RESUMEN
Unsymmetrically disubstituted metallocene derivatives, characterized as the first sandwich structure, have found interest in asymmetrical synthesis and in medicinal chemistry as well. Besides, they present a particular case of chirality. Twenty original and six commercially available molecules presenting either i) a planar chirality or ii) an asymmetrical carbon containing group or iii) being symmetrically substituted were analyzed in supercritical fluid chromatography on eleven polysaccharide-based chiral stationary phases with carbon dioxide containing 30% of methanol or 2-propanol as a co-solvent mobile phase. A basic additive, either diethylamine, triethylamine or n-butylamine was also required at 1% to the co-solvent for elution. While some of the tested chiral stationary phases provided enantioseparation for the racemates, chlorinated cellulosic phases proved to be both highly retentive and highly enantioselective towards these particular species with the highest rate of success compared to their non-chlorinated counterparts. For instance, the resolution value was equal to 14.1 for one ferrocene derivative in one-hour analysis time on cellulose tris(3,5-dichlorophenylcarbamate) column with 30% 2-propanol/1% n-butylamine while a single peak was observed under the same conditions on cellulose tris(3,5-dimethylphenylcarbamate) column. Experimental parameters were arbitrarily set at 150 bar outlet pressure, 40 °C temperature and 3 mL/min flow-rate.
Asunto(s)
Cromatografía con Fluido Supercrítico , 2-Propanol , Cromatografía Líquida de Alta Presión/métodos , Cromatografía con Fluido Supercrítico/métodos , Metalocenos , Polisacáridos/química , Solventes/química , EstereoisomerismoRESUMEN
Over the years, numerous modifications to the structure of proline have been made in order to tune its effects on bioactive compounds. Notably, the introduction of a cyclopropane ring or a fluorine atom has produced interesting results. Herein, we describe the synthesis of a proline containing fluorocyclopropane. This modified amino acid was inserted into a tripeptide, whose conformation was studied by nuclear magnetic resonance and density functional theory calculations.
RESUMEN
INTRODUCTION: With the aim of repositioning commercially available drugs for the inhibition of the anti-apoptotic myeloid cell leukemia protein, Mcl-1, implied in various cancers, five molecules, highlighted from a published theoretical screening, were selected to experimentally validate their affinity toward Mcl-1. RESULTS: A detailed NMR study revealed that only two of the five tested drugs, Torsemide and Deferasirox, interacted with Mcl-1. NMR data analysis allowed the complete characterization of the binding mode of both drugs to Mcl-1, including the estimation of their affinity for Mcl-1. Biological assays evidenced that the biological activity of Torsemide was lower as compared to the Deferasirox, which was able to efficiently and selectively inhibit the anti-apoptotic activity of Mcl-1. Finally, docking and molecular dynamics led to a 3D model for the Deferasirox:Mcl-1 complex and revealed the positioning of the drug in the Mcl-1 P2/P3 pockets as well as almost all synthetic Mcl-1 inhibitors. Interestingly, contrary to known synthetic Mcl-1 inhibitors which interact through Arg263, Deferasirox, establishes a salt bridge with Lys234. CONCLUSION: Deferasirox could be a potential candidate for drug repositioning as Mcl-1 inhibitor.
