm and are inserted into the outer membrane. The crystal structure determination of the conserved hypothetical protein LIC12922 from Leptospira interrogans revealed a two domain protein homologous to the Escherichia coli periplasmic chaperone SurA. The LIC12922 NC-domain isstructurally related to the chaperone modules of E. coli SurA and trigger factor, whereas the parvulindomain is devoid of peptidyl prolyl cis–trans isomerase activity. Phylogenetic analyses suggest a relationshipbetween LIC12922 and the chaperones PrsA, PpiD and SurA. Based on our structural and evolutionaryanalyses, we postulate that LIC12922 is a periplasmic chaperone involved in OMPs biogenesis inLeptospira spp. Since LIC12922 homologs were identified in all spirochetal genomes sequenced to date,this assumption may have implications for the OMPs biogenesis studies not only in leptospires but in the entire Phylum Spirochaetes.

Asunto(s)

Chaperonas Moleculares/análisis , Leptospira/genética , Leptospira/inmunología , Clonación de Organismos/métodos , Escherichia coli/crecimiento & desarrollo , Leptospira interrogans/genética , Spirochaetales/genética , Spirochaetales/inmunología , Vectores Genéticos/análisis , Vectores Genéticos/genética

RESUMEN

The enzymatic activity of thioredoxin reductase enzymes is endowed by at least two redox centers: a flavin and a dithiol/disulfide CXXC motif. The interaction between thioredoxin reductase and thioredoxin is generally species-specific, but the molecular aspects related to this phenomenon remain elusive. Here, we investigated the yeast cytosolic thioredoxin system, which is composed of NADPH, thioredoxin reductase (ScTrxR1), and thioredoxin 1 (ScTrx1) or thioredoxin 2 (ScTrx2). We showed that ScTrxR1 was able to efficiently reduce yeast thioredoxins (mitochondrial and cytosolic) but failed to reduce the human and Escherichia coli thioredoxin counterparts. To gain insights into this specificity, the crystallographic structure of oxidized ScTrxR1 was solved at 2.4 A resolution. The protein topology of the redox centers indicated the necessity of a large structural rearrangement for FAD and thioredoxin reduction using NADPH. Therefore, we modeled a large structural rotation between the two ScTrxR1 domains (based on the previously described crystal structure, PDB code 1F6M ). Employing diverse approaches including enzymatic assays, site-directed mutagenesis, amino acid sequence alignment, and structure comparisons, insights were obtained about the features involved in the species-specificity phenomenon, such as complementary electronic parameters between the surfaces of ScTrxR1 and yeast thioredoxin enzymes and loops and residues (such as Ser(72) in ScTrx2). Finally, structural comparisons and amino acid alignments led us to propose a new classification that includes a larger number of enzymes with thioredoxin reductase activity, neglected in the low/high molecular weight classification.

Asunto(s)

Reductasa de Tiorredoxina-Disulfuro/química , Reductasa de Tiorredoxina-Disulfuro/metabolismo , Tiorredoxinas/química , Tiorredoxinas/metabolismo , Secuencia de Aminoácidos , Sitios de Unión , Secuencia Conservada , Disulfuros/química , Flavina-Adenina Dinucleótido/metabolismo , Flavinas/química , Flavinas/metabolismo , Cinética , Modelos Moleculares , Mutagénesis , NADP/metabolismo , Unión Proteica , Conformación Proteica , Saccharomyces cerevisiae/enzimología , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Sensibilidad y Especificidad , Reductasa de Tiorredoxina-Disulfuro/genética , Tiorredoxinas/genética , Tolueno/análogos & derivados , Tolueno/química

