RESUMO
Secretory preproteins of the Sec pathway are targeted post-translationally and cross cellular membranes through translocases. During cytoplasmic transit, mature domains remain non-folded for translocase recognition/translocation. After translocation and signal peptide cleavage, mature domains fold to native states in the bacterial periplasm or traffic further. We sought the structural basis for delayed mature domain folding and how signal peptides regulate it. We compared how evolution diversified a periplasmic peptidyl-prolyl isomerase PpiA mature domain from its structural cytoplasmic PpiB twin. Global and local hydrogen-deuterium exchange mass spectrometry showed that PpiA is a slower folder. We defined at near-residue resolution hierarchical folding initiated by similar foldons in the twins, at different order and rates. PpiA folding is delayed by less hydrophobic native contacts, frustrated residues and a ß-turn in the earliest foldon and by signal peptide-mediated disruption of foldon hierarchy. When selected PpiA residues and/or its signal peptide were grafted onto PpiB, they converted it into a slow folder with enhanced in vivo secretion. These structural adaptations in a secretory protein facilitate trafficking.
Assuntos
Dobramento de Proteína , Sinais Direcionadores de Proteínas , Sinais Direcionadores de Proteínas/genética , Proteínas/metabolismo , Membrana Celular/metabolismo , Interações Hidrofóbicas e HidrofílicasRESUMO
An unambiguous description of an experiment, and the subsequent biological observation, is vital for accurate data interpretation. Minimum information guidelines define the fundamental complement of data that can support an unambiguous conclusion based on experimental observations. We present the Minimum Information About Disorder Experiments (MIADE) guidelines to define the parameters required for the wider scientific community to understand the findings of an experiment studying the structural properties of intrinsically disordered regions (IDRs). MIADE guidelines provide recommendations for data producers to describe the results of their experiments at source, for curators to annotate experimental data to community resources and for database developers maintaining community resources to disseminate the data. The MIADE guidelines will improve the interpretability of experimental results for data consumers, facilitate direct data submission, simplify data curation, improve data exchange among repositories and standardize the dissemination of the key metadata on an IDR experiment by IDR data sources.
Assuntos
Proteínas Intrinsicamente Desordenadas , Proteínas Intrinsicamente Desordenadas/química , Conformação ProteicaRESUMO
SUMMARY: SIMSApiper is a Nextflow pipeline that creates reliable, structure-informed MSAs of thousands of protein sequences faster than standard structure-based alignment methods. Structural information can be provided by the user or collected by the pipeline from online resources. Parallelization with sequence identity-based subsets can be activated to significantly speed up the alignment process. Finally, the number of gaps in the final alignment can be reduced by leveraging the position of conserved secondary structure elements. AVAILABILITY AND IMPLEMENTATION: The pipeline is implemented using Nextflow, Python3, and Bash. It is publicly available on github.com/Bio2Byte/simsapiper.
Assuntos
Proteínas , Alinhamento de Sequência , Análise de Sequência de Proteína , Software , Proteínas/química , Alinhamento de Sequência/métodos , Análise de Sequência de Proteína/métodos , Algoritmos , Sequência de Aminoácidos , Biologia Computacional/métodos , Bases de Dados de ProteínasRESUMO
α-1 acid glycoprotein (AGP) is one of the most abundant plasma proteins. It fulfills two important functions: immunomodulation, and binding to various drugs and receptors. These different functions are closely associated and modulated via changes in glycosylation and cancer missense mutations. From a structural point of view, glycans alter the local biophysical properties of the protein leading to a diverse ligand-binding spectrum. However, glycans can typically not be observed in the resolved X-ray crystallography structure of AGP due to their high flexibility and microheterogeneity, so limiting our understanding of AGP's conformational dynamics 70 years after its discovery. We here investigate how mutations and glycosylation interfere with AGP's conformational dynamics changing its biophysical behavior, by using molecular dynamics (MD) simulations and sequence-based dynamics predictions. The MD trajectories show that glycosylation decreases the local backbone flexibility of AGP and increases the flexibility of distant regions through allosteric effects. We observe that mutations near the glycosylation site affect glycan's conformational preferences. Thus, we conclude that mutations control glycan dynamics which modulates the protein's backbone flexibility directly affecting its accessibility. These findings may assist in the drug design targeting AGP's glycosylation and mutations in cancer.
