RESUMEN
The driving forces and conformational pathways leading to amphitropic protein-membrane binding and in some cases also to protein misfolding and aggregation is the subject of intensive research. In this study, a chimeric polypeptide, A-Cage-C, derived from α-Lactalbumin is investigated with the aim of elucidating conformational changes promoting interaction with bilayers. From previous studies, it is known that A-Cage-C causes membrane leakages associated with the sporadic formation of amorphous aggregates on solid-supported bilayers. Here we express and purify double-labelled A-Cage-C and prepare partially deuterated bicelles as a membrane mimicking system. We investigate A-Cage-C in the presence and absence of these bicelles at non-binding (pH 7.0) and binding (pH 4.5) conditions. Using in silico analyses, NMR, conformational clustering, and Molecular Dynamics, we provide tentative insights into the conformations of bound and unbound A-Cage-C. The conformation of each state is dynamic and samples a large amount of overlapping conformational space. We identify one of the clusters as likely representing the binding conformation and conclude tentatively that the unfolding around the central W23 segment and its reorientation may be necessary for full intercalation at binding conditions (pH 4.5). We also see evidence for an overall elongation of A-Cage-C in the presence of model bilayers.
Asunto(s)
Proteína Oncogénica pp60(v-src)/química , Fragmentos de Péptidos/química , Péptidos/química , Lactalbúmina/química , Espectroscopía de Resonancia Magnética/métodos , Proteínas de la Membrana/química , Proteínas de la Membrana/metabolismo , Membranas , Simulación de Dinámica Molecular , Proteína Oncogénica pp60(v-src)/metabolismo , Fragmentos de Péptidos/metabolismo , Péptidos/metabolismo , Unión Proteica , Conformación ProteicaRESUMEN
Identification and localization of modifications in peptides containing multiple disulfide bonds is challenging due to inefficient fragmentation in mass spectrometry (MS) analysis. In cases where MS fragmentation techniques such as electron capture dissociation (ECD), electron transfer dissociation (ETD), and ultraviolet photodissociation (UVPD) fail to achieve efficient fragmentation, off-line disulfide bond reduction techniques are typically employed prior to MS analysis. Some commonly used reducing agents include dithiothreitol (DTT) and tris(2-carboxyethyl)phosphine (TCEP). In this work, we describe the detection and identification of an unexpected impurity that formed during the reduction of Peptide A, containing multiple disulfide bonds, while using DTT or TCEP as reducing agents and acetonitrile as a co-solvent. The DTT reduced products were found to be a mixture of the expected linear Peptide A (fully reduced) and an unknown product (>50%) with a mass corresponding to linear Peptide A plus 41â¯Da ([reduced-Mâ¯+â¯41]). A series of experiments were subsequently performed to investigate the identity and origin of this impurity. Disulfide bond reduction with DTT was performed in aqueous mixtures containing acetonitrile, methanol, and deuterated acetonitrile; and with TCEP in aqueous mixtures containing acetonitrile. Additionally, glycine amino acid was used as a surrogate to investigate the mechanism. The liquid chromatography-mass spectrometry (LCMSMS) results demonstrated that the [reduced-Mâ¯+â¯41] impurity was an acetonitrile addition on the peptide's N-terminal glycine. The corresponding impurity [Mâ¯+â¯41] was also found in the native Peptide A (non-reduced), suggesting that small amounts of this impurity may also be generated during the synthesis in the upstream process steps. By understanding the formation of this process-related impurity [Mâ¯+â¯41], one could potentially reduce or eliminate its presence in Peptide A through chemical controls. Finally, this observation provides caution against using acetonitrile as a co-solvent during DTT- or TCEP-promoted reduction of peptides with an uncapped N-terminus primary amine.
Asunto(s)
Acetonitrilos/química , Disulfuros/química , Ditiotreitol/química , Péptidos/química , Fosfinas/química , Aminas/química , Cromatografía Liquida , Glicina/química , Proteína Oncogénica pp60(v-src)/química , Oxidación-Reducción , Fragmentos de Péptidos/química , Dominios Proteicos , Sustancias Reductoras/química , Espectrometría de Masa por Ionización de Electrospray , Rayos UltravioletaRESUMEN
DEAD-box proteins are an essential class of enzymes involved in all stages of RNA metabolism. The study of DEAD-box proteins is challenging in a native setting since they are structurally similar, often essential and display dosage sensitivity. Pharmacological inhibition would be an ideal tool to probe the function of these enzymes. In this work, we describe a chemical genetic strategy for the specific inactivation of individual DEAD-box proteins with small molecule inhibitors using covalent complementarity. We identify a residue of low conservation within the P-loop of the nucleotide-binding site of DEAD-box proteins and show that it can be mutated to cysteine without a substantial loss of enzyme function to generate electrophile-sensitive mutants. We then present a series of small molecules that rapidly and specifically bind and inhibit electrophile-sensitive DEAD-box proteins with high selectivity over the wild-type enzyme. Thus, this approach can be used to systematically generate small molecule-sensitive alleles of DEAD-box proteins, allowing for pharmacological inhibition and functional characterization of members of this enzyme family.
