RESUMEN
Lymphatic filariasis and its associated health hazards have taken enormous tolls especially in the tropical and sub-tropical countries round the globe. Our present work contemplates the immunomodulatory role of filarial Thioredoxin reductase (TrxR) for the survival of the parasite inside the human host. For this, the protein TrxR was purified from the filarial parasite Setaria cervi and further substantiated through specific anti-TrxR antibody raised in mice. Both commercially available anti-TrxR antibody and laboratory raised antibody produced a single band with a molecular mass of ~80 kDa on western blot. The protein is optimally active at pH 7.0 and at temperature 37 °C. This protein contains both alpha helix and beta pleated sheet with selenocysteine at its active site. The Km was found to be 2.75 ± 0.49 mM. TrxR was found to downregulate lipopolysaccharide (LPS)-induced inflammation in macrophages due to inhibition of TLR4-NF-κB pathway. The result was further supported by the downregulation of inflammasome pathway and activation of alternatively activated macrophages upon TrxR treatment. Hence this study projects insights into the importance of filarial TrxR in host-parasite interface as well as it illustrates novel therapeutic strategy towards anti-filarial drug development.
Asunto(s)
Antiinflamatorios/metabolismo , Antiinflamatorios/farmacología , Inflamación/tratamiento farmacológico , Inflamación/metabolismo , Macrófagos/metabolismo , Reductasa de Tiorredoxina-Disulfuro/metabolismo , Reductasa de Tiorredoxina-Disulfuro/farmacología , Animales , Línea Celular , Regulación hacia Abajo/efectos de los fármacos , Proteínas del Helminto/metabolismo , Inflamación/inducido químicamente , Lipopolisacáridos/farmacología , Ratones , FN-kappa B/metabolismo , Conformación Proteica en Hélice alfa/efectos de los fármacos , Conformación Proteica en Lámina beta/efectos de los fármacos , Células RAW 264.7 , Setaria (Nematodo)/efectos de los fármacos , Receptor Toll-Like 4/metabolismoRESUMEN
The assembly and maturation of human immunodeficiency virus type 1 (HIV-1) require proteolytic cleavage of the Gag polyprotein. The rate-limiting step resides at the junction between the capsid protein CA and spacer peptide 1, which assembles as a six-helix bundle (6HB). Bevirimat (BVM), the first-in-class maturation inhibitor drug, targets the 6HB and impedes proteolytic cleavage, yet the molecular mechanisms of its activity, and relatedly, the escape mechanisms of mutant viruses, remain unclear. Here, we employed extensive molecular dynamics (MD) simulations and free energy calculations to quantitatively investigate molecular structure-activity relationships, comparing wild-type and mutant viruses in the presence and absence of BVM and inositol hexakisphosphate (IP6), an assembly cofactor. Our analysis shows that the efficacy of BVM is directly correlated with preservation of 6-fold symmetry in the 6HB, which exists as an ensemble of structural states. We identified two primary escape mechanisms, and both lead to loss of symmetry, thereby facilitating helix uncoiling to aid access of protease. Our findings also highlight specific interactions that can be targeted for improved inhibitor activity and support the use of MD simulations for future inhibitor design.
Asunto(s)
Fármacos Anti-VIH/metabolismo , VIH-1/química , Succinatos/metabolismo , Triterpenos/metabolismo , Productos del Gen gag del Virus de la Inmunodeficiencia Humana/metabolismo , Simulación de Dinámica Molecular , Mutación , Ácido Fítico/metabolismo , Conformación Proteica en Hélice alfa/efectos de los fármacos , Desplegamiento Proteico/efectos de los fármacos , Productos del Gen gag del Virus de la Inmunodeficiencia Humana/química , Productos del Gen gag del Virus de la Inmunodeficiencia Humana/genéticaRESUMEN
Human calprotectin (CP, S100A8/S100A9 oligomer, MRP8/MRP14 oligomer) is an abundant innate immune protein that contributes to the host metal-withholding response. Its ability to sequester transition metal nutrients from microbial pathogens depends on a complex interplay of Ca(II) binding and self-association, which converts the αß heterodimeric apo protein into a Ca(II)-bound (αß)2 heterotetramer that displays enhanced transition metal affinities, antimicrobial activity, and protease stability. A paucity of structural data on the αß heterodimer has hampered molecular understanding of how Ca(II) binding enables CP to exert its metal-sequestering innate immune function. We report solution NMR data that reveal how Ca(II) binding affects the structure and dynamics of the CP αß heterodimer. These studies provide a structural model in which the apo αß heterodimer undergoes conformational exchange and switches between two states, a tetramerization-incompetent or "inactive" state and a tetramerization-competent or "active" state. Ca(II) binding to the EF-hands of the αß heterodimer causes the active state to predominate, resulting in self-association and formation of the (αß)2 heterotetramer. Moreover, Ca(II) binding causes local and allosteric ordering of the His3Asp and His6 metal-binding sites. Ca(II) binding to the noncanonical EF-hand of S100A9 positions (A9)D30 and organizes the His3Asp site. Remarkably, Ca(II) binding causes allosteric effects in the C-terminal region of helix αIV of S100A9, which stabilize the α-helicity at positions H91 and H95 and thereby organize the functionally versatile His6 site. Collectively, this study illuminates the molecular basis for how CP responds to high extracellular Ca(II) concentrations, which enables its metal-sequestering host-defense function.
