RESUMO
CLIC1 (NCC27) is a member of the highly conserved class of chloride ion channels that exists in both soluble and integral membrane forms. Purified CLIC1 can integrate into synthetic lipid bilayers forming a chloride channel with similar properties to those observed in vivo. The structure of the soluble form of CLIC1 has been determined at 1.4-A resolution. The protein is monomeric and structurally homologous to the glutathione S-transferase superfamily, and it has a redox-active site resembling glutaredoxin. The structure of the complex of CLIC1 with glutathione shows that glutathione occupies the redox-active site, which is adjacent to an open, elongated slot lined by basic residues. Integration of CLIC1 into the membrane is likely to require a major structural rearrangement, probably of the N-domain (residues 1-90), with the putative transmembrane helix arising from residues in the vicinity of the redox-active site. The structure indicates that CLIC1 is likely to be controlled by redox-dependent processes.
Assuntos
Canais de Cloreto/química , Cloro/química , Sequência de Aminoácidos , Sítios de Ligação , Membrana Celular/metabolismo , Cloro/metabolismo , Cisteína/química , Eletrofisiologia , Escherichia coli/metabolismo , Glutationa/metabolismo , Glutationa Transferase/metabolismo , Humanos , Bicamadas Lipídicas/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Oxirredução , Técnicas de Patch-Clamp , Mutação Puntual , Ligação Proteica , Conformação Proteica , Estrutura Terciária de Proteína , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes/metabolismo , Homologia de Sequência de AminoácidosRESUMO
The molecular interactions driving reactive center loop (RCL) insertion are of considerable interest in gaining a better understanding of the serpin inhibitory mechanism. Previous studies have suggested that interactions in the proximal hinge/breach region may be critical determinants of RCL insertion in serpins. In this study, conformational and functional changes in plasminogen activator inhibitor-2 (PAI-2) following incubation with a panel of synthetic RCL peptides indicated that the P14 residue is critical for RCL insertion, and hence inhibitory activity, in PAI-2. Only RCL peptides with a P14 threonine were able to induce the stressed to relaxed transition and abolish inhibitory activity in PAI-2, indicating that RCL insertion into beta-sheet A of PAI-2 is dependent upon this residue. The recently solved crystal structure of relaxed PAI-2 (PAI-2.RCL peptide complex) allowed detailed analysis of molecular interactions involving P14 related to RCL insertion. Of most interest is the rearrangement of hydrogen bonding around the breach region that accompanies the stressed to relaxed transition, in particular the formation of a side chain hydrogen bond between the threonine at P14 and an adjacent tyrosine on strand 2 of beta-sheet B in relaxed PAI-2. Structural alignment of known serpin sequences showed that this pairing (or the equivalent serine/threonine pairing) is highly conserved ( approximately 87%) in inhibitory serpins and may represent a general structural basis for serpin inhibitory activity.
Assuntos
Inibidor 2 de Ativador de Plasminogênio/química , Inibidor 2 de Ativador de Plasminogênio/metabolismo , Aminoácidos/química , Eletroforese em Gel de Poliacrilamida , Humanos , Ligação de Hidrogênio , Modelos Biológicos , Modelos Químicos , Modelos Moleculares , Mutação , Peptídeos/química , Ligação Proteica , Conformação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/farmacologia , Espectrometria de Fluorescência , Treonina/química , Ureia/farmacologiaRESUMO
The structure of the serpin, plasminogen activator inhibitor type-2 (PAI-2), in a complex with a peptide mimicking its reactive center loop (RCL) has been determined at 1.6-A resolution. The structure shows the relaxed state serpin structure with a prominent six-stranded beta-sheet. Clear electron density is seen for all residues in the peptide. The P1 residue of the peptide binds to a well defined pocket at the base of PAI-2 that may be important in determining the specificity of protease inhibition. The stressed-to-relaxed state (S --> R) transition in PAI-2 can be modeled as the relative motion between a quasirigid core domain and a smaller segment comprising helix hF and beta-strands s1A, s2A, and s3A. A comparison of the Ramachandran plots of the stressed and relaxed state PAI-2 structures reveals the location of several hinge regions connecting these two domains. The hinge regions cluster in three locations on the structure, ensuring a cooperative S --> R transition. We hypothesize that the hinge formed by the conserved Gly(206) on beta-strand s3A in the breach region of PAI-2 effects the S --> R transition by altering its backbone torsion angles. This torsional change is due to the binding of the P14 threonine of the RCL to the open breach region of PAI-2.