Asunto(s)
Adenosina Monofosfato/análogos & derivados , Proteína 58 DEAD Box/química , ARN Helicasas DEAD-box/química , Proteína Oncogénica pp60(v-src)/química , Proteínas de Saccharomyces cerevisiae/química , Acrilamidas/síntesis química , Acrilamidas/metabolismo , Acrilatos/síntesis química , Acrilatos/metabolismo , Adenosina Monofosfato/metabolismo , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Sitios de Unión , Clonación Molecular , Crotonatos/síntesis química , Crotonatos/metabolismo , Cristalografía por Rayos X , Proteína 58 DEAD Box/antagonistas & inhibidores , Proteína 58 DEAD Box/genética , Proteína 58 DEAD Box/metabolismo , ARN Helicasas DEAD-box/antagonistas & inhibidores , ARN Helicasas DEAD-box/genética , ARN Helicasas DEAD-box/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Expresión Génica , Humanos , Cinética , Modelos Moleculares , Proteína Oncogénica pp60(v-src)/antagonistas & inhibidores , Proteína Oncogénica pp60(v-src)/genética , Proteína Oncogénica pp60(v-src)/metabolismo , Unión Proteica , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Dominios y Motivos de Interacción de Proteínas , Receptores Inmunológicos , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/antagonistas & inhibidores , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMEN
Heat shock protein 90 (Hsp90) is an abundant molecular chaperone, involved in the folding and activation of 60% of the human kinome. The oncogenic tyrosine kinase v-Src is one of the most stringent client proteins of Hsp90, whereas its almost identical homolog c-Src is only weakly affected by the chaperone. Here, we perform atomistic molecular simulations and in vitro kinase assays to explore the mechanistic differences in the activation of v-Src and c-Src. While activation in c-Src is strictly controlled by ATP-binding and phosphorylation, we find that activating conformational transitions are spontaneously sampled in Hsp90-dependent Src mutants. Phosphorylation results in an enrichment of the active conformation and in an increased affinity for Hsp90. Thus, the conformational landscape of the mutated kinase is reshaped by a broken "control switch", resulting in perturbations of long-range electrostatics, higher activity and increased Hsp90-dependence.
Asunto(s)
Proteínas HSP90 de Choque Térmico/metabolismo , Proteína Oncogénica pp60(v-src)/química , Proteína Oncogénica pp60(v-src)/metabolismo , Pliegue de Proteína , Familia-src Quinasas/química , Familia-src Quinasas/metabolismo , Adenosina Trifosfato/metabolismo , Proteína Tirosina Quinasa CSK , Humanos , Simulación de Dinámica Molecular , Proteínas Mutantes/química , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Proteína Oncogénica pp60(v-src)/genética , Fosforilación , Unión Proteica , Conformación Proteica , Familia-src Quinasas/genéticaRESUMEN
Many proteins are anchored to lipid bilayer membranes through a combination of hydrophobic and electrostatic interactions. In the case of the membrane-bound nonreceptor tyrosine kinase Src from Rous sarcoma virus, these interactions are mediated by an N-terminal myristoyl chain and an adjacent cluster of six basic amino-acid residues, respectively. In contrast with the acyl modifications of other lipid-anchored proteins, the myristoyl chain of Src does not match the host lipid bilayer in terms of chain conformation and dynamics, which is attributed to a tradeoff between hydrophobic burial of the myristoyl chain and repulsion of the peptidic moiety from the phospholipid headgroup region. Here, we combine thermodynamic information obtained from isothermal titration calorimetry with structural data derived from (2)H, (13)C, and (31)P solid-state nuclear magnetic resonance spectroscopy to decipher the hydrophobic and electrostatic contributions governing the interactions of a myristoylated Src peptide with zwitterionic and anionic membranes made from lauroyl (C12:0) or myristoyl (C14:0) lipids. Although the latter are expected to enable better hydrophobic matching, the Src peptide partitions more avidly into the shorter-chain lipid analog because this does not require the myristoyl chain to stretch extensively to avoid unfavorable peptide/headgroup interactions. Moreover, we find that Coulombic and intrinsic contributions to membrane binding are not additive, because the presence of anionic lipids enhances membrane binding more strongly than would be expected on the basis of simple Coulombic attraction.
