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This study delves into the early aggregation process of the Aß1-40 amyloid peptide, elucidating the associated oligomers distribution. Motivated by the acknowledged role of small oligomers in the neurotoxic damage linked to Alzheimer's disease, we present an experimental protocol for preparing 26-O-acyl isoAß1-40, a modified Aß1-40 peptide facilitating rapid isomerization to the native amide form at neutral pH. This ensures seed-free solutions, minimizing experimental variability. Additionally, we demonstrate the efficacy of coupling NMR diffusion ordered spectroscopy (DOSY) with the Inverse Laplace Transform (ILT) reconstruction method, for effective characterization of early aggregation processes. This innovative approach efficiently maps oligomers distributions across a wide spectrum of initial peptide concentrations offering unique insights into the evolution of oligomers relative populations. As a proof of concept, we demonstrate the efficacy of our approach assessing the impact of Epigallocathechin gallate, a known remodeling agent of amyloid fibrils, on the oligomeric distributions of aggregated Aß1-40. The DOSY-ILT proposed approach stands as a robust and discriminating asset, providing a powerful strategy for rapidly gaining insight into potential inhibitors' impact on the aggregation process.
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Péptidos beta-Amiloides , Fragmentos de Péptidos , Péptidos beta-Amiloides/química , Péptidos beta-Amiloides/metabolismo , Fragmentos de Péptidos/química , Fragmentos de Péptidos/metabolismo , Catequina/química , Catequina/análogos & derivados , Agregado de Proteínas , Humanos , Enfermedad de Alzheimer/metabolismo , Resonancia Magnética Nuclear Biomolecular/métodos , Espectroscopía de Resonancia Magnética/métodos , Amiloide/química , Amiloide/metabolismoRESUMEN
The aberrant activation of the fibroblast growth factor 2 (FGF2)/fibroblast growth factor receptor (FGFR) signalling pathway drives severe pathologies, including cancer development and angiogenesis-driven pathologies. The perturbation of the FGF2/FGFR axis via extracellular allosteric small inhibitors is a promising strategy for developing FGFR inhibitors with improved safety and efficacy for cancer treatment. We have previously investigated the role of new extracellular inhibitors, such as rosmarinic acid (RA), which bind the FGFR-D2 domain and directly compete with FGF2 for the same binding site, enabling the disruption of the functional FGF2/FGFR interaction. To select ligands for the previously identified FGF2/FGFR RA binding site, NMR data-driven virtual screening has been performed on an in-house library of non-commercial small molecules and metabolites. A novel drug-like compound, a resorcinol derivative named RBA4 has been identified. NMR interaction studies demonstrate that RBA4 binds the FGF2/FGFR complex, in agreement with docking prediction. Residue-level NMR perturbations analysis highlights that the mode of action of RBA4 is similar to RA in terms of its ability to target the FGF2/FGFR-D2 complex, inducing perturbations on both proteins and triggering complex dissociation. Biological assays proved that RBA4 inhibited FGF2 proliferative activity at a level comparable to the previously reported natural product, RA. Identification of RBA4 chemical groups involved in direct interactions represents a starting point for further optimization of drug-like extracellular inhibitors with improved activity.
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Factor 2 de Crecimiento de Fibroblastos , Neoplasias , Humanos , Factor 2 de Crecimiento de Fibroblastos/antagonistas & inhibidores , Espectroscopía de Resonancia Magnética , Receptores de Factores de Crecimiento de Fibroblastos/antagonistas & inhibidores , Receptores de Factores de Crecimiento de Fibroblastos/metabolismo , Transducción de Señal , Resorcinoles/química , Resorcinoles/farmacologíaRESUMEN
BACKGROUND: Cells exposed to stress factors experience time-dependent variations of metabolite concentration, acting as reliable sensors of the effective concentration of drugs in solution. NMR can detect and quantify changes in metabolite concentration, thus providing an indirect estimate of drug concentration. The quantification of bactericidal molecules released from antimicrobial-treated biomedical materials is crucial to determine their biocompatibility and the potential onset of drug resistance. METHODS: Real-time NMR measurements of extracellular metabolites produced by bacteria grown in the presence of known concentrations of an antibacterial molecule (irgasan) are employed to quantify the bactericidal molecule released from antimicrobial-treated biomedical devices. Viability tests assess their activity against E. coli and S. aureus planktonic and sessile cells. AFM and contact angle measurements assisted in the determination of the mechanism of antibacterial action. RESULTS: NMR-derived concentration kinetics of metabolites produced by bacteria grown in contact with functionalized materials allows for indirectly evaluating the effective concentration of toxic substances released from the device, lowering the detection limit to the nanomolar range. NMR, AFM and contact angle measurements support a surface-killing mechanism of action against bacteria. CONCLUSIONS: The NMR based approach provides a reliable tool to estimate bactericidal molecule release from antimicrobial materials. GENERAL SIGNIFICANCE: The novelty of the proposed NMR-based strategy is that it i) exploits bacteria as sensors of the presence of bactericidal molecules in solution; ii) is independent of the chemo-physical properties of the analyte; iii) establishes the detection limit to nanomolar concentrations.
