RESUMEN
Glycerol dehydrogenase (GldA) encoded by the STM4108 gene (gldA) has been related to the synthesis of HilA, a major transcriptional regulator that is responsible for the expression of invasion genes in the human pathogen Salmonella enterica serovar Typhimurium. Single colourless crystals were obtained from a recombinant preparation of GldA overexpressed in Escherichia coli. They belonged to space group P222(1), with unit-cell parameters a = 127.0, b = 160.1, c = 665.2 A. The crystals contained a very large number of molecules in the asymmetric unit, probably 30-35. Diffraction data were collected to 3.5 A resolution using synchrotron radiation at the European Synchrotron Radiation Facility.
Asunto(s)
Salmonella enterica/enzimología , Deshidrogenasas del Alcohol de Azúcar/química , Cristalización , Cristalografía por Rayos X , HumanosRESUMEN
Survival E (SurE) protein from Campylobacter jejuni, a Gram-negative mesophile, has been overexpressed in Escherichia coli as a soluble protein, successfully purified and crystallized in two distinct crystal forms. The first form belongs to space group P2(1)2(1)2(1), with a tetramer in the asymmetric unit and unit-cell parameters a = 80.5, b = 119.0, c = 135.3 A. The second form belongs to space group C2, with unit-cell parameters a = 121.4, b = 47.1, c = 97.8 A, and contains a dimer in the asymmetric unit. Diffraction data have been collected from these crystal forms to 2.5 and 2.95 A resolution, respectively.
Asunto(s)
Proteínas Bacterianas/química , Proteínas Bacterianas/aislamiento & purificación , Campylobacter jejuni/química , Campylobacter jejuni/metabolismo , Expresión Génica , Secuencia de Aminoácidos , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Campylobacter jejuni/genética , Cristalización , Datos de Secuencia Molecular , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Difracción de Rayos XRESUMEN
1,3-Propanediol dehydrogenase (1,3-PD-DH), encoded by the dhaT gene, is a key enzyme in the dissimilation process for converting glycerol to 1,3-propanediol in the human pathogen Klebsiella pneumoniae. Single colourless crystals were obtained from a recombinant preparation of 1,3-propanediol dehydrogenase overexpressed in Escherichia coli. The crystals belong to space group P2(1), with unit-cell parameters a = 91.9, b = 226.6, c = 232.6 A, beta = 92.9 degrees. The crystals probably contain two decamers in the asymmetric unit, with a V(M) value of 3.07 A3 Da(-1) and an estimated solvent content of 59%. Diffraction data were collected to 2.7 A resolution using synchrotron radiation at the ID14-4 beamline of the European Synchrotron Radiation Facility.
Asunto(s)
Oxidorreductasas de Alcohol/química , Proteínas Bacterianas/química , Cristalografía por Rayos X/métodos , Klebsiella pneumoniae/enzimología , Alcohol Deshidrogenasa , Oxidorreductasas de Alcohol/aislamiento & purificación , Proteínas Bacterianas/aislamiento & purificación , Cristalización , Humanos , Klebsiella pneumoniae/patogenicidadRESUMEN
The cloning, expression, purification, crystallization and preliminary crystallographic analysis of glucose-1-phosphate uridylyltransferase (UgpG) from Sphingomonas elodea ATCC 31461 bound to glucose-1-phosphate are reported. Diffraction data sets were obtained from seven crystal forms in five different space groups, with highest resolutions ranging from 4.20 to 2.65 A. The phase problem was solved for a P2(1) crystal form using multiple isomorphous replacement with anomalous scattering from an osmium derivative and a SeMet derivative. The best native crystal in space group P2(1) has unit-cell parameters a = 105.5, b = 85.7, c = 151.8 A, beta = 105.2 degrees . Model building and refinement are currently under way.
