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The structural determinants of the interaction of the G-quadruplex (G4) motif found in precursor miRNA 149 (rG4) with the acridine orange derivative C8 , a G4 ligand stabilizer possessing anticancer activity, and the protein nucleolin (overexpressed in cancer cells) were investigated by Nuclear Magnetic Resonance (NMR) spectroscopy. For the rG4/C8 complex, the results revealed a strong stabilizing interaction between the aromatic core and the iodinated ring of the C8 ligand with the rG4 structure. The NMR study revealed also different interaction patterns between nucleolin and rG4 and nucleolin and rG4/C8 complex. In the absence of the ligand, rG4 establishes interactions with polar residues of the protein while for the rG4/C8 complex, these contacts are mainly established with amino acids that have hydrophobic side chains. However, nucleolin chemical shift perturbation studies in the presence of rG4 or rG4/C8 reveal the same location between domains 1 and 2 of the protein, which suggests that the rG4 and rG4/C8 complex bind in this region. This puzzling structural study opens a new framework to study rG4/ligand/nucleolin complexes that might impact the biogenesis of miRNA 149.
Assuntos
Quadruplex G , MicroRNAs , Humanos , Ligantes , Fosfoproteínas/química , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Carcinogênese , NucleolinaRESUMO
The Cellulosome is an intricate macromolecular protein complex that centralizes the cellulolytic efforts of many anaerobic microorganisms through the promotion of enzyme synergy and protein stability. The assembly of numerous carbohydrate processing enzymes into a macromolecular multiprotein structure results from the interaction of enzyme-borne dockerin modules with repeated cohesin modules present in noncatalytic scaffold proteins, termed scaffoldins. Cohesin-dockerin (Coh-Doc) modules are typically classified into different types, depending on structural conformation and cellulosome role. Thus, type I Coh-Doc complexes are usually responsible for enzyme integration into the cellulosome, while type II Coh-Doc complexes tether the cellulosome to the bacterial wall. In contrast to other known cellulosomes, cohesin types from Bacteroides cellulosolvens, a cellulosome-producing bacterium capable of utilizing cellulose and cellobiose as carbon sources, are reversed for all scaffoldins, i.e., the type II cohesins are located on the enzyme-integrating primary scaffoldin, whereas the type I cohesins are located on the anchoring scaffoldins. It has been previously shown that type I B. cellulosolvens interactions possess a dual-binding mode that adds flexibility to scaffoldin assembly. Herein, we report the structural mechanism of enzyme recruitment into B. cellulosolvens cellulosome and the identification of the molecular determinants of its type II cohesin-dockerin interactions. The results indicate that, unlike other type II complexes, these possess a dual-binding mode of interaction, akin to type I complexes. Therefore, the plasticity of dual-binding mode interactions seems to play a pivotal role in the assembly of B. cellulosolvens cellulosome, which is consistent with its unmatched complexity and size.
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Proteínas de Bactérias/metabolismo , Bacteroides/metabolismo , Proteínas de Ciclo Celular/metabolismo , Celulossomas/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Clostridiales/metabolismo , Proteínas de Bactérias/genética , Bacteroides/genética , Bacteroides/crescimento & desenvolvimento , Proteínas de Ciclo Celular/genética , Celobiose/metabolismo , Celulose/metabolismo , Proteínas Cromossômicas não Histona/genética , Clostridiales/genética , Clostridiales/crescimento & desenvolvimento , CoesinasRESUMO
The recent structural elucidation of ex vivo Drosophila Orb2 fibrils revealed a novel amyloid formed by interdigitated Gln and His residue side chains belonging to the prion-like domain. However, atomic-level details on the conformational transitions associated with memory consolidation remain unknown. Here, we have characterized the nascent conformation and dynamics of the prion-like domain (PLD) of Orb2A using a nonconventional liquid-state NMR spectroscopy strategy based on 13C detection to afford an essentially complete set of 13Cα, 13Cß, 1Hα, and backbone 13CO and 15N assignments. At pH 4, where His residues are protonated, the PLD is disordered and flexible, except for a partially populated α-helix spanning residues 55-60, and binds RNA oligos, but not divalent cations. At pH 7, in contrast, His residues are predominantly neutral, and the Q/H segments adopt minor populations of helical structure, show decreased mobility and start to self-associate. At pH 7, the His residues do not bind RNA or Ca2+, but do bind Zn2+, which promotes further association. These findings represent a remarkable case of structural plasticity, based on which an updated model for Orb2A functional amyloidogenesis is suggested.
