RESUMEN
BACKGROUND: The pleckstrin homology (PH) domain is a region of approximately 100 amino acids, defined by sequence similarity, that has been found in about 60 proteins, many of which are involved in signal transduction downstream of cell surface receptors; the function of PH domains is unknown. The only clue to the function of PH domains is the circumstantial evidence that they may link beta gamma subunits of G proteins to second messenger systems. Knowledge of the three-dimensional structures of PH domains should help to elucidate the roles they play in the proteins that contain them. RESULTS: Using homonuclear and heteronuclear magnetic resonance spectroscopy, we have determined the solution structure of the PH domain of the GTPase dynamin, one of a number of proteins that have PH domains and interact with GTP. The fold of the dynamin PH domain is composed of two antiparallel beta-sheets, which pack face-to-face at an angle of approximately 60 degrees. The first beta-sheet comprises four strands (residues 13-58) from the amino-terminal half of the protein sequence; the second beta-sheet contains three strands (residues 63-99). A single alpha-helix (residues 102-116) flanks one edge of the interface between the two sheets, parallel in orientation to the second sheet, in an alpha/beta roll motif similar to that of the B oligomer of verotoxin-1 from Escherichia coli. CONCLUSIONS: The structure of the dynamin PH domain is very similar to the recently reported structures of the pleckstrin and spectrin PH domains. This shows that, despite the low level of sequence similarity between different PH domains, they do have a characteristic polypeptide fold. On the basis of our structure, the suggestion that PH domains engage in coiled-coil interactions with G protein beta gamma subunits seems unlikely and should be re-evaluated.
Asunto(s)
Proteínas Sanguíneas/química , GTP Fosfohidrolasas/química , Fosfoproteínas , Secuencia de Aminoácidos , Secuencia Conservada , Dinaminas , Humanos , Datos de Secuencia Molecular , Estructura Secundaria de Proteína , Homología de Secuencia de AminoácidoRESUMEN
The class II phosphoinositide 3-kinases (PI3K) PI3K-C2alpha and PI3K-C2beta are two recently identified members of the large PI3K family. Both enzymes are characterized by the presence of a C2 domain at the carboxy terminus and, in vitro, preferentially utilize phosphatidylinositol and phosphatidylinositol 4-monophosphate as lipid substrates. Little is understood about how the catalytic activity of either enzyme is regulated in vivo. In this study, we demonstrate that PI3K-C2alpha and PI3K-C2beta represent two downstream targets of the activated epidermal growth factor (EGF) receptor in human carcinoma-derived A431 cells. Stimulation of quiescent cultures with EGF resulted in the rapid recruitment of both enzymes to a phosphotyrosine signaling complex that contained the EGF receptor and Erb-B2. Ligand addition also induced the appearance of a second, more slowly migrating band of PI3K-C2alpha and PI3K-C2beta immunoreactivity on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Since both PI3K enzymes can utilize Ca(2+) as an essential divalent cation in lipid kinase assays and since the catalytic activity of PI3K-C2alpha is refractory to the inhibitor wortmannin, these properties were used to confirm the recruitment of each PI3K isozyme to the activated EGF receptor complex. To examine this interaction in greater detail, PI3K-C2beta was chosen for further investigation. EGF and platelet-derived growth factor also stimulated the association of PI3K-C2beta with their respective receptors in other cells, including epithelial cells and fibroblasts. The use of EGF receptor mutants and phosphopeptides derived from the EGF receptor and Erb-B2 demonstrated that the interaction with recombinant PI3K-C2beta occurs through E(p)YL/I phosphotyrosine motifs. The N-terminal region of PI3K-C2beta was found to selectively interact with the EGF receptor in vitro, suggesting that it mediates the association of this PI3K with the receptor. However, the mechanism of this interaction remains unclear. We conclude that class II PI3K enzymes may contribute to the generation of 3' phosphoinositides following the activation of polypeptide growth factor receptors in vivo and thus mediate certain aspects of their biological activity.