Asunto(s)
Proteínas Bacterianas/química , Glucofosfatos/metabolismo , Sphingomonas/enzimología , Sphingomonas/genética , UTP-Glucosa-1-Fosfato Uridililtransferasa/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Sitios de Unión/genética , Clonación Molecular , Cristalización , Cristalografía por Rayos X/métodos , Regulación Bacteriana de la Expresión Génica , Glucofosfatos/química , Glucofosfatos/genética , Especificidad por Sustrato/genética , UTP-Glucosa-1-Fosfato Uridililtransferasa/biosíntesis , UTP-Glucosa-1-Fosfato Uridililtransferasa/genéticaRESUMEN
In this work, we present a comparative case study of "ortho-" and "meta-nitrated" catecholic inhibitors of catechol-O-methyltransferase (COMT), with regard to their interaction with the catalytic site of the enzyme and the in vitro regioselective formation of their mono-O-methyl ether metabolites. In particular, the effects of altering the attachment position of the inhibitors' side-chain substituent, within the classic nitrocatechol pharmacophore, were investigated. For this purpose, we compared two simple regioisomeric nitrocatechol-type inhibitors of COMT, BIA 3-228 and BIA 8-176, which contain the benzoyl substituent attached at the meta and ortho positions, respectively, relative to the nitro group. The two compounds were slowly O-methylated by COMT in vitro, but the particular substitution pattern of each compound was shown to have a profound impact on the regioselectivity of their O-methylation. To provide a plausible interpretation of these results, a comprehensive analysis of the protein-inhibitor interactions and of the relative chemical susceptibility to O-methylation of the catechol hydroxyl groups was performed by means of docking simulations and ab initio molecular orbital calculations. The major structural and chemical factors that determine the enzyme regioselectivity of O-methylation were identified, and the X-ray structure of the complex of COMT with S-adenosyl-l-methionine and BIA 8-176 is herein disclosed. This is the first reported structure of the soluble form of COMT complexed with a nitrocatecholic inhibitor having a bulky substituent group in adjacent position (ortho) to the nitro group. Structural and dynamic aspects of this complex are analyzed and discussed, in the context of the present study.
Asunto(s)
Benzofenonas/química , Inhibidores de Catecol O-Metiltransferasa , Inhibidores Enzimáticos/química , Nitrofenoles/química , Animales , Benzofenonas/farmacología , Sitios de Unión , Dominio Catalítico , Catecol O-Metiltransferasa/química , Catecol O-Metiltransferasa/metabolismo , Catecoles/química , Catecoles/metabolismo , Cristalización , Dimerización , Inhibidores Enzimáticos/farmacología , Metilación , Modelos Moleculares , Estructura Molecular , Nitratos/química , Nitrofenoles/farmacología , Unión Proteica , Ratas , EstereoisomerismoRESUMEN
Inhibitors of the enzyme catechol-O-methyltransferase (COMT) are used as co-adjuvants in the therapy of Parkinson's disease. A recombinant form of the soluble cytosolic COMT from rat has been co-crystallized with a new potent inhibitor, BIA 8-176 [(3,4-dihydroxy-2-nitrophenyl)phenylmethanone], by the vapour-diffusion method using PEG 6K as precipitant. Crystals diffract to 1.6 A resolution on a synchrotron-radiation source and belong to the monoclinic space group P2(1), with unit-cell parameters a = 52.77, b = 79.63, c = 61.54 A, beta = 91.14 degrees.
