RESUMO
Several snake species possess endogenous phospholipase A2 inhibitors (sbPLIs) in their blood plasma, the primary role of which is protection against an eventual presence of toxic phospholipase A2 (PLA2) from their venom glands in the circulation. These inhibitors have an oligomeric structure of, at least, three subunits and have been categorized into three classes (α, ß and γ) based on their structural features. SbγPLIs have been further subdivided into two subclasses according to their hetero or homomeric nature, respectively. Despite the considerable number of sbγPLIs described, their structures and mechanisms of action are still not fully understood. In the present study, we focused on the native structure of CNF, a homomeric sbγPLI from Crotalus durissus terrificus, the South American rattlesnake. Based on the results of different biochemical and biophysical experiments, we concluded that, while the native inhibitor occurs as a mixture of oligomers, tetrameric arrangement appears to be the predominant quaternary structure. The inhibitory activity of CNF is most likely associated with this oligomeric conformation. In addition, we suggest that the CNF tetramer has a spherical shape and that tyrosinyl residues could play an important role in the oligomerization. The carbohydrate moiety, which is present in most sbγPLIs, is not essential for the inhibitory activity, oligomerization or complex formation of the CNF with the target PLA2. A minor component, comprising no more than 16% of the sample, was identified in the CNF preparations. The amino-terminal sequence of that component is similar to the B subunits of the heteromeric sbγPLIs; however, the role played by such molecule in the functionality of the CNF, if any, remains to be determined.
Assuntos
Crotoxina/química , Glicoproteínas/química , Inibidores de Fosfolipase A2/química , Fosfolipases A2/química , Proteínas de Répteis/química , Sequência de Aminoácidos , Animais , Cromatografia em Gel , Crotalus/fisiologia , Crotoxina/antagonistas & inibidores , Crotoxina/isolamento & purificação , Glicoproteínas/isolamento & purificação , Dados de Sequência Molecular , Inibidores de Fosfolipase A2/isolamento & purificação , Fosfolipases A2/isolamento & purificação , Multimerização Proteica , Estrutura Quaternária de Proteína , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Proteínas de Répteis/isolamento & purificação , Espalhamento a Baixo Ângulo , Homologia de Sequência de Aminoácidos , América do Sul , Tirosina/química , Difração de Raios XRESUMO
Understanding the adsorption mechanisms in nanostructured polymer films has become crucial for their use in technological applications, since film properties vary considerably with the experimental conditions utilized for film fabrication. In this paper, we employ small-angle X-ray scattering (SAXS) to investigate solutions of polyanilines and correlate the chain conformations with morphological features of the nanostructured films obtained with atomic force microscopy (AFM). It is shown that aggregates formed already in solution affect the film morphology; in particular, at early stages of adsorption film morphology appears entirely governed by the chain conformation in solution and adsorption of aggregates. We also use SAXS data for modeling poly(o-ethoxyaniline) (POEA) particle shape through an ab initio procedure based on simulated annealing using the dummy atom model (DAM), which is then compared to the morphological features of POEA films fabricated with distinct pHs and doping acids. Interestingly, when the derivative POEA is doped with p-toluene sulfonic acid (TSA), the resulting films exhibit a fibrillar morphology-seen with atomic force microscopy and transmission electron microscopy-that is consistent with the cylindrical shape inferred from the SAXS data. This is in contrast with the globular morphology observed for POEA films doped with other acids.
Assuntos
Compostos de Anilina/química , Microscopia de Força Atômica/métodos , Nanoestruturas/química , Espalhamento a Baixo Ângulo , Adsorção , Modelos Moleculares , Estrutura Molecular , Nanotecnologia/métodos , Tamanho da Partícula , Teoria Quântica , Soluções/química , Propriedades de Superfície , Difração de Raios XRESUMO
BACKGROUND: Ferredoxin-NADP(H) reductases (FNRs) are flavoenzymes that catalyze the electron transfer between NADP(H) and the proteins ferredoxin or flavodoxin. A number of structural features distinguish plant and bacterial FNRs, one of which is the mode of the cofactor FAD binding. Leptospira interrogans is a spirochaete parasitic bacterium capable of infecting humans and mammals in general. Leptospira interrogans FNR (LepFNR) displays low sequence identity with plant (34% with Zea mays) and bacterial (31% with Escherichia coli) FNRs. However, LepFNR contains all consensus sequences that define the plastidic class FNRs. RESULTS: The crystal structures of the FAD-containing LepFNR and the complex of the enzyme with NADP+, were solved and compared to known FNRs. The comparison reveals significant structural similarities of the enzyme with the plastidic type FNRs and differences with the bacterial enzymes. Our small angle X-ray scattering experiments show that LepFNR is a monomeric enzyme. Moreover, our biochemical data demonstrate that the LepFNR has an enzymatic activity similar to those reported for the plastidic enzymes and that is significantly different from bacterial flavoenzymes, which display lower turnover rates. CONCLUSION: LepFNR is the first plastidic type FNR found in bacteria and, despite of its low sequence similarity with plastidic FNRs still displays high catalytic turnover rates. The typical structural and biochemical characteristics of plant FNRs unveiled for LepFNR support a notion of a putative lateral gene transfer which presumably offers Leptospira interrogans evolutionary advantages. The wealth of structural information about LepFNR provides a molecular basis for advanced drugs developments against leptospirosis.