Assuntos
Cristalografia por Raios X , Peptídeos/química , Inibidor 2 de Ativador de Plasminogênio/química , Elétrons , Escherichia coli/metabolismo , Deleção de Genes , Glicina/química , Humanos , Modelos Moleculares , Mutação , Ligação Proteica , Conformação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Serpinas/química , Treonina/químicaRESUMO
The Sm/Lsm proteins associate with small nuclear RNA to form the core of small nuclear ribonucleoproteins, required for processes as diverse as pre-mRNA splicing, mRNA degradation and telomere formation. The Lsm proteins from archaea are likely to represent the ancestral Sm/Lsm domain. Here, we present the crystal structure of the Lsm alpha protein from the thermophilic archaeon Methanobacterium thermoautotrophicum at 2.0 A resolution. The Lsm alpha protein crystallizes as a heptameric ring comprised of seven identical subunits interacting via beta-strand pairing and hydrophobic interactions. The heptamer can be viewed as a propeller-like structure in which each blade consists of a seven-stranded antiparallel beta-sheet formed from neighbouring subunits. There are seven slots on the inner surface of the heptamer ring, each of which is lined by Asp, Asn and Arg residues that are highly conserved in the Sm/Lsm sequences. These conserved slots are likely to form the RNA-binding site. In archaea, the gene encoding Lsm alpha is located next to the L37e ribosomal protein gene in a putative operon, suggesting a role for the Lsm alpha complex in ribosome function or biogenesis.
Assuntos
Proteínas Arqueais/química , Evolução Molecular , Methanobacterium/química , Ribonucleoproteínas Nucleares Pequenas/química , Sequência de Aminoácidos , Proteínas Arqueais/genética , Proteínas Arqueais/metabolismo , Sítios de Ligação , Sequência Conservada , Cristalografia por Raios X , Ordem dos Genes , Ligação de Hidrogênio , Methanobacterium/genética , Modelos Moleculares , Dados de Sequência Molecular , Estrutura Quaternária de Proteína , Estrutura Terciária de Proteína , Subunidades Proteicas , RNA/genética , RNA/metabolismo , Ribonucleoproteínas Nucleares Pequenas/genética , Ribonucleoproteínas Nucleares Pequenas/metabolismo , Proteínas Ribossômicas/genética , Alinhamento de SequênciaRESUMO
Cryptophytes are unicellular photosynthetic algae that use a lumenally located light-harvesting system, which is distinct from the phycobilisome structure found in cyanobacteria and red algae. One of the key components of this system is water-soluble phycoerythrin (PE) 545 whose expression is enhanced by low light levels. The crystal structure of the heterodimeric alpha(1)alpha(2)betabeta PE 545 from the marine cryptophyte Rhodomonas CS24 has been determined at 1.63-A resolution. Although the beta-chain structure is similar to the alpha and beta chains of other known phycobiliproteins, the overall structure of PE 545 is novel with the alpha chains forming a simple extended fold with an antiparallel beta-ribbon followed by an alpha-helix. The two doubly linked beta50/beta61 chromophores (one on each beta subunit) are in van der Waals contact, suggesting that exciton-coupling mechanisms may alter their spectral properties. Each alpha subunit carries a covalently linked 15,16-dihydrobiliverdin chromophore that is likely to be the final energy acceptor. The architecture of the heterodimer suggests that PE 545 may dock to an acceptor protein via a deep cleft and that energy may be transferred via this intermediary protein to the reaction center.