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Proteínas de Drosophila/química , Proteínas de Drosophila/metabolismo , Drosophila/fisiologia , Consolidação da Memória , Príons/química , Multimerização Proteica , Fatores de Transcrição/química , Fatores de Transcrição/metabolismo , Fatores de Poliadenilação e Clivagem de mRNA/química , Fatores de Poliadenilação e Clivagem de mRNA/metabolismo , Animais , Espectroscopia de Ressonância MagnéticaRESUMO
Interactions of glycan-specific epitopes to human lectin receptors represent novel immune checkpoints for investigating cancer and infection diseases. By employing a multidisciplinary approach that combines isothermal titration calorimetry, NMR spectroscopy, molecular dynamics simulations, and X-ray crystallography, we investigated the molecular determinants that govern the recognition of the tumour and pathogenic glycobiomarker LacdiNAc (GalNAcß1-4GlcNAc, LDN), including their comparison with the ubiquitous LacNAc epitope (Galß1-4GlcNAc, LN), by two human immune-related lectins, galectin-3 (hGal-3) and the macrophage galactose C-type lectin (hMGL). A different mechanism of binding and interactions was observed for the hGal-3/LDN and hMGL/LDN complexes, which explains the remarkable difference in the binding specificity of LDN and LN by these two lectins. The new structural clues reported herein are fundamental for the chemical design of mimetics targeting hGal-3/hMGL recognition process.
Assuntos
Lactose , Neoplasias , Epitopos , Humanos , Lactose/análogos & derivados , Polissacarídeos , Ligação ProteicaRESUMO
In the last few years, ionic liquids (ILs) have been the focus of extensive studies concerning the relationship between structure and properties and how this impacts their application. Despite a large number of studies, several topics remain controversial or not fully answered, such as: the existence of ion pairs, the concept of free volume and the effect of water and its implications in the modulation of ILs physicochemical properties. In this paper, we present a critical review of state-of-the-art literature regarding structure-property relationship of ILs, we re-examine analytical theories on the structure-property correlations and present new perspectives based on the existing data. The interrelation between transport properties (viscosity, diffusion, conductivity) of IL structure and free volume are analysed and discussed at a molecular level. In addition, we demonstrate how the analysis of microscopic features (particularly using NMR-derived data) can be used to explain and predict macroscopic properties, reaching new perspectives on the properties and application of ILs.
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Líquidos Iônicos/química , Difusão , Condutividade Elétrica , Íons , Relação Estrutura-Atividade , ViscosidadeRESUMO
The human macrophage galactose-type lectin (MGL), expressed on macrophages and dendritic cells (DCs), modulates distinct immune cell responses by recognizing N-acetylgalactosamine (GalNAc) containing structures present on pathogens, self-glycoproteins, and tumor cells. Herein, NMR spectroscopy and molecular dynamics (MD) simulations were used to investigate the structural preferences of MGL against different GalNAc-containing structures derived from the blood groupâ A antigen, the Forssman antigen, and the GM2 glycolipid. NMR spectroscopic analysis of the MGL carbohydrate recognition domain (MGL-CRD, C181-H316) in the absence and presence of methyl α-GalNAc (α-MeGalNAc), a simple monosaccharide, shows that the MGL-CRD is highly dynamic and its structure is strongly altered upon ligand binding. This plasticity of the MGL-CRD structure explains the ability of MGL to accommodate different GalNAc-containing molecules. However, key differences are observed in the recognition process depending on whether the GalNAc is part of the blood groupâ A antigen, the Forssman antigen, or GM2-derived structures. These results are in accordance with molecular dynamics simulations that suggest the existence of a distinct MGL binding mechanism depending on the context of GalNAc moiety presentation. These results afford new perspectives for the rational design of GalNAc modifications that fine tune MGL immune responses in distinct biological contexts, especially in malignancy.
