RESUMO
Collagenase is a zinc-dependent endoproteinase and is a member of the matrix metalloproteinase (MMP) family of enzymes. The MMPs participate in connective tissue remodeling events and aberrant regulation has been associated with several pathologies. The 2.4 angstrom resolution structure of the inhibited enzyme revealed that, in addition to the catalytic zinc, there is a second zinc ion and a calcium ion which play a major role in stabilizing the tertiary structure of collagenase. Despite scant sequence homology, collagenase shares structural homology with two other endoproteinases, bacterial thermolysin and crayfish astacin. The detailed description of protein-inhibitor interactions present in the structure will aid in the design of compounds that selectively inhibit individual members of the MMP family. Such inhibitors will be useful in examining the function of MMPs in pathological processes.
Assuntos
Colagenases/química , Sequência de Aminoácidos , Sítios de Ligação , Cálcio/metabolismo , Colagenases/metabolismo , Gráficos por Computador , Cristalografia por Raios X , Humanos , Ligação de Hidrogênio , Metaloproteinase 8 da Matriz , Inibidores de Metaloproteinases de Matriz , Metaloendopeptidases/química , Modelos Moleculares , Dados de Sequência Molecular , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Termolisina/química , Zinco/metabolismoRESUMO
The combined use of a rapid virtual screen of a small fragment library together with a single point enzyme assay has been used for the discovery of novel PNP inhibitors. The availability of readily soakable crystals of bovine PNP has allowed the approach to be experimentally validated by determining the crystal structure of one of the inhibitor-PNP complexes. Comparison of the experimentally determined binding mode with that predicted by the virtual screening shows them to be similar. This represents a starting point for the growth of the ligand into a higher affinity inhibitor.
Assuntos
Inibidores Enzimáticos/química , Purina-Núcleosídeo Fosforilase/antagonistas & inibidores , Purina-Núcleosídeo Fosforilase/química , Purinas/química , Baço/enzimologia , Animais , Bovinos , Cristalografia por Raios X , Avaliação Pré-Clínica de Medicamentos , Inibidores Enzimáticos/farmacologia , Conformação Proteica , Purinas/farmacologiaRESUMO
Lipase from Pseudomonas glumae has been purified and crystallized in two forms, using the hanging drop method of vapour diffusion at 4 degrees C and 15 degrees C. Both forms grew at pH 9.0 from 0.1 M-Tris buffer in the presence of 10% (v/v) acetone. Form 1 was crystallized from 27 to 29% polyethylene glycol in the presence of less than 0.5% (v/v) n-dodecyl-beta-D-glucopyranoside. Form 2 was grown from 17 to 19% ammonium sulphate in the presence of 1% n-octyl-beta-D-glucopyranoside. Form 1 is orthorhombic with space group P2(1)2(1)2(1), and cell dimensions of a = 158.1 A, b = 158.6 A, c = 63.4 A, Form 2 is tetragonal with space group P4(1)2(1)2 (or P4(3)2(1)2) and cell dimensions of a = 89.3 A, c = 180.4 A. Form 1 probably has four molecules per asymmetric unit and diffracts to at least 2.5 A. Form 2 has two molecules per asymmetric unit and diffracts to at least 3.0 A.
Assuntos
Lipase/química , Pseudomonas/enzimologia , Cristalização , Lipase/isolamento & purificação , Lipase/metabolismo , Difração de Raios XRESUMO
The chemotactic cytokine RANTES (Regulated on Activation, Normal T-cell Expressed and Secreted) is a potent chemoattractant and activator of a number of leukocytes, with a molecular mass of 8 kDa. Crystals of this protein have been grown from 100 mM sodium acetate buffer (pH 4.6) containing 200 mM magnesium acetate, with 20% (w/v) PEG 4000 and 6% (v/v) glycerol. The crystals grow as thick rods, which diffract to at least 1.8 A resolution on a rotating anode X-ray source. The crystals belong to space group p2(1)2(1)2(1) with unit cell dimensions a = 95.14 A, b = 57.58 A and c = 24.01 A with alpha = beta = gamma = 90 degrees. The asymmetric unit contains two molecules of the RANTES monomer, with a VM of 2.0 A(3)/Da.
Assuntos
Linfocinas/química , Quimiocina CCL5 , Humanos , Proteínas Recombinantes/química , Difração de Raios XRESUMO
Crystals of recombinant phosphomannose isomerase from Candida albicans have been obtained in a form suitable for X-ray diffraction analysis. The enzyme plays a key role in the biosynthesis of the mannan component of the fungal cell wall. It crystallizes in monoclinic space group C2, with cell dimensions a = 124.9 A, b = 52.9 A, c = 85.9 A and beta = 127.4 degrees. The crystals diffract to Bragg spacings beyond 1.7 A, native data have been collected to 2.4 A and a search for heavy-metal derivatives is in progress. The asymmetric unit contains one molecule of the enzyme (M(r) approximately 49,000) with a Vm of 2.3 A3/Da.