Assuntos
Eucariotos/metabolismo , Ficoeritrina/química , Gráficos por Computador , Sequência Conservada , Cristalografia por Raios X/métodos , Dimerização , Transferência de Energia , Substâncias Macromoleculares , Modelos Moleculares , Dados de Sequência Molecular , Ficobilissomas , Conformação Proteica , Estrutura Secundária de ProteínaRESUMO
BACKGROUND: Plasminogen activator inhibitor 2 (PAI-2) is a member of the serpin family of protease inhibitors that function via a dramatic structural change from a native, stressed state to a relaxed form. This transition is mediated by a segment of the serpin termed the reactive centre loop (RCL); the RCL is cleaved on interaction with the protease and becomes inserted into betasheet A of the serpin. Major questions remain as to what factors facilitate this transition and how they relate to protease inhibition. RESULTS: The crystal structure of a mutant form of human PAI-2 in the stressed state has been determined at 2.0 A resolution. The RCL is completely disordered in the structure. An examination of polar residues that are highly conserved across all serpins identifies functionally important regions. A buried polar cluster beneath betasheet A (the so-called 'shutter' region) is found to stabilise both the stressed and relaxed forms via a rearrangement of hydrogen bonds. CONCLUSIONS: A statistical analysis of interstrand interactions indicated that the shutter region can be used to discriminate between inhibitory and non-inhibitory serpins. This analysis implied that insertion of the RCL into betasheet A up to residue P8 is important for protease inhibition and hence the structure of the complex formed between the serpin and the target protease.
Assuntos
Inibidor 2 de Ativador de Plasminogênio/química , Inibidor 2 de Ativador de Plasminogênio/metabolismo , Serpinas/química , Serpinas/metabolismo , Sequência de Aminoácidos , Cristalografia por Raios X , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Dobramento de Proteína , Estrutura Secundária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Deleção de Sequência , Homologia de Sequência de AminoácidosRESUMO
The enzyme hydroxymethylbilane synthase (HMBS, E.C. 4.3.1.8) catalyzes the conversion of porphobilinogen into hydroxymethylbilane, a key intermediate for the biosynthesis of heme, chlorophylls, vitamin B12 and related macrocycles. The enzyme is found in all organisms, except viruses. The crystal structure of the selenomethionine-labelled enzyme ([SeMet]HMBS) from Escherichia coli has been solved by the multi-wavelength anomalous dispersion (MAD) experimental method using the Daresbury SRS station 9.5. In addition, [SeMet]HMBS has been studied by MAD at the Grenoble ESRF MAD beamline BM14 (BL19) and this work is described especially with respect to the use of the ESRF CCD detector. The structure at ambient temperature has been refined, the R factor being 16.8% at 2. 4 A resolution. The dipyrromethane cofactor of the enzyme is preserved in its reduced form in the crystal and its geometrical shape is in full agreement with the crystal structures of authentic dipyrromethanes. Proximal to the reactive C atom of the reduced cofactor, spherical density is seen consistent with there being a water molecule ideally placed to take part in the final step of the enzyme reaction cycle. Intriguingly, the loop with residues 47-58 is not ordered in the structure of this form of the enzyme, which carries no substrate. Direct experimental study of the active enzyme is now feasible using time-resolved Laue diffraction and freeze-trapping, building on the structural work described here as the foundation.