Asunto(s)
Inhibidores de Catecol O-Metiltransferasa , Catecol O-Metiltransferasa/química , Inhibidores Enzimáticos/química , Animales , Clonación Molecular , Cristalización , Citosol/enzimología , Escherichia coli/enzimología , Ratas , Proteínas Recombinantes/antagonistas & inhibidores , Proteínas Recombinantes/química , Sincrotrones , Difracción de Rayos XRESUMEN
The Atomic Solvation Parameter (ASP) model is one of the simplest models of solvation, in which the solvation free energy of a molecule is proportional to the solvent accessible surface area (SAS) of its atoms. However, until now this model had not been incorporated into the Self-Consistent Mean Field Theory (SCMFT) method for modelling sidechain conformations in proteins. The reason for this is that SAS is a many-body quantity and, thus, it is not obvious how to define it within the Mean Field (MF) framework, where multiple copies of each sidechain exist simultaneously. Here, we present a method for incorporating an SAS-based potential, such as the ASP model, into SCMFT. The theory on which the method is based is exact within the MF framework, that is, it does not depend on a pairwise or any other approximation of SAS. Therefore, SAS can be calculated to arbitrary accuracy. The method is computationally very efficient: only 7.6% slower on average than the method without solvation. We applied the method to the prediction of sidechain conformation, using as a test set high-quality solution structures of 11 proteins. Solvation was found to substantially improve the prediction accuracy of well-defined surface sidechains. We also investigated whether the methodology can be applied to prediction of folding free energies of protein mutants, using a set of barnase mutants. For apolar mutants, the modest correlation observed between calculated and observed folding free energies without solvation improved substantially when solvation was included, allowing the prediction of trends in the folding free energies of this type of mutants. For polar mutants, correlation was not significant even with solvation. Several other factors also responsible for the correlation were identified and analysed. From this analysis, future directions for applying and improving the present methodology are discussed.
Asunto(s)
Proteínas/química , Proteínas/genética , Algoritmos , Proteínas Bacterianas , Estabilidad de Enzimas , Modelos Químicos , Modelos Moleculares , Mutación , Conformación Proteica , Pliegue de Proteína , Ribonucleasas/química , Ribonucleasas/genética , Solventes , TermodinámicaRESUMEN
Cell metabolism relies on energy transduction usually performed by complex membrane-spanning proteins that couple different chemical processes, e.g. electron and proton transfer in proton-pumps. There is great interest in determining at the molecular level the structural details that control these energy transduction events, particularly those involving multiple electrons and protons, because tight control is required to avoid the production of dangerous reactive intermediates. Tetraheme cytochrome c(3) is a small soluble and monomeric protein that performs a central step in the bioenergetic metabolism of sulfate reducing bacteria, termed "proton-thrusting," linking the oxidation of molecular hydrogen with the reduction of sulfate. The mechano-chemical coupling involved in the transfer of multiple electrons and protons in cytochrome c(3) from Desulfovibrio desulfuricans ATCC 27774 is described using results derived from the microscopic thermodynamic characterization of the redox and acid-base centers involved, crystallographic studies in the oxidized and reduced states of the cytochrome, and theoretical studies of the redox and acid-base transitions. This proton-assisted two-electron step involves very small, localized structural changes that are sufficient to generate the complex network of functional cooperativities leading to energy transduction, while using molecular mechanisms distinct from those established for other Desulfovibrio sp. cytochromes from the same structural family.
Asunto(s)
Grupo Citocromo c/metabolismo , Transporte de Electrón , Grupo Citocromo c/química , Desulfovibrio/enzimología , Oxidación-Reducción , Conformación Proteica , TermodinámicaRESUMEN
Catechol-O-methyltransferase (COMT) is involved in the metabolism of catecholamines, catechol steroids and xenobiotic catechols. A precise knowledge of the enzyme-inhibitor structural interactions could help in the design of better inhibitors. Soluble rat COMT was expressed in Escherichia coli and the recombinant protein was crystallized with a new tight-binding inhibitor, BIA 3-335 [1-(3,4-dihydroxy-5-nitrophenyl)-3-(n-3'-trifluoromethylphenyl)piperazine-1-propanone dihydrochloride]. The crystals were obtained by the sitting-drop vapour-diffusion method using PEG 6K as a precipitant. These crystals diffracted to better than 1.9 A and belong to the trigonal space group P3(2)21. The unit-cell parameters for the crystal measured at room temperature were a = b = 51.5, c = 168.3 A; each shrank by about 1 A on freezing.