Asunto(s)
Calcio/metabolismo , Complejo de Antígeno L1 de Leucocito/metabolismo , Multimerización de Proteína/efectos de los fármacos , Elementos de Transición/metabolismo , Calgranulina A/genética , Calgranulina A/metabolismo , Calgranulina B/genética , Calgranulina B/metabolismo , Histidina/química , Humanos , Complejo de Antígeno L1 de Leucocito/genética , Metales Pesados/metabolismo , Mutación , Resonancia Magnética Nuclear Biomolecular , Unión Proteica , Conformación Proteica en Hélice alfa/efectos de los fármacos , Multimerización de Proteína/genéticaRESUMEN
Molecules inhibiting the amyloid beta (Aß) peptide aggregation and/or disaggregating mature fibrils are a promising approach for the Alzheimer's disease (AD) therapy, as the Aß fibrillation is one of the key triggers of the disease. Gallic acid (GA) is a phenolic acid with anti-amyloidogenic activity against Aß in buffered solutions. However, there is still no evidence of these properties in vivo. Given the rate of failures of AD drug development, there is a huge demand of replicating the in vivo environment in in vitro studies, thus allowing to stop earlier the study of molecules with no effect in vivo. Thus, this study aims to evaluate the effect of in vitro neuronal membranes on the GA's ability in preventing Aß1-42 aggregation and disrupting preformed fibrils. To this end, liposomes were employed to mimic the cell membrane environment. The results reveal that the lipid membranes did not affect the GA's ability in inhibiting Aß1-42 fibrillation. However, in vitro neuronal membranes modulate the GA-induced Aß fibrils disaggregation, which may be related with the moderate affinity of the compound for the lipid membrane. Even so, GA presented strong anti-amyloidogenic properties in the cell membrane-like environment. This work highlights the promising value of GA on preventing and treating AD, thus justifying its study in animal models.
Asunto(s)
Péptidos beta-Amiloides/metabolismo , Ácido Gálico/química , Liposomas/química , Fragmentos de Péptidos/metabolismo , Multimerización de Proteína/efectos de los fármacos , Enfermedad de Alzheimer/tratamiento farmacológico , Péptidos beta-Amiloides/química , Humanos , Cinética , Fragmentos de Péptidos/química , Conformación Proteica en Hélice alfa/efectos de los fármacos , Conformación Proteica en Lámina beta/efectos de los fármacosRESUMEN
The rational synthesis of trinuclear emissive organometallic complexes with two equivalent platinum(II) centres appended to the ancillary substituted 2,2'-bipyridyl ligand of the cyclometalated iridium(III) centre is reported here. The alkynyl-platinum moiety and cyclometalated iridium(III) centres have been separated through a non-conjugated CH2 -O-CH2 linkage. The emission titration with amino acids reveals that the complexes sense free amino acids. The luminescence sensing of BSA is thus attributed to the amino acid sensing ability of the complexes and confirmed by emission anisotropy and Far-UV CD spectral study. The decrease in α-helix in the CD spectra signifies the changes in the secondary structure of protein in presence of the complexes.
