RESUMEN
The dietary consumption of extra virgin olive oil (EVOO) is believed to slow the progression of Alzheimer's disease (AD) symptoms. Its protective mechanisms are unclear, but specific EVOO phenolic compounds can individually impede the aggregation of amyloid-ß (Aß) peptides and the microtubule-associated protein tau, two important pathological manifestations of AD. It is unknown, however, whether the numerous and variable phenolic compounds that are consumed in dietary EVOO can collectively alter tau and Aß aggregation as effectively as the individual compounds. The activity of these complex mixtures against Aß and tau may be moderated by competition between active and nonactive phenolic components and by extensive derivatizations and isomerization. Here, phenolic mixtures extracted from two different EVOO sources are characterized and tested for how they modulate the aggregation of Aß40 peptide and tau peptides in vitro. The chromatographic and NMR analysis of Greek and Saudi Arabian EVOO phenolic extracts reveals that they have different concentration profiles, and over 30 compounds are identified. Thioflavin T fluorescence and circular dichroism measurements show that relatively low concentrations (<20 µg/mL) of the Greek and Saudi extracts reduce the rate of Aß40 aggregation and fibril mass, despite the extracts having different phenolic profiles. By contrast, the Greek extract reduces the rate of tau aggregation only at very high phenolic concentrations (>100 µg/mL). Most compounds in the extracts bind to preformed Aß40 fibrils and release soluble Aß oligomers that are mildly toxic to SH-SY5Y cells. Much higher (500 µg/mL) extract concentrations are required to remodel tau filaments into oligomers, and a minimal binding of phenolic compounds to the preformed filaments is observed. It is concluded that EVOO extracts having different phenol profiles are similarly capable of modulating Aß40 aggregation and fibril morphology in vitro at relatively low concentrations but are less efficient at modulating tau aggregation. Over 2 M tonnes of EVOO are consumed globally each year as part of the Mediterranean diet, and the results here provide motivation for further clinical interrogation of the antiaggregation properties of EVOO as a potential protective mechanism against AD.
RESUMEN
Structures of membrane proteins determined by X-ray crystallography and, increasingly, by cryo-electron microscopy often fail to resolve the structural details of unstable or reactive small molecular ligands in their physiological sites. This work demonstrates that 13C chemical shifts measured by magic-angle spinning (MAS) solid-state NMR (SSNMR) provide unique information on the conformation of a labile ligand in the physiological site of a functional protein in its native membrane, by exploiting freeze-trapping to stabilise the complex. We examine the ribose conformation of ATP in a high affinity complex with Na,K-ATPase (NKA), an enzyme that rapidly hydrolyses ATP to ADP and inorganic phosphate under physiological conditions. The 13C SSNMR spectrum of the frozen complex exhibits peaks from all ATP ribose carbon sites and some adenine base carbons. Comparison of experimental chemical shifts with density functional theory (DFT) calculations of ATP in different conformations and protein environments reveals that the ATP ribose ring adopts an C3'-endo (N) conformation when bound with high affinity to NKA in the E1Na state, in contrast to the C2'-endo (S) ribose conformations of ATP bound to the E2P state and AMPPCP in the E1 complex. Additional dipolar coupling-mediated measurements of H-C-C-H torsional angles are used to eliminate possible relative orientations of the ribose and adenine rings. The utilization of chemical shifts to determine membrane protein ligand conformations has been underexploited to date and here we demonstrate this approach to be a powerful tool for resolving the fine details of ligand-protein interactions.