Assuntos
Neoplasias , Orosomucoide , Humanos , Glicosilação , Orosomucoide/genética , Orosomucoide/química , Orosomucoide/metabolismo , Conformação Molecular , Polissacarídeos , Neoplasias/genéticaRESUMO
Nanobodies are popular and versatile tools for structural biology. They have a compact single immunoglobulin domain organization, bind target proteins with high affinities while reducing their conformational heterogeneity and stabilize multi-protein complexes. Here we demonstrate that engineered nanobodies can also help overcome two major obstacles that limit the resolution of single-particle cryo-electron microscopy reconstructions: particle size and preferential orientation at the water-air interfaces. We have developed and characterized constructs, termed megabodies, by grafting nanobodies onto selected protein scaffolds to increase their molecular weight while retaining the full antigen-binding specificity and affinity. We show that the megabody design principles are applicable to different scaffold proteins and recognition domains of compatible geometries and are amenable for efficient selection from yeast display libraries. Moreover, we demonstrate that megabodies can be used to obtain three-dimensional reconstructions for membrane proteins that suffer from severe preferential orientation or are otherwise too small to allow accurate particle alignment.
Assuntos
Microscopia Crioeletrônica/métodos , Lipídeos/química , Complexos Multiproteicos/química , Receptores de GABA-A/química , Imagem Individual de Molécula/métodos , Análise de Célula Única/métodos , Anticorpos de Domínio Único/química , Humanos , Modelos Moleculares , Estrutura Molecular , Conformação ProteicaRESUMO
MOTIVATION: The generation of parameter files for molecular dynamics (MD) simulations of small molecules that are suitable for force fields commonly applied to proteins and nucleic acids is often challenging. The ACPYPE software and website aid the generation of such parameter files. RESULTS: ACPYPE uses OpenBabel and ANTECHAMBER to generate MD input files in Gromacs, AMBER, CHARMM, and CNS formats. It can now take a SMILES string as input, in addition to the original PDB or mol2 coordinate files, with GAFF2 support and GLYCAM force field conversion added. It can be installed locally via Anaconda, PyPI, and Docker distributions, while the web server at https://bio2byte.be/acpype/ was updated with an API, and provides visualization of results for uploaded molecules as well as a pre-generated set of 3738 drug molecules. AVAILABILITY AND IMPLEMENTATION: The web application is freely available at https://www.bio2byte.be/acpype/ and the open-source code can be found at https://github.com/alanwilter/acpype.
Assuntos
Ácidos Nucleicos , Software , Computadores , Proteínas/metabolismo , Simulação de Dinâmica MolecularRESUMO
RNA recognition motifs (RRM) are the most prevalent class of RNA binding domains in eucaryotes. Their RNA binding preferences have been investigated for almost two decades, and even though some RRM domains are now very well described, their RNA recognition code has remained elusive. An increasing number of experimental structures of RRM-RNA complexes has become available in recent years. Here, we perform an in-depth computational analysis to derive an RNA recognition code for canonical RRMs. We present and validate a computational scoring method to estimate the binding between an RRM and a single stranded RNA, based on structural data from a carefully curated multiple sequence alignment, which can predict RRM binding RNA sequence motifs based on the RRM protein sequence. Given the importance and prevalence of RRMs in humans and other species, this tool could help design RNA binding motifs with uses in medical or synthetic biology applications, leading towards the de novo design of RRMs with specific RNA recognition.
