RESUMEN
Human HIRA, ASF1a, ASF1b and CAF-1 are evolutionally conserved histone chaperones that form multiple functionally distinct chromatin-assembly complexes, with roles linked to diverse nuclear process, such as DNA replication and formation of heterochromatin in senescent cells. We report the crystal structure of an ASF1a-HIRA heterodimer and a biochemical dissection of ASF1a's mutually exclusive interactions with HIRA and the p60 subunit of CAF-1. The HIRA B domain forms an antiparallel beta-hairpin that binds perpendicular to the strands of the beta-sandwich of ASF1a, via beta-sheet, salt bridge and van der Waals contacts. The N- and C-terminal regions of ASF1a and ASF1b determine the different affinities of these two proteins for HIRA, by contacting regions outside the HIRA B domain. CAF-1 p60 also uses B domain-like motifs for binding to ASF1a, thereby competing with HIRA. Together, these studies begin to define the molecular determinants of assembly of functionally diverse macromolecular histone chaperone complexes.
Asunto(s)
Proteínas de Ciclo Celular/química , Histonas/química , Chaperonas Moleculares/química , Factores de Transcripción/química , Secuencia de Aminoácidos , Proteínas de Ciclo Celular/metabolismo , Línea Celular Tumoral , Chaperonas de Histonas , Histonas/metabolismo , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Unión Proteica , Estructura Terciaria de Proteína , Sensibilidad y Especificidad , Relación Estructura-Actividad , Factores de Transcripción/metabolismo , TransfecciónRESUMEN
In senescent cells, specialized domains of transcriptionally silent senescence-associated heterochromatic foci (SAHF), containing heterochromatin proteins such as HP1, are thought to repress expression of proliferation-promoting genes. We have investigated the composition and mode of assembly of SAHF and its contribution to cell cycle exit. SAHF is enriched in a transcription-silencing histone H2A variant, macroH2A. As cells approach senescence, a known chromatin regulator, HIRA, enters PML nuclear bodies, where it transiently colocalizes with HP1 proteins, prior to incorporation of HP1 proteins into SAHF. A physical complex containing HIRA and another chromatin regulator, ASF1a, is rate limiting for formation of SAHF and onset of senescence, and ASF1a is required for formation of SAHF and efficient senescence-associated cell cycle exit. These data indicate that HIRA and ASF1a drive formation of macroH2A-containing SAHF and senescence-associated cell cycle exit, via a pathway that appears to depend on flux of heterochromatic proteins through PML bodies.
Asunto(s)
Proteínas de Ciclo Celular/fisiología , Ciclo Celular/fisiología , Senescencia Celular/fisiología , Proteínas Cromosómicas no Histona/metabolismo , Heterocromatina/metabolismo , Histonas/metabolismo , Secuencia de Aminoácidos , Western Blotting/métodos , Recuento de Células/métodos , Línea Celular , Homólogo de la Proteína Chromobox 5 , Compensación de Dosificación (Genética) , Regulación de la Expresión Génica/fisiología , Inmunohistoquímica/métodos , Inmunoprecipitación/métodos , Indoles , Chaperonas Moleculares , Proteínas de Neoplasias/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Represoras , Factores de Tiempo , Factores de Transcripción/metabolismo , Transfección/métodos , Proteínas Supresoras de Tumor , Proteínas ras/metabolismoRESUMEN
UNLABELLED: There are many ftp or http servers storing data required for biological research. While some download applications are available, there is no user-friendly download application with a graphical interface specifically designed and adapted to meet the requirements of bioinformatics. BioDownloader is a program for downloading and updating files from ftp and http servers. It is optimized to work robustly with large numbers of files. It allows the selective retrieval of only the required files (batch downloads, multiple file masks, ls-lR file parsing, recursive search, recent updates, etc.). BioDownloader has a built-in repository containing the settings for common bioinformatics file-synchronization needs, including the Protein Data Bank (PDB) and National Center for Biotechnology Information (NCBI) databases. It can post-process downloaded files, including archive extraction and file conversions. AVAILABILITY: The program can be installed from http://dunbrack.fccc.edu/BioDownloader. The software is freely available for both non-commercial and commercial users under the BSD license.