Asunto(s)
Receptores ErbB/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Sitios de Unión , Calcio/metabolismo , Factor de Crecimiento Epidérmico/metabolismo , Factor de Crecimiento Epidérmico/farmacología , Humanos , Fosfatos/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Fosfotirosina/metabolismo , Factor de Crecimiento Derivado de Plaquetas/metabolismo , Receptor ErbB-2/metabolismo , Receptores del Factor de Crecimiento Derivado de Plaquetas/metabolismo , Células Tumorales CultivadasRESUMEN
Wortmannin at nanomolar concentrations is a potent and specific inhibitor of phosphoinositide (PI) 3-kinase and has been used extensively to demonstrate the role of this enzyme in diverse signal transduction processes. At higher concentrations, wortmannin inhibits the ataxia telangiectasia gene (ATM)-related DNA-dependent protein kinase (DNA-PKcs). We report here the identification of the site of interaction of wortmannin on the catalytic subunit of PI 3-kinase, p110alpha. At physiological pH (6.5 to 8) wortmannin reacted specifically with p110alpha. Phosphatidylinositol-4,5-diphosphate, ATP, and ATP analogs [adenine and 5'-(4-fluorosulfonylbenzoyl)adenine] competed effectively with wortmannin, while substances containing nucleophilic amino acid side chain functions had no effect at the same concentrations. This suggests that the wortmannin target site is localized in proximity to the substrate-binding site and that residues involved in wortmannin binding have an increased nucleophilicity because of their protein environment. Proteolytic fragments of wortmannin-treated, recombinant p110alpha were mapped with anti-wortmannin and anti-p110alpha peptide antibodies, thus limiting the target site within a 10-kDa fragment, colocalizing with the ATP-binding site. Site-directed mutagenesis of all candidate residues within this region showed that only the conservative Lys-802-to-Arg mutation abolished wortmannin binding. Inhibition of PI 3-kinase occurs, therefore, by the formation of an enamine following the attack of Lys-802 on the furan ring (at C-20) of wortmannin. The Lys-802-to-Arg mutant was also unable to bind FSBA and was catalytically inactive in lipid and protein kinase assays, indicating a crucial role for Lys-802 in the phosphotransfer reaction. In contrast, an Arg-916-to-Pro mutation abolished the catalytic activity whereas covalent wortmannin binding remained intact. Our results provide the basis for the design of novel and specific inhibitors of an enzyme family, including PI kinases and ATM-related genes, that play a central role in many physiological processes.
Asunto(s)
Androstadienos/farmacología , Inhibidores Enzimáticos/farmacología , Fosfotransferasas (Aceptor de Grupo Alcohol)/antagonistas & inhibidores , Adenosina Trifosfato/metabolismo , Secuencia de Aminoácidos , Cinética , Lisina/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Fosfatidilinositol 3-Quinasas , Fosfatidilinositol 4,5-Difosfato , Fosfatos de Fosfatidilinositol/metabolismo , Fosfotransferasas (Aceptor de Grupo Alcohol)/genética , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Mutación Puntual , Transducción de Señal , Especificidad por Sustrato , WortmaninaRESUMEN
Germline mutations within one of six codons of the RET proto-oncogene account for the majority of cases of multiple endocrine neoplasia (MEN) type 2A and type 2B and familial medullary thyroid carcinoma (FMTC). MEN 2A and FMTC mutations characterised thus far occur exclusively in the cysteine-rich domain of the extracellular region of RET. We now report a missense mutation in the intracellular tyrosine kinase domain of RET in the germline of a family with FMTC that does not have a cysteine codon mutation. In this family, the mutation, which alters GAG (Glu) to GAC (Asp) at codon 768, segregates with the FMTC phenotype. The same mutation was also detected in sporadic MTC but not in corresponding constitutional DNA, confirming that it is likely to be of pathological significance rather than a rare polymorphism.
Asunto(s)
Carcinoma Medular/genética , Proteínas de Drosophila , Neoplasia Endocrina Múltiple/genética , Mutación Puntual , Proteínas Proto-Oncogénicas/genética , Proto-Oncogenes/genética , Proteínas Tirosina Quinasas Receptoras/genética , Neoplasias de la Tiroides/genética , Secuencia de Bases , Codón/genética , Femenino , Humanos , Masculino , Datos de Secuencia Molecular , Linaje , Proto-Oncogenes Mas , Proteínas Proto-Oncogénicas c-retRESUMEN
The prediction of protein secondary structure (alpha-helices, beta-sheets and coil) is improved by 9% to 66% using the information available from a family of homologous sequences. The approach is based both on averaging the Garnier et al. (1978) secondary structure propensities for aligned residues and on the observation that insertions and high sequence variability tend to occur in loop regions between secondary structures. Accordingly, an algorithm first aligns a family of sequences and a value for the extent of sequence conservation at each position is obtained. This value modifies a Garnier et al. prediction on the averaged sequence to yield the improved prediction. In addition, from the sequence conservation and the predicted secondary structure, many active site regions of enzymes can be located (26 out of 43) with limited over-prediction (8 extra). The entire algorithm is fully automatic and is applicable to all structural classes of globular proteins.