RESUMEN

Shwachman-Bodian-Diamond syndrome is an autosomal recessive genetic syndrome with pleiotropic phenotypes, including pancreatic deficiencies, bone marrow dysfunctions with increased risk of myelodysplasia or leukemia, and skeletal abnormalities. This syndrome has been associated with mutations in the SBDS gene, which encodes a conserved protein showing orthologs in Archaea and eukaryotes. The Shwachman-Bodian-Diamond syndrome pleiotropic phenotypes may be an indication of different cell type requirements for a fully functional SBDS protein. RNA-binding activity has been predicted for archaeal and yeast SBDS orthologs, with the latter also being implicated in ribosome biogenesis. However, full-length SBDS orthologs function in a species-specific manner, indicating that the knowledge obtained from model systems may be of limited use in understanding major unresolved issues regarding SBDS function, namely, the effect of mutations in human SBDS on its biochemical function and the specificity of RNA interaction. We determined the solution structure and backbone dynamics of the human SBDS protein and describe its RNA binding site using NMR spectroscopy. Similarly to the crystal structures of Archaea, the overall structure of human SBDS comprises three well-folded domains. However, significant conformational exchange was observed in NMR dynamics experiments for the flexible linker between the N-terminal domain and the central domain, and these experiments also reflect the relative motions of the domains. RNA titrations monitored by heteronuclear correlation experiments and chemical shift mapping analysis identified a classic RNA binding site at the N-terminal FYSH (fungal, Yhr087wp, Shwachman) domain that concentrates most of the mutations described for the human SBDS.

Asunto(s)

Proteínas/química , Proteínas/metabolismo , ARN/metabolismo , Secuencia de Aminoácidos , Secuencia de Bases , Sitios de Unión , Cartilla de ADN/genética , Enfermedades Genéticas Congénitas/genética , Humanos , Técnicas In Vitro , Modelos Moleculares , Datos de Secuencia Molecular , Resonancia Magnética Nuclear Biomolecular , Conformación Proteica , Proteínas/genética , Ribosomas/metabolismo , Homología de Secuencia de Aminoácido , Electricidad Estática , Homología Estructural de Proteína , Síndrome , Termodinámica

RESUMEN

Disulfide oxidoreductase DsbA catalyzes disulfide bond formation in proteins secreted to the periplasm and has been related to the folding process of virulence factors in many organisms. It is among the most oxidizing of the thioredoxin-like proteins, and DsbA redox power is understood in terms of the electrostatic interactions involving the active site motif CPHC. The plant pathogen Xylella fastidiosa has two chromosomal genes encoding two oxidoreductases belonging to the DsbA family, and in one of them, the canonical motif CPHC is replaced by CPAC. Biochemical assays showed that both X. fastidiosa homologues have similar redox properties and the determination of the crystal structure of XfDsbA revealed substitutions in the active site of X. fastidiosa enzymes, which are proposed to compensate for the lack of the conserved histidine in XfDsbA2. In addition, electron density maps showed a ligand bound to the XfDsbA active site, allowing the characterization of the enzyme interaction with an 8-mer peptide. Finally, surface analysis of XfDsbA and XfDsbA2 suggests that X. fastidiosa enzymes may have different substrate specificities.

Asunto(s)

Proteína Disulfuro Isomerasas/química , Proteína Disulfuro Isomerasas/metabolismo , Xylella/enzimología , Sustitución de Aminoácidos , Dominio Catalítico , Secuencia Conservada , Cristalografía por Rayos X , Histidina/química , Familia de Multigenes , Oxidación-Reducción , Proteína Disulfuro Isomerasas/genética , Especificidad por Sustrato , Xylella/genética

RESUMEN

The human SBDS gene and its yeast ortholog SDO1 encode essential proteins that are involved in ribosome biosynthesis. SDO1 has been implicated in recycling of the ribosomal biogenesis factor Tif6p from pre-66S particles as well as in translation activation of 60S ribosomes. The SBDS protein is highly conserved, containing approximately 250 amino acid residues in animals, fungi and Archaea, while SBDS orthologs of plants and a group of protists contain an extended C-terminal region. In this work, we describe the characterization of the Trypanosoma cruzi SBDS ortholog (TcSBDS). TcSBDS co-fractionates with polysomes in sucrose density gradients, which is consistent with a role in ribosome biosynthesis. We show that TcSBDS contains a C-terminal extension of 200 amino acids that displays the features of intrinsically disordered proteins as determined by proteolytic, circular dichroism and NMR analyses. Interestingly, the C-terminal extension is responsible for TcSBDS-RNA interaction activity in electrophoretic mobility shift assays. This finding suggests that Trypanosomatidae and possibly also other organisms containing SBDS with extended C-terminal regions have evolved an additional function for SBDS in ribosome biogenesis.