Asunto(s)
Algoritmos , Biología Computacional/métodos , Sistemas de Administración de Bases de Datos , Bases de Datos Factuales , Almacenamiento y Recuperación de la Información/métodos , Programas Informáticos , Interfaz Usuario-Computador , InternetRESUMEN
BACKGROUND: The detection of unknown mutations is important in research and medicine. For this purpose, a mismatch-specific endonuclease CEL I from celery has been established as a useful tool in high throughput projects. Previously, CEL I-like activities were described only in a variety of plants and could not be expressed in an active form in bacteria. RESULTS: We describe expression of active recombinant plant mismatch endonucleases and modification of their activities. We also report the cloning of a CEL I ortholog from Spinacia oleracea (spinach) which we termed SP I nuclease. Active CEL I and SP I nucleases were expressed as C-terminal hexahistidine fusions and affinity purified from the cell culture media. Both recombinant enzymes were active in mutation detection in BRCA1 gene of patient-derived DNA. Native SP nuclease purified from spinach is unable to incise at single-nucleotide substitutions and loops containing a guanine nucleotide, but the recombinant SP I nuclease can cut at these sites. CONCLUSION: The insect cell-expressed CEL I orthologs may not be identical to their native counterparts purified from plant tissues. The present expression system should facilitate further development of CEL I-based mutation detection technologies.
Asunto(s)
Apium/enzimología , Apium/genética , Análisis Mutacional de ADN/métodos , Endonucleasas/genética , Spinacia oleracea/enzimología , Spinacia oleracea/genética , Endonucleasas/metabolismo , Ingeniería de Proteínas/métodos , Proteínas Recombinantes/genéticaRESUMEN
MOTIVATION: Modeling of protein interactions is often possible from known structures of related complexes. It is often time-consuming to find the most appropriate template. Hypothesized biological units (BUs) often differ from the asymmetric units and it is usually preferable to model from the BUs. RESULTS: ProtBuD is a database of BUs for all structures in the Protein Data Bank (PDB). We use both the PDBs BUs and those from the Protein Quaternary Server. ProtBuD is searchable by PDB entry, the Structural Classification of Proteins (SCOP) designation or pairs of SCOP designations. The database provides the asymmetric and BU contents of related proteins in the PDB as identified in SCOP and Position-Specific Iterated BLAST (PSI-BLAST). The asymmetric unit is different from PDB and/or Protein Quaternary Server (PQS) BUs for 52% of X-ray structures, and the PDB and PQS BUs disagree on 18% of entries. AVAILABILITY: The database is provided as a standalone program and a web server from http://dunbrack.fccc.edu/ProtBuD.php.
Asunto(s)
Sistemas de Administración de Bases de Datos , Bases de Datos de Proteínas , Almacenamiento y Recuperación de la Información/métodos , Proteínas/química , Proteínas/metabolismo , Análisis de Secuencia de Proteína/métodos , Interfaz Usuario-Computador , Secuencia de Aminoácidos , Datos de Secuencia Molecular , Proteínas/clasificación , Alineación de Secuencia/métodos , Homología de Secuencia de Aminoácido , Relación Estructura-ActividadRESUMEN
The general transcription factor TFIID is a multisubunit complex of TATA-binding protein (TBP) and 14 distinct TBP-associated factors (TAFs). Although TFIID constituents are required for transcription initiation of most mRNA encoding genes, the mechanism of TFIID action remains unclear. To gain insight into TFIID function, we sought to generate a proteomic catalogue of proteins specifically interacting with TFIID subunits. Toward this end, TFIID was systematically immunopurified by using polyclonal antibodies directed against each subunit, and the constellation of TBP- and TAF-associated proteins was directly identified by coupled multidimensional liquid chromatography and tandem mass spectrometry. A number of novel protein-protein associations were observed, and several were characterized in detail. These interactions include association between TBP and the RSC chromatin remodeling complex, the TAF17p-dependent association of the Swi6p transactivator protein with TFIID, and the identification of three novel subunits of the SAGA acetyltransferase complex, including a putative ubiquitin-specific protease component. Our results provide important new insights into the mechanisms of mRNA gene transcription and demonstrate the feasibility of constructing a complete proteomic interaction map of the eukaryotic transcription apparatus.
