RESUMEN
The nonpolymorphic class Ib molecule, HLA-E, primarily presents peptides from HLA class Ia leader peptides, providing an inhibitory signal to NK cells via CD94/NKG2 interactions. Although peptides of pathogenic origin can also be presented by HLA-E to T cells, the molecular basis underpinning their role in antigen surveillance is largely unknown. Here, we solved a co-complex crystal structure of a TCR with an HLA-E presented peptide (pHLA-E) from bacterial (Mycobacterium tuberculosis) origin, and the first TCR-pHLA-E complex with a noncanonically presented peptide from viral (HIV) origin. The structures provided a molecular foundation to develop a novel method to introduce cysteine traps using non-natural amino acid chemistry that stabilized pHLA-E complexes while maintaining native interface contacts between the TCRs and different pHLA-E complexes. These pHLA-E monomers could be used to isolate pHLA-E-specific T cells, with obvious utility for studying pHLA-E restricted T cells, and for the identification of putative therapeutic TCRs.
Asunto(s)
Aminoácidos , Antígenos HLA , Antígenos de Histocompatibilidad Clase I , Péptidos , Receptores de Antígenos de Linfocitos T , Antígenos HLA-ERESUMEN
BACKGROUND: Endo-ß-1,4-galactanases are glycoside hydrolases (GH) from the GH53 family belonging to the largest clan of GHs, clan GH-A. GHs are ubiquitous and involved in a myriad of biological functions as well as being widely used industrially. Endo-ß-1,4-galactanases, in particular hydrolyse galactan and arabinogalactan in pectin, a major component of the primary plant cell wall, with important functions in plant defence and application in the food and other industries. Here, we explore the family's biological diversity by characterizing the first archaeal and hyperthermophilic GH53 galactanase, and utilize it as a scaffold for engineering enzymes with different product lengths. RESULTS: A galactanase gene was identified in the genome of the anaerobic hyperthermophilic archaeon Ignisphaera aggregans, and the isolated catalytic domain expressed and characterized (IaGal). IaGal presents the typical (ßα)8 barrel structure of clan GH-A enzymes, with catalytic carboxylates at the end of the 4th and 7th barrel strands. Its activity optimum of at least 95 °C and melting point over 100 °C indicate extreme thermostability, a very advantageous property for industrial applications. If enzyme depletion is reduced, so is the need for re-addition, and thus costs. The main stabilizing features of IaGal compared to other structurally characterized members are π-π and cation-π interactions. The length of the substrate binding site-and thus produced oligosaccharide products-is intermediate compared to previously characterized galactanases. Variants inspired by the structural diversity in the GH53 family were rationally designed to shorten or extend the substrate binding groove, in order to modulate product length. Subsite-deleted variants produced shorter products than IaGal, as do the fungal galactanases inspiring the design. IaGal variants engineered with a longer binding site produced a less expected degradation pattern, though still different from that of wild-type IaGal. All variants remained extremely stable. CONCLUSIONS: We have characterized in detail the most thermophilic endo-ß-1,4-galactanase known to date and successfully engineered it to modify the degradation profile, while maintaining much of its desirable thermostability. This is an important achievement as oligosaccharide products length is an important property for industrial and natural GHs alike.