RESUMEN
Cholesterol is an essential component of eukaryotic cellular membranes that regulates the order and phase behaviour of dynamic lipid bilayers. Although cholesterol performs many vital physiological roles, hypercholesterolaemia and the accumulation of cholesterol in atherosclerotic plaques can increase the risk of coronary heart disease morbidity. The risk is mitigated by the transportation of cholesterol from peripheral tissue to the liver by high-density lipoprotein (HDL), 6-20 nm-diameter particles of lipid bilayers constrained by an annular belt of the protein apolipoprotein A-I (apoA-I). Information on the dynamics and orientation of cholesterol in HDL is pertinent to the essential role of HDL in cholesterol cycling. This work investigates whether the molecular orientation of cholesterol in HDL differs from that in the unconstrained lipid bilayers of multilamellar vesicles (MLVs). Solid-state NMR (ssNMR) measurements of dynamically-averaged 13C-13C and 13C-1H dipolar couplings were used to determine the average orientation of triple 13C-labelled cholesterol in palmitoyloleoylphosphatidylcholine (POPC) lipid bilayers in reconstituted HDL (rHDL) nanodiscs and in MLVs. Individual 13C-13C dipolar couplings were measured from [2,3,4-13C3]cholesterol in a one-dimensional NMR experiment, by using a novel application of a method to excite double quantum coherence at rotational resonance. The measured dipolar couplings were compared with average values calculated from orientational distributions of cholesterol generated using a Gaussian probability density function. The data were consistent with small differences in the average orientation of cholesterol in rHDL and MLVs, which may reflect the effects of the constrained and unconstrained lipid bilayers in the two environments. The calculated distributions of cholesterol in rHDL and MLVs that were consistent with the NMR data also agreed well with orientational distributions extracted from previous molecular dynamics simulations of HDL nanodiscs and planar POPC bilayers.
Asunto(s)
Apolipoproteína A-I , Lipoproteínas HDL , Apolipoproteína A-I/química , Apolipoproteína A-I/metabolismo , Apolipoproteína A-I/farmacología , Colesterol , Membrana Dobles de Lípidos/química , Lipoproteínas HDL/química , Lipoproteínas HDL/metabolismo , Espectroscopía de Resonancia MagnéticaRESUMEN
Drug interactions with phospholipid bilayers underpin their behaviour in cell membranes and in liposomal delivery formulations. Liposomal drug delivery in ocular medicine can overcome the physical barriers of the eye and better enable the active molecule to reach its target. Here, Raman and 19 F solid-state NMR spectroscopy are used to characterise the interactions of two ocular corticosteroid drugs, difluprednate (DFP) and fluorometholone (FML), with multilamellar vesicles of phosphatidylcholine (PC). 31 P NMR confirms that the lipid bilayer tolerates a high drug concentration (a drug: lipid molar ratio of 1 : 10). The 19 F NMR spectra of the drugs in lipid bilayers reveal that FML and DFP have different average orientations within the lipid bilayer. Raman spectra of dried lipid films reveal that PC separates from DFP but not from FML, the less lipophilic of the two drugs. This combined approach will assist the design of, and inform the development of, improved liposomal preparations.
Asunto(s)
Membrana Dobles de Lípidos , Preparaciones Farmacéuticas , Corticoesteroides , Interacciones Farmacológicas , Espectroscopía de Resonancia MagnéticaRESUMEN
A pathological signature of Alzheimer's disease (AD) is the formation of neurofibrillary tangles comprising filamentous aggregates of the microtubule associated protein tau. Tau self-assembly is accelerated by polyanions including heparin, an analogue of heparan sulfate. Tau filaments colocalize with heparan sulfate proteoglycans (HSPGs) in vivo, and HSPGs may also assist the transcellular propagation of tau aggregates. Here, we investigate the role of the sulfate moieties of heparin in the aggregation of a recombinant tau fragment Δtau187, comprising residues 255-441 of the C-terminal microtubule-binding domain. The effects that the selective removal of the N-, 2-O-, and 6-O-sulfate groups from heparin have on the kinetics of tau aggregation, aggregate morphology, and protein structure and dynamics were examined. Aggregation kinetics monitored by thioflavin T (ThT) fluorescence revealed that aggregation is considerably slower in the presence of 2-O-desulfated heparin than with N- or 6-O-desulfated heparin. Transmission electron microscopy revealed that tau filaments induced by 2-O-desulfated heparin were more slender than filaments formed in the presence of intact heparin or 6-O-desulfated heparin. The 2-O-desulfated heparin-induced filaments had more extensive regions of flexibility than the other filaments, according to circular dichroism and solid-state NMR spectroscopy. These results indicate that the sulfation pattern of heparin regulates tau aggregation, not purely though electrostatic forces but also through conformational perturbations of heparin when the 2-O-sulfate is removed. These findings may have implications for the progression of AD, as the sulfation pattern of GAGs is known to change during the aging process, which is the main risk factor for the disease.