Assuntos
Acetilgalactosamina/química , Lectinas Tipo C/metabolismo , Antígenos de Grupos Sanguíneos/química , Antígenos de Grupos Sanguíneos/metabolismo , Mapeamento de Epitopos , Humanos , Lectinas Tipo C/química , Lectinas Tipo C/genética , Ligantes , Simulação de Dinâmica Molecular , Ressonância Magnética Nuclear Biomolecular , Ligação Proteica , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificaçãoRESUMO
The preorganization and cooperation mechanism of imide-based ionic liquids reported in a recent Communication was evocated to rationalize the extremely high gravimetric CO2 capture displayed by these fluids. An analysis of the reported spectroscopic evidences together with additional experiments led to the proposition of an alternative, simpler, and feasible mechanism involving the formation of bicarbonate.
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In this work, 1-butyl-3-methylimidazolium tetrafluoroborate/water mixtures were analysed over the whole water composition (xw ) in order to study the rotational and translational behaviour of the ions. We employed a multinuclear NMR approach to determine anion/cation/water diffusion coefficients and longitudinal relaxation rates at different water content. In neat ionic liquids (IL), the cation diffuses faster than the anion, and at low xw , anions and cations share almost the same diffusion coefficient, but above a critical water concentration, the anion begins to diffuse faster than the cation. We identified this composition as approximately 10% xw where the ions share the same diffusion coefficient. We found that the water at this composition seems to have a much more dramatic effect in the rotational diffusion of the anion that decreases substantially and approaches that of the anion in the diluted IL. Translational and rotational dynamics of the ions suggest that water is first incorporated in pockets in the nanostructure of the IL allowing the ions to maintain most of the cation/anion interactions present in neat IL but already disrupting some anion/cation interactions due to preferential interaction with the anion. HOESY and NOESY data show that water displays contacts both with the cation and the anion in a positive NOE regime in contrary to the negative regime found for the cation/anion and cation/cation cross-relaxation. This is in accordance with the high relative diffusion coefficient of water and suggests that water molecules can exchange between preferential location sites that allow water to maintain contacts both with the anion and cation.
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Protein-glycan interactions as modulators for quinary structures in crowding environments were explored. The interaction between human galectin 3 (Gal-3) and distinct macromolecular crowders, such as bovine and human serum albumin (BSA and HSA), Ficoll 70 and PEG3350, was scrutinized. The molecular recognition event of the specific ligand, lactose, by Gal-3 in crowding conditions was evaluated. Gal-3 interactions were monitored by NMR analysing chemical shift perturbation (CSP) and line broadening of 1 H15 N-HSQC signals. The intensity of the Gal-3 1 H15 N-HSQC signals decreased in the presence of all crowders, due to the increase in the solution viscosity and to the formation of large protein complexes. When glycosylated containing samples of BSA and HSA were used, signal broadening was more severe than that observed in the presence of the more viscous solutions of PEG3350 and Ficoll 70. However, for the samples containing glycoproteins, the signal intensity of 1 H15 N-HSQC recovered upon addition of lactose. We show that serum proteins interact with Gal-3, through their α2,3-linked sialylgalactose moieties exposed at their surfaces, competing with lactose for the same binding site. The quinary interaction between Gal-3 and serum glycoproteins, could help to co-localize Gal-3 at the cell surface, and may play a role in adhesion and signalling functions of this protein.