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Antiinfecciosos , Staphylococcus aureus , Escherichia coli , Antibacterianos/farmacología , Antibacterianos/química , Bacterias , Antiinfecciosos/farmacologíaRESUMEN
NMR-based approaches play a pivotal role in providing insight into molecular recognition mechanisms, affording the required atomic-level description and enabling the identification of promising inhibitors of protein-protein interactions. The aberrant activation of the fibroblast growth factor 2 (FGF2)/fibroblast growth factor receptor (FGFR) signaling pathway drives several pathologies, including cancer development, metastasis formation, resistance to therapy, angiogenesis-driven pathologies, vascular diseases, and viral infections. Most FGFR inhibitors targeting the intracellular ATP binding pocket of FGFR have adverse effects, such as limited specificity and relevant toxicity. A viable alternative is represented by targeting the FGF/FGFR extracellular interactions. We previously identified a few small-molecule inhibitors acting extracellularly, targeting FGFR or FGF. We have now built a small library of natural and synthetic molecules that potentially act as inhibitors of FGF2/FGFR interactions to improve our understanding of the molecular mechanisms of inhibitory activity. Here, we provide a comparative analysis of the interaction mode of small molecules with the FGF2/FGFR complex and the single protein domains. DOSY and residue-level NMR analysis afforded insights into the capability of the potential inhibitors to destabilize complex formation, highlighting different mechanisms of inhibition of FGF2-induced cell proliferation.
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Factor 2 de Crecimiento de Fibroblastos , Neoplasias , Adenosina Trifosfato/farmacología , Comprensión , Factor 2 de Crecimiento de Fibroblastos/farmacología , Factores de Crecimiento de Fibroblastos/metabolismo , Humanos , Neoplasias/metabolismo , Receptores de Factores de Crecimiento de Fibroblastos/metabolismo , Transducción de SeñalRESUMEN
Silk fibroin (SF) is a non-pathological amyloidogenic protein prone, in solution, to the formation of amyloid-like aggregated species, displaying similarities in fibrillation kinetics with pathological amyloids, as widely reported in the literature. We show here, on the basis of different biophysical approaches (turbidity, Congo Red assays, CD, DLS and fluorescence), that fusidic acid (FA), a well-known antibiotic, acts on SF as an anti-aggregating agent in a dose-dependent manner, being also able to revert SF aggregation. FA binds to SF inducing changes in the environment of SF aromatic residues. We further provide the proof of principle that FA, already approved as drug on humans and used in ophthalmic preparations, displays its anti-aggregation properties also on lens material derived from cataract surgery and is capable of reducing aggregation. Thus it is suggested that FA can be foreseen as a therapeutic treatment for cataract and other protein aggregation disorders.
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Fibroínas , Ácido Fusídico , Amiloide/química , Proteínas Amiloidogénicas , Fibroínas/química , Fibroínas/farmacología , Humanos , Agregado de ProteínasRESUMEN
Fibroblast growth factor (FGF2)/fibroblast growth factor receptor (FGFR) signalling plays a major role both in physiology and in several pathologies, including cancer development, metastasis formation and resistance to therapy. The development of small molecules, acting extracellularly to target FGF2/FGFR interactions, has the advantage of limiting the adverse effects associated with current intracellular FGFR inhibitors. Herein, we discuss the ability of the natural compound rosmarinic acid (RA) to induce FGF2/FGFR complex dissociation. The molecular-level description of the FGF2/FGFR/RA system, by NMR spectroscopy and docking, clearly demonstrates that RA binds to the FGFR-D2 domain and directly competes with FGF2 for the same binding site. Direct and allosteric perturbations combine to destabilise the complex. The proposed molecular mechanism is validated by cellular studies showing that RA inhibits FGF2-induced endothelial cell proliferation and FGFR activation. Our results can serve as the basis for the development of new extracellular inhibitors of the FGF/FGFR pathways.
