RESUMEN
Eukaryotic translation termination is mediated by two interacting release factors, eRF1 and eRF3, which act cooperatively to ensure efficient stop codon recognition and fast polypeptide release. The crystal structures of human and Schizosaccharomyces pombe full-length eRF1 in complex with eRF3 lacking the GTPase domain revealed details of the interaction between these two factors and marked conformational changes in eRF1 that occur upon binding to eRF3, leading eRF1 to resemble a tRNA molecule. Small-angle X-ray scattering analysis of the eRF1/eRF3/GTP complex suggested that eRF1's M domain contacts eRF3's GTPase domain. Consistently, mutation of Arg192, which is predicted to come in close contact with the switch regions of eRF3, revealed its important role for eRF1's stimulatory effect on eRF3's GTPase activity. An ATP molecule used as a crystallization additive was bound in eRF1's putative decoding area. Mutational analysis of the ATP-binding site shed light on the mechanism of stop codon recognition by eRF1.
Asunto(s)
Codón de Terminación/metabolismo , Modelos Moleculares , Factores de Terminación de Péptidos/química , Proteínas de Schizosaccharomyces pombe/química , Schizosaccharomyces , Adenosina Trifosfato/metabolismo , GTP Fosfohidrolasas/metabolismo , Orden Génico , Humanos , Mutación , Factores de Terminación de Péptidos/genética , Unión Proteica , Biosíntesis de Proteínas/genética , Estructura Cuaternaria de Proteína , Estructura Terciaria de Proteína , Reproducibilidad de los Resultados , Ribosomas/metabolismo , Dispersión del Ángulo Pequeño , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismoRESUMEN
Antiaggregation drugs play an important role in therapeutic approaches for Alzheimer's disease. Although a large number of small molecules that inhibit the aggregation of the tau protein have been identified, little is known about their mode of action. Here, we reveal the mechanism and the nature of tau species that are generated by interaction of tau with the organic compound pthalocyanine tetrasulfonate (PcTS). We demonstrate that PcTS interferes with tau filament formation by targeting the protein into soluble oligomers. A combination of NMR spectroscopy, electron paramagnetic resonance, and small-angle X-ray scattering reveals that the soluble tau oligomers contain a dynamic, noncooperatively stabilized core with a diameter of 30-40 nm that is distinct from the core of tau filaments. Our results suggest that specific modulation of the conformation of tau is a viable strategy for reduction of pathogenic tau deposits.
Asunto(s)
Indoles/química , Proteínas tau/antagonistas & inhibidores , Proteínas tau/química , Electroforesis en Gel de Poliacrilamida , Humanos , Espectroscopía de Resonancia Magnética , Conformación Molecular , Espectroscopía Infrarroja por Transformada de FourierRESUMEN
The ribosomal stalk of the 60S subunit has been shown to play a crucial role in all steps of protein synthesis, but its structure and exact molecular function remain an unanswered question. In the present study, we show the low-resolution models of the solution structure of the yeast ribosomal stalk, composed of five proteins, P0-(P1-P2)(2). The model of the pentameric stalk complex determined by small-angle X-ray scattering reveals an elongated shape with a maximum length of 13 nm. The model displays three distinct lobes, which may correspond to the individual P1-P2 heterodimers anchored to the C-terminal domain of the P0 protein.
Asunto(s)
Proteínas Ribosómicas/química , Proteínas de Saccharomyces cerevisiae/química , Dispersión del Ángulo Pequeño , Difracción de Rayos X , Unión Proteica/fisiología , Multimerización de Proteína , Estructura Terciaria de Proteína , Saccharomyces cerevisiae/química , Difracción de Rayos X/métodosRESUMEN
The construction of useful functional biomolecular components not currently part of the natural repertoire is central to synthetic biology. A new light-capturing ultra-high-efficiency energy transfer protein scaffold has been constructed by coupling the chromophore centers of two normally unrelated proteins: the autofluorescent protein enhanced green fluorescent protein (EGFP) and the heme-binding electron transfer protein cytochrome b(562) (cyt b(562)). Using a combinatorial domain insertion strategy, a variant was isolated in which resonance energy transfer from the donor EGFP to the acceptor cyt b(562) was close to 100% as evident by virtually full fluorescence quenching on heme binding. The fluorescence signal of the variant was also sensitive to the reactive oxygen species H(2)O(2), with high signal gain observed due to the release of heme. The structure of oxidized holoprotein, determined to 2.75 Å resolution, revealed that the two domains were arranged side-by-side in a V-shape conformation, generating an interchromophore distance of ~17 Å (14 Å edge-to-edge). Critical to domain arrangement is the formation of a molecular pivot point between the two domains as a result of different linker sequence lengths at each domain junction and formation of a predominantly polar interdomain interaction surface. The retrospective structural analysis has provided an explanation for the basis of the observed highly efficient energy transfer through chromophore arrangement in the directly evolved protein scaffold and provides an insight into the molecular principles by which to design new proteins with coupled functions.