Assuntos
Galectina 3/química , Glicoproteínas/síntese química , Lactose/química , Animais , Sítios de Ligação , Proteínas Sanguíneas , Bovinos , Linhagem Celular , Escherichia coli , Galectina 3/isolamento & purificação , Galectinas , Glicosilação , Humanos , Ligantes , Espectroscopia de Ressonância Magnética , Polietilenoglicóis/química , Ligação Proteica , Conformação Proteica , Albumina Sérica/químicaRESUMO
BACKGROUND: G-quadruplexes (G4) are found at important genome regions such as telomere ends and oncogene promoters. One prominent strategy to explore the therapeutic potential of G4 is stabilized it with specific ligands. METHODS: We report the synthesis of new phenanthroline, phenyl and quinoline acyclic bisoxazole compounds in order to explore and evaluate the targeting to c-myc and human telomeric repeat 22AG G4 using FRET-melting, CD-melting, NMR, fluorescence titrations and FID assays. RESULTS: The design strategy has led to potent compounds (Phen-1 and Phen-2) that discriminate different G4 structures (human telomeric sequences and c-myc promoter) and selectively stabilize G4 over duplex DNA. CD studies show that Phen-2 binds and induces antiparallel topologies in 22AG quadruplex and also binds c-myc promotor, increasing their Tm in about 12°C and 30°C respectively. In contrast, Phen-1 induces parallel topologies in 22AG and c-myc, with a moderate stabilization of 4°C for both sequences. Consistent with a CD melting study, Phen-2 binds strongly (K=106 to 107M-1) to c-myc and 22AG quadruplexes. CONCLUSIONS: Phen-1 and Phen-2 discriminated among various quadruplex topologies and exhibited high selectivity for quadruplexes over duplexes. Phen-2 retains antiparallel topologies for quadruplex 22AG and does not induce conformational changes on the parallel c-myc quadruplex although Phen-1 favors the parallel topology. NMR studies also showed that the Phen-2 binds to the c-myc quadruplex via end stacking. GENERAL SIGNIFICANCE: Overall, the results suggest the importance of Phen-2 as a scaffold for the fine-tuning with substituents in order to enhance binding and stabilization to G4 structures. This article is part of a Special Issue entitled "G-quadruplex" Guest Editor: Dr. Concetta Giancola and Dr. Daniela Montesarchio.
Assuntos
Antineoplásicos/metabolismo , DNA de Neoplasias/metabolismo , Desenho de Fármacos , Quadruplex G , Guanosina/química , Oxazóis/metabolismo , Fenantrolinas/metabolismo , Proteínas Proto-Oncogênicas c-myc/genética , Telômero/metabolismo , Antineoplásicos/síntese química , Antineoplásicos/farmacologia , Sítios de Ligação , Dicroísmo Circular , DNA de Neoplasias/química , DNA de Neoplasias/efeitos dos fármacos , Transferência Ressonante de Energia de Fluorescência , Quadruplex G/efeitos dos fármacos , Humanos , Ligantes , Espectroscopia de Ressonância Magnética , Desnaturação de Ácido Nucleico , Oxazóis/síntese química , Oxazóis/farmacologia , Fenantrolinas/síntese química , Fenantrolinas/farmacologia , Regiões Promotoras Genéticas/efeitos dos fármacos , Relação Estrutura-Atividade , Telômero/química , Telômero/efeitos dos fármacos , TemperaturaRESUMO
We applied Pulsed Gradient Spin Echo diffusion with high-resolution magic angle spinning NMR to study sol-gel matrices used to encapsulate enzymes for biocatalysis (TMOS/MTMS and TMOS/BTMS) to gain insight into the local chemical microenvironment. Transport properties of solvents with different polarities (1-pentanol, acetonitrile and n-hexane) were studied through their apparent self-diffusion coefficients. The spin echo attenuation of the solvents shows two distinct diffusion domains, one with fast diffusion (Dfast ) associated with interparticle diffusion and another with slow diffusion (Dslow ) corresponding to the displacement inside the pores within the sol-gel particles. The analysis of the root mean square displacements at different diffusion times showed that the Dfast domain has a free diffusion regime in both matrices (the root mean square displacement is linearly dependent of the diffusion time), while the Dslow domain shows a different regime that depends on the matrix. We investigated the exchange regime between the two diffusion sites. In both matrices, n-hexane was in intermediate exchange between diffusion domains, while the polar solvents were in slow exchange in TMOS/BTMS and in intermediate exchange in TMOS/MTMS. Data were fitted for TMOS/BTMS with the Kärger model, and the physical parameters were obtained. The results add to the evidence that the pores are a hydrophobic environment but that the presence of some free hydrophilic groups inside the pore, as observed in the TMOS/BTMS, has a key role in slowing down the exchange of polar solvents and that this is relevant to explain previously reported enzyme activity in these materials. Copyright © 2016 John Wiley & Sons, Ltd.