RESUMEN
Tumor-associated peptide-human leukocyte antigen complexes (pHLAs) represent the largest pool of cell surface-expressed cancer-specific epitopes, making them attractive targets for cancer therapies. Soluble bispecific molecules that incorporate an anti-CD3 effector function are being developed to redirect T cells against these targets using 2 different approaches. The first achieves pHLA recognition via affinity-enhanced versions of natural TCRs (e.g., immune-mobilizing monoclonal T cell receptors against cancer [ImmTAC] molecules), whereas the second harnesses an antibody-based format (TCR-mimic antibodies). For both classes of reagent, target specificity is vital, considering the vast universe of potential pHLA molecules that can be presented on healthy cells. Here, we made use of structural, biochemical, and computational approaches to investigate the molecular rules underpinning the reactivity patterns of pHLA-targeting bispecifics. We demonstrate that affinity-enhanced TCRs engage pHLA using a comparatively broad and balanced energetic footprint, with interactions distributed over several HLA and peptide side chains. As ImmTAC molecules, these TCRs also retained a greater degree of pHLA selectivity, with less off-target activity in cellular assays. Conversely, TCR-mimic antibodies tended to exhibit binding modes focused more toward hot spots on the HLA surface and exhibited a greater degree of crossreactivity. Our findings extend our understanding of the basic principles that underpin pHLA selectivity and exemplify a number of molecular approaches that can be used to probe the specificity of pHLA-targeting molecules, aiding the development of future reagents.
Asunto(s)
Antígenos HLA/inmunología , Péptidos/inmunología , Receptores de Antígenos de Linfocitos T/inmunología , Secuencia de Aminoácidos , Anticuerpos Biespecíficos/química , Anticuerpos Biespecíficos/genética , Anticuerpos Biespecíficos/inmunología , Anticuerpos Antineoplásicos/química , Anticuerpos Antineoplásicos/genética , Anticuerpos Antineoplásicos/inmunología , Especificidad de Anticuerpos , Antígenos de Neoplasias/química , Antígenos de Neoplasias/genética , Antígenos de Neoplasias/inmunología , Línea Celular , Línea Celular Tumoral , Cristalografía por Rayos X , Antígenos HLA/química , Antígenos HLA/genética , Humanos , Indicadores y Reactivos , Modelos Moleculares , Simulación de Dinámica Molecular , Imitación Molecular/genética , Imitación Molecular/inmunología , Péptidos/química , Péptidos/genética , Receptores de Antígenos de Linfocitos T/química , Receptores de Antígenos de Linfocitos T/genética , Linfocitos T/inmunologíaRESUMEN
Bacterial phosphoinositide-specific phospholipases C (PI-PLCs) are the smallest members of the PI-PLC family, which includes much larger mammalian enzymes responsible for signal transduction as well as enzymes from protozoan parasites, yeast and plants. Eukaryotic PI-PLCs have calcium in the active site, but this is absent in the known structures of Gram-positive bacteria, where its role is instead played by arginine. In addition to their use in a number of industrial applications, the bacterial enzymes attract special interest because they can serve as convenient models of the catalytic domains of eukaryotic enzymes for in vitro activity studies. Here, the structure of a PI-PLC from Pseudomonas sp. 62186 is reported, the first from a Gram-negative bacterium and the first of a native bacterial PI-PLC with calcium present in the active site. Solution of the structure posed particular problems owing to the low sequence identity of available homologous structures. Its dependence on calcium for catalysis makes this enzyme a better model for studies of the mammalian PI-PLCs than the previously used calcium-independent bacterial PI-PLCs.
RESUMEN
The activity and substrate degradation pattern of a novel Aspergillus nidulans GH26 endo-ß-mannanase (AnMan26A) was investigated using two galactomannan substrates with varying amounts of galactopyranosyl residues. The AnMan26A was characterized in parallel with the GH26 endomannanase from Podospora anserina (PaMan26A) and three GH5 endomannanases from A. nidulans and Trichoderma reesei (AnMan5A, AnMan5C and TrMan5A). The initial rates and the maximal degree of enzymatically catalyzed conversion of locust bean gum and guar gum galactomannans were determined. The hydrolysis product profile at maximal degree of conversion was determined using DNA sequencer-Assisted Saccharide analysis in High throughput (DASH). This is the first reported use of this method for analyzing galactomannooligosaccharides. AnMan26A and PaMan26A were found to have a novel substrate degradation pattern on the two galactomannan substrates. On the highly substituted guar gum AnMan26A and PaMan26A reached 35-40% as their maximal degree of conversion whereas the three tested GH5 endomannanases only reached 8-10% as their maximal degree of conversion. α-Galactosyl-mannose was identified as the dominant degradation product resulting from AnMan26A and PaMan26A action on guar gum, strongly indicating that these two enzymes can accommodate galactopyranosyl residues in the -1 and in the +1 subsite. The degradation of α-6(4)-6(3)-di-galactosyl-mannopentaose by AnMan26A revealed accommodation of galactopyranosyl residues in the -2, -1 and +1 subsite of the enzyme. Accommodation of galactopyranosyl residues in subsites -2 and +1 has not been observed for other characterized endomannanases to date. Docking analysis of galactomannooligosaccharides in available crystal structures and homology models supported the conclusions drawn from the experimental results. This newly discovered diversity of substrate degradation patterns demonstrates an expanded functionality of fungal endomannanases, than hitherto reported.
