RESUMEN
CDK5 plays an indispensable role in the central nervous system, and its deregulation is involved in neurodegeneration. We report the crystal structure of a complex between CDK5 and p25, a fragment of the p35 activator. Despite its partial structural similarity with the cyclins, p25 displays an unprecedented mechanism for the regulation of a cyclin-dependent kinase. p25 tethers the unphosphorylated T loop of CDK5 in the active conformation. Residue Ser159, equivalent to Thr160 on CDK2, contributes to the specificity of the CDK5-p35 interaction. Its substitution with threonine prevents p35 binding, while the presence of alanine affects neither binding nor kinase activity. Finally, we provide evidence that the CDK5-p25 complex employs a distinct mechanism from the phospho-CDK2-cyclin A complex to establish substrate specificity.
Asunto(s)
Quinasas Ciclina-Dependientes/química , Proteínas del Tejido Nervioso/química , Secuencia de Aminoácidos , Animales , Sitios de Unión , Células COS , Cristalografía por Rayos X , Quinasa 5 Dependiente de la Ciclina , Quinasas Ciclina-Dependientes/metabolismo , Humanos , Immunoblotting , Modelos Moleculares , Datos de Secuencia Molecular , Proteínas del Tejido Nervioso/metabolismo , Péptidos/química , Péptidos/metabolismo , Fosforilación , Unión Proteica , Conformación Proteica , Estructura Terciaria de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Alineación de SecuenciaRESUMEN
Small G proteins are GTP-dependent molecular switches that regulate numerous cellular functions. They can be classified into homologous subfamilies that are broadly associated with specific biological processes. Cross-talk between small G-protein families has an important role in signalling, but the mechanism by which it occurs is poorly understood. The coordinated action of Arf and Rho family GTPases is required to regulate many cellular processes including lipid signalling, cell motility and Golgi function. Arfaptin is a ubiquitously expressed protein implicated in mediating cross-talk between Rac (a member of the Rho family) and Arf small GTPases. Here we show that Arfaptin binds specifically to GTP-bound Arf1 and Arf6, but binds to Rac.GTP and Rac.GDP with similar affinities. The X-ray structure of Arfaptin reveals an elongated, crescent-shaped dimer of three-helix coiled-coils. Structures of Arfaptin with Rac bound to either GDP or the slowly hydrolysable analogue GMPPNP show that the switch regions adopt similar conformations in both complexes. Our data highlight fundamental differences between the molecular mechanisms of Rho and Ras family signalling, and suggest a model of Arfaptin-mediated synergy between the Arf and Rho family signalling pathways.
Asunto(s)
Factores de Ribosilacion-ADP/metabolismo , Proteínas Adaptadoras Transductoras de Señales , Proteínas Portadoras/química , Proteínas Portadoras/metabolismo , Transducción de Señal , Proteínas de Unión al GTP rac/metabolismo , Factor 1 de Ribosilacion-ADP/genética , Factor 1 de Ribosilacion-ADP/metabolismo , Factor 6 de Ribosilación del ADP , Factores de Ribosilacion-ADP/genética , Secuencia de Aminoácidos , Unión Competitiva , Calorimetría , Proteínas Portadoras/genética , Cristalografía por Rayos X , Dimerización , Polarización de Fluorescencia , Guanosina Difosfato/metabolismo , Guanosina Trifosfato/metabolismo , Guanilil Imidodifosfato/metabolismo , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Mutación/genética , Unión Proteica , Conformación Proteica , Alineación de Secuencia , Temperatura , Volumetría , Proteínas de Unión al GTP rac/química , Proteínas de Unión al GTP rac/genéticaRESUMEN
Thermodynamics and kinetics for cyanide, azide, thiocyanate and imidazole binding to recombinant ferric Vitreoscilla sp. homodimeric hemoglobin (Vitreoscilla Hb) have been determined at pH 6.4 and 7.0, and 20.0 degrees C, in solution and in the crystalline state. Moreover, the three-dimensional structures of the diligated thiocyanate and imidazole derivatives of recombinant ferric Vitreoscilla Hb have been determined by X-ray crystallography at 1.8 A (Rfactor=19.9%) and 2.1 A (Rfactor=23.8%) resolution, respectively. Ferric Vitreoscilla Hb displays an anticooperative ligand binding behaviour in solution. This very unusual feature can only be accounted for by assuming ligand-linked conformational changes in the monoligated species, which lead to the observed 300-fold decrease in the affinity of cyanide, azide, thiocyanate and imidazole for the monoligated ferric Vitreoscilla Hb with respect to that of the fully unligated homodimer. In the crystalline state, thermodynamics for azide and imidazole binding to ferric Vitreoscilla Hb may be described as a simple process with an overall ligand affinity for the homodimer corresponding to that for diligation in solution. These data suggest that the ligand-free homodimer, observed in the crystalline state, is constrained in a low affinity conformation whose ligand binding properties closely resemble those of the monoligated species in solution. From the kinetic viewpoint, anticooperativity is reflected by the 300-fold decrease of the second-order rate constant for cyanide and imidazole binding to the monoligated ferric Vitreoscilla Hb with respect to that for ligand association to the ligand-free homodimer in solution. On the other hand, values of the first-order rate constant for cyanide and imidazole dissociation from the diligated and monoligated derivatives of ferric Vitreoscilla Hb in solution are closely similar. As a whole, ligand binding and structural properties of ferric Vitreoscilla Hb appear to be unique among all Hbs investigated to date.