Asunto(s)
Membrana Dobles de Lípidos/química , Proteína Oncogénica pp60(v-src)/química , Péptidos/química , Secuencia de Aminoácidos , Membrana Dobles de Lípidos/metabolismo , Datos de Secuencia Molecular , Ácido Mirístico/química , Proteína Oncogénica pp60(v-src)/metabolismo , Péptidos/metabolismo , Fosfolípidos/química , Unión Proteica , Estructura Terciaria de Proteína , Electricidad Estática , TermodinámicaRESUMEN
Hsp90 is a molecular chaperone involved in the activation of numerous client proteins, including many kinases. The most stringent kinase client is the oncogenic kinase v-Src. To elucidate how Hsp90 chaperones kinases, we reconstituted v-Src kinase chaperoning in vitro and show that its activation is ATP-dependent, with the cochaperone Cdc37 increasing the efficiency. Consistent with in vivo results, we find that Hsp90 does not influence the almost identical c-Src kinase. To explain these findings, we designed Src kinase chimeras that gradually transform c-Src into v-Src and show that their Hsp90 dependence correlates with compactness and folding cooperativity. Molecular dynamics simulations and hydrogen/deuterium exchange of Hsp90-dependent Src kinase variants further reveal increased transitions between inactive and active states and exposure of specific kinase regions. Thus, Hsp90 shifts an ensemble of conformations of v-Src toward high activity states that would otherwise be metastable and poorly populated.
Asunto(s)
Proteínas HSP90 de Choque Térmico/metabolismo , Proteína Oncogénica pp60(v-src)/metabolismo , Animales , Pollos , Simulación de Dinámica Molecular , Proteína Oncogénica pp60(v-src)/química , Conformación Proteica , Proteínas Recombinantes de Fusión/metabolismoRESUMEN
In proteins, proton-coupled electron transfer (PCET) can involve the transient oxidation and reduction of the aromatic amino acid tyrosine. Due to the short life time of tyrosyl radical intermediates, transient absorption spectroscopy provides an important tool in deciphering electron-transfer mechanisms. In this report, the photoionization of solution tyrosine and tyrosinate was investigated using transient, picosecond absorption spectroscopy. The results were compared to data acquired from a tyrosine-containing ß-hairpin peptide. This maquette, peptide A, is an 18-mer that exhibits π-π interaction between tyrosine (Y5) and histidine (H14). Y5 and H14 carry out an orthogonal PCET reaction when Y5 is oxidized in the mid-pH range. Photolysis of all samples (280 nm, instrument response: 360 fs) generated a solvated electron signal within 3 ps. A signal from the S1 state and a 410 nm signal from the neutral tyrosyl radical were also formed in 3 ps. Fits to S1 and tyrosyl radical decay profiles revealed biphasic kinetics with time constants of 10-50 and 400-1300 ps. The PCET reaction at pH 9 was associated with a significant decrease in the rate of tyrosyl radical and S1 decay compared to electron transfer (ET) alone (pH 11). This pH dependence was observed both in solution and peptide samples. The pH 9 reaction may occur with a sequential electron-transfer, proton-transfer (ETPT) mechanism. Alternatively, the pH 9 reaction may occur by coupled proton and electron transfer (CPET). CPET would be associated with a reorganization energy larger than that of the pH 11 reaction. Significantly, the decay kinetics of S1 and the tyrosyl radical were accelerated in peptide A compared to solution samples at both pH values. These data suggest either an increase in electronic coupling or a specific, sequence-dependent interaction, which facilitates ET and PCET in the ß hairpin.
Asunto(s)
Proteína Oncogénica pp60(v-src)/química , Fragmentos de Péptidos/química , Protones , Tirosina/química , Transporte de Electrón , Cinética , Modelos Moleculares , Fotólisis , Estructura Secundaria de ProteínaRESUMEN
The cancer drug Ruxolitinib is a potent janus kinase inhibitor approved for the treatment of the myeloproliferative neoplasms. In addition, Ruxolitinib has weak inhibitory activity against a panel of other kinases, including Src kinase. There is no structural information of Ruxolitinib binding to any kinase. In this paper, we determined the crystal structure of c-Src kinase domain in complex of Ruxolitinib at a resolution of 2.26 Å. C-Src kinase domain adopts the DFG-in active conformation upon Ruxolitinib binding, indicating Ruxolitinib is a type I inhibitor for c-Src. Ruxolitinib forms two hydrogen bonds with Met341, a water-mediated hydrogen bond with Thr338, and a number of van der Waals contacts with c-Src. Ruxolitinib was then docked into the ligand-binding pocket of a previously solved JAK1 structure. From the docking result, Ruxolitinib also binds JAK1 as a type I inhibitor, with more interactions and a higher shape complementarity with the ligand-binding pocket of JAK1 compared to that of c-Src. Since Ruxolitinib is a relatively small inhibitor and there is sizeable cavity between Ruxolitinib and c-Src ligand-binding pocket, we propose to modify Ruxolitinib to develop more potent inhibitors to c-Src.