Asunto(s)

Proteínas Protozoarias/metabolismo , Proteínas de Unión al ARN/metabolismo , ARN/metabolismo , Trypanosoma cruzi/metabolismo , Secuencia de Aminoácidos , Animales , Clonación Molecular , Datos de Secuencia Molecular , Polirribosomas/fisiología , Proteínas/química , Proteínas/genética , Proteínas/metabolismo , Proteínas Protozoarias/química , Proteínas Protozoarias/genética , Proteínas de Unión al ARN/química , Proteínas de Unión al ARN/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Ribosomas/fisiología , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Alineación de Secuencia

RESUMEN

The Brazilian Synchrotron Light Laboratory [Laboratório Nacional de Luz Síncrotron (LNLS), Campinas, SP, Brazil] is the first commissioned synchrotron light source in the southern hemisphere. The first wiggler macromolecular crystallography beamline (MX2) at the LNLS has been recently constructed and brought into operation. Here the technical design, experimental set-up, parameters of the beamline and the first experimental results obtained at MX2 are described. The beamline operates on a 2.0 T hybrid 30-pole wiggler, and its optical layout includes collimating mirror, Si(111) double-crystal monochromator and toroidal bendable mirror. The measured flux density at the sample position at 8.7 eV reaches 4.8 x 10(11) photons s(-1) mm(-2) (100 mA)(-1). The beamline is equipped with a MarResearch Desktop Beamline Goniostat (MarDTB) and 3 x 3 MarMosaic225 CCD detector, and is controlled by a customized version of the Blu-Ice software. A description of the first X-ray diffraction data sets collected at the MX2 LNLS beamline and used for macromolecular crystal structure solution is also provided.

RESUMEN

Initially identified in yeast, the exosome has emerged as a central component of the RNA maturation and degradation machinery both in Archaea and eukaryotes. Here we describe a series of high-resolution structures of the RNase PH ring from the Pyrococcus abyssi exosome, one of them containing three 10-mer RNA strands within the exosome catalytic chamber, and report additional nucleotide interactions involving positions N5 and N7. Residues from all three Rrp41-Rrp42 heterodimers interact with a single RNA molecule, providing evidence for the functional relevance of exosome ring-like assembly in RNA processivity. Furthermore, an ADP-bound structure showed a rearrangement of nucleotide interactions at site N1, suggesting a rationale for the elimination of nucleoside diphosphate after catalysis. In combination with RNA degradation assays performed with mutants of key amino acid residues, the structural data presented here provide support for a model of exosome-mediated RNA degradation that integrates the events involving catalytic cleavage, product elimination, and RNA translocation. Finally, comparisons between the archaeal and human exosome structures provide a possible explanation for the eukaryotic exosome inability to catalyze phosphate-dependent RNA degradation.

Asunto(s)

Archaea/metabolismo , Proteínas Arqueales/química , Estabilidad del ARN , ARN/química , Catálisis , Dimerización , Humanos , Modelos Biológicos , Conformación Molecular , Mutagénesis , Nucleótidos/química , Fosfatos/química , Pyrococcus abyssi/metabolismo , ARN/metabolismo , Procesamiento Postranscripcional del ARN , Sulfolobus solfataricus/metabolismo