Asunto(s)
Hemoglobinas/química , Hemoglobinas/metabolismo , Neisseriaceae/metabolismo , Cristalografía por Rayos X , Dimerización , Hemo/química , Hierro/química , Cinética , Ligandos , Modelos Moleculares , Unión Proteica , Conformación Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Soluciones , TermodinámicaRESUMEN
FlgM is an anti-sigma factor of the flagellar-specific sigma (sigma) subunit of RNA polymerase in Bacillus subtilis, and it is responsible of the coupling of late flagellar gene expression to the completion of the hook-basal body structure. We have overproduced the protein in soluble form and characterized it. FlgM forms dimers as shown by gel exclusion chromatography and native polyacrylamide gel electrophoresis and interacts in vitro with the cognate sigmaD factor. The FlgM.sigmaD complex is a stable heterodimer as demonstrated by gel exclusion chromatography, chemical cross-linking, native polyacrylamide gel electrophoresis, and isoelectric focusing. sigmaD belongs to the group of sigma factors able to bind to the promoter sequence even in the absence of core RNA polymerase. The FlgM.sigmaD complex gave a shift in a DNA mobility shift assay with a probe containing a sigmaD-dependent promoter sequence. Limited proteolysis studies indicate the presence of two structural motifs, corresponding to the N- and C-terminal regions, respectively.
Asunto(s)
Bacillus subtilis/metabolismo , Proteínas Bacterianas/genética , Secuencia de Aminoácidos , Proteínas Bacterianas/metabolismo , Secuencia de Bases , Clonación Molecular , Cartilla de ADN , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Hidrólisis , Datos de Secuencia Molecular , Unión ProteicaRESUMEN
Kinetics, thermodynamics and structural aspects of human alpha-thrombin (thrombin) inhibition by newly synthesized low molecular weight derivatives of alpha-azalysine have been investigated. The thrombin catalyzed hydrolysis of N-ethoxycarbonyl-D-Phe-Pro-alpha-azaLys p-nitrophenyl ester (Eoc-D-Phe-Pro-azaLys-ONp) and N-carbobenzoxy-Pro-alpha-azaLys p-nitrophenyl ester (Cbz-Pro-azaLys-ONp) was investigated at pH 6.2 and 21.0 degrees C, and analyzed in parallel with that of N-alpha-(N,N-dimethylcarbamoyl)-alpha-azalysine p-nitrophenyl ester (Dmc-azaLys-ONp). Decarboxylation following the enzymatic hydrolysis of these p-nitrophenyl esters gave the corresponding 1-peptidyl-2(4-aminobutyl) hydrazines (peptidyl-Abh) showing properties of thrombin competitive inhibitors. Therefore, thermodynamics for the reversible binding of D-Phe-Pro-Abh, Cbz-Pro-Abh and Dmc-Abh to thrombin was examined. These results are consistent with the minimum four-step catalytic mechanism for product inhibition of serine proteinases. Eoc-D-Phe-Pro-azaLys-ONp and Eoc-D-Phe-Pro-Abh display a sub-micromolar affinity for thrombin together with a high selectivity versus homologous plasmatic and pancreatic serine proteinases acting on cationic substrates. The three-dimensional structures of the reversible non-covalent thrombin:Eoc-D-Phe-Pro-Abh and thrombin:Cbz-Pro-Abh complexes have been determined by X-ray crystallography at 2.0 A resolution (R-factor = 0.169 and 0.179, respectively), and analyzed in parallel with that of the thrombin:Dmc-azaLys acyl-enzyme adduct. Both Eoc-D-Phe-Pro-Abh and Cbz-Pro-Abh competitive inhibitors are accommodated in the thrombin active center, spanning the region between the aryl binding site and the S1 primary specificity subsite.
