RESUMO
Congenital fibrinogen disorders are caused by mutations in genes coding for fibrinogen and may lead to various clinical phenotypes. Here, we present a functional and structural analysis of 4 novel variants located in the FGB gene coding for fibrinogen Bß chain-heterozygous missense BßY416C and BßA68S, homozygous nonsense BßY345*, and heterozygous nonsense BßW403* mutations. The cases were identified by coagulation screening tests and further investigated by various methods. Fibrin polymerization had abnormal development with decreased maximal absorbance in all patients. Plasmin-induced fibrin degradation revealed different lytic phases of BßY416C and BßW403* than those of the control. Fibrinopeptide cleavage measured by reverse phase high pressure liquid chromatography of BßA68S showed impaired release of fibrinopeptide B. Morphological properties, studied through scanning electron microscopy, differed significantly in the fiber thickness of BßY416C, BßA68S, and BßW403*, and in the fiber density of BßY416C and BßW403*. Finally, homology modeling of BßA68S showed that mutation caused negligible alternations in the protein structure. In conclusion, all mutations altered the correct fibrinogen function or structure that led to congenital fibrinogen disorders.
Assuntos
Afibrinogenemia/diagnóstico , Afibrinogenemia/genética , Fibrinogênio/química , Fibrinogênio/genética , Predisposição Genética para Doença , Mutação , Fenótipo , Adolescente , Afibrinogenemia/sangue , Idoso , Coagulação Sanguínea , Testes de Coagulação Sanguínea , Análise Mutacional de DNA , Feminino , Fibrinogênio/metabolismo , Estudos de Associação Genética , Humanos , Recém-Nascido , Masculino , Pessoa de Meia-Idade , Modelos Moleculares , Conformação Proteica , Relação Estrutura-AtividadeRESUMO
Fibrinogen, an abundant plasma glycoprotein, is involved in the final stage of blood coagulation. Decreased fibrinogen levels, which may be caused by mutations, are manifested mainly in bleeding and thrombotic disorders. Clinically relevant mutations of fibrinogen are listed in the Human Fibrinogen Database. For the αC-connector (amino acids Aα240-410, nascent chain numbering), we have extended this database, with detailed descriptions of the clinical manifestations among members of reported families. This includes the specification of bleeding and thrombotic events and results of coagulation assays. Where available, the impact of a mutation on clotting and fibrinolysis is reported. The collected data show that the Human Fibrinogen Database reports considerably fewer missense and synonymous mutations than the general COSMIC and dbSNP databases. Homozygous nonsense or frameshift mutations in the αC-connector are responsible for most clinically relevant symptoms, while heterozygous mutations are often asymptomatic. Symptomatic subjects suffer from bleeding and, less frequently, from thrombotic events. Miscarriages within the first trimester and prolonged wound healing were reported in a few subjects. All mutations inducing thrombotic phenotypes are located at the identical positions within the consensus sequence of the tandem repeats.
Assuntos
Fibrinogênio/genética , Coagulação Sanguínea/genética , Testes de Coagulação Sanguínea/métodos , Hemorragia/genética , Humanos , Mutação/genética , Trombose/genéticaRESUMO
The fusion oncoprotein BCR-ABL1 exhibits aberrant tyrosine kinase activity and it has been proposed that it deregulates signaling networks involving both transcription factors and non-coding microRNAs that result in chronic myeloid leukemia (CML). Previously, microRNA expression profiling showed deregulated expression of miR-150 and miR-155 in CML. In this study, we placed these findings into the broader context of the MYC/miR-150/MYB/miR-155/PU.1 oncogenic network. We propose that up-regulated MYC and miR-155 in CD34+ leukemic stem and progenitor cells, in concert with BCR-ABL1, impair the molecular mechanisms of myeloid differentiation associated with low miR-150 and PU.1 levels. We revealed that MYC directly occupied the -11.7 kb and -0.35 kb regulatory regions in the MIR150 gene. MYC occupancy was markedly increased through BCR-ABL1 activity, causing inhibition of MIR150 gene expression in CML CD34+ and CD34- cells. Furthermore, we found an association between reduced miR-150 levels in CML blast cells and their resistance to tyrosine kinase inhibitors (TKIs). Although TKIs successfully disrupted BCR-ABL1 kinase activity in proliferating CML cells, this treatment did not efficiently target quiescent leukemic stem cells. The study presents new evidence regarding the MYC/miR-150/MYB/miR-155/PU.1 leukemic network established by aberrant BCR-ABL1 activity. The key connecting nodes of this network may serve as potential druggable targets to overcome resistance of CML stem and progenitor cells.