RESUMEN

The conserved protein Nip7 is involved in ribosome biogenesis, being required for proper 27S pre-rRNA processing and 60S ribosome subunit assembly in Saccharomyces cerevisiae. Yeast Nip7p interacts with nucleolar proteins and with the exosome subunit Rrp43p, but its molecular function remains to be determined. Solution of the Pyrococcus abyssi Nip7 (PaNip7) crystal structure revealed a monomeric protein composed by two alpha-beta domains. The N-terminal domain is formed by a five-stranded antiparallel beta-sheet surrounded by three alpha-helices and a 310 helix while the C-terminal, a mixed beta-sheet domain composed by strands beta8 to beta12, one alpha-helix, and a 310 helix, corresponds to the conserved PUA domain (after Pseudo-Uridine synthases and Archaeosine-specific transglycosylases). By combining structural analyses and RNA interaction assays, we assessed the ability of both yeast and archaeal Nip7 orthologues to interact with RNA. Structural alignment of the PaNip7 PUA domain with the RNA-interacting surface of the ArcTGT (archaeosine tRNA-guanine transglycosylase) PUA domain indicated that in the archaeal PUA domain positively charged residues (R151, R152, K155, and K158) are involved in RNA interaction. However, equivalent positions are occupied by mostly hydrophobic residues (A/G160, I161, F164, and A167) in eukaryotic Nip7 orthologues. Both proteins can bind specifically to polyuridine, and RNA interaction requires specific residues of the PUA domain as determined by site-directed mutagenesis. This work provides experimental verification that the PUA domain mediates Nip7 interaction with RNA and reveals that the preference for interaction with polyuridine sequences is conserved in Archaea and eukaryotic Nip7 proteins.

Asunto(s)

Poli U/química , Proteínas Ribosómicas/química , Proteínas de Saccharomyces cerevisiae/química , Secuencia de Aminoácidos , Cristalografía por Rayos X , Ensayo de Cambio de Movilidad Electroforética , Modelos Moleculares , Datos de Secuencia Molecular , Oligorribonucleótidos/química , Pentosiltransferasa/química , Mapeo de Interacción de Proteínas , Estructura Terciaria de Proteína , Pyrococcus abyssi/química , Alineación de Secuencia

RESUMEN

6,7-Dimethyl-8-ribityllumazine synthase (lumazine synthase; LS) catalyzes the penultimate step in the biosynthesis of riboflavin in plants and microorganisms. This protein is known to exhibit different quaternary assemblies between species, existing as free pentamers, decamers (dimers of pentamers) and icosahedrally arranged dodecamers of pentamers. A phylogenetic analysis on eubacterial, fungal and plant LSs allowed us to classify them into two categories: Type I LSs (pentameric or icosahedral) and Type II LSs (decameric). The Rhizobiales represent an order of alpha-proteobacteria that includes, among others, the genera Mesorhizobium, Agrobacterium and Brucella. Here, we present structural and kinetic studies on several LSs from Rhizobiales. Interestingly, Mesorhizobium and Brucella encode both a Type-I LS and a Type-II LS called RibH1 and RibH2, respectively. We show that Type II LSs appear to be almost inactive, whereas Type I LSs present a highly variable catalytic activity according to the genus. Additionally, we have solved four RibH1/RibH2 crystallographic structures from the genera Mesorhizobium and Brucella. The relationship between the active-site architecture and catalytic properties in these isoenzymes is discussed, and a model that describes the enzymatic behavior is proposed. Furthermore, sequence alignment studies allowed us to extend our results to the genus Agrobacterium. Our results suggest that the selective pressure controlling the riboflavin pathway favored the evolution of catalysts with low reaction rates, since the excess of flavins in the intracellular pool in Rhizobiales could act as a negative factor when these bacteria are exposed to oxidative or nitrosative stress.

Asunto(s)

Brucella/enzimología , Complejos Multienzimáticos/química , Complejos Multienzimáticos/metabolismo , Rhizobium/enzimología , Secuencia de Aminoácidos , Sitios de Unión , Brucella/genética , Catálisis , Cristalografía por Rayos X , Cinética , Modelos Moleculares , Datos de Secuencia Molecular , Complejos Multienzimáticos/genética , Estructura Cuaternaria de Proteína , Pteridinas/metabolismo , Rhizobium/genética , Homología de Secuencia de Aminoácido , Especificidad por Sustrato