Assuntos
Candida albicans/enzimologia , Manose-6-Fosfato Isomerase/química , Cristalização , Cristalografia por Raios X , Proteínas Recombinantes/químicaRESUMO
The structure of mucor pusillus pepsin (EC 3.4.23.6), the aspartic proteinase from Mucor pusillus, has been refined to a crystallographic R-factor of 16.2% at 2.0 A resolution. The positions of 2638 protein atoms, 221 solvent atoms and a sulphate ion have been determined with an estimated root-mean-square (r.m.s.) error of 0.15 to 0.20 A. In the final model, the r.m.s. deviation from ideality for bond distances is 0.022 A, and for angle distances it is 0.050 A. Comparison of the overall three-dimensional structure with other aspartic proteinases shows that mucor pusillus pepsin is as distant from the other fungal enzymes as it is from those of mammalian origin. Analysis of a rigid body shift of residues 190 to 302 shows that mucor pusillus pepsin displays one of the largest shifts relative to other aspartic proteinases (14.4 degrees relative to endothiapepsin) and that changes have occurred at the interface between the two rigid bodies to accommodate this large shift. A new sequence alignment has been obtained on the basis of the three-dimensional structure, enabling the positions of large insertions to be identified. Analysis of secondary structure shows the beta-sheet to be well conserved whereas alpha-helical elements are more variable. A new alpha-helix hN4 is formed by a six-residue insertion between positions 131 and 132. Most insertions occur in loop regions: -5 to 1 (five residues relative to porcine pepsin): 115 to 116 (six residues); 186 to 187 (four residues); 263 to 264 (seven residues); 278 to 279 (four residues); and 326 to 332 (six residues). The active site residues are highly conserved in mucor pusillus pepsin; r.m.s. difference with rhizopuspepsin is 0.37 A for 25 C alpha atom pairs. However, residue 303, which is generally conserved as an aspartate, is changed to an asparagine in mucor pusillus pepsin, possibly influencing pH optimum. Substantial changes have occurred in the substrate binding cleft in the region of S1 and S3 due to the insertion between 115 and 116 and the rearrangement of loop 9-13. Residue Asn219 necessitates a shift in position of substrate main-chain atoms to maintain hydrogen bonding pattern. Invariant residues Asp11 and Tyr14 have undergone a major change in conformation apparently due to localized changes in molecular structure. Both these residues have been implicated in zymogen stability and activation.
Assuntos
Ácido Aspártico Endopeptidases/ultraestrutura , Proteínas Fúngicas/ultraestrutura , Mucor/enzimologia , Pepsina A/ultraestrutura , Sequência de Aminoácidos , Sítios de Ligação , Cristalografia , Ativação Enzimática , Ligação de Hidrogênio , Modelos Moleculares , Dados de Sequência Molecular , Precursores de Proteínas/ultraestrutura , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Alinhamento de Sequência , Solventes , Difração de Raios XRESUMO
The family of lipases (triacylglycerol-acyl-hydrolases EC 3.1.1.3) constitutes an interesting class of enzymes because of their ability to interact with lipid-water interfaces, their wide range of substrate specificities, and their potential industrial applications. Here we report the first crystal structure of a bacterial lipase, from Pseudomonas glumae. The structure is formed from three domains, the largest of which contains a subset of the alpha/beta hydrolase fold and a calcium site. Asp263, the acidic residue in the catalytic triad, has previously been mutated into an alanine with only a modest reduction in activity.
Assuntos
Ácido Aspártico/análise , Lipase/química , Pseudomonas/enzimologia , Sequência de Aminoácidos , Catálise , Cristalização , Dados de Sequência Molecular , Conformação ProteicaRESUMO
The first paper in this series (see previous article) described structure-activity studies of carboxamide analogues of zanamivir binding to influenza virus sialidase types A and B and showed that inhibitory activity of these compounds was much greater against influenza A enzyme. To understand the large differences in affinities, a number of protein-ligand complexes have been investigated using crystallography and molecular dynamics. The crystallographic studies show that the binding of ligands containing tertiary amide groups is accompanied by the formation of an intramolecular planar salt bridge between two amino acid residues in the active site of the enzyme. It is proposed that the unexpected strong binding of these inhibitors is a result of the burial of hydrophobic surface area and salt-bridge formation in an environment of low dielectric. In sialidase from type A virus, binding of the carboxamide moeity and salt-bridge formation have only a minor effect on the positions of the surrounding residues, whereas in type B enzyme, significant distortion of the protein is observed. The results suggest that the decreased affinity in enzyme from influenza B is directly correlated with the small changes that occur in the amino acid residue interactions accompanying ligand binding. Molecular dynamics calculations have shown that the tendency for salt-bridge formation is greater in influenza A sialidase than influenza B sialidase and that this tendency is a useful descriptor for the prediction of inhibitor potency.