Asunto(s)
Proteínas Reguladoras de la Apoptosis/efectos de los fármacos , Deferasirox/farmacología , Reposicionamiento de Medicamentos , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/antagonistas & inhibidores , Deferasirox/química , Lenalidomida/química , Lenalidomida/farmacología , Espectroscopía de Resonancia Magnética , Simulación del Acoplamiento Molecular , Simulación de Dinámica Molecular , Estructura Molecular , Oxcarbazepina/química , Oxcarbazepina/farmacología , Risperidona/química , Risperidona/farmacología , Torasemida/química , Torasemida/farmacologíaRESUMEN
Herein, we report a new class of dual binding site AChE inhibitor 4 designed to exert a central cholinergic activation thanks to a redox-activation step of a prodrug precursor 3. Starting from potent pseudo-irreversible quinolinium salts AChE inhibitors 2 previously reported, a new set of diversely substituted quinolinium salts 2a-p was prepared and assayed for their inhibitory activity against AChE. Structure-activity relationship (SAR) analysis of 2a-p coupled with molecular docking studies allowed us to determine which position of the quinolinium scaffold may be considered to anchor the phtalimide fragment presumed to interact with the peripheral anionic site (PAS). In addition, molecular docking provided insight on the linker length required to connect both quinolinium and phatlimide moieties without disrupting the crucial role of quinolinium salt moiety within the catalytic active site (CAS); namely placing the carbamate in the correct position to trigger carbamylation of the active-site serine hydroxyl. Based on this rational design, the putative dual binding site inhibitor 4 and its prodrug 3 were synthesized and subsequently evaluated in vitro against AChE. Pleasingly, whereas compound 4 showed to be a highly potent inhibitor of AChE (IC50â¯=â¯6â¯nM) and binds to AChE-PAS to the same extent as donepezil, its prodrug 3 revealed to be inactive (IC50â¯>â¯10⯵M). These preliminary results constitute one of the few examples of carbamate-based dual binding site AChE inhibitors.
Asunto(s)
Acetilcolinesterasa/metabolismo , Carbamatos/farmacología , Inhibidores de la Colinesterasa/farmacología , Diseño de Fármacos , Profármacos/farmacología , Compuestos de Quinolinio/farmacología , Péptidos beta-Amiloides/antagonistas & inhibidores , Sitios de Unión/efectos de los fármacos , Células CACO-2 , Carbamatos/química , Inhibidores de la Colinesterasa/síntesis química , Inhibidores de la Colinesterasa/química , Relación Dosis-Respuesta a Droga , Humanos , Simulación del Acoplamiento Molecular , Estructura Molecular , Fragmentos de Péptidos/antagonistas & inhibidores , Profármacos/síntesis química , Profármacos/química , Agregado de Proteínas/efectos de los fármacos , Compuestos de Quinolinio/síntesis química , Compuestos de Quinolinio/química , Relación Estructura-ActividadRESUMEN
Thioglycosides, even if rare in Nature, have gained increased interest for their biological properties. Chemical syntheses of this class of compounds have been largely studied but little has been reported on their biosynthesis. Herein, combining experiments from the different fields of enzymology, bioorganic chemistry and molecular modeling, we wish to demonstrate the versatility of the glucosyltransferase UGT74B1 and its synthetic potency for the preparation of a variety of natural and unnatural desulfoglycosinolates.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/química , Glucosiltransferasas/metabolismo , Glicósidos/biosíntesis , Arabidopsis/metabolismo , Proteínas de Arabidopsis/química , Biocatálisis , Glucosiltransferasas/química , Glicósidos/química , Estructura MolecularRESUMEN
This study was undertaken to characterize functions of the outer membrane protein OmpW, which potentially contributes to the development of colistin- and imipenem-resistance in Acinetobacter baumannii. Reconstitution of OmpW in artificial lipid bilayers showed that it forms small channels (23 pS in 1 m KCl) and markedly interacts with iron and colistin, but not with imipenem. In vivo, (55) Fe uptake assays comparing the behaviours of ΔompW mutant and wild-type strains confirmed a role for OmpW in A. baumannii iron homeostasis. However, the loss of OmpW expression did not have an impact on A. baumannii susceptibilities to colistin or imipenem.
Asunto(s)
Acinetobacter baumannii/metabolismo , Proteínas de la Membrana Bacteriana Externa/metabolismo , Colistina/metabolismo , Hierro/metabolismo , Porinas/metabolismo , Secuencia de Aminoácidos , Proteínas de la Membrana Bacteriana Externa/química , Proteínas de la Membrana Bacteriana Externa/aislamiento & purificación , Datos de Secuencia Molecular , Porinas/química , Porinas/aislamiento & purificación , Unión Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Homología de Secuencia de AminoácidoRESUMEN
Synthesis of fluorocyclopropyl building blocks, which constitute the core of various therapeutic agents against the hepatitis C virus, is described. The relevant methyl α-amino-ß-fluoro-ß-vinylcyclopropanecarboxylate has been used as a key intermediate for the total synthesis of a fluorinated analogue of Simeprevir (TMC 435), a HCV NS3/4A protease inhibitor.