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Inhibidores de la Angiogénesis/farmacología , Productos Biológicos/farmacología , Cinamatos/farmacología , Depsidos/farmacología , Factor 2 de Crecimiento de Fibroblastos/antagonistas & inhibidores , Receptores de Factores de Crecimiento de Fibroblastos/antagonistas & inhibidores , Receptores de Factores de Crecimiento de Fibroblastos/metabolismo , Inhibidores de la Angiogénesis/química , Animales , Productos Biológicos/química , Bovinos , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Cinamatos/química , Depsidos/química , Factor 2 de Crecimiento de Fibroblastos/química , Factor 2 de Crecimiento de Fibroblastos/metabolismo , Simulación del Acoplamiento Molecular , Fosforilación/efectos de los fármacos , Receptores de Factores de Crecimiento de Fibroblastos/química , Ácido RosmarínicoRESUMEN
Correction for 'Exploring exchange processes in proteins by paramagnetic perturbation of NMR spectra' by Yamanappa Hunashal et al., Phys. Chem. Chem. Phys., 2020, 22, 6247-6259, DOI: .
RESUMEN
The effect of extrinsic paramagnetic probes on NMR relaxation rates for surface mapping of proteins and other biopolymers is a widely investigated and powerful NMR technique. Here we describe a new application of those probes. It relies on the setting of the relaxation delay to generate magnetization equilibrium and off-equilibrium conditions, in order to tailor the extent of steady state signal recovery with and without the water-soluble nitroxide Tempol. With this approach it is possible to identify signals whose relaxation is affected by exchange processes and, from the relative assignments, to map the protein residues involved in association or conformational interconversion processes on a micro-to-millisecond time scale. This finding is confirmed by the comparison with the results obtained from relaxation dispersion measurements. This simple and convenient method allows preliminary inspection to highlight regions where structural or chemical exchange events are operative, in order to focus on quantitative subsequent determinations by transverse relaxation dispersion experiments or analogous NMR relaxation studies, and/or to gain insights into the predictions of calculations.
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Modelos Moleculares , Resonancia Magnética Nuclear Biomolecular , Proteínas/química , Magnetismo , Conformación ProteicaRESUMEN
Alzheimer's disease (AD) is one of the most common neurodegenerative disorders, with no cure and preventive therapy. Misfolding and extracellular aggregation of Amyloid-ß (Aß) peptides are recognized as the main cause of AD progression, leading to the formation of toxic Aß oligomers and to the deposition of ß-amyloid plaques in the brain, representing the hallmarks of AD. Given the urgent need to provide alternative therapies, natural products serve as vital resources for novel drugs. In recent years, several natural compounds with different chemical structures, such as polyphenols, alkaloids, terpenes, flavonoids, tannins, saponins and vitamins from plants have received attention for their role against the neurodegenerative pathological processes. However, only for a small subset of them experimental evidences are provided on their mechanism of action. This review focuses on those natural compounds shown to interfere with Aß aggregation by direct interaction with Aß peptide and whose inhibitory mechanism has been investigated by means of biophysical and structural biology experimental approaches. In few cases, the combination of approaches offering a macroscopic characterization of the oligomers, such as TEM, AFM, fluorescence, together with high-resolution methods could shed light on the complex mechanism of inhibition. In particular, solution NMR spectroscopy, through peptide-based and ligand-based observation, was successfully employed to investigate the interactions of the natural compounds with both soluble NMR-visible (monomer and low molecular weight oligomers) and NMR-invisible (high molecular weight oligomers and protofibrils) species. The molecular determinants of the interaction of promising natural compounds are here compared to infer the chemical requirements of the inhibitors and the common mechanisms of inhibition. Most of the data converge to indicate that the Aß regions relevant to perturb the aggregation cascade and regulate the toxicity of the stabilized oligomers, are the N-term and ß1 region. The ability of the natural aggregation inhibitors to cross the brain blood barrier, together with the tactics to improve their low bioavailability are discussed. The analysis of the data ensemble can provide a rationale for the selection of natural compounds as molecular scaffolds for the design of new therapeutic strategies against the progression of early and late stages of AD.