Assuntos
Motivo de Reconhecimento de RNA , RNA , Humanos , RNA/química , Sequência de Aminoácidos , Alinhamento de Sequência , Motivos de Nucleotídeos/genética , Ligação Proteica , Sítios de LigaçãoRESUMO
Inactive rhodopsin can absorb photons, which induces different structural transitions that finally activate rhodopsin. We have examined the change in spatial configurations and physicochemical factors that result during the transition mechanism from the inactive to the active rhodopsin state via intermediates. During the activation process, many existing atomic contacts are disrupted, and new ones are formed. This is related to the movement of Helix 5, which tilts away from Helix 3 in the intermediate state in lumirhodopsin and moves closer to Helix 3 again in the active state. Similar patterns of changing atomic contacts are observed between Helices 3 and 5 of the adenosine and neurotensin receptors. In addition, residues 220-238 of rhodopsin, which are disordered in the inactive state, fold in the active state before binding to the Gα, where it catalyzes GDP/GTP exchange on the Gα subunit. Finally, molecular dynamics simulations in the membrane environment revealed that the arrestin binding region adopts a more flexible extended conformation upon phosphorylation, likely promoting arrestin binding and inactivation. In summary, our results provide additional structural understanding of specific rhodopsin activation which might be relevant to other Class A G protein-coupled receptor proteins.
Assuntos
Receptores Acoplados a Proteínas G , Rodopsina , Animais , Bovinos , Rodopsina/química , Rodopsina/metabolismo , Conformação Proteica , Receptores Acoplados a Proteínas G/química , Simulação de Dinâmica Molecular , Arrestinas/metabolismoRESUMO
One of the most intriguing fields emerging in current molecular biology is the study of membraneless organelles formed via liquid-liquid phase separation (LLPS). These organelles perform crucial functions in cell regulation and signalling, and recent years have also brought about the understanding of the molecular mechanism of their formation. The LLPS field is continuously developing and optimizing dedicated in vitro and in vivo methods to identify and characterize these non-stoichiometric molecular condensates and the proteins able to drive or contribute to LLPS. Building on these observations, several computational tools and resources have emerged in parallel to serve as platforms for the collection, annotation and prediction of membraneless organelle-linked proteins. In this survey, we showcase recent advancements in LLPS bioinformatics, focusing on (i) available databases and ontologies that are necessary to describe the studied phenomena and the experimental results in an unambiguous way and (ii) prediction methods to assess the potential LLPS involvement of proteins. Through hands-on application of these resources on example proteins and representative datasets, we give a practical guide to show how they can be used in conjunction to provide in silico information on LLPS.
Assuntos
Bases de Dados Factuais , Modelos Químicos , Organelas/química , Proteínas/química , Proteínas/isolamento & purificaçãoRESUMO
We provide integrated protein sequence-based predictions via https://bio2byte.be/b2btools/. The aim of our predictions is to identify the biophysical behaviour or features of proteins that are not readily captured by structural biology and/or molecular dynamics approaches. Upload of a FASTA file or text input of a sequence provides integrated predictions from DynaMine backbone and side-chain dynamics, conformational propensities, and derived EFoldMine early folding, DisoMine disorder, and Agmata ß-sheet aggregation. These predictions, several of which were previously not available online, capture 'emergent' properties of proteins, i.e. the inherent biophysical propensities encoded in their sequence, rather than context-dependent behaviour (e.g. final folded state). In addition, upload of a multiple sequence alignment (MSA) in a variety of formats enables exploration of the biophysical variation observed in homologous proteins. The associated plots indicate the biophysical limits of functionally relevant protein behaviour, with unusual residues flagged by a Gaussian mixture model analysis. The prediction results are available as JSON or CSV files and directly accessible via an API. Online visualisation is available as interactive plots, with brief explanations and tutorial pages included. The server and API employ an email-free token-based system that can be used to anonymously access previously generated results.