Asunto(s)
Proteínas Fúngicas/metabolismo , Espectrometría de Masas/métodos , Factores Asociados con la Proteína de Unión a TATA , Factores de Transcripción TFII/metabolismo , Levaduras/genética , Levaduras/metabolismo , Cromatografía Liquida/métodos , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Proteínas Fúngicas/inmunología , Inmunoquímica/métodos , Mutación , Proteoma , Reproducibilidad de los Resultados , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteína de Unión a TATA-Box , Factor de Transcripción TFIID , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
PURPOSE: Gastrointestinal stromal tumors (GIST) are characterized by expressing a gain-of-function mutation in KIT, and to a lesser extent, PDGFR. Imatinib mesylate, a tyrosine kinase inhibitor, has activity against GISTs that contain oncogenic mutations of KIT. In this study, KIT and PDGFRalpha mutation status was analyzed and protein modeling approaches were used to assess the potential effect of KIT mutations in response to imatinib therapy. EXPERIMENTAL DESIGN: Genomic DNA was isolated from GIST tumors. Exons 9, 11, 13, and 17 of c-KIT and exons 12, 14, and 18 of PDGFRalpha were evaluated for oncogenic mutations. Protein modeling was used to assess mutations within the juxtamembrane region and the kinase domain of KIT. RESULTS: Mutations in KIT exons 9, 11, and 13 were identified in GISTs with the majority of changes involving the juxtamembrane region of KIT. Molecular modeling indicates that mutations in this region result in disruption of the KIT autoinhibited conformation, and lead to gain-of-function activation of the kinase. Furthermore, a novel germ-line mutation in KIT was identified that is associated with an autosomal dominant predisposition to the development of GIST. CONCLUSIONS: We have used protein modeling and structural analyses to elucidate why patients with GIST tumors containing exon 11 mutations are the most responsive to imatinib mesylate treatment. Importantly, mutations detected in this exon and others displayed constitutive activation of KIT. Furthermore, we have found tumors that are both KIT and PDGFRalpha mutation negative, suggesting that additional, yet unidentified, abnormalities may contribute to GIST tumorigenesis.
Asunto(s)
Antineoplásicos/farmacología , Análisis Mutacional de ADN , ADN de Neoplasias/análisis , Tumores del Estroma Gastrointestinal/genética , Piperazinas/farmacología , Proteínas Proto-Oncogénicas c-kit/genética , Pirimidinas/farmacología , Adulto , Anciano , Secuencia de Aminoácidos , Benzamidas , Transformación Celular Neoplásica , Cristalografía por Rayos X , Femenino , Tumores del Estroma Gastrointestinal/fisiopatología , Mutación de Línea Germinal , Humanos , Mesilato de Imatinib , Masculino , Persona de Mediana Edad , Modelos Químicos , Datos de Secuencia Molecular , Linaje , Factor de Crecimiento Derivado de Plaquetas/biosíntesis , Factor de Crecimiento Derivado de Plaquetas/genética , Conformación Proteica , Proteínas Proto-Oncogénicas c-kit/biosíntesisRESUMEN
Tumor-associated fibroblasts are functionally and phenotypically distinct from normal fibroblasts that are not in the tumor microenvironment. Fibroblast activation protein is a 95 kDa cell surface glycoprotein expressed by tumor stromal fibroblasts, and has been shown to have dipeptidyl peptidase and collagenase activity. Site-directed mutagenesis at the catalytic site of fibroblast activation protein, Ser624 --> Ala624, resulted in an approximately 100,000-fold loss of fibroblast activation protein dipeptidyl peptidase (DPP) activity. HEK293 cells transfected with wild-type fibroblast activation protein, enzymatic mutant (S624A) fibroblast activation protein, or vector alone, were inoculated subcutaneously into immunodeficient mouse to assess the contribution of fibroblast activation protein enzymatic activity to tumor growth. Overexpression of wild-type fibroblast activation protein showed growth potentiation and enhanced tumorigenicity compared with both fibroblast activation protein S624A and vector-transfected HEK293 xenografts. HEK293 cells transfected with fibroblast activation protein S624A showed tumor growth rates and tumorigenicity potential similar only to vector-transfected HEK293. In vivo assessment of fibroblast activation protein DPP activity of these tumors showed enhanced enzymatic activity of wild-type fibroblast activation protein, with only baseline levels of fibroblast activation protein DPP activity in either fibroblast activation protein S624A or vector-only xenografts. These results indicate that the enzymatic activity of fibroblast activation protein is necessary for fibroblast activation protein-driven tumor growth in the HEK293 xenograft model system. This establishes the proof-of-principle that the enzymatic activity of fibroblast activation protein plays an important role in the promotion of tumor growth, and provides an attractive target for therapeutics designed to alter fibroblast activation protein-induced tumor growth by targeting its enzymatic activity.