RESUMEN
In this study natural-based complex polyphenols, obtained through a smart synthetic approach, have been evaluated for their ability to inhibit the formation of Aß42 oligomers, the most toxic species causing synaptic dysfunction, neuroinflammation, and neuronal death leading to the onset and progression of Alzheimer's disease. In vitro neurotoxicity tests on primary hippocampal neurons have been employed to select nontoxic candidates. Solution NMR and molecular docking studies have been performed to clarify the interaction mechanism of Aß42 with the synthesized polyphenol derivatives, and highlight the sterical and chemical requirements important for their antiaggregating activity. NMR results indicated that the selected polyphenolic compounds target Aß42 oligomeric species. Combined NMR and docking studies indicated that the Aß42 central hydrophobic core, namely, the 17-31 region, is the main interaction site. The length of the peptidomimetic scaffold and the presence of a guaiacol moiety were identified as important requirements for the antiaggregating activity. In vivo experiments on an Aß42 oligomer-induced acute mouse model highlighted that the most promising polyphenolic derivative (PP04) inhibits detrimental effects of Aß42 oligomers on memory and glial cell activation. NMR kinetic studies showed that PP04 is endowed with the chemical features of true inhibitors, strongly affecting both the Aß42 nucleation and growth rates, thus representing a promising candidate to be further developed into an effective drug against neurodegenerative diseases of the amyloid type.
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Enfermedad de Alzheimer/inducido químicamente , Enfermedad de Alzheimer/tratamiento farmacológico , Péptidos beta-Amiloides/toxicidad , Modelos Animales de Enfermedad , Trastornos de la Memoria/inducido químicamente , Trastornos de la Memoria/tratamiento farmacológico , Fragmentos de Péptidos/toxicidad , Polifenoles/uso terapéutico , Enfermedad Aguda , Enfermedad de Alzheimer/metabolismo , Péptidos beta-Amiloides/química , Animales , Células Cultivadas , Inflamación/inducido químicamente , Inflamación/tratamiento farmacológico , Inflamación/metabolismo , Trastornos de la Memoria/metabolismo , Ratones , Ratones Endogámicos C57BL , Simulación del Acoplamiento Molecular/métodos , Fragmentos de Péptidos/química , Polifenoles/química , Estructura Secundaria de ProteínaRESUMEN
Rhodopsins are photoreceptive proteins using light to drive a plethora of biological functions such as vision, proton and ion pumping, cation and anion channeling, and gene and enzyme regulation. Here we combine organic synthesis, NMR structural studies, and photochemical characterization to show that it is possible to prepare a fully synthetic mimic of rhodopsin photoreceptors. More specifically, we conjugate a bile acid binding protein with a synthetic mimic of the rhodopsin protonated Schiff base chromophore to achieve a covalent complex featuring an unnatural protein host, photoswitch, and photoswitch-protein linkage with a reverse orientation. We show that, in spite of its molecular-level diversity, light irradiation of the prepared mimic fuels a photochromic cycle driven by sequential photochemical and thermal Z/E isomerizations reminiscent of the photocycles of microbial rhodopsins.
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Materiales Biomiméticos/química , Proteínas Portadoras/química , Colorantes/química , Glicoproteínas de Membrana/química , Fotorreceptores Microbianos/química , Rodopsina/química , Isomerismo , Luz , Procesos Fotoquímicos , Conformación Proteica , Protones , Relación Estructura-ActividadRESUMEN
Soluble Aß oligomers are widely recognized as the toxic forms responsible for triggering AD, and Aß receptors are hypothesized to represent the first step in a neuronal cascade leading to dementia. Cellular prion protein (PrP) has been reported as a high-affinity binder of Aß oligomers. The interactions of PrP with both Aß42 and the highly toxic N-truncated pyroglutamylated species (AßpE3-42) are here investigated, at a molecular level, by means of ThT fluorescence, NMR and TEM. We demonstrate that soluble PrP binds both Aß42 and AßpE3-42, preferentially interacting with oligomeric species and delaying fibril formation. Residue level analysis of Aß42 oligomerization process reveals, for the first time, that PrP is able to differently interact with the forming oligomers, depending on the aggregation state of the starting Aß42 sample. A distinct behavior is observed for Aß42 1-30 region and C-terminal residues, suggesting that PrP protects Aß42 N-tail from entangling on the mature NMR-invisible fibril, consistent with the hypothesis that Aß42 N-tail is the locus of interaction with PrP. PrP/AßpE3-42 interactions are here reported for the first time. All interaction data are validated and complemented by cellular tests performed on Wt and PrP-silenced neuronal cell lines, clearly showing PrP dependent Aß oligomer cell internalization and toxicity. The ability of soluble PrP to compete with membrane-anchored PrP for binding to Aß oligomers bears relevance for studies of druggable pathways.