Asunto(s)
Hidrazinas/farmacología , Oligopéptidos/farmacología , Trombina/antagonistas & inhibidores , Trombina/química , Animales , Cristalografía por Rayos X , Humanos , Cinética , TermodinámicaRESUMEN
BACKGROUND: The first hemoglobin identified in bacteria was isolated from Vitreoscilla stercoraria (VtHb) as a homodimeric species. The wild-type protein has been reported to display medium oxygen affinity and cooperative ligand-binding properties. Moreover, VtHb can support aerobic growth in Escherichia coli with impaired terminal oxidase function. This ability of VtHb to improve the growth properties of E. coli has important applications in fermentation technology, assisting the overexpression of recombinant proteins and antibiotics. Oxygen binding heme domains have been identified in chimeric proteins from bacteria and yeast, where they are covalently linked to FAD- and NAD(P)H-binding domains. We investigate here the fold, the distal heme site structure and the quaternary assembly of a bacterial hemoglobin which does not bear the typical flavohemoglobin domain organization. RESULTS: The VtHb three-dimensional structure conforms to the well known globin fold. Nevertheless, the polypeptide segment connecting helices C and E is disordered, and residues E7-E10 (defined according to the standard globin fold nomenclature) do not adopt the usual alpha-helical conformation, thus locating Gln53(E7) out of the heme pocket. Binding of azide to the heme iron introduces substantial structural perturbations in the heme distal site residues, particularly Tyr29(B10) and Pro54(E8). The quaternary assembly of homodimeric VtHb, not observed before within the globin family, is based on a molecular interface defined by helices F and H of both subunits, the two heme iron atoms being 34 A apart. CONCLUSIONS: The unusual heme distal site structure observed shows that previously undescribed molecular mechanisms of ligand stabilization are operative in VtHb. The polypeptide chain disorder observed in the CE region indicates a potential site of interaction with the FAD/NADH reductase partner, in analogy with observations in the chimeric flavohemoglobin from Alcaligenes eutrophus.
Asunto(s)
Globinas/química , Bacterias Aerobias Gramnegativas/metabolismo , Hemoglobinas/química , Oxihemoglobinas/química , Pliegue de Proteína , Estructura Secundaria de Proteína , Secuencia de Aminoácidos , Sitios de Unión , Cristalografía por Rayos X , Dimerización , Hemo/análisis , Hemoglobinas/aislamiento & purificación , Hemoglobinas/metabolismo , Sustancias Macromoleculares , Modelos Moleculares , Datos de Secuencia Molecular , Oxígeno , Oxihemoglobinas/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Homología de Secuencia de AminoácidoRESUMEN
The recombinant homodimeric hemoglobin from the strictly aerobe gram-negative bacterium Vitreoscilla stercoraria has been expressed in Escherichia coli, purified to homogeneity, and crystallized by vapor diffusion techniques, using ammonium sulfate as precipitant. The crystals belong to the monoclinic space group P2(1) and diffract to HIGH resolution. The unit cell parameters are alpha = 62.9, b = 42.5, c = 63.2 A, beta = 106.6 degrees; the asymmetric unit contains the homodimeric hemoglobin, with a volume solvent content of 42%.
Asunto(s)
Bacterias Aerobias/química , Hemoglobinas/química , Bacillus subtilis/genética , Clonación Molecular , Hemoglobinas/genética , Hemoglobinas/aislamiento & purificación , Difracción de Rayos XRESUMEN
X-ray absorption spectroscopy has been carried out on the copper K edge in oxidized and reduced bovine Cu,Zn SOD in solution and in crystalline state. The results indicate that the copper coordination geometry is unaffected by the solution or by the crystalline state of the protein, in both oxidation states. Moreover the two oxidation states of the active copper ion are reflected under, all the experimental conditions, by distinct coordination spheres around the catalytic metal, which is four-coordinated and three-coordinated in the Cu(II) and in the Cu(I) enzyme, respectively.