Asunto(s)
Conformación Proteica , Algoritmos , Secuencia de Aminoácidos , Sitios de UniónRESUMEN
Src-homology 3 (SH3) domains are small protein modules that bind to proline-rich motifs and mediate the formation of signalling complexes. SH3 domains have been implicated in the assembly of the phagocyte NADPH oxidase complex, a multicomponent enzyme responsible for the production of antimicrobial oxidants. Two components of the NADPH oxidase, p67phox and p47phox, each contain two SH3 domains and we have previously shown that the SH3 domain near the carboxyl terminus of p67phox interacts with a proline-rich region of p47phox. In order to gain an insight into the specificity of this interaction, a structural model of the p67phox SH3 domain has been produced using the known structure of the c-abl SH3 domain as a template. The model suggests that the proline-rich ligand of p47phox can bind to the SH3 domain in either of two orientations. In each orientation, the key residues of the SH3 domain that contact the ligand have been identified and altered by site-directed mutagenesis. The ability of the mutated SH3 domains to associate with p47phox from cell lysates was tested and the results provide the first evidence for the binding of a full-length protein to an SH3 domain in a reversed orientation.
Asunto(s)
Modelos Moleculares , Fosfoproteínas/química , Fosfoproteínas/metabolismo , Dominios Homologos src , Secuencia de Aminoácidos , Humanos , Ligandos , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , NADPH Oxidasas , Oligopéptidos/síntesis química , Oligopéptidos/metabolismo , Fosfoproteínas/genética , Prolina/metabolismo , Unión Proteica , Proteínas Proto-Oncogénicas c-abl/químicaRESUMEN
An unexpected structural similarity is described between the pleckstrin homology (PH) domain and verotoxin. This similarity has escaped detection primarily due to the differences in topology that exist between the two proteins. By comparing this result with two previously reported similarities for the PH domain, one with the lipocalins and another with the FK506 binding protein, we discuss the problems of measuring and assessing structural similarities.
Asunto(s)
Toxinas Bacterianas/química , Proteínas Sanguíneas/química , Fosfoproteínas , Conformación Proteica , Estructura Secundaria de Proteína , Secuencia de Aminoácidos , Sitios de Unión , Proteínas Portadoras/química , Proteínas de Unión al ADN/química , Enterotoxinas/química , Proteínas de Choque Térmico/química , Modelos Moleculares , Modelos Estructurales , Datos de Secuencia Molecular , Homología de Secuencia de Aminoácido , Toxina Shiga I , Tacrolimus/metabolismo , Proteínas de Unión a TacrolimusRESUMEN
An important consideration in the design of vaccines to prevent HIV-1 infection effective against different strains is the amino acid sequence conservation of antigenic determinants. Even one amino acid change can destroy the antigenicity of a site for the antibody or T-cell receptor. The comparisons of predicted T- and B-cell epitopes between human HIV-1, HIV-2 and monkey SIVMAC AIDS viruses are presented. The three major gene products (env, gag and pol) were examined. A number of epitopes were identical between strains of HIV-1. Our analysis highlights the problem of designing an effective HIV-1 and HIV-2 vaccine and also the problem of testing human vaccines in monkey models.
Asunto(s)
Linfocitos B/inmunología , Epítopos/inmunología , Antígenos VIH/inmunología , VIH-1/inmunología , VIH-2/inmunología , Virus de la Inmunodeficiencia de los Simios/inmunología , Linfocitos T/inmunología , Secuencia de Aminoácidos , Productos del Gen gag , Datos de Secuencia Molecular , Proteínas de los Retroviridae/inmunología , Proteínas del Envoltorio Viral/inmunologíaRESUMEN
Criteria for the design of peptide vaccines to prevent AIDS are presented. The best vaccine candidates contain both B and T lymphocyte-defined epitopes in regions conserved in sequence between viral isolates. We propose that attention should focus on proteins specified by the gag and, possibly, pol genes in addition to the env gene envelope glycoproteins being actively studied. The predictions of B- and T-epitopes are refined by consideration of secondary structure prediction and inter-isolate sequence variability to suggest peptides from env, gag and pol that would be the best vaccine candidates.