Asunto(s)
Aspergillus nidulans/enzimología , Proteínas Fúngicas/metabolismo , Mananos/metabolismo , Manosidasas/metabolismo , Aspergillus nidulans/genética , Dominio Catalítico , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Galactanos/metabolismo , Galactosa/análogos & derivados , Hidrólisis , Cinética , Mananos/química , Manosidasas/química , Manosidasas/genética , Modelos Moleculares , Gomas de Plantas/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Especificidad por SustratoRESUMEN
Microbial fermentation of renewable feedstocks into plastic monomers can decrease our fossil dependence and reduce global CO2 emissions. 3-Hydroxypropionic acid (3HP) is a potential chemical building block for sustainable production of superabsorbent polymers and acrylic plastics. With the objective of developing Saccharomyces cerevisiae as an efficient cell factory for high-level production of 3HP, we identified the ß-alanine biosynthetic route as the most economically attractive according to the metabolic modeling. We engineered and optimized a synthetic pathway for de novo biosynthesis of ß-alanine and its subsequent conversion into 3HP using a novel ß-alanine-pyruvate aminotransferase discovered in Bacillus cereus. The final strain produced 3HP at a titer of 13.7±0.3gL(-1) with a 0.14±0.0C-molC-mol(-1) yield on glucose in 80h in controlled fed-batch fermentation in mineral medium at pH 5, and this work therefore lays the basis for developing a process for biological 3HP production.
Asunto(s)
Bacillus cereus , Proteínas Bacterianas , Ácido Láctico/análogos & derivados , Ingeniería Metabólica , Saccharomyces cerevisiae , beta-Alanina-Piruvato Transaminasa , Bacillus cereus/enzimología , Bacillus cereus/genética , Proteínas Bacterianas/biosíntesis , Proteínas Bacterianas/genética , Ácido Láctico/biosíntesis , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , beta-Alanina/genética , beta-Alanina/metabolismo , beta-Alanina-Piruvato Transaminasa/biosíntesis , beta-Alanina-Piruvato Transaminasa/genéticaRESUMEN
Biologically produced 3-hydroxypropionic acid (3 HP) is a potential source for sustainable acrylates and can also find direct use as monomer in the production of biodegradable polymers. For industrial-scale production there is a need for robust cell factories tolerant to high concentration of 3 HP, preferably at low pH. Through adaptive laboratory evolution we selected S. cerevisiae strains with improved tolerance to 3 HP at pH 3.5. Genome sequencing followed by functional analysis identified the causal mutation in SFA1 gene encoding S-(hydroxymethyl)glutathione dehydrogenase. Based on our findings, we propose that 3 HP toxicity is mediated by 3-hydroxypropionic aldehyde (reuterin) and that glutathione-dependent reactions are used for reuterin detoxification. The identified molecular response to 3 HP and reuterin may well be a general mechanism for handling resistance to organic acid and aldehydes by living cells.
