RESUMO
Cutinases are serine esterases that belong to the α/ß hydrolases superfamily. The natural substrates for these enzymes are cutin and suberin, components of the plant cuticle, the first barrier in the defense system against pathogen invasion. It is well-reported that plant pathogens produce cutinases to facilitate infection. Fusarium verticillioides, one important corn pathogens, is an ascomycete upon which its cutinases are poorly explored. Consequently, the objective of this study was to perform the biochemical characterization of three precursor cutinases (FvCut1, FvCut2, and FvCut3) from F. verticillioides and to obtain structural insights about them. The cutinases were produced in Escherichia coli and purified. FvCut1, FvCut2, and FvCut3 presented optimal temperatures of 20, 40, and 35 °C, and optimal pH of 9, 7, and 8, respectively. Some chemicals stimulated the enzymatic activity. The kinetic parameters revealed that FvCut1 has higher catalytic efficiency (Kcat/Km) in the p-nitrophenyl-butyrate (p-NPB) substrate. Nevertheless, the enzymes were not able to hydrolyze polyethylene terephthalate (PET). Furthermore, the three-dimensional models of these enzymes showed structural differences among them, mainly FvCut1, which presented a narrower opening cleft to access the catalytic site. Therefore, our study contributes to exploring the diversity of fungal cutinases and their potential biotechnological applications.
Assuntos
Ascomicetos , Fusarium , Hidrolases de Éster Carboxílico/genética , Hidrolases de Éster Carboxílico/química , Fusarium/genéticaRESUMO
Resumen El modelamiento ¡n silíco ha sido de gran contribución en los procesos proteómicos, desarrollando estructuras de las secuencias proteicas ya existentes, que por motivos de altos costos y las diferentes tecnologías necesarias para el desarrollo de estas metodologías, se encuentran deficientes en el número de modelamientos de proteínas disponibles. Entre aquellas secuencias con carencia de estructura proteica se encuentra la proteína liasa organomercurial (MerB) de Pseudomonas /luorescens, importante en la resistencia al mercurio. En el presente artículo se analizó tanto estructural como funcionalmente la proteína MerB en Pseudomonas jluorescens, utilizando la herramienta de la química estructural "modelamiento por homología" mediante plataformas bioinformáticas, con el fin de obtener un modelo que represente la estructura 3D más precisa y que capturen las mejores variantes estructurales entre todas las posibles conformaciones de las proteínas en la familia. En este trabajo, se desarrolló un método comparativo de la secuencia estudiada con las reportadas en las bases de datos para las proteínas MerB del género Pseudomonas. Se propone un modelo tridimensional para la enzima (MerB) en P. jluorescens, mediante el modelamiento por homología, se muestra la caracterización en la estructura secundaria, terciaria, la caracterización del dominio catalítico y los motivos estructurales presentes.
Abstract In silico modeling has made a great contribution to proteomic processes, developing structures of the already existing protein sequences, which for reasons of high costs and the different technologies necessary for the development of these methodologies, are deficient in the number of models of available proteins. Among those sequences lacking protein structure is the organomercurial lyase (MerB) protein from Pseudomonas fluoresceins, important in mercury resistance. In this article, the MerB protein in Pseudomonas fluorescens was analyzed both structurally and functionally, using the structural chemistry tool "homology modeling" using bioinformatic platforms, in order to obtain a model that represents the most accurate 3D structure and that captures the best structural variants among all the possible conformations of the proteins in the family. In this work, a comparative method of the sequence studied with those reported in the databases for MerB proteins of the genus Pseudomonas was developed. A three-dimensional model for the enzyme (MerB) in P. fluorescens is proposed, through homology modeling, the characterization at the secondary and tertiary structure level, the characterization of the catalytic domain and the structural motifs present is shown.