Asunto(s)
Grupo Citocromo b/química , Proteínas de Escherichia coli/química , Escherichia coli/química , Colorantes Fluorescentes/química , Proteínas Fluorescentes Verdes/química , Hidrozoos/química , Animales , Cristalografía por Rayos X , Transferencia de Energía , Modelos Moleculares , Oxidación-Reducción , Estructura Terciaria de Proteína , Proteínas Recombinantes de Fusión/químicaRESUMEN
SUMMARY: Automatic methods for macromolecular structure prediction (fold recognition, de novo folding and docking programs) produce large sets of alternative models. These large model sets often include many native-like structures, which are often scored as false positives. Such native-like models can be more easily identified based on data from experimental analyses used as structural restraints (e.g. identification of nearby residues by cross-linking, chemical modification, site-directed mutagenesis, deuterium exchange coupled with mass spectrometry, etc.). We present a simple server for scoring and ranking of models according to their agreement with user-defined restraints. AVAILABILITY: FILTREST3D is freely available for users as a web server and standalone software at: http://filtrest3d.genesilico.pl/ CONTACT: iamb@genesilico.pl SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.
Asunto(s)
Modelos Moleculares , Conformación de Ácido Nucleico , Conformación Proteica , Programas Informáticos , Biología Computacional/métodosRESUMEN
BACKGROUND: Computational models of protein structure are usually inaccurate and exhibit significant deviations from the true structure. The utility of models depends on the degree of these deviations. A number of predictive methods have been developed to discriminate between the globally incorrect and approximately correct models. However, only a few methods predict correctness of different parts of computational models. Several Model Quality Assessment Programs (MQAPs) have been developed to detect local inaccuracies in unrefined crystallographic models, but it is not known if they are useful for computational models, which usually exhibit different and much more severe errors. RESULTS: The ability to identify local errors in models was tested for eight MQAPs: VERIFY3D, PROSA, BALA, ANOLEA, PROVE, TUNE, REFINER, PROQRES on 8251 models from the CASP-5 and CASP-6 experiments, by calculating the Spearman's rank correlation coefficients between per-residue scores of these methods and local deviations between C-alpha atoms in the models vs. experimental structures. As a reference, we calculated the value of correlation between the local deviations and trivial features that can be calculated for each residue directly from the models, i.e. solvent accessibility, depth in the structure, and the number of local and non-local neighbours. We found that absolute correlations of scores returned by the MQAPs and local deviations were poor for all methods. In addition, scores of PROQRES and several other MQAPs strongly correlate with 'trivial' features. Therefore, we developed MetaMQAP, a meta-predictor based on a multivariate regression model, which uses scores of the above-mentioned methods, but in which trivial parameters are controlled. MetaMQAP predicts the absolute deviation (in Angströms) of individual C-alpha atoms between the model and the unknown true structure as well as global deviations (expressed as root mean square deviation and GDT_TS scores). Local model accuracy predicted by MetaMQAP shows an impressive correlation coefficient of 0.7 with true deviations from native structures, a significant improvement over all constituent primary MQAP scores. The global MetaMQAP score is correlated with model GDT_TS on the level of 0.89. CONCLUSION: Finally, we compared our method with the MQAPs that scored best in the 7th edition of CASP, using CASP7 server models (not included in the MetaMQAP training set) as the test data. In our benchmark, MetaMQAP is outperformed only by PCONS6 and method QA_556 - methods that require comparison of multiple alternative models and score each of them depending on its similarity to other models. MetaMQAP is however the best among methods capable of evaluating just single models. We implemented the MetaMQAP as a web server available for free use by all academic users at the URL https://genesilico.pl/toolkit/
Asunto(s)
Biología Computacional , Sistemas de Administración de Bases de Datos , Metaanálisis como Asunto , Modelos Moleculares , Conformación Proteica , Proyectos de Investigación/estadística & datos numéricos , Inteligencia Artificial , Biología Computacional/métodos , Análisis Multivariante , Variaciones Dependientes del Observador , Reconocimiento de Normas Patrones Automatizadas/métodos , Proteínas/química , Control de Calidad , Análisis de RegresiónRESUMEN
MODOMICS is the first comprehensive database resource for systems biology of RNA modification. It integrates information about the chemical structure of modified nucleosides, their localization in RNA sequences, pathways of their biosynthesis and enzymes that carry out the respective reactions. MODOMICS also provides literature information, and links to other databases, including the available protein sequence and structure data. The current list of modifications and pathways is comprehensive, while the dataset of enzymes is limited to Escherichia coli and Saccharomyces cerevisiae and sequence alignments are presented only for tRNAs from these organisms. RNAs and enzymes from other organisms will be included in the near future. MODOMICS can be queried by the type of nucleoside (e.g. A, G, C, U, I, m1A, nm5s2U, etc.), type of RNA, position of a particular nucleoside, type of reaction (e.g. methylation, thiolation, deamination, etc.) and name or sequence of an enzyme of interest. Options for data presentation include graphs of pathways involving the query nucleoside, multiple sequence alignments of RNA sequences and tabular forms with enzyme and literature data. The contents of MODOMICS can be accessed through the World Wide Web at http://genesilico.pl/modomics/.