Asunto(s)
Antineoplásicos/metabolismo , Proteína Oncogénica pp60(v-src)/metabolismo , Pirazoles/metabolismo , Adenosina Trifosfato/metabolismo , Secuencia de Aminoácidos , Sitios de Unión , Simulación del Acoplamiento Molecular , Datos de Secuencia Molecular , Nitrilos , Proteína Oncogénica pp60(v-src)/química , Unión Proteica , Pirimidinas , Homología de Secuencia de AminoácidoRESUMEN
Network-biology inspired modeling of interactome data and computational chemistry have the potential to revolutionize drug discovery by complementing conventional methods. We consider asthma, a complex disease characterized by intricate molecular mechanisms, for our study. We aim to integrate prediction of potent drug targets using graph-theoretical methods and subsequent identification of small molecules capable of modulating activity of the best target. In this work, we construct the protein interactome underlying this disease: Asthma Protein Interactome (API). Using a strategy based on network analysis of the interactome, we identify a set of potential drug targets for asthma. Topologically and dynamically, v-src sarcoma (Schmidt-Ruppin A-2) viral oncogene homolog (SRC) emerges as the most central target in API. SRC is known to play an important role in promoting airway smooth muscle cell growth and facilitating migration in airway remodeling. From interactome analysis, and with the reported role in respiratory mechanisms, SRC emerges as a promising drug target for asthma. Further, we proceed to identify leads for SRC from a public database of small molecules. We predict two potential leads for SRC using ligand-based virtual screening methodology.
Asunto(s)
Antiasmáticos/química , Asma/tratamiento farmacológico , Simulación por Computador , Descubrimiento de Drogas/métodos , Proteína Oncogénica pp60(v-src)/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/química , Algoritmos , Asma/enzimología , Asma/genética , Dominio Catalítico , Estudios de Asociación Genética , Humanos , Modelos Biológicos , Simulación del Acoplamiento Molecular , Proteína Oncogénica pp60(v-src)/química , Unión Proteica , Mapeo de Interacción de Proteínas , Proteoma/química , Curva ROC , Propiedades de SuperficieRESUMEN
The adaptor protein ALIX [ALG-2 (apoptosis-linked-gene-2 product)-interacting protein X] links retroviruses to ESCRT (endosomal sorting complex required for transport) machinery during retroviral budding. This function of ALIX requires its interaction with the ESCRT-III component CHMP4 (charged multivesicular body protein 4) at the N-terminal Bro1 domain and retroviral Gag proteins at the middle V domain. Since cytoplasmic or recombinant ALIX is unable to interact with CHMP4 or retroviral Gag proteins under non-denaturing conditions, we constructed ALIX truncations and mutations to define the intrinsic mechanism through which ALIX interactions with these partner proteins are prohibited. Our results demonstrate that an intramolecular interaction between Patch 2 in the Bro1 domain and the TSG101 (tumour susceptibility gene 101 protein)-docking site in the proline-rich domain locks ALIX into a closed conformation that renders ALIX unable to interact with CHMP4 and retroviral Gag proteins. Relieving the intramolecular interaction of ALIX, by ectopically expressing a binding partner for one of the intramolecular interaction sites or by deleting one of these sites, promotes ALIX interaction with these partner proteins and facilitates ALIX association with the membrane. Ectopic expression of a GFP (green fluorescent protein)-ALIX mutant with a constitutively open conformation, but not the wild-type protein, increases EIAV (equine infectious anaemia virus) budding from HEK (human embryonic kidney)-293 cells. These findings predict that relieving the autoinhibitory intramolecular interaction of ALIX is a critical step for ALIX to participate in retroviral budding.