Asunto(s)
Evolución Molecular Dirigida/métodos , Escherichia coli/genética , Mejoramiento Genético/métodos , Glutatión/metabolismo , Ácido Láctico/análogos & derivados , Saccharomyces cerevisiae/genética , Proliferación Celular/efectos de los fármacos , Proliferación Celular/genética , Relación Dosis-Respuesta a Droga , Tolerancia a Medicamentos/genética , Escherichia coli/efectos de los fármacos , Glutatión/genética , Ácido Láctico/administración & dosificación , Saccharomyces cerevisiae/efectos de los fármacosRESUMEN
STITCH is a database of protein-chemical interactions that integrates many sources of experimental and manually curated evidence with text-mining information and interaction predictions. Available at http://stitch.embl.de, the resulting interaction network includes 390 000 chemicals and 3.6 million proteins from 1133 organisms. Compared with the previous version, the number of high-confidence protein-chemical interactions in human has increased by 45%, to 367 000. In this version, we added features for users to upload their own data to STITCH in the form of internal identifiers, chemical structures or quantitative data. For example, a user can now upload a spreadsheet with screening hits to easily check which interactions are already known. To increase the coverage of STITCH, we expanded the text mining to include full-text articles and added a prediction method based on chemical structures. We further changed our scheme for transferring interactions between species to rely on orthology rather than protein similarity. This improves the performance within protein families, where scores are now transferred only to orthologous proteins, but not to paralogous proteins. STITCH can be accessed with a web-interface, an API and downloadable files.
Asunto(s)
Bases de Datos de Proteínas , Proteínas/metabolismo , Animales , Minería de Datos , Humanos , Internet , Ratones , Preparaciones Farmacéuticas/química , Mapeo de Interacción de Proteínas , Proteínas/química , Integración de SistemasRESUMEN
Asthma exacerbations are among the most frequent causes of hospitalization during childhood, but the underlying mechanisms are poorly understood. We performed a genome-wide association study of a specific asthma phenotype characterized by recurrent, severe exacerbations occurring between 2 and 6 years of age in a total of 1,173 cases and 2,522 controls. Cases were identified from national health registries of hospitalization, and DNA was obtained from the Danish Neonatal Screening Biobank. We identified five loci with genome-wide significant association. Four of these, GSDMB, IL33, RAD50 and IL1RL1, were previously reported as asthma susceptibility loci, but the effect sizes for these loci in our cohort were considerably larger than in the previous genome-wide association studies of asthma. We also obtained strong evidence for a new susceptibility gene, CDHR3 (encoding cadherin-related family member 3), which is highly expressed in airway epithelium. These results demonstrate the strength of applying specific phenotyping in the search for asthma susceptibility genes.
Asunto(s)
Asma/genética , Cadherinas/genética , Predisposición Genética a la Enfermedad , Proteínas de la Membrana/genética , Ácido Anhídrido Hidrolasas , Asma/etiología , Proteínas Relacionadas con las Cadherinas , Cadherinas/química , Cadherinas/metabolismo , Estudios de Casos y Controles , Niño , Preescolar , Cromosomas Humanos Par 17 , Enzimas Reparadoras del ADN/genética , Proteínas de Unión al ADN/genética , Dinamarca , Femenino , Estudio de Asociación del Genoma Completo , Humanos , Proteína 1 Similar al Receptor de Interleucina-1 , Interleucina-33 , Interleucinas/genética , Masculino , Proteínas de la Membrana/química , Proteínas de la Membrana/metabolismo , Modelos Moleculares , Proteínas de Neoplasias/genética , Polimorfismo de Nucleótido Simple , Conformación Proteica , Receptores de Superficie Celular/genéticaRESUMEN