Asunto(s)
Catecol O-Metiltransferasa/química , Animales , Catecol O-Metiltransferasa/genética , Cristalización , Cristalografía por Rayos X , Inhibidores Enzimáticos/química , Conformación Proteica , Ratas , Proteínas Recombinantes/química , Especificidad por SustratoRESUMEN
A series of novel derivatives of oxcarbazepine (5), 10,11-dihydro-10-oxo-5H-dibenz/b,f/azepine-5-carboxamide was synthesised and evaluated for their anticonvulsant activity and sodium channel blocking properties. The oxime 8 was found to be the most active compound from this series, displaying greater potency than its geometric isomer 9 and exhibiting also the highest protective index value. Importantly, the metabolic profile of 8 differs from the already established dibenz/b,f/azepine-5-carboxamide drugs such as 1 and 5 which undergo rapid and complete conversion in vivo to several biologically active metabolites. In contrast 8 is metabolised to only a very minor extent leading to the conclusion that the observed anti-convulsant effect is solely attributable to 8. It is concluded that 8 may be as effective as 1 and 5 at controlling seizures and that the low toxicity and consequently high protective index should provide the compound with an improved side-effect profile.
Asunto(s)
Amidas/química , Amidas/síntesis química , Anticonvulsivantes/síntesis química , Anticonvulsivantes/farmacología , Azepinas/síntesis química , Azepinas/farmacología , Animales , Anticonvulsivantes/farmacocinética , Azepinas/farmacocinética , Encéfalo/efectos de los fármacos , Encéfalo/metabolismo , Carbamazepina/química , Carbamazepina/farmacología , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Femenino , Masculino , Ratones , Ratones Endogámicos , Conejos , Ratas , Ratas Wistar , Bloqueadores de los Canales de Sodio , Canales de Sodio/metabolismo , Especificidad de la EspecieRESUMEN
The performance of the self-consistent mean field theory (SCMFT) method for side-chain modeling, employing rotamer energies calculated with the flexible rotamer model (FRM), is evaluated in the context of comparative modeling of protein structure. Predictions were carried out on a test set of 56 model backbones of varying accuracy, to allow side-chain prediction accuracy to be analyzed as a function of backbone accuracy. A progressive decrease in the accuracy of prediction was observed as backbone accuracy decreased. However, even for very low backbone accuracy, prediction was substantially higher than random, indicating that the FRM can, in part, compensate for the errors in the modeled tertiary environment. It was also investigated whether the introduction in the FRM-SCMFT method of knowledge-based biases, derived from a backbone-dependent rotamer library, could enhance its performance. A bias derived from the backbone-dependent rotamer conformations alone did not improve prediction accuracy. However, a bias derived from the backbone-dependent rotamer probabilities improved prediction accuracy considerably. This bias was incorporated through two different strategies. In one (the indirect strategy), rotamer probabilities were used to reject unlikely rotamers a priori, thus restricting prediction by FRM-SCMFT to a subset containing only the most probable rotamers in the library. In the other (the direct strategy), rotamer energies were transformed into pseudo-energies that were added to the average potential energies of the respective rotamers, thereby creating hybrid energy-based/knowledge-based average rotamer energies, which were used by the FRM-SCMFT method for prediction. For all degrees of backbone accuracy, an optimal strength of the knowledge-based bias existed for both strategies for which predictions were more accurate than pure energy-based predictions, and also than pure knowledge-based predictions. Hybrid knowledge-based/energy-based methods were obtained from both strategies and compared with the SCWRL method, a hybrid method based on the same backbone-dependent rotamer library. The accuracy of the indirect method was approximately the same as that of the SCWRL method, but that of the direct method was significantly higher.
Asunto(s)
Conformación Proteica , Proteínas/química , Simulación por Computador , Bases de Datos Factuales , Modelos Teóricos , Reproducibilidad de los Resultados , TermodinámicaRESUMEN
The crystal structure of low-potential cytochrome c549, an extrinsic component of the photosystem II (PS II) from Synechocystis sp. PCC 6803, was obtained directly from single-wavelength 1.21 A resolution diffraction data. This is the first monodomain bis-histidinyl monoheme cytochrome c to be structurally characterized. The extended N-terminal region of c549 builds up a two-strand antiparallel beta-sheet in a hairpin motif, which extends through two molecules owing to crystal packing. Both peptide termini are involved in crystal contacts, which may explain their protrusion out of the globular fold. The C-terminus is preceded by a 9 A-long hydrophobic finger extending from a positively charged base and could be involved in PSII interactions, as well as a protruding negative patch built by a set of conserved acidic residues among c549 sequences.