Asunto(s)
Proteínas de Unión al Calcio/fisiología , Proteínas de Ciclo Celular/fisiología , Complejos de Clasificación Endosomal Requeridos para el Transporte/metabolismo , Proteínas de los Retroviridae/metabolismo , Liberación del Virus/fisiología , Proteínas de Unión al Calcio/química , Proteínas de Unión al Calcio/genética , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/genética , Membrana Celular/metabolismo , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Complejos de Clasificación Endosomal Requeridos para el Transporte/química , Complejos de Clasificación Endosomal Requeridos para el Transporte/genética , Complejos de Clasificación Endosomal Requeridos para el Transporte/fisiología , Productos del Gen gag/química , Productos del Gen gag/genética , Productos del Gen gag/metabolismo , Células HEK293 , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Virus de la Anemia Infecciosa Equina/fisiología , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Proteína Oncogénica pp60(v-src)/química , Proteína Oncogénica pp60(v-src)/genética , Proteína Oncogénica pp60(v-src)/metabolismo , Dominios Proteicos Ricos en Prolina/fisiología , Dominios y Motivos de Interacción de Proteínas , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Proteínas de los Retroviridae/química , Proteínas de los Retroviridae/genética , Factores de Transcripción/química , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Proteínas de Transporte Vesicular/metabolismoRESUMEN
Cancer cells are exposed to external and internal stresses by virtue of their unrestrained growth, hostile microenvironment, and increased mutation rate. These stresses impose a burden on protein folding and degradation pathways and suggest a route for therapeutic intervention in cancer. Proteasome and Hsp90 inhibitors are in clinical trials and a 20S proteasome inhibitor, Velcade, is an approved drug. Other points of intervention in the folding and degradation pathway may therefore be of interest. We describe a simple screen for inhibitors of protein synthesis, folding, and proteasomal degradation pathways in this paper. The molecular chaperone-dependent client v-Src was fused to firefly luciferase and expressed in HCT-116 colorectal tumor cells. Both luciferase and protein tyrosine kinase activity were preserved in cells expressing this fusion construct. Exposing these cells to the Hsp90 inhibitor geldanamycin caused a rapid reduction of luciferase and kinase activities and depletion of detergent-soluble v-Src::luciferase fusion protein. Hsp70 knockdown reduced v-Src::luciferase activity and, when combined with geldanamycin, caused a buildup of v-Src::luciferase and ubiquitinated proteins in a detergent-insoluble fraction. Proteasome inhibitors also decreased luciferase activity and caused a buildup of phosphotyrosine-containing proteins in a detergent-insoluble fraction. Protein synthesis inhibitors also reduced luciferase activity, but had less of an effect on phosphotyrosine levels. In contrast, certain histone deacetylase inhibitors increased luciferase and phosphotyrosine activity. A mass screen led to the identification of Hsp90 inhibitors, ubiquitin pathway inhibitors, inhibitors of Hsp70/Hsp40-mediated refolding, and protein synthesis inhibitors. The largest group of compounds identified in the screen increased luciferase activity, and some of these increase v-Src levels and activity. When used in conjunction with appropriate secondary assays, this screen is a powerful cell-based tool for studying compounds that affect protein synthesis, folding, and degradation.
Asunto(s)
Inhibidores Enzimáticos/farmacología , Inhibidores de Histona Desacetilasas/farmacología , Chaperonas Moleculares/antagonistas & inhibidores , Inhibidores de Proteasoma , Pliegue de Proteína/efectos de los fármacos , Inhibidores de la Síntesis de la Proteína/farmacología , Benzoquinonas/farmacología , Línea Celular Tumoral , Evaluación Preclínica de Medicamentos , Proteínas del Choque Térmico HSP40/antagonistas & inhibidores , Proteínas del Choque Térmico HSP40/metabolismo , Proteínas HSP70 de Choque Térmico/antagonistas & inhibidores , Proteínas HSP70 de Choque Térmico/genética , Proteínas HSP70 de Choque Térmico/metabolismo , Proteínas HSP90 de Choque Térmico/antagonistas & inhibidores , Proteínas HSP90 de Choque Térmico/genética , Proteínas HSP90 de Choque Térmico/metabolismo , Histona Desacetilasas/química , Histona Desacetilasas/farmacología , Humanos , Lactamas Macrocíclicas/farmacología , Luciferasas de Luciérnaga/genética , Luciferasas de Luciérnaga/metabolismo , Proteína Oncogénica pp60(v-src)/química , Proteína Oncogénica pp60(v-src)/genética , Proteína Oncogénica pp60(v-src)/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Interferencia de ARN , ARN Interferente Pequeño , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Ubiquitina/antagonistas & inhibidores , Ubiquitina/metabolismoRESUMEN
NMR spectroscopy and molecular dynamics (MD) simulations were used to probe the structure and dynamics of complexes of three phosphotyrosine-derived peptides with the Src SH2 domain in an effort to uncover a structural explanation for enthalpy-entropy compensation observed in the binding thermodynamics. The series of phosphotyrosine peptide derivatives comprises the natural pYEEI Src SH2 ligand, a constrained mimic, in which the phosphotyrosine (pY) residue is preorganized in the bound conformation for the purpose of gaining an entropic advantage to binding, and a flexible analogue of the constrained mimic. The expected gain in binding entropy of the constrained mimic was realized; however, a balancing loss in binding enthalpy was also observed that could not be rationalized from the crystallographic structures. We examined protein dynamics to evaluate whether the observed enthalpic penalty might be the result of effects arising from altered motions in the complex. (15)N-relaxation studies and positional fluctuations from molecular dynamics indicate that the main-chain dynamics of the protein show little variation among the three complexes. Root mean squared (rms) coordinate deviations vary by less than 1.5 A for all non-hydrogen atoms for the crystal structures and in the ensemble average structures calculated from the simulations. In contrast to this striking similarity in the structures and dynamics, there are a number of large chemical shift differences from residues across the binding interface, but particularly from key Src SH2 residues that interact with pY, the "hot spot" residue, which contributes about one-half of the binding free energy. Rank-order correlations between chemical shifts and ligand binding enthalpy for several pY-binding residues, coupled with available mutagenesis and calorimetric data, suggest that subtle structural perturbations (<1 A) from the conformational constraint of the pY residue sufficiently alter the geometry of enthalpically critical interactions in the binding pocket to cause the loss of binding enthalpy, leading to the observed enthalpy-entropy compensation. We find no evidence to support the premise that enthalpy-entropy compensation is an inherent property and conclude that preorganization of Src SH2 ligand residues involved in binding hot spots may eventuate in suboptimal interactions with the domain. We propose that introducing constraints elsewhere in the ligand could minimize enthalpy-entropy compensation effects. The results illustrate the utility of the NMR chemical shift to highlight small, but energetically significant, perturbations in structure that might otherwise go unnoticed in an apparently rigid protein.