Asunto(s)
Antígenos de Neoplasias/fisiología , Biomarcadores de Tumor/fisiología , Fibroblastos/metabolismo , Neoplasias/enzimología , Serina Endopeptidasas/fisiología , Alanina/química , Animales , Antígenos de Neoplasias/metabolismo , Antineoplásicos/farmacología , Sitios de Unión , Biomarcadores de Tumor/metabolismo , Western Blotting , Dominio Catalítico , Línea Celular , Línea Celular Tumoral , Proliferación Celular , ADN Complementario/metabolismo , Dipeptidil-Peptidasas y Tripeptidil-Peptidasas/metabolismo , Endopeptidasas , Citometría de Flujo , Gelatinasas , Humanos , Concentración de Iones de Hidrógeno , Inmunohistoquímica , Cinética , Proteínas de la Membrana , Ratones , Microscopía Fluorescente , Modelos Moleculares , Trasplante de Neoplasias , Serina/química , Serina Endopeptidasas/metabolismo , Factores de Tiempo , TransfecciónRESUMEN
In protein structure prediction, it is often the case that a protein segment must be adjusted to connect two fixed segments. This occurs during loop structure prediction in homology modeling as well as in ab initio structure prediction. Several algorithms for this purpose are based on the inverse Jacobian of the distance constraints with respect to dihedral angle degrees of freedom. These algorithms are sometimes unstable and fail to converge. We present an algorithm developed originally for inverse kinematics applications in robotics. In robotics, an end effector in the form of a robot hand must reach for an object in space by altering adjustable joint angles and arm lengths. In loop prediction, dihedral angles must be adjusted to move the C-terminal residue of a segment to superimpose on a fixed anchor residue in the protein structure. The algorithm, referred to as cyclic coordinate descent or CCD, involves adjusting one dihedral angle at a time to minimize the sum of the squared distances between three backbone atoms of the moving C-terminal anchor and the corresponding atoms in the fixed C-terminal anchor. The result is an equation in one variable for the proposed change in each dihedral. The algorithm proceeds iteratively through all of the adjustable dihedral angles from the N-terminal to the C-terminal end of the loop. CCD is suitable as a component of loop prediction methods that generate large numbers of trial structures. It succeeds in closing loops in a large test set 99.79% of the time, and fails occasionally only for short, highly extended loops. It is very fast, closing loops of length 8 in 0.037 sec on average.
Asunto(s)
Algoritmos , Proteínas/química , Modelos Moleculares , Péptidos Cíclicos/química , Conformación Proteica , Robótica , Homología de SecuenciaRESUMEN
Fast and accurate side-chain conformation prediction is important for homology modeling, ab initio protein structure prediction, and protein design applications. Many methods have been presented, although only a few computer programs are publicly available. The SCWRL program is one such method and is widely used because of its speed, accuracy, and ease of use. A new algorithm for SCWRL is presented that uses results from graph theory to solve the combinatorial problem encountered in the side-chain prediction problem. In this method, side chains are represented as vertices in an undirected graph. Any two residues that have rotamers with nonzero interaction energies are considered to have an edge in the graph. The resulting graph can be partitioned into connected subgraphs with no edges between them. These subgraphs can in turn be broken into biconnected components, which are graphs that cannot be disconnected by removal of a single vertex. The combinatorial problem is reduced to finding the minimum energy of these small biconnected components and combining the results to identify the global minimum energy conformation. This algorithm is able to complete predictions on a set of 180 proteins with 34342 side chains in <7 min of computer time. The total chi(1) and chi(1 + 2) dihedral angle accuracies are 82.6% and 73.7% using a simple energy function based on the backbone-dependent rotamer library and a linear repulsive steric energy. The new algorithm will allow for use of SCWRL in more demanding applications such as sequence design and ab initio structure prediction, as well addition of a more complex energy function and conformational flexibility, leading to increased accuracy.
Asunto(s)
Proteínas/química , Proteómica/métodos , Algoritmos , Biología Computacional , Simulación por Computador , Bases de Datos como Asunto , Disulfuros , Modelos Moleculares , Modelos Estadísticos , Conformación Molecular , Conformación Proteica , Estructura Secundaria de Proteína , Programas InformáticosRESUMEN
SCWRL and MolIDE are software applications for prediction of protein structures. SCWRL is designed specifically for the task of prediction of side-chain conformations given a fixed backbone usually obtained from an experimental structure determined by X-ray crystallography or NMR. SCWRL is a command-line program that typically runs in a few seconds. MolIDE provides a graphical interface for basic comparative (homology) modeling using SCWRL and other programs. MolIDE takes an input target sequence and uses PSI-BLAST to identify and align templates for comparative modeling of the target. The sequence alignment to any template can be manually modified within a graphical window of the target-template alignment and visualization of the alignment on the template structure. MolIDE builds the model of the target structure on the basis of the template backbone, predicted side-chain conformations with SCWRL and a loop-modeling program for insertion-deletion regions with user-selected sequence segments. SCWRL and MolIDE can be obtained at (http://dunbrack.fccc.edu/Software.php).