Assuntos
Espectroscopia de Ressonância Magnética/métodos , Silanos/química , Solventes/química , Acetonitrilas/química , Difusão , Hexanos/química , Interações Hidrofóbicas e Hidrofílicas , Cinética , Microscopia Eletrônica de Varredura , Modelos Moleculares , Pentanóis/química , Transição de Fase , Álcool de Polivinil/química , Porosidade , Dióxido de Silício/químicaRESUMO
A prerequisite for any rational drug design strategy is understanding the mode of protein-ligand interaction. This motivated us to explore protein-substrate interaction in Type-II NADH:quinone oxidoreductase (NDH-2) from Staphylococcus aureus, a worldwide problem in clinical medicine due to its multiple drug resistant forms. NDHs-2 are involved in respiratory chains and recognized as suitable targets for novel antimicrobial therapies, as these are the only enzymes with NADH:quinone oxidoreductase activity expressed in many pathogenic organisms. We obtained crystal and solution structures of NDH-2 from S. aureus, showing that it is a dimer in solution. We report fast kinetic analyses of the protein and detected a charge-transfer complex formed between NAD(+) and the reduced flavin, which is dissociated by the quinone. We observed that the quinone reduction is the rate limiting step and also the only half-reaction affected by the presence of HQNO, an inhibitor. We analyzed protein-substrate interactions by fluorescence and STD-NMR spectroscopies, which indicate that NADH and the quinone bind to different sites. In summary, our combined results show the presence of distinct binding sites for the two substrates, identified quinone reduction as the rate limiting step and indicate the establishment of a NAD(+)-protein complex, which is released by the quinone.
Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Quinona Redutases/química , Quinona Redutases/metabolismo , Quinonas/metabolismo , Staphylococcus aureus/enzimologia , Proteínas de Bactérias/genética , Sítios de Ligação , Cristalografia por Raios X , Descoberta de Drogas , Transporte de Elétrons , Hidroxiquinolinas/farmacologia , Cinética , Modelos Moleculares , Oxirredução , Multimerização Proteica , Quinona Redutases/antagonistas & inibidores , Quinona Redutases/genética , Staphylococcus aureus/metabolismoRESUMO
In this work, we explore the interactions between the ionic liquid 1-ethyl-3-methylimidazolim acetate and different inorganic salts belonging to two different cation families, those based on ammonium and others based on sodium. NMR and Raman spectroscopy are used to screen for changes in the molecular environment of the ions in the ionic liquid + inorganic salt mixtures as compared to pure ionic liquid. The ion self-diffusion coefficients are determined from NMR data, allowing the discussion of the ionicity values of the ionic liquid + inorganic salt mixtures calculated using different methods. Our data reveal that preferential interactions are established between the ionic liquid and ammonium-based salts, as opposed to sodium-based salts. Computational calculations show the formation of aggregates between the ionic liquid and the inorganic salt, which is consistent with the spectroscopic data, and indicate that the acetate anion of the ionic liquid establishes preferential interactions with the ammonium cation of the inorganic salts, leaving the imidazolium cation less engaged in the media.