Resumo A modelagem in silico tem dado um grande contributo para os processos proteómicos, desenvolvendo estruturas de sequências de proteínas já existentes, as quais, pelos elevados custos e pelas diferentes tecnologias necessárias ao desenvolvimento destas metodologias, são deficientes no número de modelos de proteínas disponíveis. Entre as sequências sem estrutura protéica está a proteína organomercurial liase (MerB) de Pseudomonas fluorescens, importante na resistência ao mercúrio. Neste artigo, a proteína MerB em Pseudomonas fluorescens foi analisada estrutural e funcionalmente, usando a ferramenta de química estrutural "modelagem de homologia" usando plataformas de bioinformática, a fim de obter um modelo que represente a estrutura 3D mais precisa e que capture as melhores variantes estruturais. entre todas as conformações possíveis das proteínas da família. Neste trabalho, foi desenvolvido um método comparativo da sequência estudada com aqueles relatados em bancos de dados para proteínas MerB do gênero Pseudomonas. Um modelo tridimensional para a enzima (MerB) em P. fluorescens é proposto, através de modelagem por homologia, a caracterização em nível de estrutura secundária e terciária, a caracterização do domínio catalítico e os motivos estruturais presentes são mostradas.
RESUMO
Trypsin inhibitors from tamarind seed have been studied in vitro and in preclinical studies for the treatment of obesity, its complications and associated comorbidities. It is still necessary to fully understand the structure and behaviour of these molecules. We purifed this inhibitor, sequenced de novo by MALDI-TOF/TOF, performed its homology modelling, and assessed the interaction with the trypsin enzyme through molecular dynamics (MD) simulation under physiological conditions. We identified additional 75 amino acid residues, reaching approximately 72% of total coverage. The four best conformations of the best homology modelling were submitted to the MD. The conformation n°287 was selected considering the RMSD analysis and interaction energy (-301.0128 kcal.mol-1). Residues Ile (54), Pro (57), Arg (59), Arg (63), and Glu (78) of pTTI presented the highest interactions with trypsin, and arginine residues were mainly involved in its binding mechanism. The results favour bioprospecting of this protein for pharmaceutical health applications.
Assuntos
Simulação de Dinâmica Molecular , Extratos Vegetais/farmacologia , Tamarindus/química , Inibidores da Tripsina/farmacologia , Tripsina/metabolismo , Relação Dose-Resposta a Droga , Estrutura Molecular , Extratos Vegetais/química , Extratos Vegetais/isolamento & purificação , Sementes/química , Relação Estrutura-Atividade , Inibidores da Tripsina/química , Inibidores da Tripsina/isolamento & purificaçãoRESUMO
HLA epitope analysis emerged as a strategy to determine alloimmune risk in solid organ transplantation. However, it requires not only knowledge on HLA amino acids sequences, but also on HLA three-dimensional structures. Unfortunately, the number of structures available is still unsatisfactory. This work reports the modelling of 106 heterotrimeric (alpha chainâ¯+â¯ß2Mâ¯+â¯peptide) HLA class I molecules. The models were generated by homology modelling using Modeller, refined using GalaxyRefine server, heterodimerized with Swiss-PDB Viewer and, finally, assessed as to their structural quality through Dali server. The final structures were made available through a free online database, pHLA3D (www.phla3d.com.br), developed in Ruby language using the Ruby on Rails web framework. Structural parameters were similar between refined molecules and their templates. The new database may improve HLA epitope analysis and better guide risk assessment in solid organ transplantation setting.
Assuntos
Sistemas de Gerenciamento de Base de Dados , Bases de Dados de Proteínas , Antígenos de Histocompatibilidade Classe I/química , Estrutura Secundária de Proteína , Homologia Estrutural de Proteína , Alelos , Sequência de Aminoácidos , Epitopos/imunologia , Histocompatibilidade , Humanos , Modelos Moleculares , NavegadorRESUMO
The WFDC1 gene is frequently down-regulated or lost in prostate cancer, and the encoded protein, ps20, has been implicated in epithelial cell behaviour and angiogenesis. However, ps20 remains largely uncharacterised with respect to its structure and interacting partners. This study characterised the evolution, functionality and structural characteristics of WFDC1/ps20 using phylogenetic reconstruction and other computational approaches. Bayesian phylogenetic analyses suggested that ps20 appeared in a common ancestor of deuterostomes-protostomes. The rate of evolutionary change within the coding regions of vertebrate WFDC1 genes and the synteny conservation in mammals differed from that of other vertebrate clades, indicating a possible functional diversity of ps20 homologues. A gene set enrichment analysis of the genes around WFDC1 (conserved synteny) showed functional relationships between the WFDC1, CDH13, CRISPLD2, IRF8 and TFPI2 genes. The molecular evolution of ps20 has been driven by purifying selection, particularly in the segments corresponding to exons 3 and 4, which encode the most conserved regions of the protein. A co-evolution analysis showed that residues within these regions co-vary with each other during the evolution of ps20. These results show that the regions corresponding to exons 3 and 4 are ps20-specific structure-function modules. Homology modelling of the exon 2-encoded polypeptide and subsequent dynamics calculus using a Gaussian network model showed that residues with high conformational flexibility are part of a loop region involved in protein-protein recognition, given the similarity with other serine protease inhibitors. Residues C96, R94, L105, and C66 are critical for the integrity and functionality of this ps20 region.
