RESUMEN

The 3.35 Šresolution crystal structure of a mutant form of the staphylococcal sphingomyelinase ß toxin in which a conserved hydrophobic ß-hairpin has been deleted is reported. It is shown that this mutation induces domain swapping of a C-terminal ß-strand, leading to the formation of dimers linked by a conformationally flexible hinge region. Eight dimers are seen in the asymmetric unit, exhibiting a broad spectrum of conformations trapped in place by intermolecular contacts within the crystal lattice. Furthermore, the 16 monomers within each asymmetric unit exhibit a remarkable heterogeneity in thermal factors, which can be accounted for by the varying degrees to which each monomer interacts with other molecules in the crystal. This structure provides a unique example of the challenges associated with crystallographic study of flexible proteins.

Asunto(s)

Toxinas Bacterianas/química , Staphylococcus aureus/química , Toxinas Bacterianas/genética , Cristalografía por Rayos X , Modelos Moleculares , Estructura Cuaternaria de Proteína , Estructura Terciaria de Proteína , Staphylococcus aureus/genética

RESUMEN

Biofilms are surface-associated communities of microbes encompassed by an extracellular matrix. It is estimated that 80% of all bacterial infections involve biofilm formation, but the structure and regulation of biofilms are incompletely understood. Extracellular DNA (eDNA) is a major structural component in many biofilms of the pathogenic bacterium Staphylococcus aureus, but its role is enigmatic. Here, we demonstrate that beta toxin, a neutral sphingomyelinase and a virulence factor of S. aureus, forms covalent cross-links to itself in the presence of DNA (we refer to this as biofilm ligase activity, independent of sphingomyelinase activity) producing an insoluble nucleoprotein matrix in vitro. Furthermore, we show that beta toxin strongly stimulates biofilm formation in vivo as demonstrated by a role in causation of infectious endocarditis in a rabbit model. Together, these results suggest that beta toxin cross-linking in the presence of eDNA assists in forming the skeletal framework upon which staphylococcal biofilms are established.

Asunto(s)

Toxinas Bacterianas/metabolismo , Biopelículas/crecimiento & desarrollo , Proteínas Hemolisinas/metabolismo , Nucleoproteínas/metabolismo , Esfingomielina Fosfodiesterasa/metabolismo , Staphylococcus/crecimiento & desarrollo , Animales , Catálisis , ADN Bacteriano , Endocarditis , Conejos , Staphylococcus/patogenicidad

RESUMEN

Fatty acid binding proteins (FABP) have been characterized as facilitating the intracellular solubilization and transport of long-chain fatty acyl carboxylates via noncovalent interactions. More recent work has shown that the adipocyte FABP is also covalently modified in vivo on Cys117 with 4-hydroxy-2-nonenal (4-HNE), a bioactive aldehyde linked to oxidative stress and inflammation. To evaluate 4-HNE binding and modification, the crystal structures of adipocyte FABP covalently and noncovalently bound to 4-HNE have been solved to 1.9 A and 2.3 A resolution, respectively. While the 4-HNE in the noncovalently modified protein is coordinated similarly to a carboxylate of a fatty acid, the covalent form show a novel coordination through a water molecule at the polar end of the lipid. Other defining features between the two structures with 4-HNE and previously solved structures of the protein include a peptide flip between residues Ala36 and Lys37 and the rotation of the side chain of Phe57 into its closed conformation. Representing the first structure of an endogenous target protein covalently modified by 4-HNE, these results define a new class of in vivo ligands for FABPs and extend their physiological substrates to include bioactive aldehydes.