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The identification of MUC1 tumor-associated Tn antigen (αGalpNAc1-O-Ser/Thr) has boosted the development of anticancer vaccines. Combining microarrays and saturation transfer difference NMR, we have characterized the fine-epitope mapping of a MUC1 chemical library (naked and Tn-glycosylated) toward two families of cancer-related monoclonal antibodies (anti-MUC1 and anti-Tn mAbs). Anti-MUC1 mAbs clone VU-3C6 and VU-11E2 recognize naked MUC1-derived peptides and bind GalNAc in a peptide-sequence-dependent manner. In contrast, anti-Tn mAbs clone 8D4 and 14D6 mostly recognize the GalNAc and do not bind naked MUC1-derived peptides. These anti-Tn mAbs show a clear preference for glycopeptides containing the Tn-Ser antigen rather than the Tn-Thr analogue, stressing the role of the underlying amino acid (serine or threonine) in the binding process. The reported strategy can be employed, in general, to unveil the key minimal structural features that modulate antigen-antibody recognition, with particular relevance for the development of Tn-MUC1-based anticancer vaccines.
Assuntos
Anticorpos Monoclonais/metabolismo , Vacinas Anticâncer , Epitopos/imunologia , Espectroscopia de Ressonância Magnética , Análise Serial de Proteínas , Mapeamento de Epitopos , HumanosRESUMO
The human macrophage galactose-type lectin (MGL) is a key physiological receptor for the carcinoma-associated Tn antigen (GalNAc-α-1-O-Ser/Thr) in mucins. NMR and modeling-based data on the molecular recognition features of synthetic Tn-bearing glycopeptides by MGL are presented. Cognate epitopes on the sugar and matching key amino acids involved in the interaction were identified by saturation transfer difference (STD) NMR spectroscopy. Only the amino acids close to the glycosylation site in the peptides are involved in lectin contact. Moreover, control experiments with non-glycosylated MUC1 peptides unequivocally showed that the sugar residue is essential for MGL binding, as is Ca(2+) . NMR data were complemented with molecular dynamics simulations and Corcema-ST to establish a 3D view on the molecular recognition process between Gal, GalNAc, and the Tn-presenting glycopeptides and MGL. Gal and GalNAc have a dual binding mode with opposite trend of the main interaction pattern and the differences in affinity can be explained by additional hydrogen bonds and CH-π contacts involving exclusively the NHAc moiety.
Assuntos
Antígenos Glicosídicos Associados a Tumores/metabolismo , Glicopeptídeos/metabolismo , Lectinas Tipo C/metabolismo , Mucina-1/metabolismo , Sequência de Aminoácidos , Antígenos Glicosídicos Associados a Tumores/química , Glicopeptídeos/química , Humanos , Lectinas Tipo C/química , Simulação de Dinâmica Molecular , Dados de Sequência Molecular , Mucina-1/química , Ressonância Magnética Nuclear BiomolecularRESUMO
We investigated imidazolium-based ionic liquid (IL) interactions with human serum albumin (HSA) to discern the level of cation interactions towards protein stability. STD-NMR spectroscopy was used to observe the imidazolium IL protons involved in direct binding and to identify the interactions responsible for changes in Tm as accessed by differential scanning calorimetry (DSC). Cations influence protein stability less than anions but still significantly. It was found that longer alkyl side chains of imidazolium-based ILs (more hydrophobic) are associated with a higher destabilisation effect on HSA than short-alkyl groups (less hydrophobic). The reason for such destabilisation lies on the increased surface contact area of the cation with the protein, particularly on the hydrophobic contacts promoted by the terminus of the alkyl chain. The relevance of the hydrophobic contacts is clearly demonstrated by the introduction of a polar moiety in the alkyl chain: a methoxy or alcohol group. Such structural modification reduces the degree of hydrophobic contacts with HSA explaining the lesser extent of protein destabilisation when compared to longer alkyl side chain groups: above [C2mim](+). Competition STD-NMR experiments using [C2mim](+), [C4mim](+) and [C2OHmim](+) also validate the importance of the hydrophobic interactions. The combined effect of cation and anion interactions was explored using (35)Cl NMR. Such experiments show that the nature of the cation has no influence on the anion-protein contacts, still the nature of the anion modulates the cation-protein interaction. Herein we propose that more destabilising anions are likely to be a result of a partial contribution from the cation as a direct consequence of the different levels of interaction (cation-anion pair and cation-protein).