Asunto(s)
Complejos de Coordinación/química , Colorantes Fluorescentes/química , Albúmina Sérica Bovina/análisis , Animales , Bovinos , Dicroismo Circular , Complejos de Coordinación/síntesis química , Complejos de Coordinación/metabolismo , Complejos de Coordinación/efectos de la radiación , Polarización de Fluorescencia , Colorantes Fluorescentes/síntesis química , Colorantes Fluorescentes/metabolismo , Colorantes Fluorescentes/efectos de la radiación , Iridio/química , Ligandos , Luz , Platino (Metal)/química , Unión Proteica , Conformación Proteica en Hélice alfa/efectos de los fármacos , Albúmina Sérica Bovina/química , Albúmina Sérica Bovina/metabolismoRESUMEN
Alpha-synuclein (αS) is a membrane-binding protein found predominantly in neurons and erythrocytes. The protein remains unordered in aqueous solutions but folds into an α-helical structure when bound to membranes. Besides, it gets deposited as ß-sheet rich aggregates in diseases known as synucleinopathies. The native αS has been reported to be acetylated at the N-terminus. Here, we compare the interfacial properties of the N-terminal acetylated αS (Ac-αS) with non-acetylated αS (NH2-αS) at the air-water interface. Both the protein forms are highly surface-active, with surface pressure reaching up to ~30 mN/m upon compression. The pressure-area isotherms obtained from the repeated compression-expansion cycles display large hysteresis suggesting self-assembly at higher surface pressures. The expansion isotherm is characterized by a rapid decrease in surface pressure followed by a slower transition phase starting around 15-17 mN/m. These data suggest that the compressed monolayer breaks into small clusters upon expansion, followed by these clusters' loosening. The circular dichroism spectroscopic analysis of the Blodgett-deposited films suggests the protein to be in largely α-helical conformation. The linear dichroism investigations suggest the protein to be anisotropically deposited. Blodgett deposition of the Langmuir films, therefore, is a rather simple method for preparing oriented monolayers of surface-active macromolecules.
Asunto(s)
alfa-Sinucleína/química , alfa-Sinucleína/metabolismo , Acetilación , Cromatografía/métodos , Dicroismo Circular/métodos , Membranas Artificiales , Microscopía de Fuerza Atómica/métodos , Transición de Fase , Conformación Proteica en Hélice alfa/efectos de los fármacos , Procesamiento Proteico-Postraduccional , Propiedades de Superficie , Agua/químicaRESUMEN
The effects of mulberry anthocyanins (MAs) on the digestibility of whey proteins (WP) in freshly-prepared and stored energy balls were studied. Results showed that MAs increased digestibility of the energy balls by increasing their hydrolysis-degree, soluble peptides-fractions, and decreasing their particle's size and agglomeration. To understand the mechanism of the promoting and/or inhibiting digestive effects of MAs, secondary structure alterations and binding of WP-MAs-mixtures were therefore measured. Results revealed that MAs could noncovalently/covalently interact with WP and form WP-MAs-adducts. This interaction seemed to be responsible for the alterations in the secondary structure of WP which could promote the digestibility of the energy balls subsequently. MAs also partially unfolded the structure of digested-WP through fluctuating their α-helix and ß-sheet. It was concluded that the unfolding in WP-structure induced by MAs-interactions might increase accessibility of the peptide bonds to the digestive enzymes and consequentially facilitate the protein's digestibility in the energy balls.
Asunto(s)
Antocianinas/farmacología , Digestión/efectos de los fármacos , Modelos Moleculares , Morus/química , Proteína de Suero de Leche/química , Proteína de Suero de Leche/metabolismo , Cápsulas , Glicosilación/efectos de los fármacos , Hidrólisis , Conformación Proteica en Hélice alfa/efectos de los fármacosRESUMEN
Alzheimer's disease (AD), an epidemic growing worldwide due to no effective medical aid available in the market, is a neurological disorder. AD is known to be directly associated with the toxicity of amyloid-ß (Aß) aggregates. In search of potent inhibitors of Aß aggregation, Hamilton and co-workers reported an α-helix mimetic, ADH-31, which acts as a powerful antagonist of Aß42 aggregation. To identify the key interactions between protein-ligand complexes and to gain insights into the inhibitory mechanism of ADH-31 against Aß42 aggregation, molecular dynamics (MD) simulations were performed in the present study. The MD simulations highlighted that ADH-31 showed distinct binding capabilities with residues spanning from the N-terminal to the central hydrophobic core (CHC) region of Aß42 and restricted the conformational transition of the helix-rich structure of Aß42 into another form of secondary