Assuntos
Resistencia a Medicamentos Antineoplásicos/genética , Proteínas de Fusão bcr-abl/genética , Genes myc/genética , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , MicroRNAs/genética , Adulto , Idoso , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/genética , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/genética , Regulação para Baixo/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Feminino , Regulação Leucêmica da Expressão Gênica/efeitos dos fármacos , Células HL-60 , Humanos , Células K562 , Leucemia Mielogênica Crônica BCR-ABL Positiva/tratamento farmacológico , Leucemia Mielogênica Crônica BCR-ABL Positiva/patologia , Masculino , Pessoa de Meia-Idade , Células-Tronco Neoplásicas/efeitos dos fármacos , Células-Tronco Neoplásicas/metabolismo , Inibidores de Proteínas Quinases/farmacologiaRESUMO
Protonation states of molecules significantly influence the thermodynamics and kinetics of chemical reactions. This is especially important in biochemical processes, where appropriate protonation states of amino acids control the exo/endoergicity of practically all biochemical cycles. This paper is focused on appraisal of the impact of DFT functionals and PCM solvation models on the accuracy of pKa evaluations for all proteinogenic amino acids. Eight functionals (B3LYP, PBE0, revPBE0, M06-2X, M11, M11-L, TPSSh, and ωB97X-D) and four basis sets are considered, together with four kinds of implicit solvation models when additional attention is paid to a cavity construction. An influence of nonelectrostatic contributions and Wertz's corrections on Gibbs free energy is investigated together with accuracy of provided proton solvation energy. The best model is based on the M06-2X/6-311++G**/D-PCM/UAKS computational level. The fitting procedure is utilized to improve the accuracy of the evaluated models. All of these results are also compared with values obtained from the COSMOtherm program and CCSD(T) calculations. Results for cysteine and histidine are discussed individually, as they can be found in different protonation states at neutral pH.
RESUMO
NKR-P1C is an activating immune receptor expressed on the surface of mouse natural killer cells. It has been widely used as a marker for NK cell identification in different mice strains. Recently we solved a crystal structure of the C-type lectin-like domain of a homologous protein, NKR-P1A, using X-ray crystallography and also described the strategy for rapid characterization of the protein conformation in solution. This procedure utilized chemical cross-linking, hydrogen/deuterium exchange, and molecular modeling. It was found that the solution structure differs from the crystal structure in the conformation of the loop region. The loop, detached from the protein compact core in the crystal structure, is closely attached to the core of the protein in solution. Here we present and interpret the solution structure of the C-type lectin-like domain of NKR-P1C using chemical cross-linking and molecular modeling. The validation of the model and conformation of the loop region in NKR-P1C were addressed using ion-mobility mass spectrometry.
Assuntos
Antígenos Ly/química , Antígenos Ly/metabolismo , Linfócitos/metabolismo , Espectrometria de Massas/métodos , Modelos Moleculares , Subfamília B de Receptores Semelhantes a Lectina de Células NK/química , Subfamília B de Receptores Semelhantes a Lectina de Células NK/metabolismo , Sequência de Aminoácidos , Animais , Cristalografia por Raios X/métodos , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Estrutura Secundária de ProteínaRESUMO
BACKGROUND: Extracellular vesicles are released into body fluids from the majority of, if not all, cell types. Because their secretion and specific cargo (e.g., proteins) varies according to pathology, extracellular vesicles may prove a rich source of biomarkers. However, their biological and pathophysiological functions are poorly understood in hematological malignancies. OBJECTIVE: Here, we investigated proteome changes in the exosome-rich fraction of the plasma of myelodysplastic syndrome patients and healthy donors. METHODS: Exosome-rich fraction of the plasma was isolated using ExoQuick™: proteomes were compared and statistically processed; proteins were identified by nanoLC-MS/MS and verified using the ExoCarta and QuickGO databases. Mann-Whitney and Spearman analyses were used to statistically analyze the data. 2D western blot was used to monitor clusterin proteoforms. RESULTS: Statistical analyses of the data highlighted clusterin alterations as the most significant. 2D western blot showed that the clusterin changes were caused by posttranslational modifications. Moreover, there was a notable increase in the clusterin proteoform in the exosome-rich fraction of plasma of patients with more severe myelodysplastic syndrome; this corresponded with a simultaneous decrease in their plasma. CONCLUSIONS: This specific clusterin proteoform seems to be a promising biomarker for myelodysplastic syndrome progression.