Major histocompatibility complex class II (MHCII) molecules play an important role in cell-mediated immunity. They present specific peptides derived from endosomal proteins for recognition by T helper cells. The identification of peptides that bind to MHCII molecules is therefore of great importance for understanding the nature of immune responses and identifying T cell epitopes for the design of new vaccines and immunotherapies. Given the large number of MHC variants, and the costly experimental procedures needed to evaluate individual peptide-MHC interactions, computational predictions have become particularly attractive as first-line methods in epitope discovery. However, only a few so-called pan-specific prediction methods capable of predicting binding to any MHC molecule with known protein sequence are currently available, and all of them are limited to HLA-DR. Here, we present the first pan-specific method capable of predicting peptide binding to any HLA class II molecule with a defined protein sequence. The method employs a strategy common for HLA-DR, HLA-DP and HLA-DQ molecules to define the peptide-binding MHC environment in terms of a pseudo sequence. This strategy allows the inclusion of new molecules even from other species. The method was evaluated in several benchmarks and demonstrates a significant improvement over molecule-specific methods as well as the ability to predict peptide binding of previously uncharacterised MHCII molecules. To the best of our knowledge, the NetMHCIIpan-3.0 method is the first pan-specific predictor covering all HLA class II molecules with known sequences including HLA-DR, HLA-DP, and HLA-DQ. The NetMHCpan-3.0 method is available at http://www.cbs.dtu.dk/services/NetMHCIIpan-3.0 .
Asunto(s)
Biología Computacional/métodos , Antígenos HLA-DP/inmunología , Antígenos HLA-DQ/inmunología , Antígenos HLA-DR/inmunología , Antígenos de Histocompatibilidad Clase II/inmunología , Secuencia de Aminoácidos , Análisis por Conglomerados , Antígenos HLA-DP/química , Antígenos HLA-DP/genética , Antígenos HLA-DQ/química , Antígenos HLA-DQ/genética , Antígenos HLA-DR/química , Antígenos HLA-DR/genética , Antígenos de Histocompatibilidad Clase II/clasificación , Antígenos de Histocompatibilidad Clase II/genética , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Reproducibilidad de los Resultados , Homología de Secuencia de AminoácidoRESUMEN
The mountains of data thrusting from the new landscape of modern high-throughput biology are irrevocably changing biomedical research and creating a near-insatiable demand for training in data management and manipulation and data mining and analysis. Among life scientists, from clinicians to environmental researchers, a common theme is the need not just to use, and gain familiarity with, bioinformatics tools and resources but also to understand their underlying fundamental theoretical and practical concepts. Providing bioinformatics training to empower life scientists to handle and analyse their data efficiently, and progress their research, is a challenge across the globe. Delivering good training goes beyond traditional lectures and resource-centric demos, using interactivity, problem-solving exercises and cooperative learning to substantially enhance training quality and learning outcomes. In this context, this article discusses various pragmatic criteria for identifying training needs and learning objectives, for selecting suitable trainees and trainers, for developing and maintaining training skills and evaluating training quality. Adherence to these criteria may help not only to guide course organizers and trainers on the path towards bioinformatics training excellence but, importantly, also to improve the training experience for life scientists.
Asunto(s)
Disciplinas de las Ciencias Biológicas/educación , Biología Computacional/educación , Curriculum , Minería de Datos , Sistemas de Administración de Bases de Datos , Lenguajes de Programación , Diseño de Software , EnseñanzaRESUMEN
SUMMARY: We present iAnn, an open source community-driven platform for dissemination of life science events, such as courses, conferences and workshops. iAnn allows automatic visualisation and integration of customised event reports. A central repository lies at the core of the platform: curators add submitted events, and these are subsequently accessed via web services. Thus, once an iAnn widget is incorporated into a website, it permanently shows timely relevant information as if it were native to the remote site. At the same time, announcements submitted to the repository are automatically disseminated to all portals that query the system. To facilitate the visualization of announcements, iAnn provides powerful filtering options and views, integrated in Google Maps and Google Calendar. All iAnn widgets are freely available. AVAILABILITY: http://iann.pro/iannviewer CONTACT: manuel.corpas@tgac.ac.uk.