Asunto(s)
Heparina/metabolismo , Proteínas tau/química , Proteínas tau/metabolismo , Benzotiazoles/química , Benzotiazoles/metabolismo , Heparina/química , Heparitina Sulfato/química , Heparitina Sulfato/metabolismo , Humanos , Cinética , Espectroscopía de Resonancia Magnética , Microscopía Electrónica de Transmisión , Ovillos Neurofibrilares/metabolismo , Conformación Proteica , Proteínas tau/ultraestructuraRESUMEN
It has been shown extensively that glycosaminoglycan (GAG)-protein interactions can induce, accelerate, and impede the clearance of amyloid fibrils associated with systemic and localized amyloidosis. Obtaining molecular details of these interactions is fundamental to our understanding of amyloid disease. Consequently, there is a need for analytical approaches that can identify protein conformational transitions and simultaneously characterize heparin interactions. By combining Raman spectroscopy with two-dimensional (2D) perturbation correlation moving window (2DPCMW) analysis, we have successfully identified changes in protein secondary structure during pH- and heparin-induced fibril formation of apolipoprotein A-I (apoA-I) associated with atherosclerosis. Furthermore, from the 2DPCMW, we have identified peak shifts and intensity variations in Raman peaks arising from different heparan sulfate moieties, indicating that protein-heparin interactions vary at different heparin concentrations. Raman spectroscopy thus reveals new mechanistic insights into the role of GAGs during amyloid fibril formation.
Asunto(s)
Amiloide/química , Apolipoproteína A-I/química , Heparina/química , Espectrometría Raman/métodos , Apolipoproteína A-I/genética , Apolipoproteína A-I/metabolismo , Heparina/metabolismo , Humanos , Agregado de Proteínas , Unión Proteica , Estructura Secundaria de Proteína , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificaciónRESUMEN
The biological function of high-density lipoprotein (HDL) nanoparticles, the so-called good cholesterol that is associated with a low risk of heart disease, depends on their composition, morphology, and size. The morphology of HDL particles composed of apolipoproteins, lipids and cholesterol is routinely visualised by transmission electron microscopy (TEM), but higher-resolution tools are needed to observe more subtle structural differences between particles of different composition. Here, reconstituted HDL formulations are oriented on glass substrates and solid-state 31 Pâ NMR spectroscopy is shown to be highly sensitive to the surface curvature of the lipid headgroups. The spectra report potentially functionally important differences in the morphology of different HDL preparations that are not detected by TEM. This method provides new morphological insights into HDL comprising a naturally occurring apolipoprotein A-I mutant, which may be linked to its atheroprotective properties, and holds promise as a future research tool in the clinical analysis of plasma HDL.
Asunto(s)
Lipoproteínas HDL/química , Espectroscopía de Resonancia Magnética/métodos , Nanopartículas/química , Isótopos de Fósforo/química , Colesterol/química , Microscopía Electrónica de Transmisión , Fosfatidilcolinas/químicaRESUMEN
Potential drug treatments for Alzheimer's disease (AD) may be found by identifying compounds that block the assembly of the microtubule-associated protein tau into neurofibrillar tangles associated with neuron destabilization and cell death. Here, a small library of structurally diverse compounds was screened in vitro for the ability to inhibit tau aggregation, using high-throughput synchrotron radiation circular dichroism as a novel tool to monitor the structural changes in the protein as it assembles into filaments. The catecholamine epinephrine was found to be the most effective tau aggregation inhibitor of all 88 screened compounds. Subsequently, we tested chemically similar phenolamine drugs from the ß-adrenergic receptor agonist class, using conventional circular dichroism spectroscopy, thioflavin T fluorescence, and transmission electron microscopy. Two compounds, salbutamol and dobutamine, used widely in the treatment of respiratory and cardiovascular disease, impede the aggregation of tau in vitro. Dobutamine reduces both the rate and yield of tau filament formation over 24 h; however, it has little effect on the structural transition of tau into ß-sheet structures over 24 h. Salbutamol also reduces the yield and rate of filament formation and additionally inhibits tau's structural change into ß-sheet-rich aggregates. Salbutamol has a good safety profile and a half-life that facilitates permeation through the blood-brain barrier and could represent an expediated approach to developing AD therapeutics. These results provide the motivation for the in vivo evaluation of pre-existing ß-adrenergic receptor agonists as a potential therapy for AD through the reduction of tau deposition.