RESUMEN

The 26S proteasome is a large protein complex involved in protein degradation. We have shown previously that the PSMD7/Mov34 subunit of the human proteasome contains a proteolytically resistant MPN domain. MPN domain family members comprise subunits of the proteasome, COP9-signalosome and translation initiation factor 3 complexes. Here, the crystal structure of two C-terminally truncated proteins, MPN 1-186 and MPN 1-177, were solved to 1.96 and 3.0 A resolution, respectively. MPN 1-186 is formed by nine beta-strands surrounded by three alpha-helices plus a fourth alpha-helix at the C terminus. This final alpha-helix emerges from the domain core and folds along with a symmetrically related subunit, typical of a domain swap. The crystallographic dimer is consistent with size-exclusion chromatography and DLS analysis showing that MPN 1-186 is a dimer in solution. MPN 1-186 shows an overall architecture highly similar to the previously reported crystal structure of the Archaeal MPN domain AfJAMM of Archaeoglobus fulgidus. However, previous structural and biophysical analyses have shown that neither MPN 1-186 nor full-length human Mov34 bind metal, in opposition to the zinc-binding AfJAMM structures. The zinc ligand residues observed in AfJAMM are conserved in the yeast Rpn11 proteasome and Csn5 COP-signalosome subunits, which is consistent with the isopeptidase activity described for these proteins. The results presented here show that, although the MPN domain of Mov34 shows a typical metalloprotease fold, it is unable to coordinate a metal ion. This finding and amino acid sequence comparisons can explain why the MPN-containing proteins Mov34/PSMD7, RPN8, Csn6, Prp8p and the translation initiation factor 3 subunits f and h do not show catalytic isopeptidase activity, allowing us to propose the hypothesis that in these proteins the MPN domain has a primarily structural function.

Asunto(s)

Proteínas Portadoras/química , Factores Eucarióticos de Iniciación/química , Modelos Moleculares , Zinc/metabolismo , Proteínas Adaptadoras Transductoras de Señales , Secuencia de Aminoácidos , Sitios de Unión , Complejo del Señalosoma COP9 , Proteínas Portadoras/metabolismo , Cationes Bivalentes , Cristalografía por Rayos X , Dimerización , Factores Eucarióticos de Iniciación/metabolismo , Humanos , Datos de Secuencia Molecular , Estructura Secundaria de Proteína

RESUMEN

Eukaryotic MPN domain proteins are components of the complexes proteasome lid, COP9-signalosome (CSN), and translation initiation factor 3 (eIF3). The proteasome lid Rpn11 and COP9-signalosome Csn5 subunits, which contain the conserved JAMM motif involved in zinc ion coordination, show catalytic isopeptidase activity. Homology modeling indicates that the MPN domain of Mov34 cannot coordinate a zinc ion in the same manner as catalytically active MPN domains. In this work, we show that the MPN domain of Mov34 is highly resistant to proteolysis and the major product comprises residues 9-186, which includes the conserved MPN domain. Two clones containing the MPN domain region (MPN1-177 and MPN1-186) including the eight N-terminal residues show a less pronounced band in the 220 nm region of the CD, indicating lower alpha-helical content relative to the clones lacking these residues (MPN9-177 and MPN9-186). However, clones lacking residues 1-8 show lower expression levels and thermal stability, indicating that residues 1-8 are required for proper folding and stability of this particular MPN domain.

Asunto(s)

Proteínas Portadoras/metabolismo , Factores Eucarióticos de Iniciación/metabolismo , Proteínas Adaptadoras Transductoras de Señales , Secuencia de Aminoácidos , Complejo del Señalosoma COP9 , Proteínas Portadoras/química , Proteínas Portadoras/genética , Proteínas Portadoras/farmacología , Dicroismo Circular , Factores Eucarióticos de Iniciación/química , Factores Eucarióticos de Iniciación/genética , Factores Eucarióticos de Iniciación/farmacología , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Fragmentos de Péptidos/química , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/metabolismo , Fragmentos de Péptidos/farmacología , Péptido Hidrolasas/metabolismo , Inhibidores de Proteasas/química , Inhibidores de Proteasas/metabolismo , Inhibidores de Proteasas/farmacología , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Alineación de Secuencia , Homología Estructural de Proteína