structures (coil/turn/ß-sheet). Hydrophobic contacts, hydrogen bonding and π-π interaction contribute to the strong binding between ADH-31 and Aß42 monomer. The Dictionary of Secondary Structure of Proteins (DSSP) analysis highlighted that the probability of helical content increases from 38.5% to 50.2% and the turn content reduces from 14.7% to 6.2% with almost complete loss of the ß-sheet structure (4.5% to 0%) in the Aß42 monomer + ADH-31 complex. The per-residue binding free energy analysis demonstrated that Arg5, Tyr10, His14, Gln15, Lys16, Val18, Phe19 and Lys28 residues of Aß42 are responsible for the favourable binding free energy in Aß42 monomer + ADH-31 complex, which is consistent with the 2D HSQC NMR of the Aß42 monomer that depicted a change in the chemical shift of residues spanning from Glu11 to Phe20 in the presence of ADH-31. The MD simulations highlighted the prevention of sampling of amyloidogenic ß-strand conformations in Aß42 trimer in the presence of ADH-31 as well as the ability of ADH-31 to destabilize Aß42 trimer and protofibril structures. The lower binding affinity between Aß42 trimer chains in the presence of ADH-31 highlights the destabilization of the Aß42 trimer structure. Overall, MD results highlighted that ADH-31 inhibited Aß42 aggregation by constraining Aß peptides into helical conformation and destabilized Aß42 trimer as well as protofibril structures. The present study provides a theoretical insight into the atomic level details of the inhibitory mechanism of ADH-31 against Aß42 aggregation as well as protofibril destabilization and could be implemented in the structure-based drug design of potent therapeutic agents for AD.
Asunto(s)
Péptidos beta-Amiloides/metabolismo , Niacinamida/análogos & derivados , Niacinamida/metabolismo , Fragmentos de Péptidos/metabolismo , Peptidomiméticos/metabolismo , Multimerización de Proteína/efectos de los fármacos , Secuencia de Aminoácidos , Péptidos beta-Amiloides/química , Enlace de Hidrógeno , Interacciones Hidrofóbicas e Hidrofílicas , Simulación de Dinámica Molecular , Fragmentos de Péptidos/química , Unión Proteica , Conformación Proteica en Hélice alfa/efectos de los fármacos , TermodinámicaRESUMEN
In this study, inhibitory kinetics of Nuciferine and Methyl Ganoderate extrated from Lotus Leaves and Ganoderma lucidum on Mucor miehei Lipase were studied first. The molecular structure of Nuciferine and Methyl Ganoderate were determined. The inhibitory effects of two extracts on lipase were reversible, with the IC50 values of 0.194 and 0.332 mg/mL, respectively. The inhibition kinetic analysis by Lineweaver-Burk plots showed that they were a mixed-type inhibitor of lipase, with inhibition constants KI of 0.16 and 0.29 mg/mL, and KIS of 0.36 and 0.49 mg/mL, respectively. Results of spectral analysis showed that the UV absorption and the molecule fluorescence spectrum of the lipase hydrolyzate were significantly decreased after the inhibitor was added. The molecular docking further suggested that the interaction site between the active substance and inhibitor was located in an α-helix and a ß-sheet of the lipase, and the lipase active site was interfered by the inhibitor near the cap structure. In addition, the proliferation and differentiation of 3 T3-L1 preadipocytes were inhibited by two extracts. Total triglycerides and cholesterol were significantly reduced in the cells. The results confirmed that Nuciferine and Methyl Ganoderate can be used as potential obesity treatment drugs.
Asunto(s)
Aporfinas/farmacología , Lipasa/metabolismo , Rhizomucor/efectos de los fármacos , Triterpenos/farmacología , Células 3T3-L1 , Adipocitos/efectos de los fármacos , Adipogénesis/efectos de los fármacos , Animales , Fármacos Antiobesidad/farmacología , Dominio Catalítico/efectos de los fármacos , Diferenciación Celular/efectos de los fármacos , Línea Celular , Proliferación Celular/efectos de los fármacos , Colesterol/metabolismo , Cinética , Ratones , Simulación del Acoplamiento Molecular/métodos , Obesidad/tratamiento farmacológico , Obesidad/metabolismo , Conformación Proteica en Hélice alfa/efectos de los fármacos , Conformación Proteica en Lámina beta/efectos de los fármacos , Reishi/química , Triglicéridos/metabolismoRESUMEN
Phosphodiesterase 5A1 (PDE5) is a key target for treating cardiovascular diseases and erectile dysfunction. Here, we report the crystal structure of PDE5 complexed with the sole second generation drug avanafil. Analysis of protein-drug interactions revealed the structural basis of avanafil's superior isoform selectivity. Moreover, a halogen bonding was observed between avanafil and a backbone carbonyl oxygen of an adjacent α-helix, whose contribution to inhibitory potency illustrates the feasibility of exploiting α-helix backbone in structure-based drug design.