Asunto(s)
Epítopos , VIH/análisis , Proteínas de los Retroviridae/inmunología , Secuencia de Aminoácidos , Epítopos/inmunología , VIH/inmunología , Conformación Proteica , Vacunas Virales/inmunologíaRESUMEN
Methods to predict the three-dimensional structure of a protein from its sequence are reviewed. The approaches to derive information about the local conformation from the local sequence include hydrophobicity plots, secondary structure prediction and the identification of short, functional sequence motifs. The most reliable method of tertiary structure prediction is model building from the experimentally determined coordinates of a protein with an homologous sequence. This approach is illustrated by a prediction of the three-dimensional structure of human cytochrome P450-IA1. If no known homologous structure is available, then the only approach is to suggest models for the tertiary fold of proteins by packing together predicted secondary structures. A three-dimensional model for the dimerisation of the transmembrane alpha-helices of neu, a tyrosine kinase growth factor receptor, is described. In general, structure prediction can suggest approaches for regulating protein activity that may lead to new pharmaceutical therapies for cancer.
Asunto(s)
Secuencia de Aminoácidos , Receptor ErbB-2 , Sistema Enzimático del Citocromo P-450/química , Receptores ErbB/química , Humanos , Modelos Moleculares , Conformación Proteica , Proteínas Tirosina Quinasas , Proteínas Proto-Oncogénicas/química , Relación Estructura-ActividadRESUMEN
The sequence of the major soluble protein component of the cuticle of filarial nematodes is homologous to that of bovine glutathione peroxidase, for which an X-ray structure is available. Due to the high degree of sequence identity (42%), it has been possible to build an apparently reliable three-dimensional model of the gp29 cuticular protein from Brugia spp. that will aid studies of the molecule both as a target immunogen and secreted enzyme. The modelled core of the gp29 structure is conserved compared to the bovine enzyme, consisting of a beta-sheet surrounded by alpha-helices. Experimental data showed that Brugia spp. gp29 has four subunits, and a tetrameric form of gp29 has also been modelled. The two N-linked glycosylation sites per subunit were predicted to lie on the surface of the tetramer. Most of the variation in amino acid sequence compared to that of mammalian enzymes, occurs in the surface loops, several of which are larger and more exposed in gp29. Deglycosylated gp29 was demonstrated to be immunogenic in human infection, and six likely B-cell epitopes have been predicted on the basis of a high protrusion index and sequence variability.
Asunto(s)
Antígenos Helmínticos/inmunología , Brugia Malayi/inmunología , Epítopos/inmunología , Glutatión Peroxidasa/inmunología , Proteínas del Helminto/inmunología , Sistema Linfático/parasitología , Secuencia de Aminoácidos , Animales , Sitios de Unión , Brugia Malayi/enzimología , Glicosilación , Modelos Moleculares , Datos de Secuencia Molecular , Conformación Proteica , Alineación de SecuenciaRESUMEN
Using a variety of techniques, including sequence alignment, secondary structure prediction, molecular mechanics and molecular dynamics, we have constructed a model for the three-dimensional structure of P-450arom (human aromatase) based on that of P-450cam, the only cytochrome P-450 enzyme for which the crystal structure is known. The predicted structure is found to be in good agreement with current experimental data; both direct, from site-directed mutagenesis studies, and indirect, from the consideration of the structures and activities of known substrates and inhibitors.
