RESUMO
TDP-43 is an RNA-binding protein that presents four domains comprising an N-terminal region, two RNA recognition motifs and a C-terminal region. The N-terminal domain (NTD) has a relevant role in the oligomerization and splicing activity of TDP-43. In this work, we have expressed, purified and biophysically characterized the region that includes residues 1 to 102 that contains the nuclear localization signal (residues 80-102, NLS). Furthermore, we have evaluated the oligomerization equilibrium for this protein fragment. Also, we have determined changes in the tertiary structure and its stability in a broad range of pH values by means of different spectroscopic methods. Additionally, we compared this fragment with the one that lacks the NLS employing experimental and computational methods. Finally, we evaluated the motion of dimeric forms to get insights into the conformational flexibility of this TDP-43 module in an oligomeric state. Our results suggest that this domain has a conformational plasticity in the vicinity of the single tryptophan of this domain (Trp68), which is enhanced by the presence of the nuclear localization signal. All these results help to understand the molecular features of the NTD of TDP-43.
Assuntos
Sinais de Localização Nuclear , Triptofano , Conformação Proteica , Proteínas de Ligação a DNA/metabolismoRESUMO
Gluten related-disorders have a prevalence of 1-5 % worldwide triggered by the ingestion of gluten proteins in wheat, rye, barley, and some oats. In wheat gluten, the most studied protein is gliadin, whose immunodominant 33-mer amino acid fragment remains after digestive proteolysis and accumulates in the gut mucosa. Here, we report the formation of 33-mer thin-plate superstructures using intrinsic tyrosine (Tyr) steady-state fluorescence anisotropy and cryo-TEM in combination with water tension measurements. Furthermore, we showed that fluorescence decay measurements of 33-mer intrinsic fluorophore Tyr provided information on the early stages of the formation of the thin-plate structures. Finally, conformational analysis of Tyr residues using minimalist models by molecular dynamic simulations (MD) demonstrated that changes in Tyr rotamer states depend on the oligomerization stage. Our findings further advance the understanding of the formation of the 33-mer gliadin peptide superstructures and their relation to health and disease.
Assuntos
Gliadina , Glutens , Gliadina/química , Gliadina/metabolismo , Glutens/química , Triticum , Proteínas , Peptídeos/química , Fragmentos de Peptídeos/químicaRESUMO
The activation of GABAA receptors by the neurotransmitter gamma-aminobutyric acid mediates the rapid inhibition response in the central nervous system of mammals. Many neurological and mental health disorders arise from alterations in the structure or function of these pentameric ion channels. GABAA receptors are targets for numerous drugs, including benzodiazepines, which bind to α1ß2γ2 GABAA receptors with high affinity to a site in the extracellular domain, between subunits α1 and γ2. It has been established experimentally that the binding of these drugs depends on the presence of one particular amino acid in the α1 subunit: histidine 102. However, the specific role it plays in the intermolecular interaction has not been elucidated. In this work, we applied in silico methods to understand whether certain protonation and rotamer states of α1His102 facilitate the binding of modulators. We analysed diazepam binding, a benzodiazepine, and the antagonist flumazenil to the GABAA receptor using molecular dynamics simulations and adaptive biasing force simulations. The binding free energy follows changes in the protonation state for both ligands, and rotameric states of α1His102 were specific for the different compounds, suggesting distinct preferences for positive allosteric modulators and antagonists. Moreover, in the presence of diazepam and favoured by a neutral tautomer, we identified a water molecule that links loops A, B, and C and may be relevant to the modulation mechanism.