Assuntos
Hidroximetilbilano Sintase/química , Selenometionina/química , Sítios de Ligação , Cristalografia por Raios X , Coleta de Dados , Modelos Moleculares , Conformação Proteica , Proteínas Recombinantes/química , Selênio/química , TemperaturaRESUMO
BACKGROUND: . Sulfatases catalyze the hydrolysis of sulfuric acid esters from a wide variety of substrates including glycosaminoglycans, glycolipids and steroids. There is sufficient common sequence similarity within the class of sulfatase enzymes to indicate that they have a common structure. Deficiencies of specific lysosomal sulfatases that are involved in the degradation of glycosamino-glycans lead to rare inherited clinical disorders termed mucopolysaccharidoses. In sufferers of multiple sulfatase deficiency, all sulfatases are inactive because an essential post-translational modification of a specific active-site cysteine residue to oxo-alanine does not occur. Studies of this disorder have contributed to location and characterization of the sulfatase active site. To understand the catalytic mechanism of sulfatases, and ultimately the determinants of their substrate specificities, we have determined the structure of N-acetylgalactosamine-4-sulfatase. RESULTS: . The crystal structure of the enzyme has been solved and refined at 2.5 resolution using data recorded at both 123K and 273K. The structure has two domains, the larger of which belongs to the alpha/beta class of proteins and contains the active site. The enzyme active site in the crystals contains several hitherto undescribed features. The active-site cysteine residue, Cys91, is found as the sulfate derivative of the aldehyde species, oxo-alanine. The sulfate is bound to a previously undetected metal ion, which we have identified as calcium. The structure of a vanadate-inhibited form of the enzyme has also been solved, and this structure shows that vanadate has replaced sulfate in the active site and that the vanadate is covalently linked to the protein. Preliminary data is presented for crystals soaked in the monosaccharide N-acetylgalactosamine, the structure of which forms a product complex of the enzyme. CONCLUSIONS: . The structure of N-acetylgalactosamine-4-sulfatase reveals that residues conserved amongst the sulfatase family are involved in stabilizing the calcium ion and the sulfate ester in the active site. This suggests an archetypal fold for the family of sulfatases. A catalytic role is proposed for the post-translationally modified highly conserved cysteine residue. Despite a lack of any previously detectable sequence similarity to any protein of known structure, the large sulfatase domain that contains the active site closely resembles that of alkaline phosphatase: the calcium ion in sulfatase superposes on one of the zinc ions in alkaline phosphatase and the sulfate ester of Cys91 superposes on the phosphate ion found in the active site of alkaline phosphatase.
Assuntos
Condro-4-Sulfatase/química , Lisossomos/enzimologia , Conformação Proteica , Fosfatase Alcalina/química , Sequência de Aminoácidos , Animais , Sítios de Ligação , Células CHO , Condro-4-Sulfatase/antagonistas & inibidores , Condro-4-Sulfatase/deficiência , Condro-4-Sulfatase/genética , Sequência Consenso , Cricetinae , Cristalografia por Raios X , Inibidores Enzimáticos/metabolismo , Inibidores Enzimáticos/farmacologia , Glicosilação , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Mucopolissacaridose VI/enzimologia , Mucopolissacaridose VI/genética , Família Multigênica , Mutação Puntual , Proteínas Recombinantes/antagonistas & inibidores , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Vanadatos/metabolismo , Vanadatos/farmacologiaAssuntos
Cristalografia/métodos , Proteínas/química , Proteínas/isolamento & purificação , Biofísica/tendências , Concanavalina A/química , Concanavalina A/isolamento & purificação , Cristalização , Cristalografia por Raios X , Eletroquímica , Interferometria , Luz , Substâncias Macromoleculares , Microscopia de Força Atômica , Microscopia de Vídeo , Nêutrons , Espalhamento de Radiação , Solubilidade , Síncrotrons , Ausência de PesoRESUMO
The crystal structure of a brominated oligonucleotide d(CGCG(Br)CG), chemical formula C(114)N(48)O(68)P(10)Br(2), has been analysed by multiwavelength anomalous dispersion (MAD) methods. The oligonucleotide crystallizes in space group P2(1)2(1)2(1) with a = 17.97, b = 30.98, c = 44.85 A, alpha = beta = gamma 90 degrees . Data to a resolution of 1.65 A were collected at four wavelengths about the K-absorption edge of the bromine atom (lambda(1) = 0.9323 A, a reference wavelength at the long-wavelength side of the edge; lambda(2) = 0.9192 A, at the absorption-edge inflection point; lambda(3) = 0.9185 A, at the ;white line' absorption maximum; lambda(4) = 0.8983 A, a reference wavelength at the short-wavelength side) using synchrotron radiation at Station PX9.5, SRS, Daresbury. Multiwavelength data could be collected on a single-crystal as the sample was radiation stable. Anomalous and dispersive Patterson maps were readily interpretable to give the bromine anomalous scatterer positions. Phase