Assuntos
Biomarcadores/sangue , Vesículas Extracelulares/metabolismo , Síndromes Mielodisplásicas/patologia , Proteoma/metabolismo , Proteômica/métodos , Idoso , Estudos de Casos e Controles , Cromatografia Líquida , Feminino , Humanos , Masculino , Síndromes Mielodisplásicas/metabolismo , Proteoma/análise , Espectrometria de Massas em TandemRESUMO
BACKGROUND: Leucine-rich alpha-2-glycoprotein (LRG) has been repeatedly proposed as a potential plasma biomarker for myelodysplastic syndrome (MDS). OBJECTIVE: The goal of our work was to establish the total LRG plasma level and LRG posttranslational modifications (PTMs) as a suitable MDS biomarker. METHODS: The total plasma LRG concentration was determined with ELISA, whilst the LRG-specific PTMs and their locations, were established using mass spectrometry and public mass spectrometry data re-analysis. Homology modelling and sequence analysis were used to establish the potential impact of PTMs on LRG functions via their impact on the LRG structure. RESULTS: While the results showed that the total LRG plasma concentration is not a suitable MDS marker, alterations within two LRG sites correlated with MDS diagnosis (p= 0.0011). Sequence analysis and the homology model suggest the influence of PTMs within the two LRG sites on the function of this protein. CONCLUSIONS: We report the presence of LRG proteoforms that correlate with diagnosis in the plasma of MDS patients. The combination of mass spectrometry, re-analysis of publicly available data, and homology modelling, represents an approach that can be used for any protein to predict clinically relevant protein sites for biomarker research despite the character of the PTMs being unknown.
Assuntos
Glicoproteínas , Síndromes Mielodisplásicas , Biomarcadores , Glicoproteínas/genética , Glicoproteínas/metabolismo , Humanos , Leucina/metabolismo , Espectrometria de Massas , Síndromes Mielodisplásicas/diagnóstico , Processamento de Proteína Pós-TraducionalRESUMO
Fibrinogen is an abundant blood plasma protein that, inter alia, participates in blood coagulation. It polymerizes to form a fibrin clot that is among the major components of the thrombus. Fibrinogen reactions with various reactive metabolites may induce post-translational modifications (PTMs) into the protein structure that affect the architecture and properties of fibrin clots. We reviewed the previous literature to find the positions of PTMs of fibrinogen. For 7 out of 307 reported PTMs, we used molecular dynamics simulations to characterize their effect on the behavior of the fibrinogen coiled-coil domain. Interactions of the γ-coil with adjacent chains give rise to π-helices in Aα and Bß chains of even unmodified fibrinogen. The examined PTMs suppress fluctuations of the γ-coil, which may affect the fibrinolysis and stiffness of the fibrin fibers. Citrullination of AαR104 and oxidations of γP70 and γP76 to glutamic semialdehyde unfold the α-helical structure of Aα and Bß chains. Oxidation of γM78 to methionine sulfoxide induces the formation of an α-helix in the γ-coil region. Our findings suggest that certain PTMs alter the protein secondary structure. Thus, the altered protein structure may indicate the presence of PTMs in the molecule and consequently of certain metabolites within the system.
RESUMO
Oxidative stress in humans is related to various pathophysiological processes, which can manifest in numerous diseases including cancer, cardiovascular diseases, and Alzheimer's disease. On the atomistic level, oxidative stress causes posttranslational modifications, thus inducing structural and functional changes into the proteins structure. This study focuses on fibrinogen, a blood plasma protein that is frequently targeted by reagents causing posttranslational modifications in proteins. Fibrinogen was in vitro modified by three reagents, namely sodium hypochlorite, malondialdehyde, and 3-morpholinosydnonimine that mimic the oxidative stress in diseases. Newly induced posttranslational modifications were detected via mass spectrometry. Electron microscopy was used to visualize changes in the fibrin networks, which highlight the extent of disturbances in fibrinogen behavior after exposure to reagents. We used molecular dynamics simulations to observe the impact of selected posttranslational modifications on the fibrinogen structure at the atomistic level. In total, 154 posttranslational modifications were identified, 84 of them were in fibrinogen treated with hypochlorite, 51 resulted from a reaction of fibrinogen with malondialdehyde, and 19 were caused by 3-morpholinosydnonimine. Our data reveal that the stronger reagents induce more posttranslational modifications in the fibrinogen structure than the weaker ones, and they extensively alter the architecture of the fibrin network. Molecular dynamics simulations revealed that the effect of posttranslational modifications on fibrinogen secondary structure varies from negligible alternations to serious disruptions. Among the serious disruptions is the oxidation of γR375 resulting in the release of Ca2+ ion that is necessary for appropriate fibrin fiber formation. Folding of amino acids γE72-γN77 into a short α-helix is a result of oxidation of γP76 to glutamic acid. The study describes behaviour of fibrinogen coiled-coil connecter in the vicinity of plasmin and hementin cleavage sites.
