RESUMEN
Nearly complete resonance assignment and the high-resolution NMR structure of the acyl-carrier protein from Borrelia burgdorferi, a target of the Seattle Structural Genomics Center for Infectious Disease (SSGCID) structure-determination pipeline, are reported. This protein was chosen as a potential target for drug-discovery efforts because of its involvement in fatty-acid biosynthesis, an essential metabolic pathway, in bacteria. It was possible to assign >98% of backbone resonances and >92% of side-chain resonances using multidimensional NMR spectroscopy. The NMR structure was determined to a backbone r.m.s.d. of 0.4 Å and contained four α-helices and two 3(10)-helices. A structure-homology search revealed that this protein is highly similar to the acyl-carrier protein from Aquifex aeolicus.
Asunto(s)
Proteína Transportadora de Acilo/química , Proteínas Bacterianas/química , Borrelia burgdorferi/química , Secuencia de Aminoácidos , Modelos Moleculares , Datos de Secuencia Molecular , Resonancia Magnética Nuclear Biomolecular , Estructura Terciaria de Proteína , Alineación de Secuencia , Homología Estructural de ProteínaRESUMEN
The NIAID-funded Seattle Structural Genomics Center for Infectious Disease (SSGCID) is a consortium established to apply structural genomics approaches to potential drug targets from NIAID priority organisms for biodefense and emerging and re-emerging diseases. The mission of the SSGCID is to determine approximately 400 protein structures over the next five years. In order to maximize biomedical impact, ligand-based drug-lead discovery campaigns will be pursued for a small number of high-impact targets. Here we review the center's target selection processes, which include pro-active engagement of the infectious disease research and drug therapy communities to identify drug targets, essential enzymes, virulence factors and vaccine candidates of biomedical relevance to combat infectious diseases. This is followed by a brief overview of the SSGCID structure determination pipeline and ligand screening methodology. Finally, specifics of our resources available to the scientific community are presented. Physical materials and data produced by SSGCID will be made available to the scientific community, with the aim that they will provide essential groundwork benefiting future research and drug discovery.
Asunto(s)
Academias e Institutos , Genómica/métodos , Homología Estructural de Proteína , Animales , Bioterrorismo/prevención & control , Enfermedades Transmisibles/tratamiento farmacológico , Descubrimiento de Drogas , Humanos , Ligandos , Modelos MolecularesRESUMEN
Two different analytical methods for the quality control of fluoxetine in commercial formulations have been developed and compared: a spectrofluorimetric method and a capillary zone electrophoretic (CZE) method. The fluorescence emission values were measured at lambda=293 nm when exciting at lambda=230 nm. The CZE method used an uncoated fused-silica capillary and pH 2.5 phosphate buffer as the background electrolyte. The extraction of fluoxetine from the capsules consisted of a simple one-step dissolution with methanol/water, filtration and dilution. Both methods gave satisfactory results in terms of precision; the best results were obtained for the electrophoretic method, with RSD% values always lower than 2.0%. The accuracy was assessed by means of recovery studies, which gave very good results, between 97.5 and 102.6%. Furthermore, both methods also have the advantage of being very rapid.
Asunto(s)
Fluoxetina/análisis , Cápsulas , Electroforesis Capilar , Reproducibilidad de los Resultados , Espectrometría de FluorescenciaRESUMEN
The hepatitis C virus (HCV) is the main causative agent of non-A, non-B hepatitis in humans and a major cause of mortality and morbidity in the world. Currently there is no effective treatment available for the infection caused by this virus, whose replication depends on an unusual translation-initiation mechanism. The viral RNA contains an internal ribosome-entry site (IRES) that is recognized specifically by the small ribosomal subunit and by eukaryotic initiation factor 3, and these interactions allow cap (7-methyl-guanine nucleotide)-independent initiation of viral protein synthesis. In this article, we review the structure and mechanism of translation initiation of the HCV IRES, and its potential as a target for novel antivirals.