Assuntos
Evolução Molecular , Modelos Moleculares , Filogenia , Proteínas , Humanos , Domínios Proteicos , Proteínas/química , Proteínas/genética , Homologia Estrutural de ProteínaRESUMO
γ-aminobutyric acid-type A (GABAA) receptors mediate fast synaptic inhibition in the central nervous system of mammals. They are modulated via several sites by numerous compounds, which include GABA, benzodiazepines, ethanol, neurosteroids and anaesthetics among others. Due to their potential as targets of novel drugs, a detailed knowledge of their structure-function relationships is needed. Here, we present the model of the α1ß2γ2 subtype GABAA receptor in the APO state and in complex with selected ligands, including agonists, antagonists and allosteric modulators. The model is based on the crystallographic structure of the human ß3 homopentamer GABAA receptor. The complexes were refined using atomistic molecular dynamics simulations. This allowed a broad description of the binding modes and the detection of important interactions in agreement with experimental information. From the best of our knowledge, this is the only model of the α1ß2γ2 GABAA receptor that represents altogether the desensitized state of the channel and comprehensively describes the interactions of ligands of the orthosteric and benzodiazepines binding sites in agreement with the available experimental data. Furthermore, it is able to explain small differences regarding the binding of a variety of chemically divergent ligands. Finally, this new model may pave the way for the design of focused experimental studies that will allow a deeper description of the receptor.
Assuntos
Benzodiazepinas/química , Antagonistas de Receptores de GABA-A/química , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Receptores de GABA-A/química , Sequência de Aminoácidos , Benzodiazepinas/farmacologia , Sítios de Ligação , Descoberta de Drogas/métodos , Antagonistas de Receptores de GABA-A/farmacologia , Ligação de Hidrogênio , Ligantes , Reprodutibilidade dos Testes , Relação Estrutura-AtividadeRESUMO
The human macrophage migration inhibitory factor 1 (Hu-MIF-1) is a protein involved in the inflammatory and immunology response to parasite infection. In the present study, the existence of Hu-MIF-1 from parasites have been explored by mining WormBase. A total of 35 helminths were found to have Hu-MIF-1 homologs, including some parasites of importance for public health. Physicochemical, structural, and biological properties of Hu-MIF-1 were compared with its orthologs in parasites showing that most of these are secretory proteins, with positive net charge and presence of the Cys-Xaa-Xaa-Cys motif that is critical for its oxidoreductase activity. The inhibitor-binding site present in Hu-MIF-1 is well conserved among parasite MIFs suggesting that Hu-MIF inhibitors may target orthologs in pathogens. The binding of Hu-MIF-1 to its cognate receptor CD74 was predicted by computer-assisted docking, and it resulted to be very similar to the predicted complexes formed by parasite MIFs and human CD74. More than 1 plausible conformation of MIFs in the extracellular loops of CD74 may be possible as demonstrated by the different predicted conformations of MIF orthologs in complex with CD74. Parasite MIFs in complex with CD74 resulted with some charged residues oriented to CD74, which was not observed in the Hu-MIF-1/CD74 complex. Our findings predict the binding mode of Hu-MIF-1 and orthologs with CD74, which can assist in the design of novel MIF inhibitors. Whether the parasite MIFs function specifically subvert host immune responses to suit the parasite is an open question that needs to be further investigated. Future research should lead to a better understanding of parasite MIF action in the parasite biology.