Assuntos
Fibrinogênio/química , Fibrinogênio/metabolismo , Processamento de Proteína Pós-Traducional , Humanos , Simulação de Dinâmica Molecular , Estrutura Secundária de ProteínaRESUMO
Ceramides are lipids that are involved in numerous biologically important structures (e.g., the stratum corneum and ceramide-rich platforms) and processes (e.g., signal transduction and membrane fusion), but their behavior is not fully understood. We report coarse-grain force field parameters for N-lignocerylsphingosine (ceramide NS, also known as ceramide 2) that are consistent with the Martini force field. These parameters were optimized for simulations in the gel phase and validated against atomistic simulations. Coarse-grained simulations with our parameters provide areas per lipid, membrane thicknesses, and electron density profiles that are in good agreement with atomistic simulations. Properties of the simulated membranes are compared with available experimental data. The obtained parameters were used to model the phase behavior of ceramide NS as a function of temperature and hydration. At low water content and above the main phase transition temperature, the bilayer melts into an irregular phase, which may correspond to the unstructured melted-chain phase observed in X-ray diffraction experiments. The developed parameters also reproduce the extended conformation of ceramide, which may occur in the stratum corneum. The parameters presented herein will facilitate studies on important complex functional structures such as the uppermost layer of the skin and ceramide-rich platforms in phospholipid membranes.
Assuntos
Ceramidas/química , Simulação de Dinâmica Molecular , Pele/química , Humanos , Cristais Líquidos/química , Transição de Fase , Fosfolipídeos/química , Temperatura de Transição , Água/químicaRESUMO
Ceramides are indispensable constituents of the stratum corneum (SC), the uppermost impermeable layer of human skin. Ceramides with shorter (four- to eight-carbon acyl chains) fatty acid chains increase skin and model membrane permeability, while further shortening of the chain leads to increased resistance to penetration almost as good as that of ceramides from healthy skin (24 carbons long on average). Here we address the extent to which the atomistic CHARMM36 and coarse-grain MARTINI molecular dynamics (MD) simulations reflect the skin permeability data. As a result, we observed the same bell-shaped permeability trend for water that was observed in the skin and multilayer membrane experiments for model compounds. We showed that the enhanced permeability of the short ceramides is mainly caused by the disturbance of their headgroup conformation because of their inability to accommodate the shorter lipid acyl chain into a typical hairpin conformation, which further led to their destabilization and phase separation. As MD simulations described well delicate structural features of SC membranes, they seem to be suitable for further studies of the SC superstructure, including the development of skin penetration enhancers for transdermal drug delivery and skin toxicity risk assessment studies.
Assuntos
Ceramidas/química , Ceramidas/metabolismo , Epiderme/metabolismo , Simulação de Dinâmica Molecular , Membrana Celular/química , Membrana Celular/metabolismo , Bicamadas Lipídicas/química , Bicamadas Lipídicas/metabolismo , Conformação Molecular , Permeabilidade , Água/metabolismoRESUMO
We present an extension of the Martini coarse-grained force field to glycolipids. The glycolipids considered here are the glycoglycerolipids monogalactosyldiacylglycerol (MGDG), sulfoquinovosyldiacylglycerol (SQDG), digalactosyldiacylglycerol (DGDG), and phosphatidylinositol (PI) and its phosphorylated forms (PIP, PIP2), as well as the glycosphingolipids galactosylceramide (GCER) and monosialotetrahexosylganglioside (GM1). The parametrization follows the same philosophy as was used previously for lipids, proteins, and carbohydrates focusing on the reproduction of partitioning free energies of small compounds between polar and nonpolar solvents. Bonded parameters are optimized by comparison to lipid conformations sampled with an atomistic force field, in particular with respect to the representation of the most populated states around the glycosidic linkage. Simulations of coarse-grained glycolipid model membranes show good agreement with atomistic simulations as well as experimental data available, especially concerning structural properties such as electron densities, area per lipid, and membrane thickness. Our coarse-grained model opens the way to large scale simulations of biological processes in which glycolipids are important, including recognition, sorting, and clustering of both external and membrane bound proteins.
