RESUMEN
G protein-coupled receptors (GPCRs) form the largest superfamily of membrane proteins in the human genome, and represent one of the most important classes of drug targets. Their structural studies facilitate rational drug discovery. However, atomic structures of only about 20% of human GPCRs have been solved to date. Recombinant production of GPCRs for structural studies at a large scale is challenging due to their low expression levels and stability. Therefore, in this study, we explored the efficacy of the eukaryotic system LEXSY (Leishmania tarentolae) for GPCR production. We selected the human A2A adenosine receptor (A2AAR), as a model protein, expressed it in LEXSY, purified it, and compared with the same receptor produced in insect cells, which is the most popular expression system for structural studies of GPCRs. The A2AAR purified from both expression systems showed similar purity, stability, ligand-induced conformational changes and structural dynamics, with a remarkably higher protein yield in the case of LEXSY expression. Overall, our results suggest that LEXSY is a promising platform for large-scale production of GPCRs for structural studies.
Asunto(s)
Receptor de Adenosina A2A , Receptores Acoplados a Proteínas G , Proteínas Recombinantes , Humanos , Descubrimiento de Drogas , Receptores Acoplados a Proteínas G/biosíntesis , Receptores Acoplados a Proteínas G/química , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Leishmania , Receptor de Adenosina A2A/biosíntesis , Receptor de Adenosina A2A/química , Conformación Proteica , Ligandos , Estabilidad ProteicaRESUMEN
Destabilase from the medical leech Hirudo medicinalis belongs to the family of i-type lysozymes. It has two different enzymatic activities: microbial cell walls destruction (muramidase activity), and dissolution of the stabilized fibrin (isopeptidase activity). Both activities are known to be inhibited by sodium chloride at near physiological concentrations, but the structural basis remains unknown. Here we present two crystal structures of destabilase, including a 1.1 Å-resolution structure in complex with sodium ion. Our structures reveal the location of sodium ion between Glu34/Asp46 residues, which were previously recognized as a glycosidase active site. While sodium coordination with these amino acids may explain inhibition of the muramidase activity, its influence on previously suggested Ser49/Lys58 isopeptidase activity dyad is unclear. We revise the Ser49/Lys58 hypothesis and compare sequences of i-type lysozymes with confirmed destabilase activity. We suggest that the general base for the isopeptidase activity is His112 rather than Lys58. pKa calculations of these amino acids, assessed through the 1 µs molecular dynamics simulation, confirm the hypothesis. Our findings highlight the ambiguity of destabilase catalytic residues identification and build foundations for further research of structure-activity relationship of isopeptidase activity as well as structure-based protein design for potential anticoagulant drug development.
Asunto(s)
Hirudo medicinalis , Sanguijuelas , Animales , Hirudo medicinalis/química , Muramidasa/química , Endopeptidasas/metabolismo , Sanguijuelas/metabolismo , Fibrinolíticos/uso terapéuticoRESUMEN
Alzheimer's disease (AD) is a severe neurodegenerative pathology with no effective treatment known. Toxic amyloid-ß peptide (Aß) oligomers play a crucial role in AD pathogenesis. All-d-Enantiomeric peptide D3 and its derivatives were developed to disassemble and destroy cytotoxic Aß aggregates. One of the D3-like compounds is approaching phase II clinical trials; however, high-resolution details of its disease-preventing or pharmacological actions are not completely clear. We demonstrate that peptide D3 stabilizing Aß monomer dynamically interacts with the extracellular juxtamembrane region of a membrane-bound fragment of an amyloid precursor protein containing the Aß sequence. MD simulations based on NMR measurement results suggest that D3 targets the amyloidogenic region, not compromising its α-helicity and preventing intermolecular hydrogen bonding, thus creating prerequisites for inhibition of early steps of Aß conversion into ß-conformation and its toxic oligomerization. An enhanced understanding of the D3 action molecular mechanism facilitates development of effective AD treatment and prevention strategies.
Asunto(s)
Enfermedad de Alzheimer/tratamiento farmacológico , Precursor de Proteína beta-Amiloide/metabolismo , Oligopéptidos/química , Oligopéptidos/uso terapéutico , Secuencia de Aminoácidos , Animales , Humanos , Ratones , Simulación de Dinámica Molecular , Oligopéptidos/metabolismo , Unión Proteica , EstereoisomerismoRESUMEN
Sea anemones (Actiniaria) are intensely popular objects of study in venomics. Order Actiniaria includes more than 1,000 species, thus presenting almost unlimited opportunities for the discovery of novel biologically active molecules. The venoms of cold-water sea anemones are studied far less than the venoms of tropical sea anemones. In this work, we analysed the molecular venom composition of the cold-water sea anemone Cnidopus japonicus. Two sets of NGS data from two species revealed molecules belonging to a variety of structural classes, including neurotoxins, toxin-like molecules, linear polypeptides (Cys-free), enzymes, and cytolytics. High-throughput proteomic analyses identified 27 compounds that were present in the venoms. Some of the toxin-like polypeptides exhibited novel Cys frameworks. To characterise their function in the venom, we heterologously expressed 3 polypeptides with unusual Cys frameworks (designated CjTL7, CjTL8, and AnmTx Cj 1c-1) in E. coli. Toxicity tests revealed that the CjTL8 polypeptide displays strong crustacean-specific toxicity, while AnmTx Cj 1c-1 is toxic to both crustaceans and insects. Thus, an improved NGS data analysis algorithm assisted in the identification of toxins with unusual Cys frameworks showing no homology according to BLAST. Our study shows the advantage of combining omics analysis with functional tests for active polypeptide discovery.