Asunto(s)
Aromatasa/química , Modelos Moleculares , Estructura Secundaria de Proteína , Secuencia de Aminoácidos , Aromatasa/metabolismo , Sitios de Unión , Alcanfor 5-Monooxigenasa , Sistema Enzimático del Citocromo P-450/química , Humanos , Enlace de Hidrógeno , Oxigenasas de Función Mixta/química , Datos de Secuencia Molecular , Homología de Secuencia de Aminoácido , Programas InformáticosRESUMEN
Hoechst is developing flavopiridol, a synthetic flavonoid based on an extract from an Indian plant, for the potential treatment of cancer. Flavopiridol, a cyclin-dependent kinase inhibitor, arrests cell division and causes apoptosis in non-small lung cancer cells [283660]. A phase II trial, in collaboration with the National Cancer Institute, has commenced at the University of Chicago Medical Center, which involves patients with high or intermediate-grade lymphoma or multiple myeloma [272937], [277372]. In ex vivo experiments with tumor cells from refractory chronic lymphoblastic leukemia, dose-dependent CDK2 inhibition associated with apoptotic changes was seen at concentrations greater than 100 nM of flavopiridol. In vitro pharmacokinetic studies have shown that flavopiridol undergoes hepatic biotransformation to its corresponding glucoronide by uridine diphosphate glucoronosyltransferases [283791]. Flavopiridol inhibits CDK with an IC50 value of 0.4 mM [285707]. Preclinical toxicology studies in rats and dogs demonstrated dose-related leukopenia and drug-related lesions in the thymus, spleen and bone marrow. The gastrointestinal and bone marrow toxicity was dose-limiting [178579]. Hoechst Marion Roussel expects to launch flavopiridol in the year 2001, with potential sales in excess of DM 750 million [288651].
RESUMEN
The catalytic residues of an enzyme are defined as the amino acids directly involved in chemical catalysis. They mainly act as a general acid--base, electrophilic or nucleophilic catalyst or they polarize and stabilize the transition state. An analysis of the structural features of 36 catalytic residues in 17 enzymes of known structure and with defined mechanism is reported. Residues that bind metal ions (Zn2+ and Cu2+) are considered separately. The features examined are: residue type, location in secondary structure, separation between the residues, accessibility to solvent, intra-protein electrostatic interactions, mobility as evaluated from crystallographic temperature factors, polarity of the environment and the sequence conservation between homologous enzymes of residues that were sequentially or spatially close to the catalytic residue. In general the environment of catalytic residues is similar to that of polar side chains that have low accessibility to solvent. Two algorithms have been developed to identify probable catalytic residues. Scanning an alignment of homologous enzyme sequences for peaks of sequence conservation identifies 13 out of the 16 catalytic residues with 50 residues overpredicted. When the conservation of the spatially close residues is used instead, a different set of 13 residues are identified with 47 residues overpredicted. A combination of the two algorithms identifies 11 residues with 36 residues overpredicted.
Asunto(s)
Catálisis , Enzimas , Conformación Proteica , Algoritmos , Secuencia de Aminoácidos , Aminoácidos/análisis , Sitios de Unión , Cloranfenicol O-Acetiltransferasa , Enzimas/análisis , Enlace de Hidrógeno , Iones , Metales , Modelos Moleculares , Datos de Secuencia Molecular , Movimiento (Física) , Relación Estructura-ActividadRESUMEN
A three-dimensional structure for human cytochrome P450IA1 was predicted based on the crystal coordinates of cytochrome P450cam from Pseudomonas putida. As there was only 15% residue identity between the two enzymes, additional information was used to establish an accurate sequence alignment that is a prerequisite for model building. Twelve representative eukaryotic sequences were aligned and a net prediction of secondary structure was matched against the known alpha-helices and beta-sheets of P450cam. The cam secondary structure provided a fixed main-chain framework onto which loops of appropriate length from the human P450IA1 structure were added. The model-built structure of the human cytochrome conformed to the requirements for the segregation of polar and nonpolar residues between the core and the surface. The first 44 residues of human cytochrome P450 could not be built into the model and sequence analysis suggested that residues 1-26 formed a single membrane-spanning segment. Examination of the sequences of cytochrome P450s from distinct gene families suggested specific residues that could account for the differences in substrate specificity. A major substrate for P450IA1, 3-methyl-cholanthrene, was fitted into the proposed active site and this planar aromatic molecule could be accommodated into the available cavity. Residues that are likely to interact with the haem were identified. The sequence similarity between 59 eukaryotic enzymes was represented as a dendrogram that in general clustered according to gene family. Until a crystallographic structure is available, this model-building study identifies potential residues in cytochrome P450s important in the function of these enzymes and these residues are candidates for site-directed mutagenesis.
