RESUMEN
We present a suite of programs, named CING for Common Interface for NMR Structure Generation that provides for a residue-based, integrated validation of the structural NMR ensemble in conjunction with the experimental restraints and other input data. External validation programs and new internal validation routines compare the NMR-derived models with empirical data, measured chemical shifts, distance- and dihedral restraints and the results are visualized in a dynamic Web 2.0 report. A red-orange-green score is used for residues and restraints to direct the user to those critiques that warrant further investigation. Overall green scores below ~20 % accompanied by red scores over ~50 % are strongly indicative of poorly modelled structures. The publically accessible, secure iCing webserver ( https://nmr.le.ac.uk ) allows individual users to upload the NMR data and run a CING validation analysis.
Asunto(s)
Resonancia Magnética Nuclear Biomolecular/métodos , Proteínas/química , Programas Informáticos , Modelos Moleculares , Conformación Proteica , Reproducibilidad de los Resultados , Interfaz Usuario-ComputadorRESUMEN
Several studies have shown that biomolecular NMR structures are often of lower quality when compared to crystal structures, and consequently they are often excluded from structural analyses. We present a publicly available database of re-refined NMR structures, exhibiting significantly improved quality. This database (available at http://www.cmbi.kun.nl/dress/) presents a uniformly refined and validated set of structural models that improves the value of these NMR structures as input for experimental and theoretical studies in many fields of research.
Asunto(s)
Bases de Datos de Proteínas , Modelos Moleculares , Resonancia Magnética Nuclear Biomolecular , Proteínas/química , Solventes/químicaRESUMEN
The attack of fungal cell walls by plant chitinases is an important plant defense response to fungal infection. Anti-fungal activity of plant chitinases is largely restricted to chitinases that contain a noncatalytic, plant-specific chitin-binding domain (ChBD) (also called Hevein domain). Current data confirm that the race-specific elicitor AVR4 of the tomato pathogen Cladosporium fulvum can protect fungi against plant chitinases, which is based on the presence of a novel type of ChBD in AVR4 that was first identified in invertebrates. Although these two classes of ChBDs (Hevein and invertebrate) are sequentially unrelated, they share structural homology. Here, we show that the chitin-binding sites of these two classes of ChBDs have different topologies and characteristics. The K(D), DeltaH, and DeltaS values obtained for the interaction between AVR4 and chito-oligomers are comparable with those obtained for Hevein. However, the binding site of AVR4 is larger than that of Hevein, i.e. AVR4 interacts strictly with chitotriose, whereas Hevein can also interact with the monomer N-acetylglucosamine. Moreover, binding of additional AVR4 molecules to chitin occurs through positive cooperative protein-protein interactions. By this mechanism AVR4 is likely to effectively shield chitin on the fungal cell wall, preventing the cell wall from being degraded by plant chitinases.
Asunto(s)
Proteínas Fúngicas/química , Trisacáridos/química , Regulación Alostérica , Secuencia de Aminoácidos , Sitios de Unión , Quitina/metabolismo , Cladosporium/química , Cladosporium/metabolismo , Proteínas Fúngicas/metabolismo , Modelos Moleculares , Conformación Molecular , Datos de Secuencia Molecular , Unión Proteica , Pliegue de Proteína , Termodinámica , Trisacáridos/metabolismoRESUMEN
Sin3 forms the scaffold for a multiprotein corepressor complex that silences transcription via the action of histone deacetylases. Sin3 is recruited to the DNA by several DNA binding repressors, such as the helix-loop-helix proteins of the Mad family. Here, we elaborate on the Mad-Sin3 interaction based on a binding study, solution structure, and dynamics of the PAH2 domain of mSin3 in complex to an extended Sin3 interacting domain (SID) of 24 residues of Mad1. We show that SID residues Met7 and Glu23, outside the previously defined minimal binding motif, mediate additional hydrophobic and electrostatic interactions with PAH2. On the basis of these results we propose an extended consensus sequence describing the PAH2-SID interaction specifically for the Mad family, showing that residues outside the hydrophobic core of the SID interact with PAH2 and modulate binding affinity to appropriate levels.
Asunto(s)
Proteínas de Unión al ADN/química , Proteínas Represoras , Proteínas de Saccharomyces cerevisiae , Homología de Secuencia de Aminoácido , Factores de Transcripción/química , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Animales , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice , Proteínas de Caenorhabditis elegans/química , Secuencia Conservada , Cristalografía por Rayos X , Proteínas Fúngicas/química , Histona Desacetilasas , Humanos , Proteínas de la Membrana/química , Datos de Secuencia Molecular , Familia de Multigenes , Resonancia Magnética Nuclear Biomolecular , Unión Proteica , Estructura Terciaria de Proteína , Alineación de Secuencia , Soluciones , Resonancia por Plasmón de Superficie , TermodinámicaRESUMEN
We present a CPU efficient protocol for refinement of protein structures in a thin layer of explicit solvent and energy parameters with completely revised dihedral angle terms. Our approach is suitable for protein structures determined by theoretical (e.g., homology modeling or threading) or experimental methods (e.g., NMR). In contrast to other recently proposed refinement protocols, we put a strong emphasis on consistency with widely accepted covalent parameters and computational efficiency. We illustrate the method for NMR structure calculations of three proteins: interleukin-4, ubiquitin, and crambin. We show a comparison of their structure ensembles before and after refinement in water with and without a force field energy term for the dihedral angles; crambin was also refined in DMSO. Our results demonstrate the significant improvement of structure quality by a short refinement in a thin layer of solvent. Further, they show that a dihedral angle energy term in the force field is beneficial for structure calculation and refinement. We discuss the optimal weight for the energy constant for the backbone angle omega and include an extensive discussion of meaning and relevance of the calculated validation criteria, in particular root mean square Z scores for covalent parameters such as bond lengths.