Assuntos
Diazepam/metabolismo , Flumazenil/metabolismo , Moduladores GABAérgicos/metabolismo , Antagonistas de Receptores de GABA-A/metabolismo , Receptores de GABA-A/metabolismo , Histidina/química , Humanos , Simulação de Dinâmica Molecular , Conformação Proteica , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , Prótons , Receptores de GABA-A/químicaRESUMO
In this work, we assess a previously advanced hypothesis that predicts the existence of ion channels in the capsid of small and non-enveloped icosahedral viruses. With this purpose we examine Triatoma Virus (TrV) as a case study. This virus has a stable capsid under highly acidic conditions but disassembles and releases the genome in alkaline environments. Our calculations range from a subtle sub-atomic proton interchange to the dismantling of a large-scale system representing several million of atoms. Our results provide structure-based explanations for the three roles played by the capsid to enable genome release. First, we observe, for the first time, the formation of a hydrophobic gate in the cavity along the five-fold axis of the wild-type virus capsid, which can be disrupted by an ion located in the pore. Second, the channel enables protons to permeate the capsid through a unidirectional Grotthuss-like mechanism, which is the most likely process through which the capsid senses pH. Finally, assuming that the proton leak promotes a charge imbalance in the interior of the capsid, we model an internal pressure that forces shell cracking using coarse-grained simulations. Although qualitatively, this last step could represent the mechanism of capsid opening that allows RNA release. All of our calculations are in agreement with current experimental data obtained using TrV and describe a cascade of events that could explain the destabilization and disassembly of similar icosahedral viruses.
Assuntos
Dicistroviridae/fisiologia , Dicistroviridae/ultraestrutura , Canais Iônicos/metabolismo , Animais , Capsídeo/fisiologia , Capsídeo/ultraestrutura , Biologia Computacional , Dicistroviridae/genética , Concentração de Íons de Hidrogênio , Interações Hidrofóbicas e Hidrofílicas , Modelos Biológicos , Modelos Moleculares , Simulação de Dinâmica Molecular , Prótons , Eletricidade Estática , Montagem de Vírus/fisiologiaRESUMO
The proteolytic resistant 33-mer gliadin peptide is an immunodominant fragment in gluten and responsible for the celiac disease and other gluten-related disorders. Meanwhile, the primary structure of the 33-mer is associated with the adaptive immune response in celiac patients, and the structural transformation of the 33-mer into protofilaments activates a primordial innate immune response in human macrophages. This means that accumulation, oligomerisation and structural transformation of the 33-mer could be the unknown first event that triggers the disease. Herein, we reveal the early stepwise mechanism of 33-mer oligomerisation by combining multiple computational simulations, tyrosine cross-linking, fluorescence spectroscopy and circular dichroism experiments. Our theoretical findings demonstrated that the partial charge distribution along the 33-mer molecule and the presence of glutamine that favours H-bonds between the oligomers are the driving forces that trigger oligomerisation. The high content of proline is critical for the formation of the flexible PPII secondary structure that led to a ß structure transition upon oligomerisation. Experimentally, we stabilised the 33-mer small oligomers by dityrosine cross-linking, detecting from dimers to higher molecular weight oligomers, which confirmed our simulations. The relevance of 33-mer oligomers as a trigger of the disease as well as its inhibition may be a novel therapeutic strategy for the treatment of gluten-related disorders.
RESUMO
The pentameric γ-aminobutyric acid type A receptors are ion channels activated by ligands, which intervene in the rapid inhibitory transmission in the mammalian CNS. Due to their rich pharmacology and therapeutic potential, it is essential to understand their structure and function thoroughly. This deep characterization was hampered by the lack of experimental structural information for many years. Thus, computational techniques have been extensively combined with experimental data, in order to undertake the study of γ-aminobutyric acid type A receptors and their interaction with drugs. Here, we review the exciting journey made to assess the structures of these receptors and outline major outcomes. Finally, we discuss the brand new structure of the α1ß2γ2 subtype and the amazing advances it brings to the field.
RESUMO
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
Based on the analysis of the mechanism of ligand transfer to membranes employing in vitro methods, Fatty Acid Binding Protein (FABP) family has been divided in two subgroups: collisional and diffusional FABPs. Although the collisional mechanism has been well characterized employing in vitro methods, the structural features responsible for the difference between collisional and diffusional mechanisms remain uncertain. In this work, we have identified the amino acids putatively responsible for the interaction with membranes of both, collisional and diffusional, subgroups of FABPs. Moreover, we show how specific changes in FABPs' structure could change the mechanism of interaction with membranes. We have computed protein-membrane interaction energies for members of each subgroup of the family, and performed Molecular Dynamics simulations that have shown different configurations for the initial interaction between FABPs and membranes. In order to generalize our hypothesis, we extended the electrostatic and bioinformatics analysis over FABPs of different mammalian genus. Also, our methodological approach could be used for other systems involving protein-membrane interactions.