Assuntos
Proteínas/química , Alinhamento de Sequência , Análise de Sequência de Proteína/métodos , Software , InternetRESUMO
The MobiDB database (URL: https://mobidb.org/) provides predictions and annotations for intrinsically disordered proteins. Here, we report recent developments implemented in MobiDB version 4, regarding the database format, with novel types of annotations and an improved update process. The new website includes a re-designed user interface, a more effective search engine and advanced API for programmatic access. The new database schema gives more flexibility for the users, as well as simplifying the maintenance and updates. In addition, the new entry page provides more visualisation tools including customizable feature viewer and graphs of the residue contact maps. MobiDB v4 annotates the binding modes of disordered proteins, whether they undergo disorder-to-order transitions or remain disordered in the bound state. In addition, disordered regions undergoing liquid-liquid phase separation or post-translational modifications are defined. The integrated information is presented in a simplified interface, which enables faster searches and allows large customized datasets to be downloaded in TSV, Fasta or JSON formats. An alternative advanced interface allows users to drill deeper into features of interest. A new statistics page provides information at database and proteome levels. The new MobiDB version presents state-of-the-art knowledge on disordered proteins and improves data accessibility for both computational and experimental users.
Assuntos
Bases de Dados de Proteínas , Proteínas Intrinsicamente Desordenadas/química , Algoritmos , Internet , Anotação de Sequência Molecular , Processamento de Proteína Pós-Traducional , SoftwareRESUMO
Protein phosphorylation is the most common reversible post-translational modification of proteins and is key in the regulation of many cellular processes. Due to this importance, phosphorylation is extensively studied, resulting in the availability of a large amount of mass spectrometry-based phospho-proteomics data. Here, we leverage the information in these large-scale phospho-proteomics data sets, as contained in Scop3P, to analyze and characterize proteome-wide protein phosphorylation sites (P-sites). First, we set out to differentiate correctly observed P-sites from false-positive sites using five complementary site properties. We then describe the context of these P-sites in terms of the protein structure, solvent accessibility, structural transitions and disorder, and biophysical properties. We also investigate the relative prevalence of disease-linked mutations on and around P-sites. Moreover, we assess the structural dynamics of P-sites in their phosphorylated and unphosphorylated states. As a result, we show how large-scale reprocessing of available proteomics experiments can enable a more reliable view on proteome-wide P-sites. Furthermore, adding the structural context of proteins around P-sites helps uncover possible conformational switches upon phosphorylation. Moreover, by placing sites in different biophysical contexts, we show the differential preference in protein dynamics at phosphorylated sites when compared to the nonphosphorylated counterparts.
Assuntos
Proteoma , Proteômica , Humanos , Espectrometria de Massas , Fosforilação , Processamento de Proteína Pós-Traducional , Proteoma/metabolismo , Proteômica/métodosRESUMO
MOTIVATION: High-throughput experiments are generating ever increasing amounts of various -omics data, so shedding new light on the link between human disorders, their genetic causes and the related impact on protein behavior and structure. While numerous bioinformatics tools now exist that predict which variants in the human exome cause diseases, few tools predict the reasons why they might do so. Yet, understanding the impact of variants at the molecular level is a prerequisite for the rational development of targeted drugs or personalized therapies. RESULTS: We present the updated MutaFrame webserver, which aims to meet this need. It offers two deleteriousness prediction softwares, DEOGEN2 and SNPMuSiC, and is designed for bioinformaticians and medical researchers who want to gain insights into the origins of monogenic diseases. It contains information at two levels for each human protein: its amino acid sequence and its three-dimensional structure; we used the experimental structures whenever available, and modeled structures otherwise. MutaFrame also includes higher-level information, such as protein essentiality and protein-protein interactions. It has a user-friendly interface for the interpretation of results and a convenient visualization system for protein structures, in which the variant positions introduced by the user and other structural information are shown. In this way, MutaFrame aids our understanding of the pathogenic processes caused by single-site mutations and their molecular and contextual interpretation. AVAILABILITY AND IMPLEMENTATION: Mutaframe webserver at http://mutaframe.com/. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.