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Péptidos beta-Amiloides/metabolismo , Neuronas/metabolismo , Fragmentos de Péptidos/metabolismo , Proteínas Priónicas/metabolismo , Animales , Sitios de Unión , Línea Celular , Supervivencia Celular/fisiología , Espectroscopía de Resonancia Magnética , Ratones , Unión ProteicaRESUMEN
Amyloid structures are universal structures, widely diffuse in nature. Silk, capable of forming some of the strongest tensile materials on earth represents an important example of formation of functional amyloid fibrils, a process reminiscent of the oligomerization of peptides involved in neurodegenerative diseases. The stability of silk fibroin solutions in different conditions and its transition from α-helix/random coil to ß-sheet structures, at the basis of gelation processes and fibril formation, have been here investigated and monitored employing different biophysical approaches. Silk fibroin aggregation state as a function of concentration, pH and aging has been characterized employing NMR ordered diffusion spectroscopy. The change of silk fibroin diffusion coefficient over time, which reflects the progress of oligomerization, has been monitored for silk fibroin alone and in the presence of a polycondensed aromatic dye, namely rhodamine 6G. NMR, UV and DLS measurements indicated that rhodamine specifically binds to silk fibroin with a micromolar KD. The reported data reveal, for the first time, that RHD is capable of inhibiting fibroin self-association, thus controlling ß-conformational transition at the basis of fibril formation. The described approach could be extended to further protein systems, allowing better control of the oligomerisation process.
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Fibroínas/metabolismo , Agregado de Proteínas , Rodaminas/metabolismo , Sitios de Unión , Concentración de Iones de Hidrógeno , Resonancia Magnética Nuclear Biomolecular , Unión Proteica , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Multimerización de Proteína , Estabilidad Proteica , Espectroscopía de Protones por Resonancia Magnética , Espectrofotometría Ultravioleta , Relación Estructura-ActividadRESUMEN
Ferritin is a ubiquitous nanocage protein, which can accommodate up to thousands of iron atoms inside its cavity. Aside from its iron storage function, a new role as a fatty acid binder has been proposed for this protein. The interaction of apo horse spleen ferritin (HoSF) with a variety of lipids has been here investigated through NMR spectroscopic ligand-based experiments, to provide new insights into the mechanism of ferritin-lipid interactions, and the link with iron mineralization. 1D 1 H,â diffusion (DOSY) and saturation-transfer difference (STD)â NMR experiments provided evidence for a stronger interaction of ferritin with unsaturated fatty acids compared to saturated fatty acids, detergents, and bile acids. Mineralization assays showed that oleate c aused the most efficient increase in the initial rate of iron oxidation, and the highest formation of ferric species in HoSF. The comprehension of the factors inducing a faster biomineralization is an issue of the utmost importance, given the association of ferritin levels with metabolic syndromes, such as insulin resistance and diabetes, characterized by fatty acid concentration dysregulation. The human ferritin H-chain homopolymer (HuHF), featuring ferroxidase activity, was also tested for its fatty acid binding capabilities. Assays show that oleate can bind with high affinity to HuHF, without altering the reaction rates at the ferroxidase site.
