RESUMEN
BACKGROUND: The insulin-like growth-factor-1 (IGF-1) receptor, which is widely expressed in cells that have undergone oncogenic transformation, is emerging as a novel target in cancer therapy. IGF-1-induced receptor activation results in autophosphorylation of cytoplasmic kinase domains and enhances their capability to phosphorylate downstream substrates. Structures of the homologous insulin receptor kinase (IRK) exist in an open, unphosphorylated form and a closed, trisphosphorylated form. RESULTS: We have determined the 2.1 A crystal structure of the IGF-1 receptor protein tyrosine kinase domain phosphorylated at two tyrosine residues within the activation loop (IGF-1RK2P) and bound to an ATP analog. The ligand is not in a conformation compatible with phosphoryl transfer, and the activation loop is partially disordered. Compared to the homologous insulin receptor kinase, IGF-1RK2P is trapped in a half-closed, previously unobserved conformation. Observed domain movements can be dissected into two orthogonal rotational components. CONCLUSIONS: Conformational changes upon kinase activation are triggered by the degree of phosphorylation and are crucially dependent on the conformation of the proximal end of the kinase activation loop. This IGF-1RK structure will provide a molecular basis for the design of selective antioncogenic therapeutic agents.
Asunto(s)
Receptor IGF Tipo 1/química , Receptor IGF Tipo 1/metabolismo , Adenilil Imidodifosfato/metabolismo , Secuencia de Aminoácidos , Sitios de Unión , Dominio Catalítico , Cristalización , Cristalografía por Rayos X , Activación Enzimática , Humanos , Datos de Secuencia Molecular , Fosforilación , Unión Proteica , Conformación Proteica , Estructura Terciaria de Proteína , Receptor IGF Tipo 1/biosíntesis , Especificidad por SustratoRESUMEN
Human macrophage elastase (MMP-12) is a member of the family of matrix metalloproteinases (MMPs) that plays, like other members of the family, an important role in inflammatory processes contributing to tissue remodelling and destruction. In particular, a prominent role of MMP-12 in the destruction of elastin in the lung alveolar wall and the pathogenesis of emphysema has been suggested. It is therefore an attractive therapeutic target. We describe here the crystal structure of the catalytic domain of MMP-12 in complex with a hydroxamic acid inhibitor, CGS27023A. MMP-12 adopts the typical MMP fold and binds a structural zinc ion and three calcium ions in addition to the catalytic zinc ion. The enzyme structure shows an ordered N terminus close to the active site that is identical in conformation with the superactivated form of MMP-8. The S1'-specificity pocket is large and extends into a channel through the protein, which puts MMP-12 into the class of MMPs 3, 8 and 13 with large and open specificity pockets. The two crystallographically independent molecules adopt different conformations of the S1'-loop and its neighbouring loop due to differing crystal packing environments, suggesting that flexibility or the possibility of structural adjustments of these loop segments are intrinsic features of the MMP-12 structure and probably a common feature for all MMPs. The inhibitor binds in a bidentate fashion to the catalytic zinc ion. Its polar groups form hydrogen bonds in a substrate-like manner with beta-strand sIV of the enzyme, while the hydrophobic substituents are either positioned on the protein surface and are solvent-exposed or fill the upper part of the specificity pocket. The present structure enables us to aid the design of potent and selective inhibitors for MMP-12.