Asunto(s)
Proteínas/química , Análisis de Secuencia de Proteína/métodos , Programas Informáticos , Homología Estructural de Proteína , Algoritmos , Ligandos , Modelos Moleculares , Estructura Terciaria de Proteína , Alineación de SecuenciaRESUMEN
Genotoxic stress triggers a rapid translocation of p53 to the mitochondria, contributing to apoptosis in a transcription-independent manner. Using immunopurification protocols and mass spectrometry, we previously identified the proapoptotic protein BAK as a mitochondrial p53-binding protein and showed that recombinant p53 directly binds to BAK and can induce its oligomerization, leading to cytochrome c release. In this work we describe a combination of molecular modeling, electrostatic analysis, and site-directed mutagenesis to define contact residues between BAK and p53. Our data indicate that three regions within the core DNA binding domain of p53 make contact with BAK; these are the conserved H2 alpha-helix and the L1 and L3 loop. Notably, point mutations in these regions markedly impair the ability of p53 to oligomerize BAK and to induce transcription-independent cell death. We present a model whereby positively charged residues within the H2 helix and L1 loop of p53 interact with an electronegative domain on the N-terminal alpha-helix of BAK; the latter is known to undergo conformational changes upon BAK activation. We show that mutation of acidic residues in the N-terminal helix impair the ability of BAK to bind to p53. Interestingly, many of the p53 contact residues predicted by our model are also direct DNA contact residues, suggesting that p53 interacts with BAK in a manner analogous to DNA. The combined data point to the H2 helix and L1 and L3 loops of p53 as novel functional domains contributing to transcription-independent apoptosis by this tumor suppressor protein.
Asunto(s)
Proteína p53 Supresora de Tumor/química , Proteína Destructora del Antagonista Homólogo bcl-2/química , Animales , Apoptosis , Caenorhabditis elegans , Línea Celular Tumoral , Fibroblastos/metabolismo , Humanos , Ratones , Mitocondrias/metabolismo , Modelos Biológicos , Mutagénesis Sitio-Dirigida , Unión Proteica , Conformación Proteica , Estructura Terciaria de Proteína , Proteína Destructora del Antagonista Homólogo bcl-2/metabolismoRESUMEN
Many proteins function as homo-oligomers and are regulated via their oligomeric state. For some proteins, the stoichiometry of homo-oligomeric states under various conditions has been studied using gel filtration or analytical ultracentrifugation experiments. The interfaces involved in these assemblies may be identified using cross-linking and mass spectrometry, solution-state NMR, and other experiments. However, for most proteins, the actual interfaces that are involved in oligomerization are inferred from X-ray crystallographic structures using assumptions about interface surface areas and physical properties. Examination of interfaces across different Protein Data Bank (PDB) entries in a protein family reveals several important features. First, similarities in space group, asymmetric unit size, and cell dimensions and angles (within 1%) do not guarantee that two crystals are actually the same crystal form, containing similar relative orientations and interactions within the crystal. Conversely, two crystals in different space groups may be quite similar in terms of all the interfaces within each crystal. Second, NMR structures and an existing benchmark of PDB crystallographic entries consisting of 126 dimers as well as larger structures and 132 monomers were used to determine whether the existence or lack of common interfaces across multiple crystal forms can be used to predict whether a protein is an oligomer or not. Monomeric proteins tend to have common interfaces across only a minority of crystal forms, whereas higher-order structures exhibit common interfaces across a majority of available crystal forms. The data can be used to estimate the probability that an interface is biological if two or more crystal forms are available. Finally, the Protein Interfaces, Surfaces, and Assemblies (PISA) database available from the European Bioinformatics Institute is more consistent in identifying interfaces observed in many crystal forms compared with the PDB and the European Bioinformatics Institute's Protein Quaternary Server (PQS). The PDB, in particular, is missing highly likely biological interfaces in its biological unit files for about 10% of PDB entries.