Asunto(s)
Aminoácidos Cíclicos/farmacología , Antivirales/farmacología , Ciclopropanos/farmacología , Hepacivirus/efectos de los fármacos , Inhibidores de Serina Proteinasa/farmacología , Proteínas no Estructurales Virales/antagonistas & inhibidores , Aminoácidos Cíclicos/síntesis química , Aminoácidos Cíclicos/química , Antivirales/síntesis química , Antivirales/química , Línea Celular , Ciclopropanos/síntesis química , Ciclopropanos/química , Relación Dosis-Respuesta a Droga , Hepacivirus/metabolismo , Humanos , Pruebas de Sensibilidad Microbiana , Estructura Molecular , Inhibidores de Serina Proteinasa/síntesis química , Inhibidores de Serina Proteinasa/química , Relación Estructura-Actividad , Proteínas no Estructurales Virales/metabolismoRESUMEN
The aim of this work was to develop a reliable and efficient analytical method to characterise and differentiate saxitoxin analogues (STX), including sulphated (gonyautoxins, GTX) and non-sulphated analogues. For this purpose, hydrophilic interaction liquid chromatography (HILIC) was used to separate sulphated analogues. We also resorted to ion mobility spectrometry to differentiate the STX analogues because this technique adds a new dimension of separation based on ion gas phase conformation. Positive and negative ionisation modes were used for gonyautoxins while positive ionisation mode was used for non-sulphated analogues. Subsequently, the coupling of these three complementary techniques, HILIC-IM-MS, permitted the separation and identification of STX analogues; isomer differentiation was achieved in HILIC dimension while non-sulphated analogues were separated in the IM-MS dimension. Additional structural characteristics concerning the conformation of STXs could be obtained using IM-MS measurements. Thus, the collision cross sections (CCS) of STXs are reported for the first time in the positive ionisation mode. These experimental CCSs correlated well with the calculated CCS values using the trajectory method.
Asunto(s)
Cromatografía Liquida/métodos , Espectrometría de Masas/métodos , Saxitoxina/análisis , Interacciones Hidrofóbicas e Hidrofílicas , Isomerismo , Estructura Molecular , Saxitoxina/análogos & derivados , Saxitoxina/química , Saxitoxina/aislamiento & purificación , Espectrometría de Masa por Ionización de Electrospray/métodos , Espectrometría de Masas en TándemRESUMEN
This paper reports the design and synthesis of a novel series of 8-arylpyrido[3',2':4,5]thieno[3,2-d]pyrimidin-4-amines via microwave-assisted multi-step synthesis. A common precursor of the whole series, 3-amino-5-bromothieno[2,3-b]pyridine-2-carbonitrile, was rapidly synthesized in one step from commercially-available 5-bromo-2-chloronicotinonitrile. Formylation with DMF-DMA led to (E)-N'-(5-bromo-2-cyanothieno[2,3-b]pyridin-3-yl)-N,N-dimethylformimidamide (4) which was conveniently functionalized at position 8 by palladium-catalyzed Suzuki-Miyaura cross-coupling to introduce a heteroaromatic ring. High-temperature formamide-mediated cyclization of the cyanoamidine intermediate gave seventeen 8-arylpyrido[3',2':4,5]thieno[3,2-d]pyrimidin-4-amines. The inhibitory potency of the final products was evaluated against five protein kinases (CDK5/p25, CK1δ/ε, GSK3α/ß, DYRK1A and CLK1) and revealed that 8-(2,4-dichlorophenyl)pyrido[3',2':4,5]thieno[3,2-d]pyrimidin-4-amine 1g specifically inhibits CK1δ/ε and CLK1 (220 and 88 nM, respectively) while its 7-(2,4-dichlorophenyl)pyrido[3',2':4,5]thieno[3,2-d]pyrimidin-4-amine isomer 10 showed no activity on the panel of tested kinases. Molecular modelling of 10 and 1g in the ATP binding sites of CK1δ/ε and CLK1 showed that functionalization at position 7 of pyrido[3',2':4,5]thieno[3,2-d]pyrimidin-4-amines is likely to induce a steric clash on the CK1δ/ε P-loop and thus a complete loss of inhibitory activity.