Asunto(s)
Ácidos Hidroxámicos/metabolismo , Metaloendopeptidasas/antagonistas & inhibidores , Metaloendopeptidasas/química , Inhibidores de Proteasas/metabolismo , Pirazinas , Secuencia de Aminoácidos , Sitios de Unión , Cationes Bivalentes/metabolismo , Cristalización , Cristalografía por Rayos X , Diseño de Fármacos , Humanos , Enlace de Hidrógeno , Ácidos Hidroxámicos/química , Macrófagos/enzimología , Metaloproteinasa 12 de la Matriz , Metaloendopeptidasas/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Inhibidores de Proteasas/química , Conformación Proteica , Alineación de Secuencia , Especificidad por Sustrato , Sulfonamidas , Zinc/metabolismoRESUMEN
BACKGROUND: A major current focus of pharmaceutical research is the development of selective inhibitors of the blood coagulation enzymes thrombin or factor Xa to be used as orally bioavailable anticoagulant drugs in thromboembolic disorders and in the prevention of venous and arterial thrombosis. Simultaneous direct inhibition of thrombin and factor Xa by synthetic proteinase inhibitors as a novel approach to antithrombotic therapy could result in potent anticoagulants with improved pharmacological properties. RESULTS: The binding mode of such dual specific inhibitors of thrombin and factor Xa was determined for the first time by comparative crystallography using human alpha-thrombin, human des-Gla (1--44) factor Xa and bovine trypsin as the ligand receptors. The benzamidine-based inhibitors utilize two different conformations for the interaction with thrombin and factor Xa/trypsin, which are evoked by the steric requirements of the topologically different S2 subsites of the enzymes. Compared to the unliganded forms of the proteinases, ligand binding induces conformational adjustments of thrombin and factor Xa active site residues indicative of a pronounced induced fit mechanism. CONCLUSION: The structural data reveal the molecular basis for a desired unselective inhibition of the two key components of the blood coagulation cascade. The 4-(1-methyl-benzimidazole-2-yl)-methylamino-benzamidine moieties of the inhibitors are able to fill both the small solvent accessible as well as the larger hydrophobic S2 pockets of factor Xa and thrombin, respectively. Distal fragments of the inhibitors are identified which fit into both the cation hole/aromatic box of factor Xa and the hydrophobic aryl binding site of thrombin. Thus, binding constants in the medium-to-low nanomolar range are obtained against both enzymes.
Asunto(s)
Inhibidores de Factor de Coagulación Sanguínea/farmacología , Coagulación Sanguínea/efectos de los fármacos , Inhibidores del Factor Xa , Factor Xa/química , Trombina/antagonistas & inhibidores , Trombina/química , Trombina/metabolismo , Animales , Bovinos , Cristalografía por Rayos X , Humanos , Ligandos , Modelos Moleculares , Unión Proteica , Conformación Proteica , Trombosis/prevención & control , Tripsina/metabolismoRESUMEN
The crystal structure of recombinant human GTP cyclohydrolase I was solved by Patterson search methods by using the coordinates of the Escherichia coli enzyme as a model. The human as well as bacterial enzyme were shown to contain an essential zinc ion coordinated to a His side chain and two thiol groups in each active site of the homodecameric enzymes that had escaped detection during earlier studies of the E. coli enzyme. The zinc ion is proposed to generate a hydroxyl nucleophile for attack of imidazole ring carbon atom eight of the substrate, GTP. It may also be involved in the hydrolytic release of formate from the intermediate, 2-amino-5-formylamino-6-ribosylamino-4(3H)-pyrimidinone 5'-triphosphate, and in the consecutive Amadori rearrangement of the ribosyl moiety.
Asunto(s)
GTP Ciclohidrolasa/metabolismo , Zinc/metabolismo , Secuencia de Aminoácidos , Secuencia de Bases , Cristalización , GTP Ciclohidrolasa/química , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Oligodesoxirribonucleótidos , Conformación Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Homología de Secuencia de AminoácidoRESUMEN
Three crystalline modifications (A, B, and C) of 4'-[[2-n-propyl-4-methyl-6-(1-methyl-benzimidazol-2-yl)benzi midazol-1-yl]methyl]biphenyl-2-carboxylic acid (INN name, telmisartan) have been detected and their crystal structures have been determined by single-crystal X-ray diffraction (pseudopolymorph C) and the method of simulated annealing from high-resolution X-ray powder diffraction data (polymorphs A and B). The compound is of interest because of its use as an angiotensin II receptor antagonist. Polymorph A crystallizes in space group P2(I)/c, Z = 4, with unit cell parameters a = 18.7798(3), b = 18.1043(2), and c = 8.00578(7) A, beta = 97.066(1) degrees, and V = 2701.31 A(3). Polymorph B crystallizes in space group P2(I)/a, Z = 4, with unit cell parameters a = 16.0646(5), b = 13.0909(3), and c = 13.3231(3) A, beta = 99.402(1) degrees, and V = 2764.2(1) A(3). The solvated form C crystallizes in space group C2/c, Z = 8, with unit cell parameters a = 30.990(5), b = 13.130(3), and c = 16.381(3) A, beta = 95.02(2) degrees, and V = 6639(2) A(3). For the structure solutions of polymorphs A and B, 13 degrees of freedom (3 translational, 3 orientational, 7 torsion angles) were determined in approximately 2 h of computer time, demonstrating that the crystal packing and the molecular conformation of medium-sized (MW approximately 500) pharmaceutical compounds can now be solved quickly and routinely from high-resolution X-ray powder diffraction data.