Asunto(s)
Cianobacterias/enzimología , Grupo Citocromo c/química , Secuencia de Aminoácidos , Cristalografía por Rayos X , Modelos Moleculares , Datos de Secuencia Molecular , Filogenia , Conformación Proteica , Homología de Secuencia de AminoácidoRESUMEN
The primary and three-dimensional structures of a [NiFe] hydrogenase isolated from D. desulfitricans ATCC 27774 were determined, by nucleotide analysis and single-crystal X-ray crystallography. The three-dimensional structural model was refined to R=0.167 and Rfree=0.223 using data to 1.8 A resolution. Two unique structural features are observed: the [4Fe-4S] cluster nearest the [NiFe] centre has been modified [4Fe-3S-3O] by loss of one sulfur atom and inclusion of three oxygen atoms; a three-fold disorder was observed for Cys536 which binds to the nickel atom in the [NiFe] centre. Also, the bridging sulfur atom that caps the active site was found to have partial occupancy, thus corresponding to a partly activated enzyme. These structural features may have biological relevance. In particular, the two less-populated rotamers of Cys536 may be involved in the activation process of the enzyme, as well as in the catalytic cycle. Molecular modelling studies were carried out on the interaction between this [NiFe] hydrogenase and its physiological partner, the tetrahaem cytochrome c3 from the same organism. The lowest energy docking solutions were found to correspond to an interaction between the haem IV region in tetrahaem cytochrome c3 with the distal [4Fe-4S] cluster in [NiFe] hydrogenase. This interaction should correspond to efficient electron transfer and be physiologically relevant, given the proximity of the two redox centres and the fact that electron transfer decay coupling calculations show high coupling values and a short electron transfer pathway. On the other hand, other docking solutions have been found that, despite showing low electron transfer efficiency, may give clues on possible proton transfer mechanisms between the two molecules.
Asunto(s)
Grupo Citocromo c/metabolismo , Desulfovibrio/enzimología , Hidrogenasas/química , Hidrogenasas/genética , Secuencia de Aminoácidos , Secuencia de Bases , Cristalografía por Rayos X , ADN Bacteriano , Hidrogenasas/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Unión Proteica , Conformación ProteicaRESUMEN
Bacterioferritins constitute a subfamily of heme ferritins, proteins involved in iron storage and homeostasis. The protein isolated from Desulfovibrio desulfuricans ATCC 27774 is a homodimer of mass 52 kDa. The monomers are linked by an iron-coproporphyrin group and each monomer contains a diferric center. The 24-monomer clusters found in the crystal are probably the functional particles. MAD data from cubic bacterioferritin crystals were collected at the K-shell iron edge. Preliminary phasing was performed using the positions of 23 of the 40 Fe atoms expected in the asymmetric unit. Further MAD phasing allowed the identification of individual iron sites. Clear and interpretable electron-density maps were obtained after density modification.
Asunto(s)
Proteínas Bacterianas , Grupo Citocromo b/química , Desulfovibrio/metabolismo , Ferritinas/química , Cristalización , Cristalografía por Rayos X/métodos , Grupo Citocromo b/aislamiento & purificación , Dimerización , Ferritinas/aislamiento & purificación , Peso Molecular , Conformación Proteica , Programas InformáticosRESUMEN
Desulfovibrio gigas is a strict anaerobe that contains a well-characterized metabolic pathway that enables it to survive transient contacts with oxygen. The terminal enzyme in this pathway, rubredoxin:oxygen oxidoreductase (ROO) reduces oxygen to water in a direct and safe way. The 2.5 A resolution crystal structure of ROO shows that each monomer of this homodimeric enzyme consists of a novel combination of two domains, a flavodoxin-like domain and a Zn-beta-lactamase-like domain that contains a di-iron center for dioxygen reduction. This is the first structure of a member of a superfamily of enzymes widespread in strict and facultative anaerobes, indicating its broad physiological significance.