Asunto(s)

Adipocitos/química , Aldehídos/química , Inhibidores de Cisteína Proteinasa/química , Proteínas de Unión a Ácidos Grasos/química , Animales , Cristalografía por Rayos X , Dimerización , Proteínas de Unión a Ácidos Grasos/genética , Proteínas de Unión a Ácidos Grasos/metabolismo , Ligandos , Ratones , Modelos Moleculares , Datos de Secuencia Molecular , Estrés Oxidativo , Conformación Proteica

RESUMEN

Molecular disruption of the lipid carrier AFABP/aP2 in mice results in improved insulin sensitivity and protection from atherosclerosis. Because small molecule inhibitors may be efficacious in defining the mechanism(s) of AFABP/aP2 action, a chemical library was screened and identified 1 (HTS01037) as a pharmacologic ligand capable of displacing the fluorophore 1-anilinonaphthalene 8-sulfonic acid from the lipid binding cavity. The X-ray crystal structure of 1 bound to AFABP/aP2 revealed that the ligand binds at a structurally similar position to a long-chain fatty acid. Similar to AFABP/aP2 knockout mice, 1 inhibits lipolysis in 3T3-L1 adipocytes and reduces LPS-stimulated inflammation in cultured macrophages. 1 acts as an antagonist of the protein-protein interaction between AFABP/aP2 and hormone sensitive lipase but does not activate PPARgamma in macrophage or CV-1 cells. These results identify 1 as an inhibitor of fatty acid binding and a competitive antagonist of protein-protein interactions mediated by AFABP/aP2.

Asunto(s)

Proteínas de Unión a Ácidos Grasos/antagonistas & inhibidores , Compuestos Heterocíclicos con 2 Anillos/farmacología , Inflamación/tratamiento farmacológico , Células 3T3-L1 , Animales , Ácido Butírico , Cristalografía por Rayos X , Evaluación Preclínica de Medicamentos , Compuestos Heterocíclicos con 2 Anillos/química , Inflamación/inducido químicamente , Ligandos , Macrófagos , Ratones , Estructura Molecular , Unión Proteica , Bibliotecas de Moléculas Pequeñas

RESUMEN

C-type natriuretic peptide (CNP) stimulates endochondrial ossification by activating the transmembrane guanylyl cyclase, natriuretic peptide receptor-B (NPR-B). Recently, a spontaneous autosomal recessive mutation that causes severe dwarfism in mice was identified. The mutant, called long bone abnormality (lbab), contains a single point mutation that converts an arginine to a glycine in a conserved coding region of the CNP gene, but how this mutation affects CNP activity has not been reported. Here, we determined that 30-fold to greater than 100-fold more CNP(lbab) was required to activate NPR-B as compared to wild-type CNP in whole cell cGMP elevation and membrane guanylyl cyclase assays. The reduced ability of CNP(lbab) to activate NPR-B was explained, at least in part, by decreased binding since 10-fold more CNP(lbab) than wild-type CNP was required to compete with [125I][Tyr0]CNP for receptor binding. Molecular modeling suggested that the conserved arginine is critical for binding to an equally conserved acidic pocket in NPR-B. These results indicate that reduced binding to and activation of NPR-B causes dwarfism in lbab(-/-) mice.

Asunto(s)

Enanismo/etiología , Péptido Natriurético Tipo-C/fisiología , Receptores del Factor Natriurético Atrial/fisiología , Secuencia de Aminoácidos , Animales , GMP Cíclico/metabolismo , Enanismo/genética , Ratones , Ratones Mutantes , Modelos Moleculares

RESUMEN

Beta toxin is a neutral sphingomyelinase secreted by certain strains of Staphylococcus aureus. This virulence factor lyses erythrocytes in order to evade the host immune system as well as scavenge nutrients. The structure of beta toxin was determined at 2.4-A resolution using crystals that were merohedrally twinned. This structure is similar to that of the sphingomyelinases of Listeria ivanovii and Bacillus cereus. Beta toxin belongs to the DNase I folding superfamily; in addition to sphingomyelinases, the proteins most structurally related to beta toxin include human endonuclease HAP1, Escherichia coli endonuclease III, bovine pancreatic DNase I, and the endonuclease domain of TRAS1 from Bombyx mori. Our biological assays demonstrated for the first time that beta toxin kills proliferating human lymphocytes. Structure-directed active site mutations show that biological activities, including hemolysis and lymphotoxicity, are due to the sphingomyelinase activity of the enzyme.