Asunto(s)
Inhibidores de la Enzima Convertidora de Angiotensina/química , Bencimidazoles/química , Benzoatos/química , Cristalización , Cristalografía por Rayos X , Polvos , TelmisartánRESUMEN
Pin1, a member of the parvulin family of peptidyl-prolyl cis-trans isomerases (PPIases) has been implicated in the G2-M transition of the mammalian cell cycle. Pin1 interacts with a series of mitotic phosphoproteins, including Polo-like kinase-1, Cdc25C, and Cdc27, and is thought to act as a phosphorylation-dependent PPIase for these target molecules. Pin1 recognizes phosphorylated serine-proline or threonine-proline peptide-bonds in test substrates up to 1300-fold better than in the respective unphosphorylated peptides. To test directly whether Pin1 regulates the G2-M transition and/or progression through mitosis by catalyzing phosphorylation-dependent prolyl isomerization of essential mitotic targets, we examined the consequences of Pin1 depletion, achieved by (a) overexpression of Pin1 antisense RNA, (b) overexpression of dominant-negative Pin1, and (c) by a known small-molecule Pin1-PPIase inhibitor, juglone. The results of all of the three lines of investigation show that the catalytic activity of Pin1 is essential for tumor cell survival and entry into mitosis.
Asunto(s)
Supervivencia Celular , Mitosis , Isomerasa de Peptidilprolil/metabolismo , Prolina/metabolismo , Antineoplásicos Fitogénicos/farmacología , Apoptosis , Western Blotting , Catálisis , Línea Celular , Núcleo Celular/metabolismo , Cromatina/metabolismo , Clonación Molecular , Inhibidores Enzimáticos/farmacología , Histonas/metabolismo , Humanos , Interfase , Cinética , Microscopía Fluorescente , Mutación , Peptidilprolil Isomerasa de Interacción con NIMA , Naftoquinonas/farmacología , Paclitaxel/farmacología , Isomerasa de Peptidilprolil/antagonistas & inhibidores , Isomerasa de Peptidilprolil/química , Fosforilación , Estructura Terciaria de Proteína , Proteínas Recombinantes de Fusión/metabolismo , Transfección , Células Tumorales CultivadasRESUMEN
The crystal structure of a constitutively active multiple site mutant of plasminogen activator inhibitor 1 (PAI-1) was determined and refined at a resolution of 2.7 A. The present structure comprises a dimer of two crystallographically independent PAI-1 molecules that pack by association of the residues P6 to P3 of the reactive centre loop of one molecule (A) with the edge of the main beta-sheet A of the other molecule (B).Thus, the reactive centre loop is ordered for molecule A by crystal packing forces, while for molecule B it is unconstrained by crystal packing contacts and is disordered. The overall structure of active PAI-1 is similar to the structures of other active inhibitory serpins exhibiting as the major secondary structural feature a five-stranded beta-sheet A and an intact proteinase-binding loop protruding from the one end of the elongated molecule. No preinsertion of the reactive centre loop is observed in this structure.A comparison of the present structure with the previously determined crystal structures of PAI-1 in its alternative conformations reveals that, upon cleavage of an intact form of PAI-1 or formation of latent PAI-1, the well-characterised rearrangements of the serpin secondary structural elements are accompanied by dramatic and partly unexpected conformational changes of helical and loop structures proximal to beta-sheet A. The present structure explains the stabilising effects of the mutated residues, reveals the structural cause for the observed spectroscopic differences between active and latent PAI-1, and provides new insights into possible mechanisms of stabilisation by its natural binding partner, vitronectin.