RESUMO
Raman microscopy permits structural analysis of protein crystals in situ in hanging drops, allowing for comparison with Raman measurements in solution. Nevertheless, the two methods sometimes reveal subtle differences in structure that are often ascribed to the water layer surrounding the protein. The novel method of drop-coating deposition Raman spectropscopy (DCDR) exploits an intermediate phase that, although nominally "dry," has been shown to preserve protein structural features present in solution. The potential of this new approach to bridge the structural gap between proteins in solution and in crystals is explored here with extrinsic protein PsbP of photosystem II from Spinacia oleracea. In the high-resolution (1.98 Å) x-ray crystal structure of PsbP reported here, several segments of the protein chain are present but unresolved. Analysis of the three kinds of Raman spectra of PsbP suggests that most of the subtle differences can indeed be attributed to the water envelope, which is shown here to have a similar Raman intensity in glassy and crystal states. Using molecular dynamics simulations cross-validated by Raman solution data, two unresolved segments of the PsbP crystal structure were modeled as loops, and the amino terminus was inferred to contain an additional beta segment. The complete PsbP structure was compared with that of the PsbP-like protein CyanoP, which plays a more peripheral role in photosystem II function. The comparison suggests possible interaction surfaces of PsbP with higher-plant photosystem II. This work provides the first complete structural picture of this key protein, and it represents the first systematic comparison of Raman data from solution, glassy, and crystalline states of a protein.
Assuntos
Complexo de Proteína do Fotossistema II/química , Proteínas de Plantas/química , Spinacia oleracea/química , Motivos de Aminoácidos , Sequência de Aminoácidos , Cristalografia por Raios X , Ligação de Hidrogênio , Modelos Moleculares , Dados de Sequência Molecular , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Análise Espectral RamanRESUMO
Receptor proteins at the cell surface regulate the ability of natural killer cells to recognize and kill a variety of aberrant target cells. The structural features determining the function of natural killer receptor proteins 1 (NKR-P1s) are largely unknown. In the present work, refined homology models are generated for the C-type lectin-like extracellular domains of rat NKR-P1A and NKR-P1B, mouse NKR-P1A, NKR-P1C, NKR-P1F, and NKR-P1G, and human NKR-P1 receptors. Experimental data on secondary structure, tertiary interactions, and thermal transitions are acquired for four of the proteins using Raman and infrared spectroscopy. The experimental and modeling results are in agreement with respect to the overall structures of the NKR-P1 receptor domains, while suggesting functionally significant local differences among species and isoforms. Two sequence regions that are conserved in all analyzed NKR-P1 receptors do not correspond to conserved structural elements as might be expected, but are represented by loop regions, one of which is arranged differently in the constructed models. This region displays high flexibility but is anchored by conserved sequences, suggesting that its position relative to the rest of the domain might be variable. This loop may contribute to ligand-binding specificity via a coupled conformational transition.
Assuntos
Sequência Conservada , Subfamília B de Receptores Semelhantes a Lectina de Células NK/química , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Sítios de Ligação , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Simulação de Dinâmica Molecular , Dados de Sequência Molecular , Subfamília B de Receptores Semelhantes a Lectina de Células NK/classificação , Filogenia , Estrutura Terciária de Proteína , Ratos , Alinhamento de Sequência , Espectroscopia de Infravermelho com Transformada de Fourier , Análise Espectral Raman , Homologia Estrutural de Proteína , TermodinâmicaRESUMO
Infrared and Raman spectroscopy were applied to identify restraints for the structure determination of the 20 amino acid loop between two beta-sheets of the N-terminal region of the PsbQ protein of the oxygen evolving complex of photosystem II from Spinacia oleracea by restraint-based homology modeling. One of the initial models has shown a stable fold of the loop in a 20 ns molecular dynamics simulation that is in accordance with spectroscopic data. Cleavage of the first 12 amino acids leads to a permanent drift in the root means square deviation of the protein backbone and induces major structural changes.