Asunto(s)
Bases de Datos de Ácidos Nucleicos , Procesamiento Postranscripcional del ARN , ARN de Transferencia/química , ARN de Transferencia/metabolismo , Secuencia de Bases , Escherichia coli/enzimología , Escherichia coli/genética , Internet , Nucleósidos/metabolismo , ARN Bacteriano/química , ARN Bacteriano/metabolismo , ARN de Hongos/química , ARN de Hongos/metabolismo , Saccharomyces cerevisiae/enzimología , Saccharomyces cerevisiae/genética , Alineación de Secuencia , Interfaz Usuario-ComputadorRESUMEN
The tRNA:m2(2)G10 methyltransferase of Pyrococus abyssi (PAB1283, a member of COG1041) catalyzes the N2,N2-dimethylation of guanosine at position 10 in tRNA. Boundaries of its THUMP (THioUridine synthases, RNA Methyltransferases and Pseudo-uridine synthases)--containing N-terminal domain [1-152] and C-terminal catalytic domain [157-329] were assessed by trypsin limited proteolysis. An inter-domain flexible region of at least six residues was revealed. The N-terminal domain was then produced as a standalone protein (THUMPalpha) and further characterized. This autonomously folded unit exhibits very low affinity for tRNA. Using protein fold-recognition (FR) methods, we identified the similarity between THUMPalpha and a putative RNA-recognition module observed in the crystal structure of another THUMP-containing protein (ThiI thiolase of Bacillus anthracis). A comparative model of THUMPalpha structure was generated, which fulfills experimentally defined restraints, i.e. chemical modification of surface exposed residues assessed by mass spectrometry, and identification of an intramolecular disulfide bridge. A model of the whole PAB1283 enzyme docked onto its tRNA(Asp) substrate suggests that the THUMP module specifically takes support on the co-axially stacked helices of T-arm and acceptor stem of tRNA and, together with the catalytic domain, screw-clamp structured tRNA. We propose that this mode of interactions may be common to other THUMP-containing enzymes that specifically modify nucleotides in the 3D-core of tRNA.
Asunto(s)
Proteínas Arqueales/química , Pyrococcus abyssi/enzimología , ARN de Transferencia/química , Proteínas de Unión al ARN/química , ARNt Metiltransferasas/química , Secuencia de Aminoácidos , Proteínas Arqueales/aislamiento & purificación , Proteínas Arqueales/metabolismo , Espectrometría de Masas , Modelos Moleculares , Datos de Secuencia Molecular , Pliegue de Proteína , Estructura Terciaria de Proteína , ARN de Transferencia/metabolismo , Proteínas de Unión al ARN/aislamiento & purificación , Proteínas de Unión al ARN/metabolismo , Alineación de Secuencia , ARNt Metiltransferasas/aislamiento & purificación , ARNt Metiltransferasas/metabolismoAsunto(s)
Fenotiazinas/química , Proteínas tau/antagonistas & inhibidores , Enfermedad de Alzheimer/metabolismo , Enfermedad de Alzheimer/patología , Animales , Caenorhabditis elegans/efectos de los fármacos , Caenorhabditis elegans/metabolismo , Cisteína/química , Modelos Animales de Enfermedad , Humanos , Espectroscopía de Resonancia Magnética , Azul de Metileno/química , Oxidación-Reducción , Fenotiazinas/metabolismo , Fenotiazinas/farmacología , Unión Proteica , Proteínas tau/metabolismoRESUMEN
In the course of CASP6, we generated models for all targets using a new version of the "FRankenstein's monster approach." Previously (in CASP5) we were able to build many very accurate full-atom models by selection and recombination of well-folded fragments obtained from crude fold recognition (FR) results, followed by optimization of the sequence-structure fit and assessment of alternative alignments on the structural level. This procedure was however very arduous, as most of the steps required extensive visual and manual input from the human modeler. Now, we have automated the most tedious steps, such as superposition of alternative models, extraction of best-scoring fragments, and construction of a hybrid "monster" structure, as well as generation of alternative alignments in the regions that remain poorly scored in the refined hybrid model. We have also included the ROSETTA method to construct those parts of the target for which no reasonable structures were generated by FR methods (such as long insertions and terminal extensions). The analysis of successes and failures of the current version of the FRankenstein approach in modeling of CASP6 targets reveals that the considerably streamlined and automated method performs almost as well as the initial, mostly manual version, which suggests that it may be a useful tool for accurate protein structure prediction even in the hands of nonexperts.