Asunto(s)
Inhibidor 1 de Activador Plasminogénico/química , Inhibidor 1 de Activador Plasminogénico/metabolismo , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Sitios de Unión , Biopolímeros/química , Biopolímeros/metabolismo , Cristalización , Cristalografía por Rayos X , Dimerización , Modelos Moleculares , Datos de Secuencia Molecular , Mutación/genética , Inhibidor 1 de Activador Plasminogénico/genética , Estructura Secundaria de Proteína , Inhibidores de Serina Proteinasa/química , Inhibidores de Serina Proteinasa/genética , Inhibidores de Serina Proteinasa/metabolismo , Termodinámica , Vitronectina/metabolismoRESUMEN
GTP cyclohydrolase I catalyzes the conversion of GTP to dihydroneopterin triphosphate. The replacement of histidine 179 by other amino acids affords mutant enzymes that do not catalyze the formation of dihydroneopterin triphosphate. However, some of these mutant proteins catalyze the conversion of GTP to 2-amino-5-formylamino-6-ribofuranosylamino-4(3H)-pyrimidinone 5'-triphosphate as shown by multinuclear NMR analysis. The equilibrium constant for the reversible conversion of GTP to the ring-opened derivative is approximately 0.1. The wild-type enzyme converts the formylamino pyrimidine derivative to dihydroneopterin triphosphate; the rate is similar to that observed with GTP as substrate. The data support the conclusion that the formylamino pyrimidine derivative is an intermediate in the overall reaction catalyzed by GTP cyclohydrolase I.
Asunto(s)
GTP Ciclohidrolasa/metabolismo , Guanosina Trifosfato/metabolismo , Pteridinas/metabolismo , Nucleótidos de Pirimidina/metabolismo , Dominio Catalítico/genética , Escherichia coli/enzimología , GTP Ciclohidrolasa/genética , Histidina/genética , Modelos Químicos , Mutación , Neopterin/análogos & derivados , Resonancia Magnética Nuclear BiomolecularRESUMEN
The enzyme 6-pyruvoyl tetrahydropterin synthase (PTPS) catalyses the second step in the de novo biosynthesis of tetrahydrobiopterin, the conversion of dihydroneopterin triphosphate to 6-pyruvoyl tetrahydropterin. The Zn and Mg-dependent reaction includes a triphosphate elimination, a stereospecific reduction of the N5-C6 double bond and the oxidation of both side-chain hydroxyl groups. The crystal structure of the inactive mutant Cys42Ala of PTPS in complex with its natural substrate dihydroneopterinetriphosphate was determined at 1.9 A resolution. Additionally, the uncomplexed enzyme was refined to 2.0 A resolution. The active site of PTPS consists of the pterin-anchoring Glu A107 neighboured by two catalytic motifs: a Zn(II) binding site and an intersubunit catalytic triad formed by Cys A42, Asp B88 and His B89. In the free enzyme the Zn(II) is in tetravalent co-ordination with three histidine ligands and a water molecule. In the complex the water is replaced by the two substrate side-chain hydroxyl groups yielding a penta-co-ordinated Zn(II) ion. The Zn(II) ion plays a crucial role in catalysis. It activates the protons of the substrate, stabilizes the intermediates and disfavours the breaking of the C1'C2' bond in the pyruvoyl side-chain. Cys A42 is activated by His B89 and Asp B88 for proton abstraction from the two different substrate side-chain atoms C1', and C2'. Replacing Ala A42 in the mutant structure by the wild-type Cys by modelling shows that the C1' and C2' substrate side-chain protons are at equal distances to Cys A42 Sgamma. The basicity of Cys A42 may be increased by a catalytic triad His B89 and Asp B88. The active site of PTPS seems to be optimised to carry out proton abstractions from two different side-chain C1' and C2' atoms, with no obvious preference for one of them. Kinetic studies with dihydroneopterin monophosphate reveal that the triphosphate moiety of the substrate is necessary for enzyme specifity.