Asunto(s)
Disciplinas de las Ciencias Biológicas , Programas Informáticos , Aniversarios y Eventos Especiales , Congresos como Asunto , InternetRESUMEN
Funding bodies are increasingly recognizing the need to provide graduates and researchers with access to short intensive courses in a variety of disciplines, in order both to improve the general skills base and to provide solid foundations on which researchers may build their careers. In response to the development of 'high-throughput biology', the need for training in the field of bioinformatics, in particular, is seeing a resurgence: it has been defined as a key priority by many Institutions and research programmes and is now an important component of many grant proposals. Nevertheless, when it comes to planning and preparing to meet such training needs, tension arises between the reward structures that predominate in the scientific community which compel individuals to publish or perish, and the time that must be devoted to the design, delivery and maintenance of high-quality training materials. Conversely, there is much relevant teaching material and training expertise available worldwide that, were it properly organized, could be exploited by anyone who needs to provide training or needs to set up a new course. To do this, however, the materials would have to be centralized in a database and clearly tagged in relation to target audiences, learning objectives, etc. Ideally, they would also be peer reviewed, and easily and efficiently accessible for downloading. Here, we present the Bioinformatics Training Network (BTN), a new enterprise that has been initiated to address these needs and review it, respectively, to similar initiatives and collections.
Asunto(s)
Biología Computacional/educación , Redes Comunitarias , Humanos , Investigadores/educaciónRESUMEN
As bioinformatics becomes increasingly central to research in the molecular life sciences, the need to train non-bioinformaticians to make the most of bioinformatics resources is growing. Here, we review the key challenges and pitfalls to providing effective training for users of bioinformatics services, and discuss successful training strategies shared by a diverse set of bioinformatics trainers. We also identify steps that trainers in bioinformatics could take together to advance the state of the art in current training practices. The ideas presented in this article derive from the first Trainer Networking Session held under the auspices of the EU-funded SLING Integrating Activity, which took place in November 2009.
Asunto(s)
Biología Computacional/educación , Investigación/educación , Enseñanza/métodosRESUMEN
Protein annotation provides a condensed and systematic view on the function of individual proteins. It has traditionally dealt with sorting proteins into functional categories, which for example has proven to be successful for the comparison of different species. However, if we are to understand the differences between many individuals of the same species-humans in particular - the focus needs be on the functional impact of individual residue variation. To fulfil the promises of personal genomics, we need to start asking not only what is in a genome but also how millions of small differences between individual genomes affect protein function and in turn human health.
Asunto(s)
Genómica/métodos , Proteínas/genética , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Animales , Genoma Humano/genética , Humanos , Datos de Secuencia Molecular , Proteínas/química , Proteínas/metabolismoRESUMEN
CD4 positive T helper cells control many aspects of specific immunity. These cells are specific for peptides derived from protein antigens and presented by molecules of the extremely polymorphic major histocompatibility complex (MHC) class II system. The identification of peptides that bind to MHC class II molecules is therefore of pivotal importance for rational discovery of immune epitopes. HLA-DR is a prominent example of a human MHC class II. Here, we present a method, NetMHCIIpan, that allows for pan-specific predictions of peptide binding to any HLA-DR molecule of known sequence. The method is derived from a large compilation of quantitative HLA-DR binding events covering 14 of the more than 500 known HLA-DR alleles. Taking both peptide and HLA sequence information into account, the method can generalize and predict peptide binding also for HLA-DR molecules where experimental data is absent. Validation of the method includes identification of endogenously derived HLA class II ligands, cross-validation, leave-one-molecule-out, and binding motif identification for hitherto uncharacterized HLA-DR molecules. The validation shows that the method can successfully predict binding for HLA-DR molecules-even in the absence of specific data for the particular molecule in question. Moreover, when compared to TEPITOPE, currently the only other publicly available prediction method aiming at providing broad HLA-DR allelic coverage, NetMHCIIpan performs equivalently for alleles included in the training of TEPITOPE while outperforming TEPITOPE on novel alleles. We propose that the method can be used to identify those hitherto uncharacterized alleles, which should be addressed experimentally in future updates of the method to cover the polymorphism of HLA-DR most efficiently. We thus conclude that the presented method meets the challenge of keeping up with the MHC polymorphism discovery rate and that it can be used to sample the MHC "space," enabling a highly efficient iterative process for improving MHC class II binding predictions.