Assuntos
Biologia Computacional , Exoma , Humanos , Software , Proteínas , Mutação de Sentido IncorretoRESUMO
Nuclear magnetic resonance (NMR) spectroscopy data provides valuable information on the behaviour of proteins in solution. The primary data to determine when studying proteins are the per-atom NMR chemical shifts, which reflect the local environment of atoms and provide insights into amino acid residue dynamics and conformation. Within an amino acid residue, chemical shifts present multi-dimensional and complexly cross-correlated information, making them difficult to analyse. The ShiftCrypt method, based on neural network auto-encoder architecture, compresses the per-amino acid chemical shift information in a single, interpretable, amino acid-type independent value that reflects the biophysical state of a residue. We here present the ShiftCrypt web server, which makes the method readily available. The server accepts chemical shifts input files in the NMR Exchange Format (NEF) or NMR-STAR format, executes ShiftCrypt and visualises the results, which are also accessible via an API. It also enables the "biophysically-based" pairwise alignment of two proteins based on their ShiftCrypt values. This approach uses Dynamic Time Warping and can optionally include their amino acid code information, and has applications in, for example, the alignment of disordered regions. The server uses a token-based system to ensure the anonymity of the users and results. The web server is available at www.bio2byte.be/shiftcrypt.
Assuntos
Ressonância Magnética Nuclear Biomolecular/métodos , Proteínas/química , Software , Aminoácidos/química , Redes Neurais de Computação , Desnaturação Proteica , Dobramento de Proteína , Desdobramento de ProteínaRESUMO
MOTIVATION: Protein beta-aggregation is an important but poorly understood phenomena involved in diseases as well as in beneficial physiological processes. However, while this task has been investigated for over 50 years, very little is known about its mechanisms of action. Moreover, the identification of regions involved in aggregation is still an open problem and the state-of-the-art methods are often inadequate in real case applications. RESULTS: In this article we present AgMata, an unsupervised tool for the identification of such regions from amino acidic sequence based on a generalized definition of statistical potentials that includes biophysical information. The tool outperforms the state-of-the-art methods on two different benchmarks. As case-study, we applied our tool to human ataxin-3, a protein involved in Machado-Joseph disease. Interestingly, AgMata identifies aggregation-prone residues that share the very same structural environment. Additionally, it successfully predicts the outcome of in vitro mutagenesis experiments, identifying point mutations that lead to an alteration of the aggregation propensity of the wild-type ataxin-3. AVAILABILITY AND IMPLEMENTATION: A python implementation of the tool is available at https://bitbucket.org/bio2byte/agmata. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.
Assuntos
Doença de Machado-Joseph , Proteínas , Sequência de Aminoácidos , Ataxina-3 , HumanosRESUMO
Intrinsically disordered proteins are proteins whose native functional states represent ensembles of highly diverse conformations. Such ensembles are a challenge for quantitative structure comparisons because their conformational diversity precludes optimal superimposition of the atomic coordinates necessary for deriving common similarity measures such as the root mean-square deviation of these coordinates. Here, we introduce superimposition-free metrics that are based on computing matrices of the Cα-Cα distance distributions within ensembles and comparing these matrices between ensembles. Differences between two matrices yield information on the similarity between specific regions of the polypeptide, whereas the global structural similarity is captured by the root mean-square difference between the medians of the Cα-Cα distance distributions of two ensembles. Together, our metrics enable rigorous investigations of structure-function relationships in conformational ensembles of intrinsically disordered proteins derived using experimental restraints or by molecular simulations and for proteins containing both structured and disordered regions.
Assuntos
Proteínas Intrinsicamente Desordenadas , Benchmarking , Peptídeos , Conformação ProteicaRESUMO
Protein phosphorylation is a key post-translational modification in many biological processes and is associated to human diseases such as cancer and metabolic disorders. The accurate identification, annotation, and functional analysis of phosphosites are therefore crucial to understand their various roles. Phosphosites are mainly analyzed through phosphoproteomics, which has led to increasing amounts of publicly available phosphoproteomics data. Several resources have been built around the resulting phosphosite information, but these are usually restricted to the protein sequence and basic site metadata. What is often missing from these resources, however, is context, including protein structure mapping, experimental provenance information, and biophysical predictions. We therefore developed Scop3P: a comprehensive database of human phosphosites within their full context. Scop3P integrates sequences (UniProtKB/Swiss-Prot), structures (PDB), and uniformly reprocessed phosphoproteomics data (PRIDE) to annotate all known human phosphosites. Furthermore, these sites are put into biophysical context by annotating each phosphoprotein with per-residue structural propensity, solvent accessibility, disordered probability, and early folding information. Scop3P, available at https://iomics.ugent.be/scop3p, presents a unique resource for visualization and analysis of phosphosites and for understanding of phosphosite structure-function relationships.