Asunto(s)
Hígado/enzimología , Liasas de Fósforo-Oxígeno/química , Animales , Sitios de Unión , Biopterinas/análogos & derivados , Biopterinas/biosíntesis , Cristalografía por Rayos X , Cinética , Modelos Moleculares , Estructura Molecular , Mutación/genética , Estructura Secundaria de Proteína , Ratas , Zinc/químicaRESUMEN
The structure-activity relationships in two series of hypoglycemic benzoic acid derivatives (5, 6) were investigated. Series 5 resulted from meglitinide (3) when the 2-methoxy was replaced by an alkyleneimino residue. Maximum activity was observed with the cis-3, 5-dimethyl-piperidino (5h) and the octamethyleneimino (5l) residues. Series 6 resulted from the meglitinide analogon 4 bearing an inversed amido function when the 2-methoxy, the 5-fluoro, and the alpha-methyl residue were replaced by a 2-piperidino, a 5-hydrogen, and a larger alpha-alkyl residue, respectively. An alkoxy residue ortho to the carboxy group further increased activity and duration of action in the rat. The most active racemic compound, 6al (R4 = isobutyl; R = ethoxy), turned out to be 12 times more active than the sulfonylurea (SU) glibenclamide (1). Activity was found to reside predominantly in the (S)-enantiomers. Compared with the SUs 1 and 2 (glimepiride), the most active enantiomer, (S)-6al (AG-EE 623 ZW; repaglinide; ED50 = 10 micro/kg po), is 25 and 18 times more active. Repaglinide turned out to be a useful therapeutic for type 2 diabetic patients; approval was granted recently by the FDA and the EMEA. From investigations on the pharmacophoric groups in compounds of type 5 and 6, it was concluded that in addition to the two already known-the acidic group (COOH; SO2NH) and the amidic spacer (CONH; NHCO)-the ortho residue R1 (alkyleneimino; alkoxy; oxo) must be regarded as a third one. A general pharmacophore model suitable for hypoglycemic benzoic acid derivatives, SUs, and sulfonamides is proposed (Figure 6). Furthermore, from superpositions of low-energy conformations (LECs) of 1, 2, and (S)-6al, it was concluded that a common binding conformation (LEC II; Figure 10B) may exist and that differences in binding to the SU receptor and in the mechanism of insulin release between repaglinide and the two SUs may be due to specific hydrophobic differences.
Asunto(s)
Transportadoras de Casetes de Unión a ATP , Benzoatos/farmacología , Carbamatos/farmacología , Hipoglucemiantes/farmacología , Piperidinas/farmacología , Canales de Potasio de Rectificación Interna , Administración Oral , Animales , Benzoatos/síntesis química , Benzoatos/química , Benzoatos/metabolismo , Glucemia/metabolismo , Carbamatos/síntesis química , Carbamatos/química , Carbamatos/metabolismo , Cristalografía por Rayos X , Femenino , Gliburida/química , Gliburida/metabolismo , Gliburida/farmacología , Hipoglucemiantes/síntesis química , Hipoglucemiantes/química , Hipoglucemiantes/metabolismo , Modelos Moleculares , Conformación Molecular , Piperidinas/síntesis química , Piperidinas/química , Piperidinas/metabolismo , Canales de Potasio/metabolismo , Ratas , Ratas Wistar , Receptores de Droga/metabolismo , Estereoisomerismo , Relación Estructura-Actividad , Compuestos de Sulfonilurea/química , Compuestos de Sulfonilurea/metabolismo , Compuestos de Sulfonilurea/farmacología , Receptores de SulfonilureasRESUMEN
This study investigated the diastereoselective synthesis of three dipeptide templates 1, 2 and 3, which may be regarded as conformationally restricted analogs of H-Gly-Xaa-OH, in which Xaa constitutes an aromatic amino acid. Bond formation between alpha-C of Gly and the aromatic moiety was achieved by proton-catalyzed intramolecular electrophilic aromatic substitution. The absolute configuration of the dipeptide templates was determined by single-crystal X-ray crystallography or by nuclear Overhauser enhancement measurements. A protective group strategy was elaborated to allow their incorporation into peptide sequences by liquid phase as well as by solid-phase peptide synthesis. The templates were used to generate an enkephalin analog 15, a modified peptidic neurokinin antagonist 20 and two dermorphin derivatives (24 and 33). Molecular dynamic simulations with 15 and 20 revealed the preference for a turn-like motif for 15. The biological activity, as investigated by respective receptor binding and functional assays, was strongly diminished with all four derivatives, indicating that their receptor-relevant molecular geometries lie outside the examined conformational space.