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Ácidos Grasos Insaturados/química , Ferritinas/química , Hierro/metabolismo , Animales , Apoproteínas/química , Apoproteínas/metabolismo , Ceruloplasmina/química , Ceruloplasmina/metabolismo , Cromatografía en Gel , Dicroismo Circular , Dispersión Dinámica de Luz , Ferritinas/metabolismo , Caballos , Humanos , Hierro/química , Ligandos , Espectroscopía de Resonancia Magnética , Concentración Osmolar , Unión ProteicaRESUMEN
Aß peptides, the main protein components of Alzheimer's disease (AD) plaques, derive from a proteolytic cleavage of the amyloid precursor protein. Due to heterogeneous cleavage sites, a series of Aß peptides, including the major and widely studied species Aß1-40 (Aß40) and Aß1-42 (Aß42), are produced. In addition to the C-terminal heterogeneity of Aß peptides, significant amounts of N-terminal truncated (Aß3-42) and pyroglutamate-modified amyloid-ß peptides (AßpE3-42) have been identified in AD affected brains and shown to be more cytotoxic than unmodified Aß peptides. Little is known about the properties of their mixtures with Aß42. Nuclear Magnetic Resonance spectroscopy is here employed to investigate the interaction of N-truncated peptides with Aß42 at different molar ratios. We highlight the critical concentration of N-truncated forms influencing the aggregation kinetics of Aß42. We provide evidence, at residue level, that the C-terminal region of Aß42 is the locus of transient specific interactions with highly aggregation prone N-truncated alloforms.
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Péptidos beta-Amiloides/química , Péptidos beta-Amiloides/metabolismo , Fragmentos de Péptidos/química , Fragmentos de Péptidos/metabolismo , Humanos , Espectroscopía de Resonancia MagnéticaRESUMEN
Electrospinning is here used for the first time to prepare nanofibers including a host/guest complex in a keratin/poly(ethylene oxide) matrix. The host is a lipid binding protein and the guest is an insoluble bactericidal molecule, irgasan, bound within the protein internal cavity. The obtained nanofibers, characterized by scanning electron microscopy, exhibit excellent antibacterial activity toward Gram positive and negative bacteria, even with a moderate protein/irgasan cargo. Solution NMR studies, employed to provide molecular information on the cargo system, points to a micromolar affinity, compatible with both the electrospinning process and slow guest release. The versatility of the carrier protein, capable of interacting with a variety of druggable hydrophobic molecules, is exploitable for the development of innovative biomedical devices, whose properties can be tuned by the selected guest.
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Antibacterianos/farmacología , Proteínas Portadoras/metabolismo , Lípidos/química , Glicoproteínas de Membrana/metabolismo , Nanofibras/química , Nanotecnología/métodos , Animales , Carbanilidas/farmacología , Pollos , Escherichia coli/efectos de los fármacos , Pruebas de Sensibilidad Microbiana , Nanofibras/ultraestructura , Espectroscopía de Protones por Resonancia Magnética , Ovinos , Soluciones , Staphylococcus aureus/efectos de los fármacos , HumectabilidadRESUMEN
The rapid development of novel nanoscale materials for applications in biomedicine urges an improved characterization of the nanobio interfaces. Nanoparticles exhibit unique structures and properties, often different from the corresponding bulk materials, and the nature of their interactions with biological systems remains poorly characterized. Solution NMR spectroscopy is a mature technique for the investigation of biomolecular structure, dynamics, and intermolecular associations, however its use in protein-nanoparticle interaction studies remains scarce and highly challenging, particularly due to unfavorable hydrodynamic properties of most nanoscale assemblies. Nonetheless, recent efforts demonstrated that a number of NMR observables, such as chemical shifts, signal intensities, amide exchange rates and relaxation parameters, together with newly designed saturation transfer experiments, could be successfully employed to characterize the orientation, structure and dynamics of proteins adsorbed onto nanoparticle surfaces. This review provides the first survey and critical assessment of the contributions from solution NMR spectroscopy to the study of transient interactions between proteins and both inorganic (gold, silver, and silica) and organic (polymer, carbon and lipid based) nanoparticles. This article is part of a Special Issue entitled: Physiological Enzymology and Protein Functions.