Asunto(s)
Histidina , Superóxido Dismutasa/química , Absorciometría de Fotón , Animales , Bovinos , Cobre , Cristalización , Imidazoles , Oxidación-Reducción , Soluciones , Superóxido Dismutasa/metabolismo , ZincRESUMEN
Binding of the paramagnetic N,N"-bis(m-boroxyphenylcarbamoylmethyl)-diethylenetriamine-N,N', N"-triacetic acid Gd(III) [sequence: see text] complex (GdBB) to chymotrypsin, chymotrypsinogen, trypsin, trypsinogen and pancreatic elastase has been investigated by 1H-NMR relaxometry, between pH 6.0 and 8.5, at 25.0 degrees C. Values of Ki for the competitive inhibition of serine proteinases by GdBB are in excellent agreement with values of Kd obtained by 1H-NMR relaxometry, suggesting that the substrate and the paramagnetic complex bind to the same region. Moreover, 1H-NMR relaxometry allowed to determine values of Kd for GdBB binding to chymotrypsinogen and trypsinogen, both devoid of catalytic activity. The increase of the water proton relaxation rate upon GdBB binding to serine (pro)enzymes may be useful in the design of novel functional contrast agents for magnetic resonance imaging.
Asunto(s)
Boro , Medios de Contraste , Compuestos Organometálicos , Páncreas/enzimología , Ácido Pentético/análogos & derivados , Inhibidores de Serina Proteinasa/química , Quimotripsina/metabolismo , Quimotripsinógeno/metabolismo , Gadolinio DTPA , Espectroscopía de Resonancia Magnética , Elastasa Pancreática/metabolismo , Tripsina/metabolismo , Tripsinógeno/metabolismoRESUMEN
Azide, cyanide, fluoride, imidazole, and pyridine binding to ferric and ferrous native horse heart cytochrome c and to its carboxymethylated derivative has been investigated, from the thermodynamic viewpoint, at pH 7.5 and 25.0 degrees C. Ligand affinity for ferric and ferrous carboxymethylated cytochrome c is higher by about 30- and 400-fold, respectively, than that observed for the native protein. The results here reported: (i) allow the estimation, for the first time, of the ligand-independent free energy associated with the heme-iron sixth coordination bond in ferric and ferrous native cytochrome c, which turns out to be +8.4 kJ mol-1 and +14.6 kJ mol-1, at 25.0 degrees C, respectively, and (ii) suggest an interplay between redox, structural, ligand binding, and recognition properties of cytochrome c.
Asunto(s)
Grupo Citocromo c/química , Compuestos Férricos/química , Compuestos Ferrosos/química , Animales , Azidas/química , Cianuros/química , Transporte de Electrón , Fluoruros/química , Caballos , Imidazoles/química , Metilación , Miocardio/enzimología , Oxidación-Reducción , Unión Proteica , Piridinas/químicaRESUMEN
The X-ray crystal structures of the aquo-met and cyano-met derivatives of the loggerhead sea turtle (Caretta caretta) myoglobin have been determined at 2.0 A resolution (R = 0.182, and 0.178, respectively). The results here reported, representing the first reptile globin solved by X-ray crystallography, have been analyzed in parallel with data for related monomeric hemoproteins, and indicate a strong overall structural similarity between the loggerhead sea turtle and mammalian myoglobins, reflected by the 63% amino acid identity of their primary structures. The root-mean-square deviation between the entire polypeptide backbones of loggerhead sea turtle and sperm whale myoglobins, after structure superposition, is 0.83 A. Upon cyanide binding to the protein distal site, the iron-bound water molecule present in the aquo-met form is displaced by the incoming ligand. Cyanide is oriented towards the inner part of the heme distal site forming a Fe-C-N angle of 133 degrees.
Asunto(s)
Hemo/química , Metamioglobina/análogos & derivados , Metamioglobina/química , Conformación Proteica , Tortugas/metabolismo , Animales , Azidas/metabolismo , Sitios de Unión , Cristalografía por Rayos X , Cianuros/metabolismo , Mamíferos/metabolismo , Modelos Moleculares , Unión Proteica , Estructura Terciaria de Proteína , Reptiles/metabolismo , Homología de Secuencia de Aminoácido , Especificidad de la Especie , Agua/químicaRESUMEN
The X-ray crystal structure of the formate derivative of ferric loggerhead sea turtle (Caretta caretta) Mb has been determined at 2.0 A resolution (R = 0.164) by difference Fourier techniques. Formate, sitting in the central part of the heme distal site, is coordinated to the heme iron as unidentate ligand, through the O1 oxygen atom, and is hydrogen bonded to the distal His64(E7) NE2 atom through O2. Thermodynamics for formate binding to ferric loggerhead sea turtle Mb, sperm whale Mb, Aplysia limacina Mb, as well as to the VR and VRS mutants of sperm whale Mb were obtained between pH 4.5 and 8.5, at 20.0 degrees C. These results, representing the first structure of a ferric hemoprotein:formate complex solved by X-ray crystallography, outline the role of amino acid residues at positions E7, F8 and E10 in modulating ligand binding properties of oxygen carrying proteins.