Asunto(s)
Antivirales/farmacología , Hepacivirus/efectos de los fármacos , Hepacivirus/metabolismo , Ribosomas/metabolismo , Secuencia de Bases , Hepacivirus/crecimiento & desarrollo , Hepacivirus/patogenicidad , Humanos , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , Iniciación de la Cadena Peptídica Traduccional , ARN Viral/química , ARN Viral/genética , Ribosomas/efectos de los fármacosRESUMEN
Researchers' increasing awareness of the essential role played by RNA in many biological processes and in the progression of disease makes the discovery of new RNA targets an emerging field in drug discovery. Since most existing pharmacologically active compounds bind proteins, RNA provides nearly untapped opportunities for pharmacological development. The elucidation of the structure of the ribosome and other cellular and viral RNA motifs creates the opportunity for discovering new drug-like compounds that inhibit RNA function. However, further advances in understanding the chemistry and structure of RNA recognition are needed before these promises are fulfilled.
Asunto(s)
Diseño de Fármacos , ARN Bacteriano/efectos de los fármacos , ARN Viral/efectos de los fármacos , Antibacterianos/química , Antibacterianos/farmacología , Biología Computacional , Conformación de Ácido Nucleico , Ribosomas/química , Ribosomas/efectos de los fármacos , Relación Estructura-ActividadRESUMEN
The past few years have witnessed remarkable progress in knowledge of the structure and function of RNA-binding proteins and their RNA complexes. X-ray crystallography and NMR spectroscopy have provided structures for all major classes of RNA-binding proteins, both alone and complexed with RNA. New computational and experimental tools have provided unprecedented insight into the molecular basis of RNA recognition.
Asunto(s)
Sitios de Unión , Proteínas de Unión al ARN/química , ARN/química , Secuencias de Aminoácidos , Cristalografía por Rayos X , Espectroscopía de Resonancia Magnética , Modelos Moleculares , ARN Bicatenario/químicaRESUMEN
The eukaryotic cap and poly(A) tail binding proteins, eIF4E and Pab1p, play important roles in the initiation of protein synthesis. The recent structures of the complex of eIF4E bound to the methylated guanosine (cap) found at the 5'end of messenger RNA (mRNA), the complex of eIF4E bound to peptide fragments of two related translation factors (eIF4G and 4E-BP1), and the complex of the N-terminal fragment of Pab1p bound to polyadenylate RNA have revealed that eIF4E and Pab1p contain at least two distinct functional surfaces. One surface is used for binding mRNA, and the other for binding proteins involved in translation initiation.
Asunto(s)
Factores de Iniciación de Péptidos/química , Factores de Iniciación de Péptidos/metabolismo , Biosíntesis de Proteínas/fisiología , Proteínas de Unión al ARN/química , Proteínas de Unión al ARN/metabolismo , Secuencia de Aminoácidos , Animales , Células Eucariotas , Factor 4E Eucariótico de Iniciación , Guanosina Difosfato/análogos & derivados , Guanosina Difosfato/metabolismo , Modelos Moleculares , Poli A/metabolismo , Proteínas de Unión a Poli(A) , Unión Proteica , Conformación ProteicaRESUMEN
Exonic and intronic mutations in the tau gene cause familial frontotemporal dementia and parkinsonism linked to chromosome 17. Here, we describe a new mutation, consisting of a C-to-T transition at position +12 of the intron following exon 10 of the tau gene in the Kumamoto pedigree, showing frontotemporal dementia. The mutation caused a marked reduction in melting temperature of the tau exon 10-splicing regulatory element RNA and a large increase in exon 10-containing transcripts. Brain tissue from affected individuals showed an abnormal preponderance of exon 10-containing transcripts that was reflected at the protein level by an overproduction of tau isoforms with four microtubule-binding repeats. Immunostaining revealed the presence of tau aggregates in degenerating neurons and glial cells. Isolated tau filaments had a twisted ribbon-like morphology and were made of hyperphosphorylated four-repeat tau isoforms. The additional mutation located dose to the splice-donor site of the intron following exon 10 of the tau gene supports the view that intronic mutations exercize their pathogenic effect by destabilizing RNA secondary structure.