calculations to 1.65 A, resolution, using all four wavelengths, gave a figure of merit of 0.825 for 2454 reflections. The electron-density map was readily interpretable showing excellent connectivity for the sugar/phosphate backbone and each base was easily characterized. The two nucleotide strands paired up as expected in an antiparallel Watson-Crick-type manner. The structure was refined to 1.65 A using all the data (R-factor = 17.0% based on 3151 reflections, with a data-to-parameter ratio of 2.6). In addition to the four-wavelength analysis, a variety of other phasing strategies, and the associated quality of the resulting electron-density maps, were compared. These included use of either of the reference wavelength data sets in the two possible three-wavelength phasing combinations to assess their relative effectiveness. Moreover, the time dependence upon measuring the Bijvoet differences and its effect upon phasing was also investigated. Finally, the use of only two wavelengths, including Friedel pairs, is demonstrated (the theoretical minimum case); this is of particular interest when considering overall beam time needs and is clearly a feasible experimental strategy, as shown here.
RESUMO
The solution of the cubic crystal form (a = 167.8 A) of concanavalin A complexed with the monosaccharide methyl alpha-D-glucopyranoside is described. The space group has been determined as I2(1)3 rather than I23. The use of cadmium to replace cobalt at the transition metal-ion binding site and to replace calcium at its binding site proved to be crucial to the successful solution of the crystal structure. The relatively small isomorphous signals of 21 e(-) for the replacement of cobalt and 28 e(-) for the replacement of calcium, yielded interpretable difference Patterson maps. The electron-density map calculated in space group I2(1)3 at 5.4 A resolution, based on phases derived from single- and double-substituted cadmium differences, revealed a classical concanavalin A tetramer of 222 point symmetry, as seen in all the known crystal structures of concanavalin A. Rigid-body refinement at 3.6 A using the refined coordinates of saccharide-free concanavalin A converged to an R factor of 27.4%. A molecular-replacement analysis, consistent with this crystal structure, and initial experiences in the incorrect space group I23 are described as these also prove to be instructive.
RESUMO
We have obtained two additional crystal forms of the metal-dependent class II fructose-1,6-bisphosphate aldolase from Escherichia coli. Crystals in the shape of elongated plates have unit-cell dimensions a = 73.4, b = 120.0, c = 190.1 A, orthorhombic space group P2(1)2(1)2(1). Monoclinic prisms have unit-cell dimensions a = 67.7, b = 104.3, c = 52.8 A, beta = 105 degrees, space group P2(1). Diffraction to slightly better than 3.0 A, has been observed for both forms using in-house and synchrotron facilities. These crystal forms may aid the structure solution of this enzyme by presenting additional forms for heavy-atom derivatization. These forms have multiple copies of the enzyme in the asymmetric unit and averaging methods might also be useful in the analysis.
RESUMO
BACKGROUND: The flexibility of DNA enables it to adopt three interconvertible types of duplex termed the A-, B- and Z-forms. It can also produce hairpin loops, triplex structures and guanine-rich quadruplex structures. Conformational flexibility assists in the tight packaging of DNA, for example in chromosomes. This is important given the large quantity of genetic information that must be packaged efficiently. Moreover, the ability of DNA to specifically self-associate or interact with complementary sequences is fundamental to many biological processes. Structural studies provide information about DNA conformation and DNA-DNA interactions and suggest features that might be relevant to how the molecule performs its biological role. RESULTS: We have characterized the structure of a synthetic heptanucleotide that folds into a novel loop structure. The loop is stabilized by association with a cation, by intra-strand hydrogen bonds between guanine and cytosine that are distinct from the normal Watson-Crick hydrogen bonds, and by van der Waals interactions. Two loops associate through the formation of four G.C pairs that exhibit pronounced base-stacking interactions. The formation of a symmetric A.A base pair further stabilizes loop dimerization. Stacking of the A.A pair on a symmetry-related A.A pairing assists the formation of a four-stranded assembly. A T.T pairing is also observed between symmetry-related loops. CONCLUSIONS: This analysis provides a rare example of an experimentally determined non-duplex DNA structure. It provides conformational detail relevant to the tight packaging or folding of a DNA strand and illustrates how a cation might modulate phosphate-phosphate repulsion in a tightly packed structure. The observation of base quartets involving G.C base pairs suggests a further structure to be considered in DNA-DNA interactions. The structure also provides detailed geometries for A.A and T.T base pairs.