Assuntos
Ferredoxina-NADP Redutase/química , Flavina-Adenina Dinucleotídeo/química , Flavina-Adenina Dinucleotídeo/metabolismo , Leptospira interrogans/enzimologia , NADP/metabolismo , Sequência de Aminoácidos , Ácido Aspártico/química , Bactérias/enzimologia , Sítios de Ligação , Cristalografia por Raios X , Escherichia coli/genética , Ferredoxina-NADP Redutase/isolamento & purificação , Ferredoxina-NADP Redutase/metabolismo , Ligação de Hidrogênio , Concentração de Íons de Hidrogênio , Cinética , Modelos Moleculares , Dados de Sequência Molecular , Plantas/enzimologia , Ligação Proteica , Conformação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Espalhamento a Baixo Ângulo , Homologia de Sequência de Aminoácidos , Temperatura , Difração de Raios XRESUMO
The orphan receptor nerve growth factor-induced B (NGFI-B) is a member of the nuclear receptor's subfamily 4A (Nr4a). NGFI-B was shown to be capable of binding both as a monomer to an extended half-site containing a single AAAGGTCA motif and also as a homodimer to a widely separated everted repeat, as opposed to a large number of nuclear receptors that recognize and bind specific DNA sequences predominantly as homo- and/or heterodimers. To unveil the structural organization of NGFI-B in solution, we determined the quaternary structure of the NGFI-B LBD by a combination of ab initio procedures from small-angle X-ray scattering (SAXS) data and hydrogen-deuterium exchange followed by mass spectrometry. Here we report that the protein forms dimers in solution with a radius of gyration of 2.9 nm and maximum dimension of 9.0 nm. We also show that the NGFI-B LBD dimer is V-shaped, with the opening angle significantly larger than that of classical dimer's exemplified by estrogen receptor (ER) or retinoid X receptor (RXR). Surprisingly, NGFI-B dimers formation does not occur via the classical nuclear receptor dimerization interface exemplified by ER and RXR, but instead, involves an extended surface area composed of the loop between helices 3 and 4 and C-terminal fraction of the helix 3. Remarkably, the NGFI-B dimer interface is similar to the dimerization interface earlier revealed for glucocorticoid nuclear receptor (GR), which might be relevant to the recognition of cognate DNA response elements by NGFI-B and to antagonism of NGFI-B-dependent transcription exercised by GR in cells.
Assuntos
Proteínas de Ligação a DNA/química , Receptores Citoplasmáticos e Nucleares/química , Receptores de Esteroides/química , Fatores de Transcrição/química , Dicroísmo Circular , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/isolamento & purificação , Dimerização , Espectrometria de Massas , Modelos Biológicos , Modelos Moleculares , Membro 1 do Grupo A da Subfamília 4 de Receptores Nucleares , Estrutura Secundária de Proteína , Receptores Citoplasmáticos e Nucleares/genética , Receptores Citoplasmáticos e Nucleares/isolamento & purificação , Receptores de Glucocorticoides/química , Receptores de Esteroides/genética , Receptores de Esteroides/isolamento & purificação , Espalhamento a Baixo Ângulo , Soluções , Fatores de Transcrição/genética , Fatores de Transcrição/isolamento & purificaçãoRESUMO
The septins are a conserved family of guanosine-5'-triphosphate (GTP)-binding proteins. In mammals they are involved in a variety of cellular processes, such as cytokinesis, exocytosis, and vesicle trafficking. Specifically, SEPT4 has also been shown to be expressed in both human colorectal cancer and malignant melanoma, as well as being involved in neurodegenerative disorders. However, many of the details of the modes of action of septins in general remain unclear, and little is known of their detailed molecular architecture. Here, we define explicitly and characterize the domains of human SEPT4. Regions corresponding to the N-terminal, GTPase, and C-terminal domains as well as the latter two together were successfully expressed in Escherichia coli in soluble form and purified by affinity and size-exclusion chromatographies. The purified domains were analyzed by circular dichroism spectroscopy, fluorescence spectroscopy, dynamic light scattering, and small-angle X-ray scattering, as well as with bioinformatics tools. Of the three major domains that comprise SEPT4, the N-terminal domain contains little regular secondary structure and may be intrinsically unstructured. The central GTPase domain is a mixed alpha/beta structure, probably based on an open beta sheet. As defined here, it is catalytically active and forms stable homodimers in vitro. The C-terminal domain also forms homodimers and can be divided into two regions, the second of which is alpha-helical and consistent with a coiled-coil structure. These studies should provide a useful basis for future biophysical studies of SEPT4, including the structural basis for their involvement in diseases such as cancer and neurodegenerative disorders.