Asunto(s)
Sistema Enzimático del Citocromo P-450/química , Secuencia de Aminoácidos , Alcanfor 5-Monooxigenasa , Simulación por Computador , Sistema Enzimático del Citocromo P-450/genética , Humanos , Oxigenasas de Función Mixta/química , Oxigenasas de Función Mixta/genética , Modelos Moleculares , Datos de Secuencia Molecular , Conformación Proteica , Alineación de Secuencia , Homología de Secuencia de Ácido Nucleico , Especificidad por SustratoRESUMEN
The dithiol trypanothione, novel to trypanosomatids and analogous to glutathione in mammalian systems, has been shown to interact with anti-trypanocidal trivalent arsenical drugs forming a stable adduct, MelT. This adduct is a competitive inhibitor of the flavoprotein trypanothione reductase, responsible for maintaining intracellular trypanothione in the reduced form. Since trypanothione reductase and the analogous glutathione reductase both contain catalytically active sulphydryl groups we have examined the ability of several arsenicals to differentially inhibit these enzymes. Melarsen oxide [p-(4,6-diamino-s-triazin-2-yl)aminophenylarsenoxide] potently inhibits both enzymes in two stages, the first being essentially complete within 1 min, the second being time dependent, exhibiting saturable pseudo-first-order kinetics with kinact of 14.3 x 10(-4) s-1 and 1.06 x 10(-4) s-1 and Ki of 17.2 microM and 9.6 microM for trypanothione reductase and glutathione reductase, respectively. Inhibition requires prior reduction of the enzyme by NADPH and can be reversed by excess dithiols or prevented by MelT in the case of trypanothione reductase. In both cases a time-dependent loss of the characteristic charge-transfer absorbance band at 530 nm is observed upon addition of arsenical to pre-reduced enzyme, which with excess NADPH leads to a spectrum resembling the EH4 form and is accompanied by an increased ability to reduce molecular oxygen. A model for inhibition is proposed where, first, free arsenical and previously reduced enzyme immediately establish an equilibrium with an inactive monothioarsane enzyme-inhibitor complex involving the interchange cysteine distal to the FAD; second, a subsequent rearrangement about the sulphur-arsenic bond leads to the binding of the arsenical to the charge-transfer cysteine, proximal to the FAD, forming a more stable dithioarsane complex. Molecular modelling suggests that the differences in kinetic behaviour of the two enzymes can be attributed to structural features of their respective disulphide-binding sites. Incubation of reduced trypanothione reductase with excess dihydrotrypanothione and melarsen oxide prevents direct inhibition of the enzyme, suggesting that dihydrotrypanothione acts as a protectant in vivo, preventing the direct modification of trypanothione reductase by sequestering the arsenical as MelT.
Asunto(s)
Arsenicales/farmacología , Crithidia fasciculata/enzimología , Eritrocitos/enzimología , Glutatión Reductasa/antagonistas & inhibidores , NADH NADPH Oxidorreductasas/antagonistas & inhibidores , Tripanocidas/farmacología , Animales , Arsenicales/química , Sitios de Unión , Unión Competitiva , Disulfuros/metabolismo , Glutatión/análogos & derivados , Glutatión/farmacología , Glutatión Reductasa/química , Humanos , Cinética , Modelos Moleculares , NADH NADPH Oxidorreductasas/química , NADP/farmacología , Relación Estructura-ActividadRESUMEN
The enzyme cytochrome P450(17 alpha) catalyses two key steps in the biosynthesis of the androgens from pregnanes: the 17 alpha hydroxylation step and the subsequent 17-20 lyase reaction. Using a variety of techniques, including sequence alignment, secondary structure prediction, molecular mechanics and molecular dynamics, we have constructed a model for the three-dimensional structure of P450(17 alpha) based on that of P450cam, the only cytochrome P450 enzyme for which the crystal structure is known. The model suggests the possibility of two modes of binding of steroid substrates at the active site, perhaps reflecting the dual functionality of the enzyme.