Asunto(s)
Entropía , Simulación de Dinámica Molecular , Resonancia Magnética Nuclear Biomolecular , Dominios Homologos src , Ligandos , Proteína Oncogénica pp60(v-src)/química , Proteína Oncogénica pp60(v-src)/metabolismo , Unión ProteicaRESUMEN
Analysis of the posttranslational modification of proteins, such as phosphorylation,might yield misleading results due to the presence of other proteins with similar electrophoreticproperties that coimmunoprecipitate with the target protein. The aim ofthe present work was to develop a reliable, easy and economical technique to completelyisolate a protein from its complex. Here we present a new assay developed tofully isolate proteins from macromolecular complexes that consists of an initialSDS/PAGE (under reducing conditions), which isolates the target protein, followedby transfer of the proteins to a buffer, from which the target protein is recaptured byconventional immunoprecipitation. This technique, that we have termed ProteinComplex Immunological Separation Assay (ProCISA), successfully separated proteinsof different sizes, such as pp60Src and the IP3 receptor (IP3R), from their complexes.We show that ProCISA allows the investigation of the tyrosine phosphorylationstate of isolated proteins. This technique could also be used to study other posttranslationalmodifications without risk of misleading results resulting from contaminationwith other proteins of similar electrophoretic mobility which complex with theprotein of interest (AU)
No disponible
Asunto(s)
Humanos , Animales , Electroforesis en Gel de Poliacrilamida/métodos , Inmunoprecipitación/métodos , Complejos Multiproteicos/aislamiento & purificación , Proteínas/aislamiento & purificación , Western Blotting/métodos , Receptores de Inositol 1,4,5-Trifosfato/química , Receptores de Inositol 1,4,5-Trifosfato/aislamiento & purificación , Proteína Oncogénica pp60(v-src)/química , Trombina/química , Complejos Multiproteicos/química , Proteína Oncogénica pp60(v-src)/aislamiento & purificación , Proteína Oncogénica pp60(v-src)/metabolismo , Activación Plaquetaria , Procesamiento Proteico-Postraduccional , Trombina/aislamiento & purificación , Trombina/metabolismoRESUMEN
SH2 domains provide fundamental recognition sites in tyrosine kinase-mediated signaling pathways which, when aberrant, give rise to disease states such as cancer, diabetes, and immune deficiency. Designing specific inhibitors that target the SH2 domain-binding site, however, have presented a major challenge. Despite well over a decade of intensive research, clinically useful SH2 domain inhibitors have yet to become available. A better understanding of the structural, dynamic, and thermodynamic contributions to ligand binding of individual SH2 domains will provide some insight as to whether inhibitor development is possible. We report the first high resolution solution structure of the apo-v-Src SH2 domain. This is accompanied by the analysis of backbone dynamics and pK(a) values within the apo- and peptide-bound states. Our results indicate that the phosphotyrosine (pY) pocket is tightly structured and hence not adaptable to exogenous ligands. On the other hand, the pocket which accommodates residues proximal and C-terminal of the pY (pY + 3) or so-called specificity determining region, is a large dynamic-binding surface. This appears to allow a high level of promiscuity in binding. Binding of a series of synthetic, phosphotyrosyl, peptidomimetic compounds designed to explore interactions in the pY + 3 pocket further demonstrates the ability of the SH2 domain to accommodate diverse ligands. The thermodynamic parameters of these interactions show dramatic enthalpy/entropy compensation. These data suggest that the v-Src SH2 domain does not have a highly specific secondary-binding site, which clearly presents a major hurdle to design selective inhibitors.