Asunto(s)
Modelos Moleculares , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Pirimidinas/farmacología , Relación Dosis-Respuesta a Droga , Humanos , Estructura Molecular , Inhibidores de Proteínas Quinasas/síntesis química , Inhibidores de Proteínas Quinasas/química , Proteínas Serina-Treonina Quinasas/metabolismo , Pirimidinas/síntesis química , Pirimidinas/química , Relación Estructura-ActividadRESUMEN
We reported the use of ion mobility (IM) combined with mass spectrometry (MS) as an analytical tool to investigate low generation polyamidoanine (PAMAM) dendrimers. This analytical approach has been employed to separate ions of defective structures with different charge state but exactly the same m/z value. Tandem mass spectrometry (MS/MS) after IM separation allowed a comprehensive structural characterization of defective dendrimers. In addition, IM was used to evaluate the collision cross-sections of ions of perfect dendrimers. They showed a good correlation with calculated collision cross-sections obtained by the trajectory method (TM) and were also consistent with dimensions reported by other established analytical methods.
RESUMEN
Original mixed selectors were synthesized by coupling a single L-valine diamide moiety on permethylated ß-cyclodextrin. The structures of the new selectors were designed to limit the interactions between the L-valine derivative and cyclodextrin by removing the amino acid moiety from the cyclodextrin cavity by means of an amide linkage on mono-6-amino permethylated ß-CD or the insertion of a carboxymethyl group. The accessibility of the amino acid group moiety was thus facilitated. The new mixed selectors exhibited better enantioselectivity than Chirasil-L-Val for half (selector based on mono-6-amino permethylated ß-CD) or more (selector with the carboxymethyl group) of the 41 amino acid derivatives. Molecular modeling confirmed that these results could be attributed to an increase in the distance between the chiral center of the amino acid and the cyclodextrin cavity allowing better access of the amino acid moiety. These new mixed chiral selectors demonstrated a novel enantioselective capability with the successful separation of more than 90 racemic mixtures among the 105 chiral compounds tested. These mixed selectors exhibited enhanced enantioselectivity in comparison to binary selectors previously described with respect to both enantiomer resolution and the number of separated chiral compounds. Moreover, an improvement of the enantioseparation factors compared to the corresponding 'parent phases' for the amino acid derivatives was observed in many cases. These mixed selectors should therefore be considered some of the most versatile selectors for chiral gas chromatography.
Asunto(s)
Aminoácidos/química , Cromatografía de Gases/instrumentación , Ciclodextrinas/química , Alcoholes/química , Espectroscopía de Resonancia Magnética , Compuestos Orgánicos/química , EstereoisomerismoRESUMEN
OBJECTIVES: In the context of the increasing worldwide occurrence of imipenem-resistant Acinetobacter baumannii strains, we investigated a possible porin-mediated mechanism relating to the carbapenem resistance-associated outer membrane protein, CarO. The aim of this study was to determine whether this porin may be a diffusion pathway for carbapenems in A. baumannii. METHODS: By analysing and comparing the sequences of CarO with protein databanks, we identified two major groups of sequences that we named CarOa and CarOb. We overproduced in Escherichia coli, extracted, purified by affinity chromatography and refolded in Triton X-100 rCarO from both groups. Their functional properties were investigated and compared by reconstitution in planar lipid bilayers. RESULTS: This functional study showed that rCarOa and rCarOb exhibit identical single-channel conductances (i.e. 20 pS in 1 M KCl) and similar poor cationic selectivity. Both channels were not specific towards meropenem and glutamic acid and poorly specific towards arginine, but they presented a marked specificity towards imipenem. From the calculated binding constants, we highlight that the CarOb channel was twice as specific as the CarOa channel for this antibiotic. Moreover, the CarOa channel could facilitate ornithine diffusion when the CarOb channel would not. CONCLUSIONS: We provide here the first evidence that CarO channels possess an imipenem (but not meropenem) binding site, and that their specificities depend on their primary structure. Any decrease in CarO expression would thus reduce the susceptibility of A. baumannii to this antibiotic.