Assuntos
Acetamidas/química , Antivirais/química , Inibidores Enzimáticos/química , Vírus da Influenza A/enzimologia , Vírus da Influenza B/enzimologia , Neuraminidase/química , Piranos/química , Ácidos Siálicos/química , Acetamidas/metabolismo , Acetamidas/farmacologia , Antivirais/metabolismo , Antivirais/farmacologia , Sítios de Ligação , Cristalografia por Raios X , Inibidores Enzimáticos/metabolismo , Inibidores Enzimáticos/farmacologia , Guanidinas , Modelos Moleculares , Neuraminidase/antagonistas & inibidores , Neuraminidase/metabolismo , Conformação Proteica , Piranos/metabolismo , Piranos/farmacologia , Ácidos Siálicos/metabolismo , Ácidos Siálicos/farmacologia , ZanamivirAssuntos
Colagenases/química , Conformação Proteica , Sequência de Aminoácidos , Sequência de Bases , Sítios de Ligação , Humanos , Ligação de Hidrogênio , Metaloproteinase 8 da Matriz , Inibidores de Metaloproteinases de Matriz , Metaloendopeptidases/antagonistas & inibidores , Modelos Estruturais , Dados de Sequência Molecular , Oligopeptídeos , Proteínas Recombinantes/antagonistas & inibidores , Proteínas Recombinantes/químicaRESUMO
The lipase produced by Pseudomonas glumae is monomeric in the crystalline state and has a serine protease-like catalytic triad; Ser87-His285-Asp263. The largest domain of the protein resembles closely a subset of the frequently observed alpha/beta-hydrolase fold and contains a well-defined calcium site. This paper describes structural analysis of this protein, focusing on (i) structural comparison with the lipase from Geotrichum candidum, (ii) the probable nature of the conformational change involved in substrate binding and (iii) structural variations amongst the family of Pseudomonas lipases. This analysis reveals similarities between P. glumae lipase and G. candidum lipase involving secondary structural elements of the hydrolase core and the loops carrying the catalytic serine and histidine residues. A possible functional equivalence has also been identified between parts of the two molecules thought to be involved in a conformational change. In addition, determination of the structure of P. glumae lipase has allowed rationalization of previously reported protein engineering experiments, which succeeded in improving the stability of the enzyme with respect to proteolysis.
Assuntos
Proteínas de Bactérias/química , Hidrolases de Éster Carboxílico/química , Pseudomonas/enzimologia , Sequência de Aminoácidos , Cristalografia por Raios X , Geotrichum/enzimologia , Dados de Sequência Molecular , Conformação ProteicaRESUMO
The binding modes of four active site-directed, acylating inhibitors of human alpha-thrombin have been determined using X-ray crystallography. These inhibitors (GR157368, GR166081, GR167088, and GR179849) are representatives of a series utilizing a novel 5, 5-trans-lactone template to specifically acylate Ser195 of thrombin, resulting in an acyl complex. In each case the crystal structure of the complex reveals a binding mode which is consistent with the formation of a covalent bond between the ring-opened lactone of the inhibitor and residue Ser195. Improvements in potency and selectivity of these inhibitors for thrombin are rationalized on the basis of the observed protein/inhibitor interactions identified in these complexes. Occupation of the thrombin S2 and S3 pockets is shown to be directly correlated with improved binding and a degree of selectivity. The binding mode of GR179849 to thrombin is compared with the thrombin/PPACK complex [Bode, W., Turk, D., and Karshikov, A. (1992) Protein Sci. 1, 426-471] as this represents the archetypal binding mode for a thrombin inhibitor. This series of crystal structures is the first to be reported of synthetic, nonpeptidic acylating inhibitors bound to thrombin and provides details of the molecular recognition features that resulted in nanomolar potency.