Asunto(s)
Diseño de Fármacos , Proteína Oncogénica pp60(v-src)/química , Proteína Oncogénica pp60(v-src)/metabolismo , Amidas/química , Secuencia de Aminoácidos , Sitios de Unión , Calorimetría , Enlace de Hidrógeno , Concentración de Iones de Hidrógeno , Ligandos , Espectroscopía de Resonancia Magnética , Datos de Secuencia Molecular , Péptidos/química , Fosfotirosina/química , Unión Proteica , Estructura Secundaria de Proteína , Soluciones , Termodinámica , Dominios Homologos srcRESUMEN
The cytoplasmic domain of LRP1 contains two NPXY motifs that have been shown to interact with signaling proteins. In previous work, we showed that Tyr(4507) in the distal NPXY motif is phosphorylated by v-Src, whereas denaturation of the protein was required for phosphorylation of Tyr(4473) in the membraneproximal NPXY motif. Amide H/D exchange studies reveal that the distal NPXY motif is fully solvent-exposed, whereas the proximal one is not. Phosphopeptide mapping combined with in vitro and in vivo kinase experiments show that Tyr(4473) can be phosphorylated, but only if Tyr(4507) is phosphorylated or substituted with glutamic acid. Amide H/D exchange experiments indicate that solvent accessibility increases across the entire LRP1 cytoplasmic region upon phosphorylation at Tyr(4507); in particular the NPXY(4473) motif becomes much more exposed. This differential phosphorylation is functionally relevant: binding of Snx17, which is known to bind at the proximal NPXY motif, is inhibited by phosphorylation at Tyr(4473). Conversely, Shp2 binds most strongly when both of the NPXY motifs in LRP1 are phosphorylated.
Asunto(s)
Proteína 1 Relacionada con Receptor de Lipoproteína de Baja Densidad/metabolismo , Secuencias de Aminoácidos/fisiología , Humanos , Proteína 1 Relacionada con Receptor de Lipoproteína de Baja Densidad/química , Proteína 1 Relacionada con Receptor de Lipoproteína de Baja Densidad/genética , Proteína Oncogénica pp60(v-src)/química , Proteína Oncogénica pp60(v-src)/genética , Proteína Oncogénica pp60(v-src)/metabolismo , Mapeo Peptídico/métodos , Fosforilación , Proteína Tirosina Fosfatasa no Receptora Tipo 11/química , Proteína Tirosina Fosfatasa no Receptora Tipo 11/genética , Proteína Tirosina Fosfatasa no Receptora Tipo 11/metabolismo , Nexinas de Clasificación , Tirosina/química , Tirosina/genética , Tirosina/metabolismo , Proteínas de Transporte Vesicular/química , Proteínas de Transporte Vesicular/genética , Proteínas de Transporte Vesicular/metabolismoRESUMEN
Analysis of the posttranslational modification of proteins, such as phosphorylation, might yield misleading results due to the presence of other proteins with similar electrophoretic properties that coimmunoprecipitate with the target protein. The aim of the present work was to develop a reliable, easy and economical technique to completely isolate a protein from its complex. Here we present a new assay developed to fully isolate proteins from macromolecular complexes that consists of an initial SDS/PAGE (under reducing conditions), which isolates the target protein, followed by transfer of the proteins to a buffer, from which the target protein is recaptured by conventional immunoprecipitation. This technique, that we have termed "Protein Complex Immunological Separation Assay" (ProCISA), successfully separated proteins of different sizes, such as pp60Src and the IP3 receptor (IP3R), from their complexes. We show that ProCISA allows the investigation of the tyrosine phosphorylation state of isolated proteins. This technique could also be used to study other posttranslational modifications without risk of misleading results resulting from contamination with other proteins of similar electrophoretic mobility which complex with the protein of interest.
Asunto(s)
Electroforesis en Gel de Poliacrilamida/métodos , Inmunoprecipitación/métodos , Complejos Multiproteicos/aislamiento & purificación , Proteínas/aislamiento & purificación , Animales , Western Blotting , Humanos , Receptores de Inositol 1,4,5-Trifosfato/química , Receptores de Inositol 1,4,5-Trifosfato/aislamiento & purificación , Complejos Multiproteicos/química , Proteína Oncogénica pp60(v-src)/química , Proteína Oncogénica pp60(v-src)/aislamiento & purificación , Activación Plaquetaria , Procesamiento Proteico-Postraduccional , TrombinaRESUMEN
Discovery of small molecule inhibitors of protein-protein interactions is a major challenge to pharmaceutical development. Fragment-based approaches have begun to be widely adopted as an effective way of exploring chemical space on a protein surface with reduced library size. On completion of a fragment screen, the subsequent selection of appropriate "hit" molecules for development is a key decision point. Thermodynamic parameters can be used in this decision process. In this work, a fragment identification protocol based on a virtual fragment analysis and selection followed by 19F NMR screening was directed at the phosphotyrosine binding site of the Src SH2 domain. Three new ligands were identified. Isothermal titration calorimetry was used to provide thermodynamic parameters for the physiologically relevant ligand and the selected fragments. One of these fragments possesses a highly favorable enthalpic contribution to complex formation compared to other fragments and to the physiologically relevant ligand suggesting that it would make a good candidate for compound development.