Assuntos
Modelos Químicos , Modelos Moleculares , Complexo de Proteínas do Centro de Reação Fotossintética/química , Complexo de Proteínas do Centro de Reação Fotossintética/ultraestrutura , Complexo de Proteína do Fotossistema II/química , Complexo de Proteína do Fotossistema II/ultraestrutura , Proteínas de Plantas/química , Proteínas de Plantas/ultraestrutura , Spinacia oleracea/química , Sequência de Aminoácidos , Simulação por Computador , Dados de Sequência Molecular , Complexo de Proteínas do Centro de Reação Fotossintética/análise , Complexo de Proteína do Fotossistema II/análise , Proteínas de Plantas/análise , Conformação Proteica , Estrutura Secundária de ProteínaRESUMO
The structural stability of the large cytoplasmic domain (H(4)-H(5) loop) of mouse alpha(1) subunit of Na(+)/K(+) ATPase (L354-I777), the number and the location of its binding sites for 2'-3'-O-(trinitrophenyl) adenosine 5'-triphosphate (TNP-ATP) and p-nitrophenylphosphate (pNPP) were investigated. C- and N-terminal shortening revealed that neither part of the phosphorylation (P)-domain are necessary for TNP-ATP binding. There is no indication of a second ATP site on the P-domain of the isolated loop, even though others reported previously of its existence by TNP-N(3)ADP affinity labeling of the full enzyme. Fluorescein isothiocyanate (FITC)-anisotropy measurements reveal a considerable stability of the nucleotide (N)-domain suggesting that it may not undergo a substantial conformational change upon ATP binding. The FITC modified loop showed only slightly diminished phosphatase activity, most likely due to a pNPP site on the N-domain around N398 whose mutation to D reduced the phosphatase activity by 50%. The amino acids forming this pNPP site (M384, L414, W411, S400, S408) are conserved in the alpha(1-4) isoforms of Na(+)/K(+) ATPase, whereas N398 is only conserved in the vertebrates' alpha(1) subunit. The phosphatase activity of the isolated H(4)-H(5) loop was neither inhibited by ATP, nor affected by mutation of D369, which is phosphorylated in native Na(+)/K(+) ATPase.
Assuntos
4-Nitrofenilfosfatase/metabolismo , Trifosfato de Adenosina/análogos & derivados , Trifosfato de Adenosina/metabolismo , Rim/enzimologia , Proteínas Tirosina Fosfatases/metabolismo , ATPase Trocadora de Sódio-Potássio/química , ATPase Trocadora de Sódio-Potássio/metabolismo , Sequência de Aminoácidos , Animais , Sítios de Ligação , Citoplasma/enzimologia , Estabilidade Enzimática , Amarelo de Eosina-(YS)/metabolismo , Corantes Fluorescentes/metabolismo , Glutationa Transferase/genética , Glutationa Transferase/metabolismo , Camundongos , Dados de Sequência Molecular , Mutação/genética , Fosforilação , Ligação Proteica , Estrutura Terciária de Proteína , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes de Fusão/metabolismo , Homologia de Sequência de Aminoácidos , Relação Estrutura-Atividade , Especificidade por Substrato , SuínosRESUMO
A model of the helical part of the human MT2 melatonin (hMT2) receptor, a member of the G protein-coupled receptors superfamily has been generated, based on the structure of bovine rhodopsin. Modeling has been combined with site-directed mutagenesis to investigate the role of the specific amino acid residues within the transmembrane domains (TM) numbers V, VI and VII of hMT2 receptor in the interaction with 2-iodomelatonin. Saturation binding assays with 2-iodomelatonin demonstrated that the substitution V204A (TMV) resulted in total loss of binding while the mutation V205A had no effect. The replacement of F209 with alanine led to a significant decrease in the Bmax value of receptor binding while mutations V205A and F209A also within TM V did not significantly change binding properties of the hMT2 receptor. In the case of TM VI, the substitution G271T caused substantial decrease in 2-iodomelatonin binding to the hMT2 receptor. The change L272A (TM VI) as well as mutation Y298A within TM VII completely abolished ligand binding to the receptor. These data suggest that several new amino acid residues within TM V, VI and VII are involved in ligand-MT2 receptor interaction.