Asunto(s)
Desulfovibrio/enzimología , Oxidorreductasas/química , Oxidorreductasas/metabolismo , Oxígeno/metabolismo , Secuencia de Aminoácidos , Sitios de Unión , Cristalografía por Rayos X , Desulfovibrio/genética , Dimerización , Flavodoxina/química , Hierro/metabolismo , Ligandos , Modelos Moleculares , Datos de Secuencia Molecular , Oxidación-Reducción , Oxidorreductasas/genética , Filogenia , Estructura Terciaria de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Alineación de Secuencia , Agua/metabolismo , beta-Lactamasas/químicaRESUMEN
The crystal structures of the human androgen receptor (hAR) and human progesterone receptor ligand-binding domains in complex with the same ligand metribolone (R1881) have been determined. Both three-dimensional structures show the typical nuclear receptor fold. The change of two residues in the ligand-binding pocket between the human progesterone receptor and hAR is most likely the source for the specificity of R1881 to the hAR. The structural implications of the 14 known mutations in the ligand-binding pocket of the hAR ligand-binding domains associated with either prostate cancer or the partial or complete androgen receptor insensitivity syndrome were analyzed. The effects of most of these mutants could be explained on the basis of the crystal structure.
Asunto(s)
Metribolona/metabolismo , Receptores Androgénicos/metabolismo , Congéneres de la Testosterona/metabolismo , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Sitios de Unión , Cristalografía por Rayos X , Humanos , Ligandos , Masculino , Modelos Químicos , Modelos Moleculares , Conformación Molecular , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Neoplasias de la Próstata/metabolismo , Conformación Proteica , Receptores Androgénicos/genéticaRESUMEN
Cinnamomin (CIN) belongs to a family of 10 kDa proteins designated as elicitins. Some of these proteins induce a hypersensitive response in diverse plant species, leading to resistance against fungal and bacterial plant pathogens. CIN was crystallized by the vapour-diffusion method using either ammonium sulfate or polyethyleneglycol (PEG) as precipitants in solutions buffered at around pH 7. These crystals are isomorphous and belong to the triclinic space group, with unit-cell parameters a = 31.69, b = 36. 99, c = 44.09 A, alpha = 76.86, beta = 84.41, gamma = 80.26 degrees. A frozen crystal diffracted X-rays beyond 1.45 A resolution on a synchrotron-radiation source.
Asunto(s)
Proteínas Fúngicas/química , Phytophthora/química , Proteínas/química , Proteínas Algáceas , Cristalización , Cristalografía por Rayos X , Proteínas Fúngicas/aislamiento & purificación , Modelos Moleculares , Pichia , Conformación Proteica , Proteínas/aislamiento & purificación , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/aislamiento & purificación , Proteínas Inactivadoras de Ribosomas Tipo 2RESUMEN
A monomeric nine-haem cytochrome c (9Hcc) with 292 amino acid residues was isolated from cells of the sulfate- and nitrate-reducing bacterium Desulfovibrio desulfuricans ATCC 27774 grown under both nitrate- and sulfate-respiring conditions. The nucleotide sequence encoding the 292 residues was determined, allowing the correction of about 10% of the previous primary structure, determined from 1.8 A electron density maps. The refinement at 1.8 A resolution of the structural model was completed, giving an R-value of 16.5%. The nine haem groups are arranged into two tetrahaem clusters, located at both ends of the molecule, with Fe-Fe distances and local protein fold very similar to tetrahaem cytochromes c3, and the extra haem is located asymmetrically between the two regions. The new primary sequence determination confirmed the 39% sequence homology found between this cytochrome and the C-terminal region (residues 229-514) of the high-molecular-weight cytochrome c (Hmc) from D. vulgaris Hildenborough, providing strong evidence of structural similarity between 9Hcc and the C-terminal region of Hmc. The interaction between 9Hcc and the tetrahaem cytochrome c3 from the same organism was studied by modelling methods, and the results suggest that a specific interaction is possible between haem 4 of tetrahaem cytochrome c3 and haem 1 or haem 2 of 9Hcc, in agreement with previous kinetic experiments which showed the catalytic effect of the tetrahaem cytochrome c3 upon the reduction of 9Hcc by the [NiFe] hydrogenase from D. desulfuricans ATCC 27774. These studies suggest a role for 9Hcc as part of the assembly of redox proteins involved in recycling the molecular hydrogen released by the cell as a result of substrate oxidation.