Asunto(s)
Simulación por Computador , Resonancia Magnética Nuclear Biomolecular/métodos , Proteína Oncogénica pp60(v-src)/química , Proteína Oncogénica pp60(v-src)/metabolismo , Fosfotirosina/química , Fosfotirosina/metabolismo , Inhibidores de Proteínas Quinasas/química , Bases de Datos de Proteínas , Evaluación Preclínica de Medicamentos , Enlace de Hidrógeno , Ligandos , Modelos Moleculares , Proteína Oncogénica pp60(v-src)/antagonistas & inhibidores , Unión Proteica , Termodinámica , Dominios Homologos srcRESUMEN
We have studied the role of bound interface water molecules on the prediction of the thermodynamics of SH2 domain binding to tyrosyl phosphopeptides using a method based on accessible surface area buried upon association. We studied three phosphopeptide ligands, which have been shown by Lubman and Waksman (J Mol Biol;328:655, 2003) and Davidson et al. (JACS;124:205, 2002) to have similar binding free energies but very different thermodynamic signatures. The thermodynamic model is semiempirical and applies to the crystal structure of the SH2 domain-bound forms. We explored all possible combinations of bound interfacial waters. We show that the model does not predict the binding thermodynamics of either ligand. However, we identified the empirical formula describing the heat capacity change as the source of the problem. Indeed, systematic exploration of heat capacity change values between 0 and -300 cal/mol deg results in a sharp distribution of the number of ligand/SH2/water-subset structures that provide binding thermodynamics similar to experimental values. The heat capacity change values at which the distributions peak are different for each peptide. This prompted us to experimentally determine the heat capacity change for each of the peptides and we found them to coincide with the values of the peaks. The implications of such findings are discussed.
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Biología Computacional/métodos , Termodinámica , Agua/química , Dominios Homologos src , Ligandos , Modelos Moleculares , Proteína Oncogénica pp60(v-src)/química , Fosfopéptidos/química , Fosfotirosina/química , Unión Proteica , Conformación ProteicaRESUMEN
Regions of protein-tyrosine phosphatase (PTP) 1B that are distant from the active site yet affect inhibitor binding were identified by a novel library screen. This screen was based on the observation that expression of v-Src in yeast leads to lethality, which can be rescued by the coexpression of PTP1B. However, this rescue is lost when yeast are grown in the presence of PTP1B inhibitors. To identify regions of PTP1B (amino acids 1-400, catalytic domain plus 80-amino acid C-terminal tail) that can affect the binding of the difluoromethyl phosphonate (DFMP) inhibitor 7-bromo-6-difluoromethylphosphonate 3-naphthalenenitrile, a library coexpressing PTP1B mutants and v-Src was generated, and the ability of yeast to grow in the presence of the inhibitor was evaluated. PTP1B inhibitor-resistant mutations were found to concentrate on helix alpha7 and its surrounding region, but not in the active site. No resistant amino acid substitutions were found to occur in the C-terminal tail, suggesting that this region has little effect on active-site inhibitor binding. An in-depth characterization of a resistant substitution localizing to region alpha7 (S295F) revealed that this change minimally affected enzyme catalytic activity, but significantly reduced the potency of a panel of structurally diverse DFMP PTP1B inhibitors. This loss of inhibitor potency was found to be due to the difluoro moiety of these inhibitors because only the difluoro inhibitors were shifted. For example, the inhibitor potency of a monofluorinated or non-fluorinated analog of one of these DFMP inhibitors was only minimally affected. Using this type of library screen, which can scan the nearly full-length PTP1B sequence (catalytic domain and C-terminal tail) for effects on inhibitor binding, we have been able to identify novel regions of PTP1B that specifically affect the binding of DFMP inhibitors.
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Proteínas Fúngicas/química , Nitrilos/farmacología , Organofosfonatos/farmacología , Proteínas Tirosina Fosfatasas/antagonistas & inhibidores , Sitio Alostérico , Sitios de Unión , Catálisis , Dominio Catalítico , Cartilla de ADN/química , Inhibidores Enzimáticos/farmacología , Biblioteca de Genes , Concentración 50 Inhibidora , Cinética , Modelos Moleculares , Modelos Estadísticos , Mutación , Proteína Oncogénica pp60(v-src)/química , Organofosfonatos/química , Plásmidos/metabolismo , Unión Proteica , Conformación Proteica , Estructura Terciaria de Proteína , Proteína Tirosina Fosfatasa no Receptora Tipo 1 , Factores de TiempoRESUMEN
v-Src is a non-receptor protein tyrosine kinase involved in many signal transduction pathways and closely related to the activation and development of cancers. We present here the expression, purification, and bioactivity of a GST (glutathione S-transferase)-fused v-Src from a bacterial expression system. Different culture conditions were examined in an isopropyl beta-D-thiogalactopyranoside (IPTG)-regulated expression, and the fused protein was purified using GSH (glutathione) affinity chromatography. ELISA (enzyme-linked immunosorbent assay) was employed to determine the phosphorylation kinase activity of the GST-fused v-Src. This strategy seems to be more promising than the insect cell system or other eukaryotic systems employed in earlier Src expression.