Asunto(s)
Fosfodiesterasas de Nucleótidos Cíclicos Tipo 5/química , Inhibidores de Fosfodiesterasa 5/farmacología , Pirimidinas/farmacología , Cristalografía por Rayos X , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 5/metabolismo , Diseño de Fármacos , Humanos , Simulación del Acoplamiento Molecular , Conformación Proteica/efectos de los fármacos , Conformación Proteica en Hélice alfa/efectos de los fármacos , Isoformas de Proteínas/química , Isoformas de Proteínas/metabolismoRESUMEN
Protein-protein interactions (PPIs), many of which are dominated by α-helical recognition domains, play key roles in many essential cellular processes, and the dysregulation of these interactions can cause detrimental effects. For instance, aberrant PPIs involving the Bcl-2 protein family can lead to several diseases including cancer, neurodegenerative diseases, and diabetes. Interactions between Bcl-2 pro-life proteins, such as Mcl-1, and pro-death proteins, such as Bim, regulate the intrinsic pathway of apoptosis. p53, a tumor-suppressor protein, also has a pivotal role in apoptosis and is negatively regulated by its E3 ubiquitin ligase HDM2. Both Mcl-1 and HDM2 are upregulated in numerous cancers, and, interestingly, there is crosstalk between both protein pathways. Recently, synergy has been observed between Mcl-1 and HDM2 inhibitors. Towards the development of new anticancer drugs, we herein describe a polypharmacology approach for the dual inhibition of Mcl-1 and HDM2 by employing three densely functionalized isoxazoles, pyrazoles, and thiazoles as mimetics of key α-helical domains of their partner proteins.
Asunto(s)
Antineoplásicos/farmacología , Diseño de Fármacos , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/antagonistas & inhibidores , Neoplasias/tratamiento farmacológico , Proteínas Proto-Oncogénicas c-mdm2/antagonistas & inhibidores , Antineoplásicos/química , Relación Dosis-Respuesta a Droga , Humanos , Isoxazoles/química , Isoxazoles/farmacología , Estructura Molecular , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/química , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/metabolismo , Neoplasias/metabolismo , Unión Proteica/efectos de los fármacos , Conformación Proteica en Hélice alfa/efectos de los fármacos , Proteínas Proto-Oncogénicas c-mdm2/química , Proteínas Proto-Oncogénicas c-mdm2/metabolismo , Pirazoles/química , Pirazoles/farmacología , Relación Estructura-Actividad , Tiazoles/química , Tiazoles/farmacologíaRESUMEN
α-Synuclein (αsyn) is a cytosolic intrinsically disordered protein (IDP) known to fold into an α-helical structure when binding to membrane lipids, decreasing protein aggregation. Model membrane enable elucidation of factors critically affecting protein folding/aggregation, mostly using either small unilamellar vesicles (SUVs) or nanodiscs surrounded by membrane scaffold proteins (MSPs). Yet SUVs are mechanically strained, while MSP nanodiscs are expensive. To test the impact of lipid particle size on α-syn structuring, while overcoming the limitations associated with the lipid particles used so far, we compared the effects of large unilamellar vesicles (LUVs) and lipid-bilayer nanodiscs encapsulated by diisobutylene/maleic acid copolymer (DIBMA) on αsyn secondary-structure formation, using human-, elephant- and whale -αsyn. Our results confirm that negatively charged lipids induce αsyn folding in h-αsyn and e-αsyn but not in w-αsyn. When a mixture of zwitterionic and negatively charged lipids was used, no increase in the secondary structure was detected at 45⯰C. Further, our results show that DIBMA/lipid particles (DIBMALPs) are highly suitable nanoscale membrane mimics for studying αsyn secondary-structure formation and aggregation, as folding was essentially independent of the lipid/protein ratio, in contrast with what we observed for LUVs having the same lipid compositions. This study reveals a new and promising application of polymer-encapsulated lipid-bilayer nanodiscs, due to their excellent efficiency in structuring disordered proteins such as αsyn into nontoxic α-helical structures. This will contribute to the unravelling and modelling aspects concerning protein-lipid interactions and α-helix formation by αsyn, paramount to the proposal of new methods to avoid protein aggregation and disease.