Assuntos
Mapeamento de Epitopos , Interações Hidrofóbicas e Hidrofílicas , Imidazóis/química , Líquidos Iônicos/química , Albumina Sérica/química , Eletricidade Estática , Cátions/química , Humanos , Estabilidade ProteicaRESUMO
Non-catalytic cellulosomal CBMs (carbohydrate-binding modules) are responsible for increasing the catalytic efficiency of cellulosic enzymes by selectively putting the substrate (a wide range of poly- and oligo-saccharides) and enzyme into close contact. In the present study we carried out an atomistic rationalization of the molecular determinants of ligand specificity for a family 11 CBM from thermophilic Clostridium thermocellum [CtCBM11 (C. thermocellum CBM11)], based on a NMR and molecular modelling approach. We have determined the NMR solution structure of CtCBM11 at 25°C and 50°C and derived information on the residues of the protein that are involved in ligand recognition and on the influence of the length of the saccharide chain on binding. We obtained models of the CtCBM11-cellohexaose and CtCBM11-cellotetraose complexes by docking in accordance with the NMR experimental data. Specific ligand-protein CH-π and Van der Waals interactions were found to be determinant for the stability of the complexes and for defining specificity. Using the order parameters derived from backbone dynamics analysis in the presence and absence of ligand and at 25°C and 50°C, we determined that the protein's backbone conformational entropy is slightly positive. This data in combination with the negative binding entropy calculated from ITC (isothermal titration calorimetry) studies supports a selection mechanism where a rigid protein selects a defined oligosaccharide conformation.
Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Metabolismo dos Carboidratos , Clostridium thermocellum/metabolismo , Oligossacarídeos/química , Proteínas de Bactérias/genética , Sítios de Ligação , Calorimetria , Celulose/análogos & derivados , Celulose/química , Celulose/metabolismo , Entropia , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Oligossacarídeos/metabolismo , Conformação Proteica , Tetroses/química , Tetroses/metabolismoRESUMO
Staphylococcus aureus is an opportunistic pathogen that has emerged as a major public health threat due to the increased incidence of its drug resistance. S. aureus presents a remarkable capacity to adapt to different niches due to the plasticity of its energy metabolism. In this work, we investigated the energy metabolism of S. aureus, focusing on the alternative NADH:quinone oxidoreductases, NDH-2s. S. aureus presents two genes encoding NDH-2s (NDH-2A and NDH-2B) and lacks genes coding for Complex I, the canonical respiratory NADH:quinone oxidoreductase. This observation makes the action of NDH-2s crucial for the regeneration of NAD+ and, consequently, for the progression of metabolism. Our study involved the comprehensive biochemical characterization of NDH-2B and the exploration of the cellular roles of NDH-2A and NDH-2B, utilizing knockout mutants (Δndh-2a and Δndh-2b). We show that NDH-2B uses NADPH instead of NADH, does not establish a charge-transfer complex in the presence of NADPH, and its reduction by this substrate is the catalytic rate-limiting step. In the case of NDH-2B, the reduction of the flavin is inherently slow, and we suggest the establishment of a charge transfer complex between NADP+ and FADH2, as previously observed for NDH-2A, to slow down quinone reduction and, consequently, prevent the overproduction of reactive oxygen species, which is potentially unnecessary. Furthermore, we observed that the lack of NDH-2A or NDH-2B impacts cell growth, volume, and division differently. The absence of these enzymes results in distinct metabolic phenotypes, emphasizing the unique cellular roles of each NDH-2 in energy metabolism.IMPORTANCEStaphylococcus aureus is an opportunistic pathogen, posing a global challenge in clinical medicine due to the increased incidence of its drug resistance. For this reason, it is essential to explore and understand the mechanisms behind its resistance, as well as the fundamental biological features such as energy metabolism and the respective players that allow S. aureus to live and survive. Despite its prominence as a pathogen, the energy metabolism of S. aureus remains underexplored, with its respiratory enzymes often escaping thorough investigation. S. aureus bioenergetic plasticity is illustrated by its ability to use different respiratory enzymes, two of which are investigated in the present study. Understanding the metabolic adaptation strategies of S. aureus to bioenergetic challenges may pave the way for the design of therapeutic approaches that interfere with the ability of the pathogen to successfully adapt when it invades different niches within its host.