Asunto(s)
Demencia/genética , Intrones/genética , Mutación Puntual , Proteínas tau/genética , Encéfalo/patología , Química Encefálica/genética , Análisis Mutacional de ADN , Demencia/patología , Detergentes , Exones/genética , Salud de la Familia , Femenino , Calor , Humanos , Masculino , Microscopía Electrónica , Persona de Mediana Edad , Linaje , Empalme del ARN/fisiología , ARN Mensajero/análisis , Secuencias Reguladoras de Ácidos Nucleicos/genética , Sarcosina/análogos & derivados , Solubilidad , Proteínas tau/análisis , Proteínas tau/ultraestructuraRESUMEN
The major packaging signal of human immunodeficiency virus type 1 (HIV-1) RNA has been localised to the region 3' to the major splice donor within the leader sequence. Secondary structural studies for this region of the HIV-1 genome have shown the existence of a stem-loop structure capped by a purine-rich tetraloop. Extensive mapping data presented here lead to the complete characterisation of the structure of the stem-loop, including a new purine-rich internal loop in the lower part of the structure and the previously established GGAG tetraloop at its tip. Biochemical analysis reveals that both internal loop and tetraloop are primary sites for interaction with Gag polyprotein, and that binding of Gag protein leads to a conformational change which alters the RNA structure. NMR spectroscopy has been used to determine the three-dimensional structure of this complete stem-loop structure. The structural analysis reveals a significant difference between the apical part of the stem-loop structure, which adopts a well-defined conformation, and the purine-rich internal loop, which is instead very flexible. In contrast to what is generally observed for internal loop structures in RNA, this region of the encapsidation signal adopts a structure lacking stable interstrand interactions capable of stabilising a unique conformation. We suggest that the stem-loop structure represents a nucleation site for Gag protein binding, and that the protein exploits the flexibility of the internal loop to initiate the unwinding of the structure with successive addition of Gag molecules interacting with the RNA and each other through conserved I (interaction) domains.
Asunto(s)
Productos del Gen gag/metabolismo , VIH-1/genética , Conformación de Ácido Nucleico , ARN Viral/química , ARN Viral/metabolismo , Ensamble de Virus/genética , Secuencia de Aminoácidos , Emparejamiento Base/genética , Sitios de Unión , Secuencia Conservada/genética , Genoma Viral , VIH-1/fisiología , Modelos Moleculares , Datos de Secuencia Molecular , Resonancia Magnética Nuclear Biomolecular , Docilidad , Unión Proteica , Purinas/metabolismo , Estabilidad del ARN , ARN Viral/genética , ADN Polimerasa Dirigida por ARN/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Ribonucleasas/metabolismoRESUMEN
The double-stranded RNA-binding domain (dsRBD) is a common RNA-binding motif found in many proteins involved in RNA maturation and localization. To determine how this domain recognizes RNA, we have studied the third dsRBD from Drosophila Staufen. The domain binds optimally to RNA stem-loops containing 12 uninterrupted base pairs, and we have identified the amino acids required for this interaction. By mutating these residues in a staufen transgene, we show that the RNA-binding activity of dsRBD3 is required in vivo for Staufen-dependent localization of bicoid and oskar mRNAs. Using high-resolution NMR, we have determined the structure of the complex between dsRBD3 and an RNA stem-loop. The dsRBD recognizes the shape of A-form dsRNA through interactions between conserved residues within loop 2 and the minor groove, and between loop 4 and the phosphodiester backbone across the adjacent major groove. In addition, helix alpha1 interacts with the single-stranded loop that caps the RNA helix. Interactions between helix alpha1 and single-stranded RNA may be important determinants of the specificity of dsRBD proteins.
Asunto(s)
Proteínas de Drosophila , Proteínas de Unión al ARN/metabolismo , ARN/metabolismo , Secuencia de Aminoácidos , Animales , Sitios de Unión , Drosophila , Datos de Secuencia Molecular , Mutación , Unión Proteica , Proteínas de Unión al ARN/química , Proteínas de Unión al ARN/genética , Alineación de Secuencia , TransfecciónRESUMEN
The status of the poly(A) tail at the 3'-end of mRNAs controls the expression of numerous genes in response to developmental and extracellular signals. Poly(A) tail regulation requires cooperative binding of two human U1A proteins to an RNA regulatory region called the polyadenylation inhibition element (PIE). When bound to PIE RNA, U1A proteins also bind to the enzyme responsible for formation of the mature 3'-end of most eukaryotic mRNAs, poly(A) polymerase (PAP). The NMR structure of the 38 kDa complex formed between two U1A molecules and PIE RNA shows that binding cooperativity depends on helix C located at the end of the RNA-binding domain and just adjacent to the PAP-interacting domain of U1A. Since helix C undergoes a conformational change upon RNA binding, the structure shows that binding cooperativity and interactions with PAP occur only when U1A is bound to its cognate RNA. This mechanism ensures that the activity of PAP enzyme, which is essential to the cell, is only down regulated when U1A is bound to the U1A mRNA.