Asunto(s)
Antígenos HLA-DR/metabolismo , Mapeo de Interacción de Proteínas/métodos , Algoritmos , Alelos , Secuencia de Aminoácidos/fisiología , Sitios de Unión/genética , Sitios de Unión/inmunología , Bases de Datos de Proteínas , Antígenos HLA-DR/genética , Antígenos HLA-DR/inmunología , Humanos , Complejo Mayor de Histocompatibilidad/genética , Datos de Secuencia Molecular , Valor Predictivo de las Pruebas , Unión Proteica/inmunología , Reproducibilidad de los Resultados , Alineación de Secuencia , Análisis de Secuencia de ProteínaRESUMEN
Disulfide bond formation in the endoplasmic reticulum is catalyzed by enzymes of the protein disulfide-isomerase family that harbor one or more thioredoxin-like domains. We recently discovered the transmembrane protein TMX3, a thiol-disulfide oxidoreductase of the protein disulfide-isomerase family. Here, we show that the endoplasmic reticulum-luminal region of TMX3 contains three thioredoxin-like domains, an N-terminal redox-active domain (named a) followed by two enzymatically inactive domains (b and b'). Using the recombinantly expressed TMX3 domain constructs a, ab, and abb', we compared structural stability and enzymatic properties. By structural and biophysical methods, we demonstrate that the reduced a domain has features typical of a globular folded domain that is, however, greatly destabilized upon oxidization. Importantly, interdomain stabilization by the b domain renders the a domain more resistant toward chemical denaturation and proteolysis in both the oxidized and reduced form. In combination with molecular modeling studies of TMX3 abb', the experimental results provide a new understanding of the relationship between the multidomain structure of TMX3 and its function as a redox enzyme. Overall, the data indicate that in addition to their role as substrate and co-factor binding domains, redox-inactive thioredoxin-like domains also function in stabilizing neighboring redox-active domains.
Asunto(s)
Retículo Endoplásmico/metabolismo , Proteína Disulfuro Isomerasas/metabolismo , Secuencia de Aminoácidos , Sitios de Unión , Humanos , Espectroscopía de Resonancia Magnética , Modelos Biológicos , Conformación Molecular , Datos de Secuencia Molecular , Oxidación-Reducción , Pliegue de Proteína , Estructura Terciaria de Proteína , Proteínas Recombinantes/química , Homología de Secuencia de Aminoácido , Relación Estructura-ActividadRESUMEN
BACKGROUND: Binding of peptides to Major Histocompatibility Complex (MHC) molecules is the single most selective step in the recognition of pathogens by the cellular immune system. The human MHC class I system (HLA-I) is extremely polymorphic. The number of registered HLA-I molecules has now surpassed 1500. Characterizing the specificity of each separately would be a major undertaking. PRINCIPAL FINDINGS: Here, we have drawn on a large database of known peptide-HLA-I interactions to develop a bioinformatics method, which takes both peptide and HLA sequence information into account, and generates quantitative predictions of the affinity of any peptide-HLA-I interaction. Prospective experimental validation of peptides predicted to bind to previously untested HLA-I molecules, cross-validation, and retrospective prediction of known HIV immune epitopes and endogenous presented peptides, all successfully validate this method. We further demonstrate that the method can be applied to perform a clustering analysis of MHC specificities and suggest using this clustering to select particularly informative novel MHC molecules for future biochemical and functional analysis. CONCLUSIONS: Encompassing all HLA molecules, this high-throughput computational method lends itself to epitope searches that are not only genome- and pathogen-wide, but also HLA-wide. Thus, it offers a truly global analysis of immune responses supporting rational development of vaccines and immunotherapy. It also promises to provide new basic insights into HLA structure-function relationships. The method is available at http://www.cbs.dtu.dk/services/NetMHCpan.