Assuntos
Proteínas de Bactérias , NAD , Quinona Redutases , Staphylococcus aureus , Staphylococcus aureus/genética , Staphylococcus aureus/enzimologia , Staphylococcus aureus/metabolismo , NAD/metabolismo , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/genética , Quinona Redutases/metabolismo , Quinona Redutases/genética , NADP/metabolismo , Metabolismo Energético , OxirreduçãoRESUMO
Abnormal trinucleotide repeat expansions alter protein conformation causing malfunction and contribute to a significant number of incurable human diseases. Scarce structural insights available on disease-related homorepeat expansions hinder the design of effective therapeutics. Here, we present the dynamic structure of human PHOX2B C-terminal fragment, which contains the longest polyalanine segment known in mammals. The major α-helical conformation of the polyalanine tract is solely extended by polyalanine expansions in PHOX2B, which are responsible for most congenital central hypoventilation syndrome cases. However, polyalanine expansions in PHOX2B additionally promote nascent homorepeat conformations that trigger length-dependent phase transitions into solid condensates that capture wild-type PHOX2B. Remarkably, HSP70 and HSP90 chaperones specifically seize PHOX2B alternative conformations preventing phase transitions. The precise observation of emerging polymorphs in expanded PHOX2B postulates unbalanced phase transitions as distinct pathophysiological mechanisms in homorepeat expansion diseases, paving the way towards the search of therapeutics modulating biomolecular condensates in central hypoventilation syndrome.
Assuntos
Proteínas de Homeodomínio , Fatores de Transcrição , Animais , Humanos , Proteínas de Homeodomínio/metabolismo , Fatores de Transcrição/metabolismo , Peptídeos/genética , Peptídeos/química , Hipoventilação/genética , Hipoventilação/congênito , Mutação , Mamíferos/metabolismoRESUMO
Acetylcholinesterase (AChE) inhibition is one of the most currently available therapies for the management of Alzheimer's disease (AD) symptoms. In this context, NMR spectroscopy binding studies were accomplished to explain the inhibition of AChE activity by Salvia sclareoides extracts. HPLC-MS analyses of the acetone, butanol and water extracts eluted with methanol and acidified water showed that rosmarinic acid is present in all the studied samples and is a major constituent of butanol and water extracts. Moreover, luteolin 4'-O-glucoside, luteolin 3',7-di-O-glucoside and luteolin 7-O-(6''-O-acetylglucoside) were identified by MS(2) and MS(3) data acquired during the LC-MS(n) runs. Quantification of rosmarinic acid by HPLC with diode-array detection (DAD) showed that the butanol extract is the richest one in this component (134â µg mg(-1) extract). Saturation transfer difference (STD) NMR spectroscopy binding experiments of S. sclareoides crude extracts in the presence of AChE in buffer solution determined rosmarinic acid as the only explicit binder for AChE. Furthermore, the binding epitope and the AChE-bound conformation of rosmarinic acid were further elucidated by STD and transferred NOE effect (trNOESY) experiments. As a control, NMR spectroscopy binding experiments were also carried out with pure rosmarinic acid, thus confirming the specific interaction and inhibition of this compound against AChE. The binding site of AChE for rosmarinic acid was also investigated by STD-based competition binding experiments using Donepezil, a drug currently used to treat AD, as a reference. These competition experiments demonstrated that rosmarinic acid does not compete with Donepezil for the same binding site. A 3D model of the molecular complex has been proposed. Therefore, the combination of the NMR spectroscopy based data with molecular modelling has permitted us to detect a new binding site in AChE, which could be used for future drug development.