Asunto(s)
Albuterol , Enfermedad de Alzheimer , Agonistas Adrenérgicos beta , Albuterol/farmacología , Dicroismo Circular , Humanos , Receptores Adrenérgicos , Proteínas tauRESUMEN
NMR measurements of 19F chemical shift anisotropy and 1H-19F dipolar couplings provide unprecedented information on the molecular orientations of two fluorine-containing statin drugs within the heterogeneous environment of reconstituted high-density lipoprotein (rHDL) nanoparticles, a drug delivery system under clinical investigation.
Asunto(s)
Inhibidores de Hidroximetilglutaril-CoA Reductasas/química , Lipoproteínas/química , Nanopartículas/química , Sistemas de Liberación de Medicamentos , Flúor , Espectroscopía de Resonancia Magnética , Modelos Moleculares , Estructura Molecular , Tamaño de la Partícula , Propiedades de SuperficieRESUMEN
With amyloid diseases poised to become a major health burden in countries with aging populations, diagnostic molecules that aid the detection of amyloid in vitro and in vivo are of considerable clinical value. Understanding how such ligands recognize their amyloid targets would help to design diagnostics that target specific amyloid types associated with a particular disease, but methods to provide comprehensive information are underdeveloped. Here, solid-state NMR is used to determine the molecular orientation of the amyloid diagnostic 1-fluoro-2,5-bis[( E)-3-carboxy-4-hydroxystyryl]-benzene (FSB) when bound to fibrils of the Alzheimer's amyloid-ß polypeptide aligned on a planar substrate. The 19F NMR spectrum of the aligned complex reveals that FSB is oriented approximately parallel with the fibril long axis and bridges four hydrogen-bonded ß-sheets. In addition to providing atomic details to aid the design of amyloid-specific diagnostics, this approach will also illuminate the molecular mechanisms of accessory molecules in amyloid disease.
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Péptidos beta-Amiloides/metabolismo , Amiloide/metabolismo , Colorantes Fluorescentes/metabolismo , Fragmentos de Péptidos/metabolismo , Estirenos/metabolismo , Isótopos de Carbono , Fluorescencia , Colorantes Fluorescentes/química , Ligandos , Espectroscopía de Resonancia Magnética/métodos , Simulación del Acoplamiento Molecular , Isótopos de Nitrógeno , Unión Proteica , Estirenos/químicaRESUMEN
Fluorine is often incorporated into the aromatic moieties of synthetic bioactive molecules such as pharmaceuticals and disease diagnostics in order to alter their physicochemical properties. Fluorine substitution may increase a molecule's lipophilicity, thereby enabling its diffusion across cell membranes to enhance bioavailability or to exert a direct physiological effect from within the lipid bilayer. Understanding the structure, dynamics and orientation of fluoroaromatic molecules in lipid bilayers can provide useful insight into the effect of fluorine on their mode of action, and their interactions with membrane-embedded targets or efflux proteins. Here we demonstrate that NMR measurements of 19F chemical shift anisotropy combined with 1H-19F dipolar coupling measurements together report on the average orientation of a lipophilic fluoroaromatic molecule, 4-(6-fluorobenzo[d]thiazol-2-yl)aniline (FBTA), rapidly rotating within a lipid bilayer. The 19F chemical shift tensor orientation in the molecular frame was calculated by density functional theory and corroborated by 1H-19F PISEMA NMR. It was then possible to analyse the line shapes of proton-coupled and proton-decoupled 19F spectra of FBTA in chain perdeuterated dimyristoylphosphatidylcholine (DMPC-d54) bilayers to restrict the average axis of molecular reorientation of FBTA in the bilayer to a limited range orientations. This approach, which exploits the high sensitivity and gyromagnetic ratios of 19F and 1H, will be useful for comparing the membrane properties of related bioactive fluoroaromatic compounds.