Assuntos
Fosfoproteínas , Processamento de Proteína Pós-Traducional , Sequência de Aminoácidos , Bases de Dados de Proteínas , Humanos , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , FosforilaçãoRESUMO
MOTIVATION: Eukaryotic cells contain different membrane-delimited compartments, which are crucial for the biochemical reactions necessary to sustain cell life. Recent studies showed that cells can also trigger the formation of membraneless organelles composed by phase-separated proteins to respond to various stimuli. These condensates provide new ways to control the reactions and phase-separation proteins (PSPs) are thus revolutionizing how cellular organization is conceived. The small number of experimentally validated proteins, and the difficulty in discovering them, remain bottlenecks in PSPs research. RESULTS: Here we present PSPer, the first in-silico screening tool for prion-like RNA-binding PSPs. We show that it can prioritize PSPs among proteins containing similar RNA-binding domains, intrinsically disordered regions and prions. PSPer is thus suitable to screen proteomes, identifying the most likely PSPs for further experimental investigation. Moreover, its predictions are fully interpretable in the sense that it assigns specific functional regions to the predicted proteins, providing valuable information for experimental investigation of targeted mutations on these regions. Finally, we show that it can estimate the ability of artificially designed proteins to form condensates (r=-0.87), thus providing an in-silico screening tool for protein design experiments. AVAILABILITY AND IMPLEMENTATION: PSPer is available at bio2byte.com/psp. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.
Assuntos
Proteínas de Ligação a RNA/metabolismo , Organelas , Príons , ProteomaRESUMO
Soluble functional proteins may transform into insoluble amyloid fibrils that deposit in a variety of tissues. Amyloid formation is a hallmark of age-related degenerative disorders. Perhaps surprisingly, amyloid fibrils can also be beneficial and are frequently exploited for diverse functional roles in organisms. Here we introduce AmyPro, an open-access database providing a comprehensive, carefully curated collection of validated amyloid fibril-forming proteins from all kingdoms of life classified into broad functional categories (http://amypro.net). In particular, AmyPro provides the boundaries of experimentally validated amyloidogenic sequence regions, short descriptions of the functional relevance of the proteins and their amyloid state, a list of the experimental techniques applied to study the amyloid state, important structural/functional/variation/mutation data transferred from UniProt, a list of relevant PDB structures categorized according to protein states, database cross-references and literature references. AmyPro greatly improves on similar currently available resources by incorporating both prions and functional amyloids in addition to pathogenic amyloids, and allows users to screen their sequences against the entire collection of validated amyloidogenic sequence fragments. By enabling further elucidation of the sequential determinants of amyloid fibril formation, we hope AmyPro will enhance the development of new methods for the precise prediction of amyloidogenic regions within proteins.
Assuntos
Proteínas Amiloidogênicas/química , Bases de Dados de Proteínas , Interface Usuário-ComputadorRESUMO
The MobiDB (URL: mobidb.bio.unipd.it) database of protein disorder and mobility annotations has been significantly updated and upgraded since its last major renewal in 2014. Several curated datasets for intrinsic disorder and folding upon binding have been integrated from specialized databases. The indirect evidence has also been expanded to better capture information available in the PDB, such as high temperature residues in X-ray structures and overall conformational diversity. Novel nuclear magnetic resonance chemical shift data provides an additional experimental information layer on conformational dynamics. Predictions have been expanded to provide new types of annotation on backbone rigidity, secondary structure preference and disordered binding regions. MobiDB 3.0 contains information for the complete UniProt protein set and synchronization has been improved by covering all UniParc sequences. An advanced search function allows the creation of a wide array of custom-made datasets for download and further analysis. A large amount of information and cross-links to more specialized databases are intended to make MobiDB the central resource for the scientific community working on protein intrinsic disorder and mobility.