Asunto(s)
Lípidos de la Membrana/química , Polímeros/farmacología , Liposomas Unilamelares/química , alfa-Sinucleína/química , Alquenos/química , Alquenos/farmacología , Humanos , Proteínas Intrínsecamente Desordenadas/química , Membrana Dobles de Lípidos/química , Maleatos/química , Maleatos/farmacología , Proteínas de la Membrana/química , Polímeros/química , Agregado de Proteínas/efectos de los fármacos , Conformación Proteica en Hélice alfa/efectos de los fármacos , Pliegue de Proteína/efectos de los fármacos , Estructura Secundaria de Proteína/efectos de los fármacosRESUMEN
Cellulose nanocrystals (CNC) were successfully prepared from wheat bran, and their effects on the gelling properties and microstructure of heat-induced whey protein isolate (WPI) gels were investigated. The results showed that the water holding capacity, gel strength, viscoelasticity, and thermal stability of the composite gels were improved by increasing the CNC concentration from 0 to 1.0 % (w/v). The incorporation of CNC restricted water mobility and facilitated conformation conversion of the secondary structure from α-helix to ß-sheet. CNC has good compatibility with the protein matrixes at relatively low concentrations. At higher CNC concentrations, the agglomerated CNC can serve as an active dehydrating agent to absorb moisture in the protein matrixes, which promotes unfolding and cross-linking of the protein molecules. Moreover, the active filling effects of CNC contributed to the formation of a compact and homogeneous gel structure. Therefore, naturally sourced CNC is suggested as a potential gel modifier in food industry.
Asunto(s)
Celulosa/química , Nanopartículas/química , Conformación Proteica/efectos de los fármacos , Proteína de Suero de Leche/química , Fibras de la Dieta , Industria de Alimentos , Geles/química , Calor , Humanos , Conformación Proteica en Hélice alfa/efectos de los fármacos , Conformación Proteica en Lámina beta/efectos de los fármacos , Agua/química , Proteína de Suero de Leche/ultraestructuraRESUMEN
Urea at sufficiently high concentration unfolds the secondary structure of proteins leading to denaturation. In contrast, choline chloride (ChCl) and urea, in 1 : 2 molar ratio, form a deep eutectic mixture, a liquid at room temperature, protecting proteins from denaturation. In order to get a microscopic picture of this phenomenon, we perform extensive all-atom molecular dynamics simulations on a model protein, HP-36. Based on our calculation of Kirkwood-Buff integrals, we analyze the relative accumulation of urea and ChCl around the protein. Additional insights are drawn from the translational and rotational dynamics of solvent molecules and hydrogen bond auto-correlation functions. In the presence of urea, water shows slow subdiffusive dynamics around the protein owing to a strong interaction of water with the backbone atoms. Urea also shows subdiffusive motion. The addition of ChCl further slows down the dynamics of urea, restricting its accumulation around the protein backbone. Adding to this, choline cations in the first solvation shell of the protein show the strongest subdiffusive behavior. In other words, ChCl acts as a nano-crowder by excluding urea from the protein backbone and thereby slowing down the dynamics of water around the protein. This prevents the protein from denaturation and makes it structurally rigid, which is supported by the smaller radius of gyration and root mean square deviation values of HP-36.
Asunto(s)
Colina/química , Proteínas de Neurofilamentos/química , Fragmentos de Péptidos/química , Desnaturalización Proteica/efectos de los fármacos , Solventes/química , Urea/química , Secuencia de Aminoácidos , Enlace de Hidrógeno , Simulación de Dinámica Molecular , Conformación Proteica en Hélice alfa/efectos de los fármacosRESUMEN
Dibutyl phthalate has been illegally used in beverages and directly affects the human health. Herein, the interaction occurred between dibutyl phthalate and bovine serum albumin was studied. The experimental results demonstrated that dibutyl phthalate could bind to bovine serum albumin and statically quench the intrinsic fluorescence of this protein. Circular dichroism measurements proved that the binding of dibutyl phthalate would lead to an obvious decrease of α-helix content in the bovine serum albumin. Molecular docking analysis clarified the fluorescence quenching mechanism, size distribution and zeta potential variation, conformational change of BSA, the site marker competitive fluorescence quenching and the interaction mechanism of dibutyl phthalate to bovine serum albumin. This work provided a useful information for the binding of dibutyl phthalate to protein.