Asunto(s)
Acinetobacter baumannii/genética , Acinetobacter baumannii/metabolismo , Antibacterianos/farmacología , Proteínas de la Membrana Bacteriana Externa/química , Proteínas de la Membrana Bacteriana Externa/metabolismo , Carbapenémicos/farmacología , Farmacorresistencia Bacteriana/fisiología , Porinas/genética , Acinetobacter baumannii/efectos de los fármacos , Algoritmos , Proteínas de la Membrana Bacteriana Externa/genética , Sitios de Unión/efectos de los fármacos , Clonación Molecular , Bases de Datos Genéticas , Escherichia coli/metabolismo , Imipenem/farmacología , Ionóforos/química , Membrana Dobles de Lípidos , Pliegue de Proteína , Proteínas Recombinantes/química , Relación Estructura-Actividad , Especificidad por SustratoRESUMEN
Like all retroviruses, human immunodeficiency virus type 1 (HIV-1) undergoes reverse transcription during its replication cycle. The cellular cofactors potentially involved in this process still remain to be identified. We show here that A-kinase anchoring protein 149 (AKAP149) interacts with HIV-1 reverse transcriptase (RT) in both the yeast two-hybrid system and human cells. The AKAP149 binding site has been mapped to the RNase H domain of HIV-1 RT. AKAP149 silencing by RNA interference in HIV-1-infected cells inhibited viral replication at the reverse transcription step. We selected single-point mutants of RT defective for AKAP149 binding and demonstrated that mutant G462R, despite retaining significant intrinsic RT activity in vitro, failed to carry out HIV-1 reverse transcription correctly in infected cells. This suggests that the interaction between RT and AKAP149 in infected cells may play an important role in HIV-1 reverse transcription.
Asunto(s)
Proteínas de Anclaje a la Quinasa A/metabolismo , Transcriptasa Inversa del VIH/metabolismo , Transcripción Reversa/fisiología , Proteínas de Anclaje a la Quinasa A/química , Proteínas de Anclaje a la Quinasa A/genética , Secuencia de Aminoácidos , Animales , Sitios de Unión , Línea Celular , Transcriptasa Inversa del VIH/química , Transcriptasa Inversa del VIH/genética , VIH-1/enzimología , VIH-1/genética , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Mutagénesis , Unión Proteica , Estructura Terciaria de Proteína , Interferencia de ARN , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Técnicas del Sistema de Dos Híbridos , Virión/metabolismo , Replicación Viral/fisiologíaRESUMEN
To replicate, human immunodeficiency virus, type 1 (HIV-1) needs to integrate a cDNA copy of its RNA genome into a chromosome of the host cell, a step controlled by the viral integrase (IN) protein. Viral integration involves the participation of several cellular proteins. SNF5/Ini1, a subunit of the SWI/SNF chromatin remodeling complex, was the first cofactor identified to interact with IN. We report here that SNF5/Ini1 interferes with early steps of HIV-1 replication. Inhibition of SNF5/Ini1 expression by RNA interference increases HIV-1 replication. Using quantitative PCR, we show that both the 2-long terminal repeat circle and integrated DNA forms accumulate upon SNF5/Ini1 knock down. By yeast two-hybrid assay, we screened a library of HIV-1 IN random mutants obtained by PCR random mutagenesis using SNF5/Ini1 as prey. Two different mutants of interaction, IN E69G and IN K71R, were impaired for SNF5/Ini1 interaction. The E69G substitution completely abolished integrase catalytic activity, leading to a replication-defective virus. On the contrary, IN K71R retained in vitro integrase activity. K71R substitution stimulates viral replication and results in higher infectious titers. Taken together, these results suggest that, by interacting with IN, SNF5/Ini1 interferes with early steps of HIV-1 infection.