Asunto(s)
Biología Computacional/métodos , Antígenos HLA-A/química , Antígenos HLA-B/química , Péptidos/metabolismo , Programas Informáticos , Sitios de Unión , Antígenos HLA-A/metabolismo , Antígenos HLA-B/metabolismo , Humanos , Péptidos/químicaRESUMEN
A high-resolution structure of the human MHC-I molecule HLA-A*1101 is presented in which it forms a complex with a sequence homologue of a peptide that occurs naturally in hepatitis B virus DNA polymerase. The sequence of the bound peptide is AIMPARFYPK, while that of the corresponding natural peptide is LIMPARFYPK. The peptide does not make efficient use of the middle E pocket for binding, which leads to a rather superficial and exposed binding mode for the central peptide residues. Despite this, the peptide binds with high affinity (IC50 of 31 nM).
Asunto(s)
ADN Polimerasa Dirigida por ADN/química , Antígenos HLA-A/química , Oligopéptidos/química , Fragmentos de Péptidos/química , Secuencia de Aminoácidos , Cristalización , Antígeno HLA-A11 , Virus de la Hepatitis B/química , Humanos , Enlace de Hidrógeno , Unión Proteica , Conformación ProteicaRESUMEN
MHC class I molecules govern human cytotoxic T cell responses. Their specificity determines which peptides they sample from the intracellular protein environment and then present to human cytotoxic T cells. More than 1100 different MHC class I proteins have been found in human populations and it would be a major undertaking to address each of these specificities individually. Based upon their peptide binding specificity, they are currently subdivided into 12 supertypes. Several of these HLA supertypes have not yet been described at the structural level. To support a comprehensive understanding of human immune responses, the structure of at least one member of each supertype should be determined. Here, the structures of two immunogenic peptide-HLA-B*1501 complexes are described. The structure of HLA-B*1501 in complex with a peptide (LEKARGSTY, corresponding to positions 274-282 in the Epstein-Barr virus nuclear antigen-3A) was determined to 2.3 A resolution. The structure of HLA-B*1501 in complex with a peptide (ILGPPGSVY) derived from human ubiquitin-conjugating enzyme-E2 corresponding to positions 91-99 was solved to 1.8 A resolution. Mutual comparisons of these two structures with structures from other HLA supertypes define and explain the specificity of the P2 and P9 peptide anchor preferences in the B62 HLA supertype. The P2 peptide residue binds to the B-pocket in HLA-B*1501. This pocket is relatively large because of the small Ser67 residue located at the bottom. The peptide proximal part of the B-pocket is hydrophobic, which is consistent with P2 anchor residue preference for Leu. The specificity of the B-pocket is determined by the Met45, Ile66 and Ser67 residues. The apex of the B-pocket is hydrophilic because of the Ser67 residue. The P9 peptide residue binds to the F-pocket in HLA-B*1501. The residues most important for the specificity of this pocket are Tyr74, Leu81, Leu95, Tyr123 and Trp147. These residues create a hydrophobic interior in the F-pocket and their spatial arrangement makes the pocket capable of containing large, bulky peptide side chains. Ser116 is located at the bottom of the F-pocket and makes the bottom of this pocket hydrophilic. Ser116, may act as a hydrogen-bonding partner and as such is a perfect place for binding of a Tyr9 peptide residue. Thus, based on structure information it is now possible to explain the peptide sequence specificity of HLA-B*1501 as previously determined by peptide binding and pool sequencing experiments.