Asunto(s)
Dibutil Ftalato/metabolismo , Plastificantes/metabolismo , Albúmina Sérica Bovina/metabolismo , Animales , Sitios de Unión , Bovinos , Simulación del Acoplamiento Molecular , Unión Proteica , Conformación Proteica en Hélice alfa/efectos de los fármacos , Albúmina Sérica Bovina/químicaRESUMEN
RATIONALE: Interactions of drug molecules and proteins play important roles in physiological and pathological processes in vivo. It is of significance to establish a reliable strategy for studying protein-drug ligand interactions and would be helpful for the design and screening of new drugs in pharmacological research. METHODS: The interactions between four indole alkaloids (IAs) extracted from Ophiorrhiza japonica (O. japonica) and myoglobin (Mb) protein were investigated using a multi-spectrometric and computational method of native electrospray ionization mass spectrometry (native ESI-MS), hydrogen/deuterium exchange mass spectrometry (HDX-MS), circular dichroism (CD) and molecular docking (MD). RESULTS: The IA-bound Mb complexes were analyzed using native ESI-MS, with the obtained protein-to-ligand stoichiometry at 1:1, 1:2 and 1:3. Binding constants were measured according to the interpretation of MS spectra. MD complemented MS measurements, probing the binding sites and modes of the four IAs to Mb. Analyses involving CD and HDX-MS demonstrated that exposure to IAs could affect the conformation of Mb by decreasing the α-helix content and made Mb more susceptible to HDX at the backbone. CONCLUSIONS: A new MS-based integrated analysis method has been developed to successfully study the interactions of Mb and IAs extracted from O. japonica. The experimental and calculation results have good consistency, revealing all of the four IA molecules could bind to Mb to form 1:1, 1:2 and 1:3 Mb-IA complexes. The order of binding ability of these IAs to Mb was ophiorrhine B > compound C > ophiorrhine A > compound D. CD and HDX-MS results indicated that binding with IAs destabilizes Mb. HDX-MS analysis suggests that Mb becomes more susceptible to HDX, indicating that binding with IAs destabilizes the structure of Mb. In addition, the interaction with IAs affected the overall structure of Mb, ascribed to the decrease of α-helix content and less folding of the backbone.
Asunto(s)
Alcaloides Indólicos/farmacología , Mioglobina/metabolismo , Extractos Vegetales/farmacología , Rubiaceae/química , Animales , Dicroismo Circular , Caballos , Alcaloides Indólicos/química , Simulación del Acoplamiento Molecular , Mioglobina/química , Extractos Vegetales/química , Conformación Proteica en Hélice alfa/efectos de los fármacos , Espectrometría de Masa por Ionización de ElectrosprayRESUMEN
Chelerythrine (CHE), a benzophenanthridine alkaloid, is usually used as a nutritional and functional additive in variety of health foods. However, it should be paid enough attention because of its potential toxicity to human health. In this work, the binding mechanism of CHE with bovine serum albumin (BSA) was systematically investigated with spectroscopic approaches. The results showed that the mixture of BSA with CHE could spontaneously cause the formation of BSA-CHE complex through electrostatic interaction under simulative physiological conditions (0.01â¯molâ¯L-1 Tris-HCl, 0.015â¯molâ¯L-1 NaCl, pHâ¯=â¯7.4). Site marker competitive displacement experiments exhibited that CHE was primarily bound to the hydrophobic pocket of the site II (subdomain IIIA) of BSA. It has been reported that the binding of small functional molecules to serum albumins remarkably impacts their absorption, distribution, metabolism, conformation, and excretion features. Therefore, this study might be helpful for human to have an in-depth understanding of the biological effect of CHE in vivo and guide human to take it safely and reasonably.