Assuntos
DNA/ultraestrutura , Modelos Moleculares , Conformação de Ácido Nucleico , Composição de Bases , Sequência de Bases , Cristalização , Ligação de Hidrogênio , Dados de Sequência MolecularRESUMO
The three-dimensional structure of the complex between methyl alpha-D-mannopyranoside and concanavalin A has been refined at 2.0 A resolution. Diffraction data were recorded from a single crystal (space group P2(1)2(1)2(1), a = 123.7, b = 128.6, c = 67.2 A) using synchrotron radiation at a wavelength of 1.488 A. The final model has good geometry and an R factor of 19.9% for 58 871 reflections (82% complete), within the resolution limits of 8 to 2 A, with F > 1.0sigma(F). The asymmetric unit contains four protein subunits arranged as a dimer of dimers with approximate 222 point symmetry. Each monomer binds one saccharide molecule. Each sugar is bound to the protein by hydrogen bonds and van der Waals contacts. Although the four subunits are not crystallographically equivalent, the protein-saccharide interactions are nearly identical in each of the four binding sites. The differences that do occur between the four sites are in the structure of the water network which surrounds each saccharide; these networks are involved in crystal packing. The structure of the complex is compared with a refined saccharide-free concanavalin A structure. The saccharide-free structure is composed of crystallographically identical subunits, again assembled as a dimer of dimers, but with exact 222 symmetry. In the saccharide complex the tetramer association is different in that the monomers tend to separate resulting in fewer intersubunit interactions. The average temperature factor of the mannoside complex is considerably higher than that of the saccharide-free protein. The binding site in the saccharide-free structure is occupied by three ordered water molecules and the side chain of Asp71 from a neighbouring molecule in the crystal. These occupy positions similar to those of the four saccharide hydroxyls which are hydrogen bonded to the site. Superposition of the saccharide-binding site from each structure shows that the major changes on binding involve expulsion of these ordered solvents and the reorientation of the side chain of Tyrl00. Overall the surface accessibility of the saccharide decreases from 370 to 100 A(2) when it binds to the protein. This work builds upon the earlier studies of Derewenda et al. [Derewenda, Yariv, Helliwell, Kalb (Gilboa), Dodson, Papiz, Wan & Campbell (1989). EMBO J. 8, 2198-2193] at 2.9 A resolution, which was the first detailed study of lectin-saccharide interactions.
RESUMO
The molecular structures of cobalt- and nickel-substituted concanavalin A have been refined at 1.6 and 2.0 A resolution, respectively. Both metal derivatives crystallize in space group I222 with approximate cell dimensions a = 89, b = 87 and c = 63 A and one monomer in the asymmetric unit. The final R factor for Co-substituted concanavalin A is 17.8% for 29 211 reflections with F > 1.0sigma(F) between 8.0 and 1.6 A. For Ni-substituted concanavalin A the final R factor is 15.9% for 16 128 reflections with F > 1.0sigma(F) between 8.0 and 2.0 A resolution. Both structures contain a transition-metal binding site and a calcium-binding site but, unlike Cd-substituted concanavalin A, do not have a third metal-binding site. The Co-substituted concanavalin A structure diffracts to the highest resolution of any concanavalin A structure reported to date. A comparison of the structures of Ni-, Co-, Cd-substituted and native concanavalin A gives an indication of coordinate errors, which is a useful baseline for comparisons with saccharide complexes of concanavalin A described in other work. We also give a detailed account of multiple conformations which were found for five side-chain residues.