Asunto(s)
Péptidos Cíclicos/química , Péptidos Cíclicos/síntesis química , Conformación Proteica , Cristalografía por Rayos X , Relación Estructura-Actividad , Moldes GenéticosRESUMEN
The Met121Glu azurin mutant has been crystallized and the structure determined at a resolution of 2.3 A. In the crystal structure a carboxyl oxygen of Met121Glu is coordinated to the metal at a distance of 2.2 A. Single-crystal resonance Raman spectroscopy was used to show that the glutamic acid residue in the copper site was in the protonated state. Titration of this residue gives rise to a number of unusual, pH-dependent properties: as the pH is increased from 4 to 8, the S(Cys)-Cu ligand-to-metal charge transfer bands are blue shifted and their intensity ratio is reversed, the EPR signal changes from type 1 copper to a new form of protein-bound copper, and the redox potential changes from 370 to 180 mV. The spectroscopic changes in this pH interval are consistent with a two-state model. From the pH dependence of the optical and EPR spectra, pKa = 5.0 for the glutamic acid in the oxidized protein was determined.
Asunto(s)
Azurina/química , Pseudomonas aeruginosa/química , Azurina/genética , Sitios de Unión , Cobre/química , Cristalografía por Rayos X , Espectroscopía de Resonancia por Spin del Electrón , Ácido Glutámico/química , Ácido Glutámico/genética , Concentración de Iones de Hidrógeno , Metionina/química , Metionina/genética , Modelos Moleculares , Espectrofotometría , Espectrometría RamanRESUMEN
The complex organic chemistry involved in the transformation of GTP to tetrahydrobiopterin is catalysed by only three enzymes: GTP cyclohydrolase I, 6-pyruvoyltetrahydropterin synthase and sepiapterin reductase. The committing reaction step from GTP to dihydroneopterin triphosphate is catalysed by GTP cyclohydrolase I and requires no cofactor. 6-Pyruvoyl tetrahydropterin synthase, a Zn-dependent metalloprotein, transforms dihydroneopterin triphosphate into 6-pyruvoyltetrahydropterin in the presence of Mg(II). Sepiapterin reductase is a NADPH-dependent short-chain dehydrogenase which reduces 6-pyruvoyltetrahydropterin to BH4. Here we review the structural and mechanistic information on the biosynthetic pathway from GTP to BH4 on the basis of the recently determined crystal structures of CYH and PTPS.
Asunto(s)
Oxidorreductasas de Alcohol/metabolismo , Biopterinas/análogos & derivados , GTP Ciclohidrolasa/metabolismo , Guanosina Trifosfato/metabolismo , Liasas de Fósforo-Oxígeno , Animales , Biopterinas/metabolismo , Humanos , Conformación ProteicaRESUMEN
The crystal structure of cobalt-substituted azurin from Pseudomonas aeruginosa has been determined to final crystallographic R value of 0.175 at 1.9 A resolution. There are four molecules in the asymmetric unit in the structure, and these four molecules are packed as a dimer of dimers. The dimer packing is very similar to that of the wild-type Pseudomonas aeruginosa azurin dimer. Replacement of the native copper by the cobalt ion has only small effects on the metal binding site presumably because of the existence of an extensive network of hydrogen bonds in its immediate neighborhood. Some differences are obvious, however. In wild-type azurin the copper atom occupies a distorted trigonal bipyramidal site, while cobalt similar to zinc and nickel occupy a distorted tetrahedral site, in which the distance to the Met121,S(delta) atom is increased to 3.3-3.5 A and the distance to the carbonyl oxygen of Gly45 has decreased to 2.1-2.4 A. The X-band EPR spectrum of the high-spin Co(II) in azurin is well resolved (apparent g values gx' = 5.23; gy' = 3.83; gz' = 1.995, and hyperfine splittings Ax' = 31; Ay' = 20-30; Az' = 53 G) and indicates that the ligand field is close to axial.