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Compuestos de Anilina/química , Membrana Dobles de Lípidos/química , Compuestos Orgánicos/química , Tiazoles/química , Anisotropía , Difusión , Dimiristoilfosfatidilcolina/química , Flúor , Hidrógeno , Interacciones Hidrofóbicas e Hidrofílicas , Espectroscopía de Resonancia Magnética , Modelos Moleculares , Estructura MolecularRESUMEN
Amyloid deposits of WT apolipoprotein A-I (apoA-I), the main protein component of high-density lipoprotein, accumulate in atherosclerotic plaques where they may contribute to coronary artery disease by increasing plaque burden and instability. Using CD analysis, solid-state NMR spectroscopy, and transmission EM, we report here a surprising cooperative effect of heparin and the green tea polyphenol (-)-epigallocatechin-3-gallate (EGCG), a known inhibitor and modulator of amyloid formation, on apoA-I fibrils. We found that heparin, a proxy for glycosaminoglycan (GAG) polysaccharides that co-localize ubiquitously with amyloid in vivo, accelerates the rate of apoA-I formation from monomeric protein and associates with insoluble fibrils. Mature, insoluble apoA-I fibrils bound EGCG (KD = 30 ± 3 µm; Bmax = 40 ± 3 µm), but EGCG did not alter the kinetics of apoA-I amyloid assembly from monomer in the presence or absence of heparin. EGCG selectively increased the mobility of specific backbone and side-chain sites of apoA-I fibrils formed in the absence of heparin, but the fibrils largely retained their original morphology and remained insoluble. By contrast, fibrils formed in the presence of heparin were mobilized extensively by the addition of equimolar EGCG, and the fibrils were remodeled into soluble 20-nm-diameter oligomers with a largely α-helical structure that were nontoxic to human umbilical artery endothelial cells. These results argue for a protective effect of EGCG on apoA-I amyloid associated with atherosclerosis and suggest that EGCG-induced remodeling of amyloid may be tightly regulated by GAGs and other amyloid co-factors in vivo, depending on EGCG bioavailability.
Asunto(s)
Amiloide/química , Apolipoproteína A-I/química , Catequina/análogos & derivados , Heparina/química , Catequina/química , Humanos , Espectroscopía de Resonancia Magnética , Estructura Secundaria de ProteínaRESUMEN
The Aß peptide forms extracellular plaques associated with Alzheimer's disease. In addition to protein fibrils, amyloid plaques also contain non-proteinaceous components, including glycosaminoglycans (GAGs). We have shown previously that the GAG low-molecular-weight heparin (LMWH) binds to Aß40 fibrils with a three-fold-symmetric (3Q) morphology with higher affinity than Aß40 fibrils in alternative structures, Aß42 fibrils, or amyloid fibrils formed from other sequences. Solid-state NMR analysis of the GAG-3Q fibril complex revealed an interaction site at the corners of the 3Q fibril structure, but the origin of the binding specificity remained obscure. Here, using a library of short heparin polysaccharides modified at specific sites, we show that the N-sulfate or 6-O-sulfate of glucosamine, but not the 2-O-sulfate of iduronate within heparin is required for 3Q binding, indicating selectivity in the interactions of the GAG with the fibril that extends beyond general electrostatic complementarity. By creating 3Q fibrils containing point substitutions in the amino acid sequence, we also show that charged residues at the fibril three-fold apices provide the majority of the binding free energy, while charged residues elsewhere are less critical for binding. The results indicate, therefore, that LMWH binding to 3Q fibrils requires a precise molecular complementarity of the sulfate moieties on the GAG and charged residues displayed on the fibril surface. Differences in GAG binding to fibrils with distinct sequence and/or structure may thus contribute to the diverse etiology and progression of amyloid diseases.