Asunto(s)
Benzofenantridinas/metabolismo , Albúmina Sérica Bovina/metabolismo , Animales , Sitios de Unión , Bovinos , Dicroismo Circular , Unión Proteica , Conformación Proteica en Hélice alfa/efectos de los fármacos , Espectrometría de Fluorescencia , TermodinámicaRESUMEN
Globular proteins are typically unfolded by SDS to form protein-decorated micelle-like structures. Several proteins have been shown subsequently to refold by addition of the nonionic surfactant octaethylene glycol monododecyl ether (C12E8). Thus SDS converts ß-lactoglobulin, which has mainly ß-sheet secondary structure, into a state rich in α-helicality, while addition of C12E8 leads to refolding and recovery of the original ß-sheet structure. Here we extend these studies to the large ß-sheet-rich cellulase Cel7b from Humicola insolens whose enzymatic activity provides a very sensitive refolding parameter. The enzymes widespread usage in the detergent industry makes it an obvious model system for protein-surfactant interactions. SDS-unfolding and subsequent refolding using C12E8 were investigated at pHâ¯4.2 using near- and far-UV circular dichroism (CD), small-angle X-ray scattering (SAXS), isothermal titration calorimetry (ITC), size-exclusion chromatography (SEC) and activity measurements. The Cel7b:SDS complex can be described as a random configuration of 3-4 connected core-shell structures in which the protein is converted to a mainly α-helical secondary structure. Addition of C12E8 recovers almost all the secondary structure, part of the tertiary structure, about 50% of the activity and dissociates part of the protein population completely from detergent micelles. The lack of complete refolding may be due to charge neutralisation of Cel7b by SDS, kinetically trapping the enzyme into aggregated structures. In support of this, aggregates did not form when C12E8 was first mixed with Cel7b followed by addition of SDS. Formation of such aggregates may be a general phenomenon hampering quantitative refolding from the SDS-denatured state.
Asunto(s)
Celulasa/química , Desplegamiento Proteico/efectos de los fármacos , Dodecil Sulfato de Sodio/farmacología , Tensoactivos/farmacología , Calorimetría , Celulasa/efectos de los fármacos , Dicroismo Circular , Cinética , Polietilenglicoles/farmacología , Conformación Proteica/efectos de los fármacos , Conformación Proteica en Hélice alfa/efectos de los fármacos , Conformación Proteica en Lámina beta/efectos de los fármacos , Desnaturalización Proteica/efectos de los fármacos , Pliegue de Proteína/efectos de los fármacos , Estructura Secundaria de Proteína/efectos de los fármacos , Dispersión del Ángulo Pequeño , Sordariales/enzimología , Tensoactivos/química , Difracción de Rayos XRESUMEN
The repressive states of nuclear receptors (i.e., apo or bound to antagonists or inverse agonists) are poorly defined, despite the fact that nuclear receptors are a major drug target. Most ligand bound structures of nuclear receptors, including peroxisome proliferator-activated receptor γ (PPARγ), are similar to the apo structure. Here we use NMR, accelerated molecular dynamics and hydrogen-deuterium exchange mass spectrometry to define the PPARγ structural ensemble. We find that the helix 3 charge clamp positioning varies widely in apo and is stabilized by efficacious ligand binding. We also reveal a previously undescribed mechanism for inverse agonism involving an omega loop to helix switch which induces disruption of a tripartite salt-bridge network. We demonstrate that ligand binding can induce multiple structurally distinct repressive states. One state recruits peptides from two different corepressors, while another recruits just one, providing structural evidence of ligand bias in a nuclear receptor.
Asunto(s)
Proteínas Co-Represoras/metabolismo , PPAR gamma/metabolismo , Péptidos/metabolismo , Anilidas/farmacología , Benzamidas/farmacología , Sitios de Unión/efectos de los fármacos , Sitios de Unión/genética , Espectrometría de Masas de Intercambio de Hidrógeno-Deuterio , Ligandos , Espectroscopía de Resonancia Magnética , Simulación de Dinámica Molecular , Mutagénesis Sitio-Dirigida , PPAR gamma/agonistas , PPAR gamma/antagonistas & inhibidores , PPAR gamma/ultraestructura , Conformación Proteica en Hélice alfa/efectos de los fármacos , Conformación Proteica en Hélice alfa/genética , Piridinas/farmacología , Rosiglitazona/farmacologíaRESUMEN
Multiple subtypes of dopamine receptors within the GPCR superfamily regulate neurological processes through various downstream signaling pathways. A crucial question about the dopamine receptor family is what structural features determine the subtype-selectivity of potential drugs. Here, we report the 3.5-angstrom crystal structure of mouse dopamine receptor D4 (DRD4) complexed with a subtype-selective antagonist, L745870. Our structure reveals a secondary binding pocket extended from the orthosteric ligand-binding pocket to a DRD4-specific crevice located between transmembrane helices 2 and 3. Additional mutagenesis studies suggest that the antagonist L745870 prevents DRD4 activation by blocking the relative movement between transmembrane helices 2 and 3. These results expand our knowledge of the molecular basis for the physiological functions of DRD4 and assist new drug design.