Asunto(s)
Azurina/química , Azurina/metabolismo , Cobalto/metabolismo , Espectroscopía de Resonancia por Spin del Electrón/métodos , Pseudomonas aeruginosa/química , Azurina/genética , Sitios de Unión , Cobalto/química , Cobre/química , Cobre/metabolismo , Cristalografía por Rayos X , Metales/metabolismo , Modelos Moleculares , Mutación , Níquel/química , Níquel/metabolismo , Conformación Proteica , Relación Estructura-Actividad , Zinc/química , Zinc/metabolismoRESUMEN
The X-ray crystal structures of two metal ligand mutants of azurin from Pseudomonas aeruginosa have been solved. In both mutants (His117Gly and His46Gly azurin) one of the copper coordinating histidine residues is replaced by a glycine, creating an empty space in the coordination sphere of the copper ion. The crystal structure of His117Gly azurin at 2.4 A resolution showed that this mutant had undergone partial oxidation at the disulfide bridge between Cys3 and Cys26 and full oxidation at the copper ligand Cys112. There is no copper present in the crystallized form and the bulky group of the oxidized cysteine at position 112 causes large structural rearrangements in the protein structure, especially in the loops connecting the beta-sheets. In the structure of the wild-type holo-azurin from P. aeruginosa the hydrophobic patch is important for the packing of the azurin molecules into dimers which then arrange into tetramers. The completely different packing of the apo-His117Gly mutant can be explained by the disruption of the hydrophobic patch area by the mutation-induced main-chain conformational change of residues 112 to 115. The structure of apo-His46Gly azurin at 2.5 A resolution is the same as the wild-type structure except for the immediate environment at the site of the mutation. In the His46Gly structure water molecules are found at positions that in the wild-type structure are occupied by the imidazole ring of His46 and the copper ion. The imidazole ring of His117 is shifted by about 1 A towards the surface of the protein, similar to that observed for 50% of the molecules in the wild-type apo-azurin structure. This shift causes a slight rearrangement of the monomers within the tetramer such that one local dyad becomes a crystallographic dyad parallel to the c-axis. This leads to a change in the space group from P2(1)2(1)2(1) to P2(1)2(1)2.
Asunto(s)
Azurina/química , Azurina/genética , Mutación , Pseudomonas aeruginosa/química , Azurina/metabolismo , Cristalografía por Rayos X , Modelos Moleculares , Conformación ProteicaRESUMEN
The cross vectors of the native Patterson map are shown to exhibit non-crystallographic symmetry in the case of local axes parallel to one another. This information can be used to determine the translation component of such axes. A program is described to search for this cross vector, and is tested on low-resolution data from crystals of the tetradecameric GroEL molecule, the decameric GTP cyclohydrolase I and the tetradecameric proteosome. For GroEL, the function produces a packing arrangement optimal for sevenfold symmetry, and is in agreement with the dimensions of the molecule as given by electron microscopy data and the recently determined crystal structure. Positioning of local axes is confirmed by two high-resolution crystal structure analyses: the fivefold axis in cyclohydrolase I and the sevenfold axis in the proteosome. Implications for the location of heavy-atom positions are discussed for these two cases.