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Péptidos beta-Amiloides/metabolismo , Amiloide/metabolismo , Heparina de Bajo-Peso-Molecular/metabolismo , Fragmentos de Péptidos/metabolismo , Péptidos beta-Amiloides/genética , Análisis Mutacional de ADN , Humanos , Espectroscopía de Resonancia Magnética , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Fragmentos de Péptidos/genética , Unión ProteicaRESUMEN
Peptides derived from apolipoprotein A-I (apoA-I), the main component of high-density lipoprotein (HDL), constitute the main component of amyloid deposits that colocalize with atherosclerotic plaques. Here we investigate the molecular details of full-length, lipid-deprived apoA-I after assembly into insoluble aggregates under physiologically relevant conditions known to induce aggregation in vitro. Unmodified apoA-I is shown to remain soluble at pH 7 for at least 3 days, retaining its native α-helical-rich structure. Upon acidification to pH 4, apoA-I rapidly assembles into insoluble nonfibrillar aggregates lacking the characteristic cross-ß features of amyloid. In the presence of heparin, the rate and thioflavin T responsiveness of the aggregates formed at pH 4 increase and short amyloid-like fibrils are observed, which give rise to amyloid-characteristic X-ray reflections at 4.7 and 10 Å. Solid-state nuclear magnetic resonance (SSNMR) and synchrotron radiation circular dichroism spectroscopy of fibrils formed in the presence of heparin show they retain some α-helical characteristics together with new ß-sheet structures. Interestingly, SSNMR indicates a similar molecular structure of aggregates formed in the absence of heparin at pH 6 after oxidation of the three methionine residues, although their morphology is rather different from that of the heparin-derived fibrils. We propose a model for apoA-I aggregation in which perturbations of a four-helix bundle-like structure, induced by interactions of heparin or methionine oxidation, cause the partially helical N-terminal residues to disengage from the remaining, intact helices, thereby allowing self-assembly via ß-strand associations.
Asunto(s)
Proteínas Amiloidogénicas/química , Apolipoproteína A-I/química , Heparina/química , Metionina/química , Agregado de Proteínas , Proteínas Amiloidogénicas/metabolismo , Proteínas Amiloidogénicas/ultraestructura , Apolipoproteína A-I/metabolismo , Apolipoproteína A-I/ultraestructura , Benzotiazoles , Clonación Molecular , Escherichia coli/genética , Escherichia coli/metabolismo , Expresión Génica , Heparina/metabolismo , Humanos , Peróxido de Hidrógeno/química , Concentración de Iones de Hidrógeno , Metionina/metabolismo , Oxidación-Reducción , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/ultraestructura , Espectrometría de Fluorescencia , Tiazoles/químicaRESUMEN
Cardiotonic steroids (CTS) are clinically important drugs for the treatment of heart failure owing to their potent inhibition of cardiac Na(+), K(+)-ATPase (NKA). Bufadienolides constitute one of the two major classes of CTS, but little is known about how they interact with NKA. We report a remarkable stereoselectivity of NKA inhibition by native 3ß-hydroxy bufalin over the 3α-isomer, yet replacing the 3ß-hydroxy group with larger polar groups in the same configuration enhances inhibitory potency. Binding of the two (13)C-labelled glycosyl diastereomers to NKA were studied by solid-state NMR (SSNMR), which revealed interactions of the glucose group of the 3ß- derivative with the inhibitory site, but much weaker interactions of the 3α- derivative with the enzyme. Molecular docking simulations suggest that the polar 3ß-groups are closer to the hydrophilic amino acid residues in the entrance of the ligand-binding pocket than those with α-configuration. These first insights into the stereoselective inhibition of NKA by bufadienolides highlight the important role of the hydrophilic moieties at C3 for binding, and may explain why only 3ß-hydroxylated bufadienolides are present as a toxic chemical defence in toad venom.
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Bufanólidos/química , Bufanólidos/farmacología , Glicósidos Cardíacos/farmacología , ATPasa Intercambiadora de Sodio-Potasio/antagonistas & inhibidores , Animales , Bufanólidos/síntesis química , Bufonidae , Isótopos de Carbono , Espectroscopía de Resonancia Magnética con Carbono-13 , Glicósidos Cardíacos/química , Cromatografía Líquida de Alta Presión , Cristalografía por Rayos X , Simulación del Acoplamiento Molecular , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , Estereoisomerismo , TemperaturaRESUMEN
The amyloid plaques associated with Alzheimer's disease (AD) comprise fibrillar amyloid-ß (Aß) peptides as well as non-protein factors including glycosaminoglycan (GAG) polysaccharides. GAGs affect the kinetics and pathway of Aß self-assembly and can impede fibril clearance; thus, they may be accessory molecules in AD. Here we report the first high-resolution details of GAG-Aß fibril interactions from the perspective of the saccharide. Binding analysis indicated that the GAG proxy heparin has a remarkably high affinity for Aß fibrils with 3-fold cross-sectional symmetry (3Q). Chemical synthesis of a uniformly (13)C-labeled octasaccharide heparin analogue enabled magic-angle spinning solid-state NMR of the GAG bound to 3Q fibrils, and measurements of dynamics revealed a tight complex in which all saccharide residues are restrained without undergoing substantial conformational changes. Intramolecular (13)C-(15)N dipolar dephasing is consistent with close (<5 Å) contact between GAG anomeric position(s) and one or more histidine residues in the fibrils. These data provide a detailed model for the interaction between 3Q-seeded Aß40 fibrils and a major non-protein component of AD plaques, and they reveal that GAG-amyloid interactions display a range of affinities that critically depend on the precise details of the fibril architecture.