Asunto(s)
Proteínas de Unión al ADN/fisiología , VIH-1/metabolismo , Factores de Transcripción/fisiología , Replicación Viral , Catálisis , Proliferación Celular , Proteínas Cromosómicas no Histona , ADN/química , Células HeLa , Humanos , Mutación , Unión Proteica , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Recombinación Genética , Proteína SMARCB1 , Técnicas del Sistema de Dos HíbridosRESUMEN
The human immunodeficiency virus type 1 (HIV-1) Vpu enhances viral particle release and, its interaction with the ubiquitin ligase SCF-beta-TrCP triggers the HIV-1 receptor CD4 degradation by the proteasome. The interaction between beta-TrCP protein and ligands containing the phosphorylated DpSGXXpS motif plays a key role for the development of severe disease states, such as HIV or cancer. This study examines the binding and conformation of phosphopeptides (P1, LIERAEDpSG and P2, EDpSGNEpSE) from HIV protein Vpu to beta-TrCP with the objective of defining the minimum length of peptide needed for effective binding. The screening step can be analyzed by NMR spectroscopy, in particular, saturation transfer NMR methods clearly identify the residues in the peptide that make direct contact with beta-TrCP protein when bound. An analysis of saturation transfer difference (STD) spectra provided clear evidence that the two peptides efficiently bound beta-TrCP receptor protein. To better characterize the ligand-protein interaction, the bound conformation of the phosphorylated peptides was determined using transferred NOESY methods, which gave rise to a well-defined structure. P1 and P2 can fold in a bend arrangement for the DpSG motif, showing the protons identified by STD-NMR as exposed in close proximity at the molecule surface. Ser phosphorylation allows electrostatic interaction and hydrogen bond with the amino acids of the beta-TrCP binding pocket. The upstream LIER hydrophobic region was also essential in binding to a hydrophobic pocket of the beta-TrCP WD domain. These findings are in good agreement with a recently published X-ray structure of a shorter beta-Catenin fragment with the beta-TrCP complex.
Asunto(s)
VIH-1/metabolismo , Proteínas del Virus de la Inmunodeficiencia Humana/química , Proteínas del Virus de la Inmunodeficiencia Humana/metabolismo , Fosfopéptidos/química , Fosfopéptidos/metabolismo , Proteínas Reguladoras y Accesorias Virales/química , Proteínas Reguladoras y Accesorias Virales/metabolismo , Proteínas con Repetición de beta-Transducina/química , Proteínas con Repetición de beta-Transducina/metabolismo , Secuencia de Aminoácidos , Sitios de Unión , VIH-1/química , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Ligandos , Espectroscopía de Resonancia Magnética , Datos de Secuencia Molecular , Unión Proteica , Estructura Terciaria de Proteína , Secuencias Repetitivas de AminoácidoRESUMEN
beta-TrCP is the F-box protein component of an Skp1/Cul1/F-box (SCF)-type ubiquitin ligase complex. Biochemical studies have suggested that beta-TrCP targets the oncogenic protein beta-catenin for ubiquitination and followed by proteasome degradation. To further elucidate the basis of this interaction, a complex between a 32-residue peptide from beta-catenin containing the phosphorylated motif DpSGXXpS (P-beta-Cat17-48) and beta-TrCP was studied using Saturation Transfer Difference (STD) Nuclear Magnetic Resonance (NMR) experiments. These experiments make it possible to identify the binding epitope of a ligand at atomic resolution. An analysis of STD spectra provided clear evidence that only a few of the 32 residues receive the largest saturation transfer. In particular, the amide protons of the residues in the phosphorylated motif appear to be in close contact to the amino acids of the beta-TrCP binding pocket. The amide and aromatic protons of the His24 and Trp25 residues also receive a significant saturation transfer. These findings are in keeping with a recently published x-ray structure of a shorter beta-catenin fragment with the beta-TrCP1-Skp1 complex and with the earlier findings from mutagenesis and activity assays. To better characterize the ligand-protein interaction, the bound conformation of the phosphorylated beta-catenin peptide was obtained using TRansfer Nuclear Overhauser Effect SpectroscopY (TRNOESY) experiments. Finally, we obtained the bound structure of the phosphorylated peptide showing the protons identified by STD NMR as exposed in close proximity to the molecule surface.