RESUMEN

Organic hydroperoxide resistance proteins (Ohr) belong to a family of proteins that possess thiol-dependent peroxidase activity endowed by reactive cysteine residues able to reduce peroxides. The crystal structure of Ohr from Xylella fastidiosa in complex with polyethylene glycol, providing insights into enzyme-substrate interactions is described herein. In addition, crystallographic studies, molecular modeling and biochemical assays also indicated that peroxides derived from long chain fatty acids could be the biological substrates of Ohr. Because different oxidation states of the reactive cysteine were present in the Ohr structures from X. fastidiosa, Pseudomonas aeruginosa and Deinococcus radiodurans it was possible to envisage a set of snapshots along the coordinate of the enzyme-catalyzed reaction. The redox intermediates of X. fastidiosa Ohr observed in the crystals were further characterized in solution by electrospray ionization mass spectrometry and by biochemical approaches. In this study, the formation of an intramolecular disulfide bond and oxidative inactivation through the formation of a sulfonic acid derivative was unequivocally demonstrated for the first time. Because Ohr proteins are exclusively present in bacteria, they may represent promising targets for therapeutical drugs. In this regard, the structural and functional analyses of Ohr presented here might be very useful.

Asunto(s)

Proteínas Bacterianas/química , Estructura Terciaria de Proteína , Xylella , Animales , Proteínas Bacterianas/metabolismo , Sitios de Unión , Cristalografía por Rayos X , Cisteína/metabolismo , Ácidos Grasos/química , Modelos Moleculares , Oxidación-Reducción , Polietilenglicoles/química , Unión Proteica , Xylella/química , Xylella/enzimología

RESUMEN

Rab GTPases constitute the largest family of small monomeric GTPases, including over 60 members in humans. These GTPases share conserved residues related to nucleotide binding and hydrolysis, and main sequence divergences lie in the carboxyl termini. They cycle between inactive (GDP-bound) and active (GTP-bound) forms and the active site regions, termed Switch I and II, undergo the larger conformational changes between the two states. The Rab11 subfamily members, comprising Rab11a, Rab11b, and Rab25, act in recycling of proteins from the endosomes to the plasma membrane, in transport of molecules from the trans-Golgi network to the plasma membrane and in phagocytosis. In this work, we describe Rab11b-GDP and Rab11b-GppNHp crystal structures solved to 1.55 and 1.95 angstroms resolution, respectively. Although Rab11b shares 90% amino acid identity to Rab11a, its crystal structure shows critical differences relative to previously reported Rab11a structures. Inactive Rab11a formed dimers with unusually ordered Switch regions and missing the magnesium ion at the nucleotide binding site. In this work, inactive Rab11b crystallized as a monomer showing a flexible Switch I and a magnesium ion which is coordinated by four water molecules, the phosphate beta of GDP (beta-P) and the invariant S25. S20 from the P-loop and S42 from the Switch I are associated to GTP hydrolysis rate. In the active structures, S20 interacts with the gamma-P oxygen in Rab11b-GppNHp but does not in Rab11a-GppNHp and the Q70 side chain is found in different positions. In the Rab11a-GTPgammaS structure, S40 is closer to S25 and S42 does not interact with the gamma-P oxygen. These differences indicate that the Rab11 isoforms may possess different GTP hydrolysis rates. In addition, the Switch II of inactive Rab11b presents a 3(10)-helix (residues 69-73) that disappears upon activation. This 3(10)-helix is not found in the Rab11a-GDP structure, which possesses a longer alpha2 helix, spanning from residue 73 to 82 alpha-helix 5.

Asunto(s)

Cristalografía por Rayos X/métodos , Proteínas de Unión al GTP rab/química , Adenosina Difosfato/química , Secuencia de Aminoácidos , Sitios de Unión , Dimerización , Vectores Genéticos , Aparato de Golgi/metabolismo , Guanosina Difosfato/química , Guanosina Trifosfato/química , Humanos , Hidrólisis , Modelos Moleculares , Conformación Molecular , Datos de Secuencia Molecular , Unión Proteica , Isoformas de Proteínas , Estructura Secundaria de Proteína