Asunto(s)
Escherichia coli/enzimología , GTP Ciclohidrolasa/química , GTP Ciclohidrolasa/metabolismo , Secuencia de Aminoácidos , Sitios de Unión , Secuencia Conservada , Cristalografía por Rayos X , Cistina , Nucleótidos de Desoxiguanina/metabolismo , Sustancias Macromoleculares , Mutagénesis Sitio-Dirigida , Estructura Secundaria de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismoRESUMEN
The blue copper protein azurin from Pseudomonas aeruginosa contains a single Trp residue that is believed to be involved in the inducible intramolecular electron transfer from a disulphide group to the copper centre. This residue shows in fluorescence spectra the highest energy emission of tryptophan-containing compounds at room temperature, which is explained by its rigid and highly hydrophobic environment. In order to investigate the role of the Trp residue in electron transfer and the influence of its environment, two mutations (17S and F110S) were introduced that were thought to increase the polarity and the mobility in its environment. The crystal structures of these mutants were solved at 2.2 A and 2.3 A resolution, respectively. These provide a structural basis for the changes observed in fluorescence spectra compared with the wild-type protein. We conclude from our results that these changes are not caused by a change in the dynamics of the Trp residue itself, but exclusively by an increased effective dielectric constant of the microenvironment of Trp48 and by changes in mobility of the mutated residues.
Asunto(s)
Azurina/química , Mutación , Conformación Proteica , Pseudomonas aeruginosa/química , Azurina/genética , Cristalografía por Rayos X , Transporte de Electrón , Isoleucina/fisiología , Estructura Molecular , Fenilalanina/fisiología , Serina/química , Triptófano/química , Agua/químicaRESUMEN
Four naturally occurring mutants with single amino acid alterations in human 6-pyruvoyltetrahydropterin synthase (PTPS) were overexpressed and characterized in vitro. The corresponding DNA mutations were found in patients with hyperphenylalaninemia and monoamine neurotransmitter insufficiency due to lack of the tetrahydrobiopterin biosynthetic enzyme PTPS. To predict the structure of the mutant enzymes, computer modeling was performed based on the solved three-dimensional structure of the homohexameric rat enzyme. One mutant (delta V57) is incorrectly folded and thus unstable in vitro and in vivo, while a second mutant (P87L) has substantial activity but enhanced sensitivity to local unfolding. Two other mutants, R16C and R25Q, form stable homomultimers and exhibit significant activity in vitro but no activity in COS-1 cells. In vivo 32P labeling showed that wild-type PTPS, P87L, and R25Q are phosphorylated, while R16C is not modified. This strongly suggests that the serine 19 within the consensus sequence for various kinases, RXXS, is the site of modification. Our results demonstrate that PTPS undergoes protein phosphorylation and requires additional, not yet identified post-translational modification(s) for its in vivo function.
Asunto(s)
Oxidorreductasas de Alcohol/química , Fenilalanina/sangre , Liasas de Fósforo-Oxígeno , Oxidorreductasas de Alcohol/deficiencia , Oxidorreductasas de Alcohol/fisiología , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Células Cultivadas , Humanos , Datos de Secuencia Molecular , Mutación , Fosforilación , Pliegue de Proteína , Procesamiento Proteico-Postraduccional , Ratas , Proteínas Recombinantes/biosíntesisRESUMEN
GTP cyclohydrolase I of Escherichia coli is a torus-shaped homodecamer with D5 symmetry and catalyzes a complex ring expansion reaction conducive to the formation of dihydroneopterin triphosphate from GTP. The x-ray structure of a complex of the enzyme with the substrate analog, dGTP, bound at the active site was determined at a resolution of 3 A. In the decamer, 10 equivalent active sites are present, each of which contains a 10-A deep pocket formed by surface areas of 3 adjacent subunits. The substrate forms a complex hydrogen bond network with the protein. Active site residues were modified by site-directed mutagenesis, and enzyme activities of the mutant proteins were measured. On this basis, a mechanism of the enzyme-catalyzed reaction is proposed. Cleavage of the imidazole ring is initiated by protonation of N7 by His-179 followed by the attack of water at C8 of the purine system. Cystine Cys-110 Cys-181 may be involved in this reaction step. Opening of the imidazole ring may be in concert with cleavage of the furanose ring to generate a Schiff's base from the glycoside. The gamma-phosphate of GTP may be involved in the subsequent Amadori rearrangement of the carbohydrate side chain by activating the hydroxyl group of Ser-135.