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Modelos Moleculares , Proteínas/química , Solventes/química , Agua/química , Dimetilsulfóxido/química , Interleucina-4/química , Estructura Molecular , Resonancia Magnética Nuclear Biomolecular , Proteínas de Plantas/química , Ubiquitina/químicaRESUMEN
Biomolecular structures provide the basis for many studies in several research areas such as homology modelling, structure-based drug design and functional genomics. It is an important prerequisite that the structure is reliable in terms of accurate description of the experimental data, and in terms of good quality of local- and overall geometry. Recent surveys indicate that structures solved by NMR-spectroscopy normally are of lower precision than high-resolution X-ray structures. Here, we present a refinement protocol that improves the quality of protein structures determined by NMR-spectroscopy to the level of those determined by high resolution X-ray crystallography in terms of local geometry. The protocol was tested on experimental data of the proteins IL4 and Ubiquitin and on simulated data of the protein Crambin. In almost all aspects, the protocol yielded better results in terms of accuracy and precision. Independent validation of the results for Ubiquitin, using residual dipolar couplings, indicates that the ensemble of NMR structure is substantially improved by the protocol.
Asunto(s)
Resonancia Magnética Nuclear Biomolecular/métodos , Conformación Proteica , Animales , Cristalografía por Rayos X , Bases de Datos de Proteínas , Interleucina-4/química , Proteínas de Plantas/química , Reproducibilidad de los Resultados , Ubiquitina/químicaRESUMEN
The PDZ domains of the protein tyrosine phosphatase PTP-BL mediate interactions by binding to specific amino acid sequences in target proteins. The solution structure of the second PDZ domain of PTP-BL, PDZ2, displays a compact fold with six beta strands and two alpha-helices. A unique feature of this domain compared to the canonical PDZ fold is an extended flexible loop at the base of the binding pocket, termed L1, that folds back onto the protein backbone, a feature that is shared by both the murine and human orthologues. The structure of PDZ2 differs significantly from the orthologous human structure. A comparison of structural quality indicators clearly demonstrates that the PDZ2 ensemble is statistically more reasonable than that of the human orthologue. The analysis of (15)N relaxation data for PDZ2 shows a normal pattern, with more rigid secondary structures and more flexible loop structures. Close to the binding pocket, Leu85 and Thr88 display greater mobility when compared to surrounding residues. Peptide binding studies demonstrated a lack of interaction between murine PDZ2 and the C terminus of the murine Fas/CD95 receptor, suggesting that the Fas/CD95 receptor is not an in vivo target for PDZ2. In addition, PDZ2 specifically binds the C termini of both human Fas/CD95 receptor and the RIL protein, despite RIL containing a non-canonical PDZ-interacting sequence of E-x-V. A model of PDZ2 with the RIL peptide reveals that the PDZ2 binding pocket is able to accommodate the bulkier side-chain of glutamic acid while maintaining crucial protein to peptide hydrogen bond interactions.
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Proteínas Tirosina Fosfatasas/química , Proteínas Tirosina Fosfatasas/metabolismo , Secuencia de Aminoácidos , Animales , Sitios de Unión , Secuencia Conservada , Evolución Molecular , Humanos , Enlace de Hidrógeno , Espectroscopía de Resonancia Magnética , Ratones , Modelos Moleculares , Datos de Secuencia Molecular , Docilidad , Unión Proteica , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Proteína Tirosina Fosfatasa no Receptora Tipo 13 , Alineación de Secuencia , Relación Estructura-ActividadRESUMEN
The human Y-box protein 1 (YB-1) is a member of the Y-box protein family, a class of proteins involved in transcriptional and translational regulation of a wide range of genes. Here, we report the solution structure of the cold-shock domain (CSD) of YB-1, which is thought to be responsible for nucleic acid binding. It is the first structure solved of a eukaryotic member of the cold-shock protein family and consists of a closed five-stranded anti-parallel beta-barrel capped by a long flexible loop. The structure of CSD is similar to the OB-fold and a comparison with bacterial cold-shock proteins shows that its structural properties are conserved from bacteria to man. Our data suggest the presence of a DNA-binding site consisting of a patch of positively charged and aromatic residues on the surface of the beta-barrel. Further, it is shown that CSD, which has a preference for binding single-stranded pyrimidine-rich sequences, binds weakly and hardly specifically to DNA. Binding affinities reported for intact YB-1 indicate that domains other than the CSD play a role in DNA binding of YB-1.