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Péptidos beta-Amiloides/química , Péptidos beta-Amiloides/metabolismo , Glicosaminoglicanos/metabolismo , Fragmentos de Péptidos/química , Fragmentos de Péptidos/metabolismo , Modelos Moleculares , Resonancia Magnética Nuclear Biomolecular , Estructura Secundaria de ProteínaRESUMEN
This paper addresses the question of long-range interactions between the intramembranous cation binding sites and the cytoplasmic nucleotide binding site of the ubiquitous ion-transporting Na,K-ATPase using (13)C cross-polarization magic-angle spinning (CP-MAS) solid-state nuclear magnetic resonance. High-affinity ATP binding is induced by the presence of Na(+) as well as of Na-like substances such as Tris(+), and these ions are equally efficient promoters of nucleotide binding. CP-MAS analysis of bound ATP with Na,K-ATPase purified from pig kidney membranes reveals subtle differences in the nucleotide interactions within the nucleotide site depending on whether Na(+) or Tris(+) is used to induce binding. Differences in chemical shifts for ATP atoms C1' and C5' observed in the presence of Na(+) or Tris(+) suggest alterations in the residues surrounding the bound nucleotide, hydrogen bonding, and/or conformation of the ribose ring. This is taken as evidence of a long-distance communication between the Na(+)-filled ion sites in the membrane interior and the nucleotide binding site in the cytoplasmic domain and reflects the first conformational change ultimately leading to phosphorylation of the enzyme. Stopped-flow fluorescence measurements with the nucleotide analogue eosin show that the dissociation rate constant for eosin is larger in Tris(+) than in Na(+), giving kinetic evidence of the difference in structural effects of Na(+) and Tris(+). According to the recent crystal structure of the E1·AlF4(-)·ADP·3Na(+) form, the coupling between the ion binding sites and the nucleotide side is mediated by, among others, the M5 helix.
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Adenosina Trifosfato/metabolismo , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , Sodio/metabolismo , Adenosina Difosfato/metabolismo , Animales , Sitios de Unión , Cationes Monovalentes/metabolismo , Modelos Moleculares , Resonancia Magnética Nuclear Biomolecular , Potasio/metabolismo , Unión Proteica , ATPasa Intercambiadora de Sodio-Potasio/química , PorcinosRESUMEN
NMR relaxation enhancement by paramagnetic metals provides powerful restraints on the three-dimensional structures of proteins in solution, and this approach has recently been utilized in several NMR structural investigations of proteins in the solid-state. Here we utilize paramagnetic relaxation enhancement (PRE) by Mn(2+) with cross-polarization magic-angle spinning (CP-MAS) solid-state NMR to investigate the interaction of a membrane-embedded protein the Na,K-ATPase (NKA) with a cardiotonic steroid inhibitor. The inhibitor, a diacetonide derivate of the cardiac glycoside ouabain, with (13)C labelled acetonide groups in the rhamnose sugar and steroid moieties ([(13)C2]ODA), is 1000-fold less potent than the parent compound. It is shown that the (13)C CP-MAS solid-state NMR spectra of the NKA-[(13)C2]ODA complex exhibit distinct signals for the two (13)C labels of the inhibitor when bound to the ouabain site of membrane-embedded NKA. Recent crystal structures of NKA indicate that the catalytic α-subunit binds a single Mn(2+) in a transmembrane site close to the high-affinity ouabain site. Here, complexation of NKA with Mn(2+) broadens the resonance line from the rhamnose group substantially more than the steroid peak, indicating that the rhamnose group is closer to the Mn(2+) site than is the steroid group. These observations agree with computational molecular docking simulations and are consistent with ODA adopting an inverted orientation compared to ouabain in the cardiac glycoside site, with the modified rhamnose group drawn toward the transmembrane centre of the protein. This work demonstrates that PRE can provide unique information on the positions and orientations of ligands within their binding pockets of transmembrane proteins.