Asunto(s)
Formiatos/metabolismo , Mioglobina/metabolismo , Animales , Cristalografía por Rayos X , Concentración de Iones de Hidrógeno , Mutación , Mioglobina/química , Mioglobina/genética , Unión Proteica , Termodinámica , TortugasRESUMEN
We describe a second Italian family with primary Lipoprotein Lipase deficiency. A new mutation in exon 8 causes a Leu365- > Val change resulting in severe mass reduction and loss of enzyme activity. We suggest that this change interferes with the correct folding and stability of the protein and impairs the assembly of the active homodimer. The procedures applied are useful to screen a large sample of population for genetic variants and allow the clear identification of asymptomatic heterozygous subjects at risk from atherosclerosis disease.
Asunto(s)
Leucina , Lipoproteína Lipasa/deficiencia , Lipoproteína Lipasa/genética , Mutación Puntual , Valina , Adulto , Secuencia de Aminoácidos , Animales , ADN/sangre , ADN/genética , Exones , Familia , Femenino , Variación Genética , Humanos , Italia , Lipoproteína Lipasa/metabolismo , Masculino , Mamíferos , Datos de Secuencia Molecular , Linaje , Reacción en Cadena de la Polimerasa/métodos , Homología de Secuencia de AminoácidoRESUMEN
Values of the association equilibrium constant (Ka) for the binding of the native and of the cyanogen bromide-cleaved bovine basic pancreatic trypsin inhibitor (native BPTI and [Hse lactone-52]-52,53-seco-BPTI, respectively) to neuraminidase-treated porcine pancreatic beta-kallikrein-B (kallikrein) and bovine alpha-chymotrypsin (chymotrypsin) have been determined between pH 4.0 and 9.0, at 20.0 degrees C. Over the whole pH range explored, native BPTI and [Hse lactone-52]-52,53-seco-BPTI show the same affinity for kallikrein. On the other hand, the affinity of [Hse lactone-52]-52,53-seco-BPTI for chymotrypsin is higher, around neutrality, than that found for native BPTI by about one order of magnitude, converging in the acidic pH limb. The simplest mechanism accounting for the observed data implies that, on lowering the pH from 9.0 to 4.0, (i) the decrease in affinity for the binding of native BPTI to kallikrein and chymotrypsin, as well as for the association of [Hse lactone-52]-52,53-seco-BPTI to kallikrein, reflects the acidic pK shift, upon inhibitor association, of a single ionizing group; and (ii) the decrease of Ka values for [Hse lactone-52]-52,53-seco-BPTI binding to chymotrypsin appears to be modulated by the acidic pK shift, upon inhibitor association, of two non-equivalent proton-binding residues. On the basis of the stereochemistry of the serine proteinase/inhibitor contact region(s), these data indicate that long-range structural changes in [Hse lactone-52]-52,53-seco-BPTI are energetically linked to the chymotrypsin:inhibitor complex formation. This observation represents an important aspect for the mechanism of molecular recognition and regulation in BPTI.
Asunto(s)
Aprotinina/análogos & derivados , Aprotinina/metabolismo , Quimotripsina/metabolismo , Homoserina/análogos & derivados , Calicreínas/metabolismo , Animales , Aprotinina/química , Bovinos , Quimotripsina/química , Homoserina/química , Homoserina/metabolismo , Concentración de Iones de Hidrógeno , Calicreínas/química , Calicreínas/efectos de los fármacos , Cinética , Modelos Moleculares , Neuraminidasa/farmacología , Unión Proteica , Conformación Proteica , Relación Estructura-Actividad , Porcinos , TermodinámicaRESUMEN
Branching observation, color and emulsionability of colonies, growth on BHI-agar, Mc Conkey-agar, Sabouraud, Mannitol Salt-agar, galattosidase, degradation of polyethylene glicol in 7H-10 medium, drug sensitivity to antituberculous drugs and semi-synthetic penicillins are reactions able to differentiate the rhodochrous complex from pathogenic pigmented Mycobacteria and Nocardia. Numerical taxonomy performed among 14 strains belonging to rhodochrous complex and 17 strains of Nocardia revealed a distinct separation of the two species and the non omogeneity of rhodochrous complex.