Assuntos
Inibidores de Serina Proteinase/química , Trombina/antagonistas & inibidores , Trombina/química , Sítios de Ligação , Cristalografia por Raios X , Humanos , Lactonas/síntese química , Lactonas/química , Substâncias Macromoleculares , Modelos Moleculares , Inibidores de Serina Proteinase/síntese química , Relação Estrutura-Atividade , Moldes Genéticos , Triterpenos/síntese química , Triterpenos/químicaRESUMO
Phosphomannose isomerase (PMI) is an essential enzyme in the early steps of the protein glycosylation pathway in both prokaryotes and eukaryotes. Lack of the enzyme is lethal for fungal organisms and it is thus a potential fungicidal target. To facilitate the solution of the three-dimensional structure of the enzyme from the pathogen Candida albicans, we have produced the recombinant selenomethionine-labelled enzyme (SeMet-PMI). DL41, a methionine auxotroph Escherichia coli strain, was transformed with a PMI expression plasmid and grown on an enriched selenomethionine-containing medium to high-cell densities. The SeMet-PMI protein has been purified and found by amino acid analysis to have its methionine residues replaced by selenomethionine residues. Electrospray mass spectroscopy showed a major species of 49,063 +/- 10 Da for SeMet-PMI compared to 48,735 +/- 6 Da for the normal recombinant enzyme, accounting for the incorporation of seven selenomethionine residues. SeMet-PMI crystallised isomorphously with the normal PMI protein and the crystals diffract to 0.23 nm. Kinetic characterisation of SeMet-PMI showed that its Km for the substrate mannose-6-phosphate was fourfold higher than that of its methionine-containing counterpart. The inhibition constant for zinc ions was also increased by a similar factor. However, the Vmax was unaltered. These results suggested that one or more methionine residues must be in close proximity to the substrate-binding pocket in the active site, rendering substrate access more difficult compared to the normal enzyme. This hypothesis was confirmed by the finding of four methionine residues lying along one wall of the active site.
Assuntos
Manose-6-Fosfato Isomerase/biossíntese , Proteínas Recombinantes/biossíntese , Selenometionina/metabolismo , Sítios de Ligação , Cinética , Manose-6-Fosfato Isomerase/antagonistas & inibidores , Manose-6-Fosfato Isomerase/química , Zinco/farmacologiaRESUMO
Phosphomannose isomerase (PMI) catalyses the reversible isomerization of fructose-6-phosphate (F6P) and mannose-6-phosphate (M6P). Absence of PMI activity in yeasts causes cell lysis and thus the enzyme is a potential target for inhibition and may be a route to antifungal drugs. The 1.7 A crystal structure of PMI from Candida albicans shows that the enzyme has three distinct domains. The active site lies in the central domain, contains a single essential zinc atom, and forms a deep, open cavity of suitable dimensions to contain M6P or F6P The central domain is flanked by a helical domain on one side and a jelly-roll like domain on the other.
Assuntos
Candida albicans/enzimologia , Manose-6-Fosfato Isomerase/química , Metaloproteínas/química , Zinco/química , Sítios de Ligação , Candida albicans/genética , Simulação por Computador , Cristalografia por Raios X , Manose-6-Fosfato Isomerase/genética , Modelos Moleculares , Conformação Proteica , Estrutura Secundária de Proteína , Proteínas Recombinantes/química , Especificidade da EspécieRESUMO
Inhibitors of the conversion of angiotensinogen to the vasoconstrictor angiotensin II have considerable value as antihypertensive agents. For example, captopril and enalapril are clinically useful as inhibitors of angiotensin-converting enzyme. This has encouraged intense activity in the development of inhibitors of kidney renin, which is a very specific aspartic proteinase catalysing the first and rate limiting step in the conversion of angiotensinogen to angiotensin II. The most effective inhibitors such as H-142 and L-363,564 have used non-hydrolysable analogues of the proposed transition state, and partial sequences of angiotensinogen (Table 1). H-142 is effective in lowering blood pressure in humans but has no significant effect on other aspartic proteinases such as pepsin in the human body (Table 1). At present there are no crystal structures available for human or mouse renins although three-dimensional models demonstrate close structural similarity to other spartic proteinases. We have therefore determined by X-ray analysis the three-dimensional structures of H-142 and L-363,564 complexed with the aspartic proteinase endothiapepsin, which binds these inhibitors with affinities not greatly different from those measured against human renin (Table 1). The structures of these complexes and of that between endothiapepsin and the general aspartic proteinase inhibitor, H-256 (Table 1) define the common hydrogen bonding schemes that allow subtle differences in side-chain orientations and in the positions of the transition state analogues with respect to the active-site aspartates.