Asunto(s)
Inhibidores Enzimáticos/farmacología , Ouabaína/farmacología , ATPasa Intercambiadora de Sodio-Potasio/antagonistas & inhibidores , Inhibidores Enzimáticos/química , Ligandos , Espectroscopía de Resonancia Magnética , Conformación Molecular , Simulación del Acoplamiento Molecular , Ouabaína/química , ATPasa Intercambiadora de Sodio-Potasio/química , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , Relación Estructura-ActividadRESUMEN
Aortic medial amyloid (AMA) is the most common localized human amyloid, occurring in virtually all of the Caucasian population over the age of 50. The main protein component of AMA, medin, readily assembles into amyloid-like fibrils in vitro. Despite the prevalence of AMA, little is known about the self-assembly mechanism of medin or the molecular architecture of the fibrils. The amino acid sequence of medin is strikingly similar to the sequence of the Alzheimer disease (AD) amyloid-ß (Aß) polypeptides around the structural turn region of Aß, where mutations associated with familial, early onset AD, have been identified. Asp(25) and Lys(30) of medin align with residues Asp(23) and Lys(28) of Aß, which are known to form a stabilizing salt bridge in some fibril morphologies. Here we show that substituting Asp(25) of medin with asparagine (D25N) impedes assembly into fibrils and stabilizes non-cytotoxic oligomers. Wild-type medin, by contrast, aggregates into ß-sheet-rich amyloid-like fibrils within 50 h. A structural analysis of wild-type fibrils by solid-state NMR suggests a molecular repeat unit comprising at least two extended ß-strands, separated by a turn stabilized by a Asp(25)-Lys(30) salt bridge. We propose that Asp(25) drives the assembly of medin by stabilizing the fibrillar conformation of the peptide and is thus reminiscent of the influence of Asp(23) on the aggregation of Aß. Pharmacological comparisons of wild-type medin and D25N will help to ascertain the pathological significance of this poorly understood protein.
Asunto(s)
Antígenos de Superficie/metabolismo , Aorta/metabolismo , Ácido Aspártico/metabolismo , Proteínas de la Leche/metabolismo , Secuencia de Aminoácidos , Antígenos de Superficie/química , Ácido Aspártico/química , Dicroismo Circular , Humanos , Proteínas de la Leche/química , Datos de Secuencia Molecular , Homología de Secuencia de AminoácidoRESUMEN
Five new cardenolide lactates (15) and one new dioxane double linked cardenolide glycoside (17) along with 15 known compounds (616 and 1821) were isolated from the ornamental milkweed Asclepias curassavica. Their structures were elucidated by extensive spectroscopic methods (IR, UV, MS, 1D- and 2D-NMR). The molecular structures and absolute configurations of 13 and 17 were further confirmed by single-crystal X-ray diffraction analysis. Simultaneous isolation of dioxane double linked cardenolide glycosides (1721) and cardenolide lactates (15) provided unique chemotaxonomic markers for this genus. Compounds 121 were evaluated for the inhibitory activities against DU145 prostate cancer cells. The dioxane double linked cardenolide glycosides showed the most potent cytotoxic effect followed by normal cardenolides and cardenolide lactates, while the C21 steroids were non-cytotoxic. Enzymatic assay established a correlation between the cytotoxic effects in DU145 cancer cells and the Ki for the inhibition of Na(+),K(+)-ATPase. Molecular docking analysis revealed relatively strong H-bond interactions between the bottom of the binding cavity and compounds 18 or 20, and explained why the dioxane double linked cardenolide glycosides possessed higher inhibitory potency on Na(+),K(+)-ATPase than the cardenolide lactate.