RESUMO
The X-ray crystal structure of the enzyme trypanothione reductase, isolated from the trypanosomatid organism Crithidia fasciculata, has been solved by molecular replacement. The search model was the crystal structure of human glutathione reductase that shares approximately 40% sequence identity. The trypanosomal enzyme crystallizes in the tetragonal space group P4(1) with unit cell lengths of a = 128.9 A and c = 92.3 A. The asymmetric unit consists of a homodimer of approximate molecular mass 108 kDa. We present the structural detail of the active site as derived from the crystallographic model obtained at an intermediate stage of the analysis using diffraction data to 2.8 A resolution with an R-factor of 23.2%. This model has root-mean-square deviations from ideal geometry of 0.026 A for bond lengths and 4.7 degrees for bond angles. The trypanosomid enzyme assumes a similar biological function to glutathione reductase and, although similar in topology to human glutathione reductase, has an enlarged active site and a number of amino acid differences, steric and electrostatic, which allows it to process only the unique substrate trypanothione and not glutathione. This protein represents a prime target for chemotherapy of several debilitating tropical diseases caused by protozoan parasites belonging to the genera Trypanosoma and Leishmania. The structural differences between the parasite and host enzymes and their substrates thus provides a rational basis for the design of new drugs active against trypanosomes. In addition, our model explains the results of site-directed mutagenesis experiments, carried out on recombinant trypanothione reductase and glutathione reductases, designed by consideration of the crystal structure of human glutathione reductase.
Assuntos
Glutationa/análogos & derivados , NADH NADPH Oxirredutases/ultraestrutura , Espermidina/análogos & derivados , Sequência de Aminoácidos , Animais , Sítios de Ligação , Crithidia fasciculata/enzimologia , Cristalografia , Desenho de Fármacos , Glutationa/metabolismo , Glutationa Redutase/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Alinhamento de Sequência , Especificidade da Espécie , Espermidina/metabolismo , Relação Estrutura-Atividade , Especificidade por Substrato , Trypanosoma/enzimologia , Difração de Raios XRESUMO
We have reproducibly crystallized the metal-dependent Class II fructose-1,6-bisphosphate aldolase from Escherichia coli. Crystals in the shape of truncated hexagonal bipyramids have unit cell dimensions of a = b = 78.4 A, c = 290.6 A and are suitable for a detailed structural analysis. The space group has been identified as P6(1)22 or enantiomorph. Data sets to approximately 2.9 A resolution have been recorded using both the Rigaku R-AXIS IIc image plate area detector coupled to a copper target rotating anode X-ray source and using the MAR image plate systems with synchrotron radiation at the EMBL outstation DESY in Hamburg, and at S.R.S. Daresbury. Diffraction beyond 2.5 A has been observed when large freshly grown crystals are used with the synchrotron beam. A data set to this resolution has been collected. Several putative heavy-atom derivative data sets have also been measured using synchrotron radiation facilities and analysis of these data sets is in progress.
Assuntos
Frutose-Bifosfato Aldolase/química , Isoenzimas/química , Cristalização , Escherichia coli/enzimologia , Frutose-Bifosfato Aldolase/isolamento & purificação , Isoenzimas/isolamento & purificação , Conformação Proteica , Difração de Raios X/métodosRESUMO
We have obtained well-ordered single crystals of the flavoenzyme trypanothione reductase from Crithidia fasciculata. The crystals are tetragonal rods with unit cell dimensions a = 128.6 A, c = 92.5 A. The diffraction pattern corresponds to a primitive lattice. Laue class 4/m. Diffraction to better than 2.4 A has been recorded at the Daresbury Synchrotron. The accurate elucidation of the three-dimensional structure of this enzyme is required to support the rational design of compounds active against a variety of tropical diseases caused by trypanosomal parasites.