Asunto(s)
Proteínas del Citoesqueleto/química , Espectroscopía de Resonancia Magnética/métodos , Transactivadores/química , Proteínas con Repetición de beta-Transducina/química , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Cristalografía por Rayos X , Glutatión Transferasa/metabolismo , Histidina/química , Humanos , Concentración de Iones de Hidrógeno , Ligandos , Modelos Moleculares , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Péptidos/química , Fosforilación , Unión Proteica , Conformación Proteica , Pliegue de Proteína , Estructura Terciaria de Proteína , Protones , Proteínas Recombinantes de Fusión/química , Programas Informáticos , Factores de Tiempo , Triptófano/química , Rayos X , beta CateninaRESUMEN
The conformational preferences of a 22-amino acid peptide (LIDRLIERAEDpSGNEpSEGEISA) that mimics the phosphorylated HIV-1-encoded virus protein U (Vpu) antigen have been investigated by NMR spectroscopy. Degradation of HIV receptor CD4 by the proteasome, mediated by the HIV-1 protein Vpu, is crucial for the release of fully infectious virions. Phosphorylation of Vpu at sites Ser52 and Ser56 on the DSGXXS motif is required for the interaction of Vpu with the ubiquitin ligase SCF(beta)(-TrCP) which triggers CD4 degradation by the proteasome. This motif is conserved in several signaling proteins known to be degraded by the proteasome. The interaction of the P-Vpu(41-62) peptide with its monoclonal antibody has been studied by transferred nuclear Overhauser effect NMR spectroscopy (TRNOESY) and saturation transfer difference NMR (STD NMR) spectroscopy. The peptide was found to adopt a bend conformation upon binding to the antibody; the peptide residues (Asp51-pSer56) forming this bend are recognized by the antibody as demonstrated by STD NMR experiments. The three-dimensional structure of P-Vpu(41-62) in the bound conformation was determined by TRNOESY spectra; the peptide adopts a compact structure in the presence of mAb with formation of several bends around Leu45 and Ile46 and around Ile60 and Ser61, with a tight bend created by the DpS(52)GNEpS(56) motif. STD NMR studies provide evidence for the existence of a conformational epitope containing tandem repeats of phosphoserine motifs. The peptide's epitope is predominantly located in the large bend and in the N-terminal segment, implicating bidentale association. These findings are in excellent agreement with a recently published NMR structure required for the interaction of Vpu with the SCF(beta)(-TrCP) protein.