Asunto(s)
Biología Computacional/métodos , Modelos Estadísticos , Proteínas/química , Biología Computacional/estadística & datos numéricos , Cristalización , Cristalografía por Rayos X , Bases de Datos de Proteínas , Dimerización , Espectroscopía de Resonancia Magnética , Modelos Moleculares , Unión Proteica , Conformación Proteica , Estructura Secundaria de ProteínaRESUMEN
For over a decade, p130Cas/BCAR1, HEF1/NEDD9/Cas-L, and Efs/Sin have defined the Cas (Crk-associated substrate) scaffolding protein family. Cas proteins mediate integrin-dependent signals at focal adhesions, regulating cell invasion and survival; at least one family member, HEF1, regulates mitosis. We here report a previously undescribed novel branch of the Cas protein family, designated HEPL (for HEF1-Efs-p130Cas-like). The HEPL branch is evolutionarily conserved through jawed vertebrates, and HEPL is found in some species lacking other members of the Cas family. The human HEPL mRNA and protein are selectively expressed in specific primary tissues and cancer cell lines, and HEPL maintains Cas family function in localization to focal adhesions, as well as regulation of FAK activity, focal adhesion integrity, and cell spreading. It has recently been demonstrated that upregulation of HEF1 expression marks and induces metastasis, whereas high endogenous levels of p130Cas are associated with poor prognosis in breast cancer, emphasizing the clinical relevance of Cas proteins. Better understanding of the complete protein family should help inform prediction of cancer incidence and prognosis.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/fisiología , Proteína Sustrato Asociada a CrK/fisiología , Quinasa 1 de Adhesión Focal/fisiología , Proteínas Adaptadoras Transductoras de Señales/química , Proteínas Adaptadoras Transductoras de Señales/genética , Secuencia de Aminoácidos , Línea Celular , Movimiento Celular/fisiología , Cromosomas Humanos Par 20/genética , Clonación Molecular , Proteína Sustrato Asociada a CrK/química , Proteína Sustrato Asociada a CrK/genética , Femenino , Adhesiones Focales/fisiología , Expresión Génica , Humanos , Masculino , Modelos Moleculares , Datos de Secuencia Molecular , Neoplasias/fisiopatología , Filogenia , Embarazo , Estructura Secundaria de Proteína , ARN Mensajero/genética , ARN Mensajero/metabolismo , Homología de Secuencia de Aminoácido , Distribución Tisular , Dominios Homologos srcRESUMEN
Factor XI (FXI) is a homodimeric blood coagulation protein. Each monomer comprises four tandem apple-domain repeats (A1-A4) and a serine protease domain. We report here the NMR solution structure of the A4 domain (residues 272-361), which mediates formation of the disulfide-linked FXI dimer. A4 exhibits characteristic features of the plasminogen apple nematode domain family, including a five-stranded beta-sheet flanked by an alpha-helix on one side and a two-stranded beta-sheet on the other. In addition, the solution structure reveals a second alpha-helix at the C terminus. Comparison with a recent crystal structure of full-length FXI, combined with molecular modeling, suggests that the C-terminal helix is formed only upon proteolytic activation. The newly formed helix disrupts interdomain contacts and reorients the catalytic domains, bringing the active sites into close proximity. This hypothesis is supported by small-angle x-ray scattering and electron microscopy data, which indicate that FXI activation is accompanied by a major change in shape. The results are consistent with biochemical evidence that activated FXI cleaves its substrate at two positions without release of an intermediate.
Asunto(s)
Precursores Enzimáticos/química , Factor XI/química , Fragmentos de Péptidos/química , Sitios de Unión , Dimerización , Activación Enzimática , Precursores Enzimáticos/metabolismo , Espectroscopía de Resonancia Magnética , Modelos Moleculares , Conformación Proteica , Estructura Terciaria de Proteína , SolucionesRESUMEN
UNLABELLED: Molecular Integrated Development Environment (MolIDE) is an integrated application designed to provide homology modeling tools and protocols under a uniform, user-friendly graphical interface. Its main purpose is to combine the most frequent modeling steps in a semi-automatic, interactive way, guiding the user from the target protein sequence to the final three-dimensional protein structure. The typical basic homology modeling process is composed of building sequence profiles of the target sequence family, secondary structure prediction, sequence alignment with PDB structures, assisted alignment editing, side-chain prediction and loop building. All of these steps are available through a graphical user interface. MolIDE's user-friendly and streamlined interactive modeling protocol allows the user to focus on the important modeling questions, hiding from the user the raw data generation and conversion steps. MolIDE was designed from the ground up as an open-source, cross-platform, extensible framework. This allows developers to integrate additional third-party programs to MolIDE. AVAILABILITY: http://dunbrack.fccc.edu/molide/molide.php CONTACT: rl_dunbrack@fccc.edu.