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Nanopartículas/química , Resonancia Magnética Nuclear Biomolecular/métodos , Estructura Terciaria de Proteína , Proteínas/química , Medición de Intercambio de Deuterio/métodos , Cinética , Modelos Químicos , Modelos Moleculares , Unión Proteica , Proteínas/metabolismo , SolucionesRESUMEN
Primary bile acids, differing in hydroxylation pattern, are synthesized from cholesterol in the liver and, once formed, can undergo extensive enzyme-catalysed glycine/taurine conjugation, giving rise to a complex mixture, the bile acid pool. Composition and concentration of the bile acid pool may be altered in diseases, posing a general question on the response of the carrier (bile acid binding protein) to the binding of ligands with different hydrophobic and steric profiles. A collection of NMR experiments (H/D exchange, HET-SOFAST, ePHOGSY NOESY/ROESY and (15) N relaxation measurements) was thus performed on apo and five different holo proteins, to monitor the binding pocket accessibility and dynamics. The ensemble of obtained data could be rationalized by a statistical approach, based on chemical shift covariance analysis, in terms of residue-specific correlations and collective protein response to ligand binding. The results indicate that the same residues are influenced by diverse chemical stresses: ligand binding always induces silencing of motions at the protein portal with a concomitant conformational rearrangement of a network of residues, located at the protein anti-portal region. This network of amino acids, which do not belong to the binding site, forms a contiguous surface, sensing the presence of the bound lipids, with a signalling role in switching protein-membrane interactions on and off.
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Ácidos y Sales Biliares/química , Ácidos y Sales Biliares/metabolismo , Proteínas Portadoras/química , Proteínas Portadoras/metabolismo , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/metabolismo , Animales , Proteínas Aviares/química , Proteínas Aviares/metabolismo , Bioestadística , Pollos , Histidina/química , Técnicas In Vitro , Ligandos , Modelos Moleculares , Resonancia Magnética Nuclear Biomolecular , Unión Proteica , Conformación Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismoRESUMEN
Fatty acid binding proteins (FABPs) act as intracellular carriers of lipid molecules, and play a role in global metabolism regulation. Liver FABP (L-FABP) is prominent among FABPs for its wide ligand repertoire, which includes long-chain fatty acids as well as bile acids (BAs). In this work, we performed a detailed molecular- and atomic-level analysis of the interactions established by human L-FABP with nine BAs to understand the binding specificity for this important class of cholesterol-derived metabolites. Protein-ligand complex formation was monitored using heteronuclear NMR, steady-state fluorescence spectroscopy, and mass spectrometry. BAs were found to interact with L-FABP with dissociation constants in the narrow range of 0.6-7 µm; however, the diverse substitution patterns of the sterol nucleus and the presence of side-chain conjugation resulted in complexes endowed with various degrees of conformational heterogeneity. Trihydroxylated BAs formed monomeric complexes in which single ligand molecules occupied similar internal binding sites, based on chemical-shift perturbation data. Analysis of NMR line shapes upon progressive addition of taurocholate indicated that the binding mechanism departed from a simple binary association equilibrium, and instead involved intermediates along the binding path. The co-linear chemical shift behavior observed for L-FABP complexes with cholate derivatives added insight into conformational dynamics in the presence of ligands. The observed spectroscopic features of L-FABP/BA complexes, discussed in relation to ligand chemistry, suggest possible molecular determinants of recognition, with implications regarding intracellular BA transport. Our findings suggest that human L-FABP is a poorly selective, universal BA binder.
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Ácidos y Sales Biliares/química , Proteínas de Unión a Ácidos Grasos/química , Sitios de Unión , Humanos , Hidroxilación , Modelos Moleculares , Resonancia Magnética Nuclear Biomolecular , Unión Proteica , Estructura Secundaria de Proteína , Espectrometría de Masa por Ionización de ElectrosprayRESUMEN
Intracellular lipid binding proteins (iLBPs) are a family of evolutionarily related small cytoplasmic proteins implicated in the transcellular transport of lipophilic ligands. Subfamily-II iLBPs include the liver fatty acid binding protein (L-FABP), and the ileal and the liver and ileal bile acid binding proteins (L-BABP and I-BABP). Atomic-level investigations during the past 15-20 years have delivered relevant information on bile acid binding by this protein group, revealing unique features including binding cooperativity, promiscuity, and site selectivity. Using NMR spectroscopy and other biophysical techniques, our laboratories have contributed to an understanding of the molecular determinants of some of these properties and their generality among proteins from different animal species. We focused especially on formation of heterotypic complexes, considering the mixed compositions of physiological bile acid pools. Experiments performed with synthetic bile acid derivatives showed that iLBPs could act as targets for cell-specific contrast agents and, more generally, as effective carriers of amphiphilic drugs. This review collects the major findings related to bile salt interactions with iLBPs aiming to provide keys for a deeper understanding of protein-mediated intracellular bile salt trafficking.