Asunto(s)
Modelos Moleculares , Neoplasias de la Próstata/tratamiento farmacológico , Esteroide 17-alfa-Hidroxilasa/química , Secuencia de Aminoácidos , Sitios de Unión , Diseño de Fármacos , Humanos , Masculino , Datos de Secuencia Molecular , Conformación Proteica , Homología de Secuencia de Ácido NucleicoRESUMEN
Src homology 2 (SH2) domains are small protein modules of approximately 100 amino acids that are found in many proteins involved in intracellular signal transduction. They mediate protein-protein interactions and modulate enzyme activity by their ability to bind to specific sequence patterns that contain a phosphorylated tyrosine. As the three-dimensional structures of the phosphatidylinositol (PI) 3-kinase, Lck, Src and Abl SH2 domains have been shown to be similar, we have modelled other SH2 domains that show distinct sequence specificity to allow comparative analysis of SH2-phosphopeptide interactions. The SH2 domains of PLC gamma-Nterm., Nck, Grb2, GAP and Abl have been model-built with high-affinity phosphopeptides fitted into the putative binding sites. For each SH2 domain a detailed analysis of the peptide-protein interaction was performed. It is apparent that specificity is mainly conferred by three to five residues downstream from the phosphotyrosine residue (Y*), especially, although not exclusively, peptide position Y* + 3. The SH2 pocket that binds the Y* + 3 residue is mainly composed of three sections: part of strand beta E going into loop EF, part of alpha B and loop BG. The residues that constitute the Y* + 3 binding pocket show variability that seems to determine which amino acid binds preferentially. Residue position beta E4 seems to play a vital role in the SH2 specificity. This study shows that the development of modelling protocols for SH2 domains whose structure has not been determined can prove very useful in predicting which residues are involved in conferring the affinity and binding specificity of these domains towards distinct phosphotyrosine-containing sequences.
Asunto(s)
Fosfopéptidos/metabolismo , Dominios Homologos src , Secuencia de Aminoácidos , Sitios de Unión , Modelos Moleculares , Datos de Secuencia Molecular , Fosfopéptidos/química , Conformación Proteica , Homología de Secuencia de AminoácidoRESUMEN
Phosphoinositide 3-kinases (PI3Ks) are dual specificity lipid and protein kinases. While the lipid-dependent PI3K downstream signaling is well characterized, little is known about PI3K protein kinase signaling and structural determinants of lipid substrate specificity across the various PI3K classes. Here we show that sequences C-terminal to the PI3K ATP-binding site determine the lipid substrate specificity of the class IA PI3Kalpha (p85/p110alpha). Transfer of such activation loop sequences from class II PI3Ks, class III PI3Ks, and a related mammalian target of rapamycin (FRAP) into p110alpha turns the lipid substrate specificity of the resulting hybrid protein into that of the donor protein, while leaving the protein kinase activity unaffected. All resulting hybrids lacked the ability to produce phosphatidylinositol 3,4,5-trisphosphate in intact cells. Amino acid substitutions and structure modeling showed that two conserved positively charged (Lys and Arg) residues in the activation loop are crucial for the functionality of class I PI3Ks as phosphatidylinositol 4,5-bisphosphate kinases. By transient transfecion of 293 cells, we show that p110alpha hybrids, although unable to support lipid-dependent PI3K signaling, such as activation of protein kinase B/Akt and p70(S6k), retain the capability to associate with and phosphorylate insulin receptor substrate-1, with the same specificity and higher efficacy than wild type PI3Kalpha. Our data lay the basis for the understanding of the class I PI3K substrate selectivity and for the use of PI3Kalpha hybrids to dissect PI3Kalpha function as lipid and protein kinase.
Asunto(s)
Fosfatidilinositol 3-Quinasas/química , Fosfatidilinositol 3-Quinasas/metabolismo , Transducción de Señal/fisiología , Adenosina Trifosfato/metabolismo , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Androstadienos/farmacología , Animales , Sitios de Unión , Células COS , Línea Celular , Chlorocebus aethiops , Secuencia Conservada , Activación Enzimática , Inhibidores Enzimáticos/farmacología , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Fosfatidilinositol 3-Quinasas/genética , Mutación Puntual , Conformación Proteica , Proteínas Quinasas/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Sirolimus/farmacología , Programas Informáticos , Especificidad por Sustrato , Transfección , WortmaninaRESUMEN
The pyruvate-stimulated adenylate cyclase from Brevibacterium liquefaciens produces up to 450 microM cyclic AMP in the culture medium when the bacterium is grown on glucose and alanine. In this paper we report the cloning, expression and sequencing of the gene for this enzyme. Residues were identified, within the C-terminal domain, which are conserved in adenylate and guanylate cyclase sequences from eukaryotes and in the adenylate cyclase of the prokaryote Rhizobium meliloti. We have also identified a sequence of 30 residues near the N-terminus of the protein which is homologous to part of the regulatory domain of the cellular homologues of the oncogenes fes and fps; this sequence is also present in the avian Fujinami sarcoma virus fps gene.