Asunto(s)
Biología Computacional/métodos , Proteómica/métodos , Algoritmos , Automatización , Simulación por Computador , Computadores , Bases de Datos de Proteínas , Modelos Moleculares , Conformación Proteica , Pliegue de Proteína , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Reproducibilidad de los Resultados , Alineación de Secuencia , Programas InformáticosRESUMEN
Intrinsically disordered proteins (IDPs) are notoriously challenging to study both experimentally and computationally. The structure of IDPs cannot be described by a single conformation but must instead be described as an ensemble of interconverting conformations. Atomistic simulations are increasingly used to obtain such IDP conformational ensembles. Here, we have compared the IDP ensembles generated by eight all-atom empirical force fields against primary small-angle X-ray scattering (SAXS) and NMR data. Ensembles obtained with different force fields exhibit marked differences in chain dimensions, hydrogen bonding, and secondary structure content. These differences are unexpectedly large: changing the force field is found to have a stronger effect on secondary structure content than changing the entire peptide sequence. The CHARMM 22* ensemble performs best in this force field comparison: it has the lowest error in chemical shifts and J-couplings and agrees well with the SAXS data. A high population of left-handed α-helix is present in the CHARMM 36 ensemble, which is inconsistent with measured scalar couplings. To eliminate inadequate sampling as a reason for differences between force fields, extensive simulations were carried out (0.964 ms in total); the remaining small sampling uncertainty is shown to be much smaller than the observed differences. Our findings highlight how IDPs, with their rugged energy landscapes, are highly sensitive test systems that are capable of revealing force field deficiencies and, therefore, contributing to force field development.
Asunto(s)
Simulación por Computador , Proteínas Intrínsecamente Desordenadas , Proteínas Intrínsecamente Desordenadas/química , Espectroscopía de Resonancia Magnética , Conformación Proteica , Dispersión del Ángulo PequeñoRESUMEN
Emerging high-throughput techniques for the characterization of protein and protein-complex structures yield noisy data with sparse information content, placing a significant burden on computation to properly interpret the experimental data. One such technique uses cross-linking (chemical or by cysteine oxidation) to confirm or select among proposed structural models (e.g., from fold recognition, ab initio prediction, or docking) by testing the consistency between cross-linking data and model geometry. This paper develops a probabilistic framework for analyzing the information content in cross-linking experiments, accounting for anticipated experimental error. This framework supports a mechanism for planning experiments to optimize the information gained. We evaluate potential experiment plans using explicit trade-offs among key properties of practical importance: discriminability, coverage, balance, ambiguity, and cost. We devise a greedy algorithm that considers those properties and, from a large number of combinatorial possibilities, rapidly selects sets of experiments expected to discriminate pairs of models efficiently. In an application to residue-specific chemical cross-linking, we demonstrate the ability of our approach to plan experiments effectively involving combinations of cross-linkers and introduced mutations. We also describe an experiment plan for the bacteriophage lambda Tfa chaperone protein in which we plan dicysteine mutants for discriminating threading models by disulfide formation. Preliminary results from a subset of the planned experiments are consistent and demonstrate the practicality of planning. Our methods provide the experimenter with a valuable tool (available from the authors) for understanding and optimizing cross-linking experiments.
Asunto(s)
Biología Computacional , Modelos Moleculares , Proteínas/química , Algoritmos , Reactivos de Enlaces Cruzados/química , Disulfuros/química , Genómica , Probabilidad , Conformación Proteica , Proyectos de InvestigaciónRESUMEN
Proteins of the Heterochromatin Protein 1 (HP1) family are regulators of chromatin structure and genome function in eukaryotes. Post-translational modifications expand the repertoire of the chemical diversity of HP1 proteins and regulate their activity. Here, we investigated the effect of phosphorylation by Casein kinase 2 (CK2) on the structure, dynamics and binding activity of human HP1ß. We show that Ser89 in the hinge region is the most effective substrate, followed by Ser175 at the C-terminal tail. Phosphorylation at these sites results in localized conformational changes in HP1ß that do not compromise the ability of the protein to bind chromatin.