Assuntos
Antígenos de Diferenciação de Linfócitos B/química , Antígenos de Histocompatibilidade Classe II/química , Fatores Inibidores da Migração de Macrófagos/química , Parasitos/metabolismo , Homologia de Sequência de Aminoácidos , Animais , Sequência Conservada , Humanos , Modelos Moleculares , Filogenia , Estrutura Terciária de Proteína , Subunidades Proteicas/metabolismo , Alinhamento de Sequência , Eletricidade Estática , Homologia Estrutural de ProteínaRESUMO
ABSTRACT The apicomplexan parasite Theileria parva, the causative agent of ECF, is an important pathogen affecting both domestic and wild animals, causing major economic losses in the world. Problems such as high cost of drugs, development of resistance, and absence of effective vaccines prevent effective combating of the pathogen. Thus, it is necessary to explore new targets for affordable and higher therapeutic value drugs. 1-Deoxy-D-xylulose-5-phosphate reductoisomerase (DXR) in the non-mevalonate isoprene biosynthesis pathway is vital to the organism and therefore has been selected as a target for developing antitheilerial drugs. In this study, the 3D structure of TpDXR was identified by template-based in silico homology modelling method, the constructed model was validated and structurally analysed, and possible ligand binding pockets were identified for the first time in the literature. A reliable 3D model for TpLDH was modelled by using 3AU9 chain 'A' Plasmodium falciparum as a template. The obtained result showed that the model has a good resolution structure with 86.768 overall quality factor and a -9.15 z-score for TpDXR. The present study promises the possibility of exploiting new and safe inhibitors using the structure-based drug design that is effective against ECF through docking studies.
RESUMO
Tuberculosis (TB) is the second leading cause of human mortality from infectious diseases worldwide. The WHO reported 1.3 million deaths and 8.6 million new cases of TB in 2012. Mycobacterium tuberculosis (M. tuberculosis), the infectious bacteria that causes TB, is encapsulated by a thick and robust cell wall. The innermost segment of the cell wall is comprised of peptidoglycan, a layer that is required for survival and growth of the pathogen. Enzymes that catalyse biosynthesis of the peptidoglycan are essential and are therefore attractive targets for discovery of novel antibiotics as humans lack similar enzymes making it possible to selectively target bacteria only. In this paper, we have reviewed the structures and functions of enzymes GlmS, GlmM, GlmU, MurA, MurB, MurC, MurD, MurE and MurF from M. tuberculosis that are involved in peptidoglycan biosynthesis. In addition, we report homology modelled 3D structures of those key enzymes from M. tuberculosis of which the structures are still unknown. We demonstrated that natural substrates can be successfully docked into the active sites of the GlmS and GlmU respectively. It is therefore expected that the models and the data provided herein will facilitate translational research to develop new drugs to treat TB.
Assuntos
Antituberculosos/farmacologia , Desenho de Fármacos , Mycobacterium tuberculosis/efeitos dos fármacos , Peptidoglicano/biossíntese , Proteínas de Bactérias/fisiologia , Humanos , Modelos Moleculares , Terapia de Alvo Molecular/métodos , Mycobacterium tuberculosis/enzimologia , Mycobacterium tuberculosis/metabolismo , Relação Quantitativa Estrutura-AtividadeRESUMO
Calpain-10 (CAPN10) is a cysteine protease that is activated by intracellular calcium (Ca(2+)) and known to be involved in diseases such as cancer, heart attack, and stroke. A role for the CAPN10 gene in diabetes mellitus type II was recently identified. Hyper activation of the enzyme initiates a series of destructive cycles that can cause irreversible damage to cells. The development of inhibitors may be useful as therapeutic agents for a number of calpainopathies. In this paper, we have used the homology modelling technique to determine the 3D structure of calpain-10 from Homo sapiens. The model of calpain-10 obtained by homology modelling suggests that its active site is conserved among family members and the main interactions are similar to those observed for µ-calpain. Structural analysis revealed that there are small differences in the charge distribution and molecular surface of the enzyme. These differences are probably less dependent on calcium for calpain-10 than they are for µ-calpain. In addition, the ion pair Cys(-)/His(+) formation was observed using of Molecular Dynamics (MD) simulations that were based upon hybrid quantum mechanical/molecular mechanical (QM/MM) approaches. Finally, the binding of the SNJ-1715 inhibitor to calpain-10 was investigated in order to further understand the mechanism of inhibition of calpain-10 by this inhibitor at the molecular level.