Asunto(s)
Poli A/metabolismo , ARN Mensajero/metabolismo , Proteínas de Unión al ARN/metabolismo , Secuencias Reguladoras de Ácidos Nucleicos/genética , Ribonucleoproteína Nuclear Pequeña U1/química , Ribonucleoproteína Nuclear Pequeña U1/metabolismo , Regiones no Traducidas 3'/química , Regiones no Traducidas 3'/genética , Regiones no Traducidas 3'/metabolismo , Regulación Alostérica , Secuencia de Aminoácidos , Secuencia de Bases , Sitios de Unión , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Peso Molecular , Resonancia Magnética Nuclear Biomolecular , Conformación de Ácido Nucleico , Polinucleotido Adenililtransferasa/antagonistas & inhibidores , Polinucleotido Adenililtransferasa/metabolismo , Unión Proteica , Estructura Secundaria de Proteína , Procesamiento Postranscripcional del ARN/genética , ARN Mensajero/química , ARN Mensajero/genética , Proteínas de Unión al ARN/química , Relación Estructura-Actividad , Especificidad por SustratoRESUMEN
Drug-like molecules that bind RNA with sequence selectivity would provide valuable tools to elucidate gene expression pathways and new avenues to the treatment of degenerative and chronic conditions. Efforts at discovering such agents have been hampered, until recently, by the limited knowledge of RNA recognition principles. Several recent structures of aminoglycoside-RNA complexes have begun to reveal the structural basis for RNA-drug recognition. However, the absence of suitable chemical scaffolds known to bind the RNA major groove, where specificity could be provided by the diversity of functional groups exposed on the RNA bases, has represented a major obstacle. Here we report an investigation of the structural basis for recognition of an RNA stem-loop by neomycin, a naturally occurring aminoglycoside antibiotic. We found that neomycin binds the RNA stem-loop that regulates alternative splicing of exon 10 within the gene coding for human tau protein. Mutations within this splicing regulatory element destabilise the RNA structure and cause frontotemporal dementia and Parkinsonism linked to chromosome 17 (FTDP-17), an autosomal dominant condition leading to neurodegeneration and death. The three-dimensional structure of the RNA-neomycin complex shows interaction of the drug in the major groove of the short RNA duplex, where familial mutations cluster. Analysis of the structure shows how aminoglycosides and related drugs bind to the RNA major groove, adding to our understanding of the principles of drug-RNA recognition.
Asunto(s)
Antibacterianos/metabolismo , Exones/genética , Conformación de Ácido Nucleico , Empalme del ARN/genética , ARN/metabolismo , Proteínas tau/genética , Antibacterianos/química , Antibacterianos/farmacología , Secuencia de Bases , Sitios de Unión , Cromosomas Humanos Par 17/genética , Framicetina/química , Framicetina/metabolismo , Framicetina/farmacología , Humanos , Enlace de Hidrógeno , Modelos Moleculares , Mutación/genética , Resonancia Magnética Nuclear Biomolecular , Conformación de Ácido Nucleico/efectos de los fármacos , Desnaturalización de Ácido Nucleico/efectos de los fármacos , Trastornos Parkinsonianos/genética , ARN/química , ARN/genética , Estabilidad del ARN/efectos de los fármacos , Secuencias Reguladoras de Ácidos Nucleicos/genética , Electricidad Estática , TermodinámicaRESUMEN
The G x U wobble base pair is a fundamental unit of RNA secondary structure that is present in nearly every class of RNA from organisms of all three phylogenetic domains. It has comparable thermodynamic stability to Watson-Crick base pairs and is nearly isomorphic to them. Therefore, it often substitutes for G x C or A x U base pairs. The G x U wobble base pair also has unique chemical, structural, dynamic and ligand-binding properties, which can only be partially mimicked by Watson-Crick base pairs or other mispairs. These features mark sites containing G x U pairs for recognition by proteins and other RNAs and allow the wobble pair to play essential functional roles in a remarkably wide range of biological processes.