Asunto(s)
Quinasa de la Caseína II/química , Proteínas Cromosómicas no Histona/química , Procesamiento Proteico-Postraduccional , Secuencia de Aminoácidos , Sitios de Unión , Homólogo de la Proteína Chromobox 5 , Proteínas Cromosómicas no Histona/metabolismo , Secuencia de Consenso , Heterocromatina/metabolismo , Humanos , Cinética , Modelos Moleculares , Datos de Secuencia Molecular , Resonancia Magnética Nuclear Biomolecular , Fosforilación , Unión ProteicaRESUMEN
The development of molecular descriptions of intrinsically disordered proteins (IDPs) is essential for elucidating conformational transitions that characterize common neurodegenerative disorders. We use nuclear magnetic resonance, small angle scattering, and molecular ensemble approaches to characterize the IDPs Tau and α-synuclein. Ensemble descriptions of IDPs are highly underdetermined due to the inherently large number of degrees of conformational freedom compared with available experimental measurements. Using extensive cross-validation we show that five different types of independent experimental parameters are predicted more accurately by selected ensembles than by statistical coil descriptions. The improvement increases in regions whose local sampling deviates from statistical coil, validating the derived conformational description. Using these approaches we identify enhanced polyproline II sampling in aggregation-nucleation sites, supporting suggestions that this region of conformational space is important for aggregation.
Asunto(s)
Espectroscopía de Resonancia Magnética/métodos , Proteínas de la Membrana/química , alfa-Sinucleína/química , Algoritmos , Humanos , Proteínas Intrínsecamente Desordenadas/química , Enfermedades Neurodegenerativas/metabolismo , Prolina , Pliegue de Proteína , Estructura Terciaria de Proteína , Dispersión de RadiaciónRESUMEN
BACKGROUND: Protein DataBank file format is used for the majority of biomolecular data available today. Haskell is a lazy functional language that enjoys a high-level class-based type system, a growing collection of useful libraries and a reputation for efficiency. FINDINGS: I present a fast library for processing biomolecular data in the Protein Data Bank format. I present benchmarks indicating that this library is faster than other frequently used Protein Data Bank parsing programs. The proposed library also features a convenient iterator mechanism, and a simple API modeled after BioPython. CONCLUSION: I set a new standard for convenience and efficiency of Protein Data Bank processing in a Haskell library, and release it to open source.
Asunto(s)
Proteínas/genética , Interfaz Usuario-Computador , Bases de Datos Factuales , Biblioteca de Péptidos , Proteínas/químicaRESUMEN
We propose a novel fragment assembly method for low-resolution modeling of RNA and show how it may be used along with small-angle X-ray solution scattering (SAXS) data to model low-resolution structures of particles having as many as 12 independent secondary structure elements. We assessed this model-building procedure by using both artificial data on a previously proposed benchmark and publicly available data. With the artificial data, SAXS-guided models show better similarity to native structures than ROSETTA decoys. The publicly available data showed that SAXS-guided models can be used to reinterpret RNA structures previously deposited in the Protein Data Bank. Our approach allows for fast and efficient building of de novo models of RNA using approximate secondary structures that can be readily obtained from existing bioinformatic approaches. We also offer a rigorous assessment of the resolving power of SAXS in the case of small RNA structures, along with a small multimetric benchmark of the proposed method.
Asunto(s)
Modelos Moleculares , ARN/química , Algoritmos , Secuencia de Bases , Simulación por Computador , Humanos , Secuencias Invertidas Repetidas , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , Dispersión del Ángulo Pequeño , Programas Informáticos , Difracción de Rayos XRESUMEN
New developments in the program package ATSAS (version 2.4) for the processing and analysis of isotropic small-angle X-ray and neutron scattering data are described. They include (i) multiplatform data manipulation and display tools, (ii) programs for automated data processing and calculation of overall parameters, (iii) improved usage of high- and low-resolution models from other structural methods, (iv) new algorithms to build three-dimensional models from weakly interacting oligomeric systems and complexes, and (v) enhanced tools to analyse data from mixtures and flexible systems. The new ATSAS release includes installers for current major platforms (Windows, Linux and Mac OSX) and provides improved indexed user documentation. The web-related developments, including a user discussion forum and a widened online access to run ATSAS programs, are also presented.