Assuntos
Angiotensinogênio/análogos & derivados , Endopeptidases/metabolismo , Oligopeptídeos/metabolismo , Renina/antagonistas & inibidores , Angiotensinogênio/metabolismo , Ácido Aspártico Endopeptidases , Cristalização , Ligação de Hidrogênio , Conformação Molecular , Inibidores de Proteases , Conformação Proteica , Difração de Raios XRESUMO
Humans are one of the few species that produce large amounts of catecholamine sulfates, and they have evolved a specific sulfotransferase, SULT1A3 (M-PST), to catalyze the formation of these conjugates. An orthologous protein has yet to be found in other species. To further our understanding of the molecular basis for the unique substrate selectivity of this enzyme, we have solved the crystal structure of human SULT1A3, complexed with 3'-phosphoadenosine 5'-phosphate (PAP), at 2.5 A resolution and carried out quantitative structure-activity relationship (QSAR) analysis with a series of phenols and catechols. SULT1A3 adopts a similar fold to mouse estrogen sulfotransferase, with a central five-stranded beta-sheet surrounded by alpha-helices. SULT1A3 is a dimer in solution but crystallized with a monomer in the asymmetric unit of the cell, although dimer interfaces were formed by interaction across crystallographic 2-fold axes. QSAR analysis revealed that the enzyme is highly selective for catechols, and catecholamines in particular, and that hydrogen bonding groups and lipophilicity (cLogD) strongly influenced K(m). We also investigated further the role of Glu(146) in SULT1A3 using site-directed mutagenesis and showed that it plays a key role not only in defining selectivity for dopamine but also in preventing many phenolic xenobiotics from binding to the enzyme.
Assuntos
Arilsulfotransferase/química , Alanina/química , Substituição de Aminoácidos , Arilsulfotransferase/metabolismo , Cristalografia por Raios X , Dimerização , Ácido Glutâmico/química , Humanos , Cinética , Modelos Moleculares , Conformação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Relação Estrutura-Atividade , Especificidade por SubstratoRESUMO
X-ray studies on semi-synthetic human insulin have shown that it crystallizes in the rhombohedral space group R3 and is nearly isomorphous with 2 Zn pig insulin. Precession photographs of crystals of human and pig insulins show observable changes in the intensity patterns. Crystallographic analysis and refinement of semi-synthetic human insulin at 1.9 A resolution have shown that its molecular structure is very like that of pig insulin except at the C-terminus of the B chain where the change in sequence occurs. We also report the results of a high resolution crystallographic study of human insulins from different origins. The X-ray diffraction patterns of three non-pancreatic human insulins are indistinguishable from each other and from pancreatic human insulin. Refinement of the structures of the non-pancreatic human insulins has shown that they are identical within the limits of experimental error.
Assuntos
Insulina , Animais , Cristalização , Análise de Fourier , Humanos , Pâncreas/análise , Suínos , Difração de Raios XRESUMO
High-throughput screening of methanolic extracts from the leaves of the plant Lantana camara identified potent inhibitors of human alpha-thrombin, which were shown to be 5,5-trans-fused cyclic lactone euphane triterpenes [O'Neill et al. (1998) J. Nat. Prod. (submitted for publication)]. Proflavin displacement studies showed the inhibitors to bind at the active site of alpha-thrombin and alpha-chymotrypsin. Kinetic analysis of alpha-thrombin showed tight-binding reversible competitive inhibition by both compounds, named GR133487 and GR133686, with respective kon values at pH 8.4 of 1.7 x 10(6) s-1 M-1 and 4.6 x 10(6) s-1 M-1. Electrospray ionization mass spectrometry of thrombin/inhibitor complexes showed the tight-bound species to be covalently attached, suggesting acyl-enzyme formation by reaction of the active-site Ser195 with the trans-lactone carbonyl. X-ray crystal structures of alpha-thrombin/GR133686 (3.0 A resolution) and alpha-thrombin/GR133487 (2.2 A resolution) complexes showed continuous electron density between Ser195 and the ring-opened lactone carbonyl, demonstrating acyl-enzyme formation. Turnover of inhibitor by alpha-thrombin was negligible and mass spectrometry of isolated complexes showed that reversal of inhibition occurs by reformation of the trans-lactone from the acyl-enzyme. The catalytic triad appears undisrupted and the inhibitor carbonyl occupies the oxyanion hole, suggesting the observed lack of turnover is due to exclusion of water for deacylation. The acyl-enzyme inhibitor hydroxyl is properly positioned for nucleophilic attack on the ester carbonyl and therefore relactonization; furthermore, the higher resolution structure of alpha-thrombin/GR133487 shows this hydroxyl to be effectively superimposable with the recently proposed deacylating water for peptide substrate hydrolysis [Wilmouth, R. C., et al. (1997) Nat. Struct.Biol. 4, 456-462], suggesting the alpha-thrombin/GR133487 complex may be a good model for this reaction.