Asunto(s)
Emparejamiento Base/genética , Guanina/química , Conformación de Ácido Nucleico , ARN/química , Uracilo/química , Enlace de Hidrógeno , Modelos Moleculares , Estructura Molecular , ARN/metabolismo , TermodinámicaRESUMEN
The ribonucleoprotein (RNP) domain is one of the most common eukaryotic protein domains, and is found in many proteins involved in recognition of a wide variety of RNAs. Two structures of RNA complexes of human U1A protein have revealed important aspects of RNP-RNA recognition, but have also raised intriguing questions concerning how RNP domains discriminate between different RNAs. In this work, we extend the investigation of U1A-RNA recognition by comparing the dynamics of U1A protein both free and in complex with RNA. We have also investigated the trimolecular complex between two U1A proteins and the complete polyadenylation inhibition element to study the effect of RNA-dependent protein-protein interactions on protein conformational flexibility. We report that changes in backbone dynamics upon complex formation identify regions of the protein where conformational exchange processes are quenched in the RNA-bound conformation. Furthermore, amino acids whose side-chains experience significant changes in conformational flexibility coincide with residues particularly important for the specificity of the U1A protein/RNA interaction. This study adds a new dimension to the description of the coordinated changes in structure and dynamics that are critical to define the biological specificity of U1A and other RNP proteins.
Asunto(s)
Proteínas de Unión al ARN , ARN/metabolismo , Ribonucleoproteína Nuclear Pequeña U1/química , Ribonucleoproteína Nuclear Pequeña U1/metabolismo , Secuencia de Aminoácidos , Sitios de Unión , Deuterio , Humanos , Técnicas In Vitro , Sustancias Macromoleculares , Modelos Moleculares , Datos de Secuencia Molecular , Isótopos de Nitrógeno , Unión Proteica , Conformación Proteica , Estructura Terciaria de Proteína , Ribonucleoproteína Nuclear Pequeña U1/genética , TermodinámicaRESUMEN
The fidelity of protein synthesis depends on specific tRNA aminoacylation by aminoacyl-tRNA synthetase enzymes, which in turn depends on the recognition of the identity of particular nucleotides and structural features in the substrate tRNA. These features generally reside within the acceptor helix, the anticodon stem-loop, and in some systems the variable pocket of the tRNA. In the alanine system, fidelity is ensured by a G.U wobble base pair located at the third position within the acceptor helix of alanine tRNA. We have investigated the activity of mutant alanine tRNAs to explore the mechanism of enzyme recognition. Here we show that the mismatched pair C-C is an excellent substitute for G.U in alanine-tRNA-knockout cells. A structural investigation by NMR spectroscopy of the C-C RNA acceptor end reveals that the two cytosines are intercalated into the helix, and that C-C exists in multiple conformations. Structural heterogeneity also is present in the wild-type G.U RNA, whereas inactive Watson-Crick helices are structurally rigid. The correlation between functional and structural data suggests that the G.U pair provides a distinctive structure and a point of deformability that allow the tRNA acceptor end to fit into the active site of the alanyl-tRNA synthetase. Fidelity is ensured because noncognate and inactive mutant tRNAs are bound in the active site in an incorrect conformation that reduces enzymatic activity.
Asunto(s)
Conformación de Ácido Nucleico , Biosíntesis de Proteínas , ARN de Transferencia de Alanina/química , ARN de Transferencia de Alanina/metabolismo , ARN/química , Acilación , Adenina/metabolismo , Emparejamiento Base , Western Blotting , Catálisis , Citosina/metabolismo , Escherichia coli/genética , Guanina/metabolismo , Espectroscopía de Resonancia Magnética , Datos de Secuencia Molecular , Mutagénesis , Uracilo/metabolismoRESUMEN
Coding region and intronic mutations in the tau gene cause frontotemporal dementia and parkinsonism linked to chromosome 17. Intronic mutations and some missense mutations increase splicing in of exon 10, leading to an increased ratio of four-repeat to three-repeat tau isoforms. Secondary structure predictions have led to the proposal that intronic mutations and one missense mutation destabilize a putative RNA stem-loop structure located close to the splice-donor site of the intron after exon 10. We have determined the three-dimensional structure of this tau exon 10 splicing regulatory element RNA by NMR spectroscopy. We show that it forms a stable, folded stem-loop structure whose thermodynamic stability is reduced by frontotemporal dementia and parkinsonism linked to chromosome 17 mutations and increased by compensatory mutations. By exon trapping, the reduction in thermodynamic stability is correlated with increased splicing in of exon 10.