Asunto(s)
Anticuerpos Monoclonales/metabolismo , Sitios de Unión de Anticuerpos , Mapeo Epitopo , VIH-1 , Fragmentos de Péptidos/metabolismo , Proteínas Reguladoras y Accesorias Virales/metabolismo , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Mapeo Epitopo/métodos , Proteínas del Virus de la Inmunodeficiencia Humana , Modelos Moleculares , Datos de Secuencia Molecular , Resonancia Magnética Nuclear Biomolecular/métodos , Fragmentos de Péptidos/inmunología , Fosforilación , Unión Proteica , Conformación Proteica , Proteínas Reguladoras y Accesorias Virales/química , Proteínas Reguladoras y Accesorias Virales/inmunologíaRESUMEN
A protein-protein association regulated by phosphorylation of serine is examined by NMR studies. Degradation of the HIV receptor CD4 by the proteasome, mediated by the HIV-1 protein Vpu, is crucial for the release of fully infectious virions. Phosphorylation of Vpu at two sites, Ser52 and Ser56, on the motif DSGXXS is required for the interaction of Vpu with the ubiquitin ligase SCF-betaTrCP which triggers CD4 degradation by the proteasome. This motif is conserved in several signaling proteins known to be degraded by the proteasome. To elucidate the basis of beta-TrCP recognition, the bound conformation of the P-Vpu(41-62) peptide was determined by using NMR and MD. The TRNOE intensities provided distance constraints which were used in simulated annealing. The beta-TrCP-bound structure of P-Vpu was found to be similar to the structure of the free peptide in solution and to the structure recognized by its antibody. Residues 50-57 formed a bend while the phosphate groups are pointing away. The binding fragment was studied by STD-NMR spectroscopy. The phosphorylated motif DpS(52)GNEpS(56) was found to make intimate contact with beta-TrCP, and pSer52 displays the strongest binding effect. It is suggested that Ser phosphorylation allows protein-protein association by electrostatic stabilization: an obvious negative binding region of Vpu was recognizable by positive residues (Arg and Lys) of the WD domain of beta-TrCP. The Ile46 residue was also found essential for interaction with the beta-TrCP protein. Leu45 and Ile46 side chains lie in close proximity to a hydrophobic pocket of the WD domain.
Asunto(s)
VIH-1/química , Proteínas Reguladoras y Accesorias Virales/química , Proteínas con Repetición de beta-Transducina/química , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Antígenos CD4/metabolismo , Mapeo Epitopo/métodos , VIH-1/metabolismo , Proteínas del Virus de la Inmunodeficiencia Humana , Datos de Secuencia Molecular , Resonancia Magnética Nuclear Biomolecular/métodos , Fragmentos de Péptidos/química , Fragmentos de Péptidos/metabolismo , Fosforilación , Unión Proteica , Conformación Proteica , Estructura Terciaria de Proteína , Termodinámica , Proteínas Reguladoras y Accesorias Virales/metabolismo , Proteínas con Repetición de beta-Transducina/metabolismoRESUMEN
Degradation of the HIV receptor CD4 by the proteasome, mediated by the HIV-1 protein Vpu, is crucial for the release of fully infectious virions. To promote CD4 degradation Vpu has to be phosphorylated on a motif DSGXXS, which is conserved in several signalling proteins known to be degraded by the proteasome upon phosphorylation. Such phosphorylation is required for the interaction of Vpu with the ubiquitin ligase SCF-beta-TrCP that triggers CD4 degradation by the proteasome. In the present work, we used two peptides of 22 amino acids between residues 41 and 62 of Vpu. Vpu41-62 was predicted to form an alpha-helix-flexible-alpha-helix including the phosphorylation motif DS52GNES56 and Vpu_P41-62 was phosphorylated at the two sites Ser52 and Ser56. We analysed the conformational change induced by the phosphorylation of this peptide on the residues Ser52 and Ser56. Homo- and heteronuclear NMR techniques were used to assess the structural influence of phosphorylation. The spectra of the free peptides, Vpu_P41-62 and Vpu41-62, in both H2O (at pH 3.5 and 7.2) and a 1:1 mixture of H2O and trifluoroethanol were completely assigned by a combined application of several two-dimensional proton NMR methods. Analysis of the short- and medium-range NOE connectivities and of the secondary chemical shifts indicated that the peptide segment (42-49) shows a less well-defined helix propensity. The Vpu_P41-62 domain of residues 50-62 forms a loop with the phosphate group pointing away, a short beta-strand and a flexible extended 'tail' of residues 60-62. Residues 50-60 exhibit alpha-proton NMR secondary chemical shift changes from random coil toward more beta-like structure with the combined (temperature, solvent and pH) NMR and molecular calculation experiments. Differences in this molecular region 50-62 suggest that conformational changes of Vpu_P play an important role in Vpu_P-induced degradation of CD4 molecules.