Asunto(s)
Grupo Citocromo c/química , Desulfovibrio/enzimología , Secuencia de Aminoácidos , Secuencia de Bases , Simulación por Computador , Cristalografía por Rayos X , Grupo Citocromo c/metabolismo , Hemo , Modelos Moleculares , Datos de Secuencia Molecular , Conformación Proteica , Análisis de SecuenciaRESUMEN
Correct alignment of the sequence of a target protein with those of homologues of known three-dimensional structure is a key step in comparative modeling. Usually an iterative approach that takes account of the local and overall structural features is required. We describe such an approach that exploits databases of structural alignments of homologous proteins (HOMSTRAD, http:/(/)www-cryst.bioc.cam.ac.uk/ approximately homstrad) and protein superfamilies (CAMPASS, http:/(/)www-cryst.bioc.cam.ac.uk/ approximately campass), in which structure-based alignments are analyzed and formatted with the program JOY (http:/(/)www-cryst.bioc.cam.ac.uk/ approximately joy) to reveal conserved local structural features. The databases facilitate the recognition of a family or superfamily, they assist in the selection of useful parent structures, they are helpful in alignment of the target sequences with the parent set, and are useful for deriving relationships that can be used in validating models. In the iterative approach, a model is constructed on the basis of the proposed sequence alignment and this is then reexpressed in the JOY format and realigned with the parent set. This is repeated until the model and sequence alignment is optimized. We examine the case for comparison and use of multiple structures of family members, rather than a single parent structure. We use the targets attempted by our group in CASP3 to assess the value of such procedures.
Asunto(s)
Modelos Moleculares , Proteínas/química , Algoritmos , Secuencia de Aminoácidos , Animales , Bases de Datos como Asunto , Internet , Datos de Secuencia Molecular , Porinas/química , Ribonucleasas/química , Alineación de SecuenciaRESUMEN
The respiratory chain of the thermohalophilic bacterium Rhodothermus marinus contains a novel complex III and a high potential iron-sulfur protein (HiPIP) as the main electron shuttle (Pereira et al., Biochemistry 38 (1999) 1268-1275 and 1276-1283). In this paper, one of the terminal oxidases expressed in this bacterium is extensively characterised. It is a caa3-type oxidase, isolated with four subunits (apparent molecular masses of 42, 19 and 15 kDa and a C-haem containing subunit of 35 kDa), which has haems of the A(s) type. This oxidase is capable of using TMPD and horse heart cytochrome c as substrates, but has a higher turnover with HiPIP, being the first example of a HiPIP:oxygen oxidoreductase. The oxidase has unusually low reduction potentials of 260 (haem C), 255 (haem A) and 180 mV (haem A3). Subunit I of R. marinus caa3 oxidase has an overall significant homology with the subunits I of the COX type oxidases, namely the metal binding sites and most residues considered to be functionally important for proton uptake and pumping (K- and D-channels). However, a major difference is present: the putative essential glutamate (E278 in Paraccocus denitrificans) of the D-channel is missing in the R. marinus oxidase. Homology modelling of the R. marinus oxidase shows that the phenol group of a tyrosine residue may occupy a similar spatial position as the glutamate carboxyl, in relation to the binuclear centre. Moreover, sequence comparisons reveal that several enzymes lacking that glutamate have a conserved substitution pattern in helix VI: -YSHPXV- instead of -XGHPEV-. These observations are discussed in terms of the mechanisms for proton uptake and it is suggested that, in these enzymes, tyrosine may play the role of the glutamate in the proton channel.