Asunto(s)

Toxinas Bacterianas/química , Toxinas Bacterianas/metabolismo , Proteínas Hemolisinas/química , Proteínas Hemolisinas/metabolismo , Esfingomielina Fosfodiesterasa/química , Esfingomielina Fosfodiesterasa/metabolismo , Staphylococcus aureus/química , Secuencia de Aminoácidos , Toxinas Bacterianas/genética , Toxinas Bacterianas/farmacología , Sitios de Unión , Proliferación Celular/efectos de los fármacos , Proliferación Celular/efectos de la radiación , Células Cultivadas , Relación Dosis-Respuesta a Droga , Proteínas Hemolisinas/genética , Proteínas Hemolisinas/farmacología , Humanos , Linfocitos/efectos de los fármacos , Modelos Moleculares , Datos de Secuencia Molecular , Conformación Proteica , Esfingomielina Fosfodiesterasa/genética , Esfingomielina Fosfodiesterasa/farmacología , Staphylococcus aureus/genética

RESUMEN

In many bacteria expression of lateral gene transfer and of virulence factors is controlled by cell-cell signalling systems. Molecular interactions of microbial signal molecules with their cognate receptors are not well understood. For the Enterococcus faecalis conjugative plasmid pCF10, the PrgX protein serves as a molecular switch controlling expression of conjugation and virulence genes encoded by the plasmid. The induction state of a pCF10-carrying donor cell is determined by the ratio of two signalling peptides, cCF10 pheromone and iCF10 inhibitor. Recent analysis of PrgX/cCF10 interactions suggests a mechanism for conversion to the induced state. However, the means by which iCF10 peptide antagonizes cCF10 activity is unclear, and it has been suggested that inhibitor peptides block import of pheromone peptides. We now show that both of these peptides interact with the same binding pocket of PrgX, but they differentially alter the conformation of the protein and its oligomerization state, resulting in opposing biological activities.

Asunto(s)

Proteínas Bacterianas/fisiología , Conjugación Genética , Enterococcus faecalis/fisiología , Oligopéptidos/fisiología , Feromonas/fisiología , Señales de Clasificación de Proteína/fisiología , ADN Bacteriano , Enterococcus faecalis/química , Enterococcus faecalis/genética , Regulación Bacteriana de la Expresión Génica , Transferencia de Gen Horizontal , Genes Bacterianos , Operón Lac , Modelos Moleculares , Mutación , Oligopéptidos/antagonistas & inhibidores , Oligopéptidos/genética , Feromonas/antagonistas & inhibidores , Feromonas/genética , Plásmidos , Unión Proteica , Receptores de Péptidos/fisiología

RESUMEN

Many bacterial activities, including expression of virulence factors, horizontal genetic transfer, and production of antibiotics, are controlled by intercellular signaling using small molecules. To date, understanding of the molecular mechanisms of peptide-mediated cell-cell signaling has been limited by a dearth of published information about the molecular structures of the signaling components. Here, we present the molecular structure of PrgX, a DNA- and peptide-binding protein that regulates expression of the conjugative transfer genes of the Enterococcus faecalis plasmid pCF10 in response to an intercellular peptide pheromone signal. Comparison of the structures of PrgX and the PrgX/pheromone complex suggests that pheromone binding destabilizes PrgX tetramers, opening a 70-bp pCF10 DNA loop required for conjugation repression.

Asunto(s)

Conjugación Genética/fisiología , Enterococcus faecalis/química , Enterococcus faecalis/fisiología , Receptores de Feromonas/química , Receptores de Feromonas/metabolismo , Atractivos Sexuales/química , Atractivos Sexuales/metabolismo , Secuencia de Bases , Cristalografía por Rayos X , ADN Bacteriano/química , ADN Bacteriano/metabolismo , Dimerización , Modelos Moleculares , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , Unión Proteica , Estructura Cuaternaria de Proteína

RESUMEN

The structure of a cell surface enzyme from a gram-positive pathogen has been determined to 2-A resolution. Gram-positive pathogens have a thick cell wall to which proteins and carbohydrate are covalently attached. Streptococcal C5a peptidase (SCP), is a highly specific protease and adhesin/invasin. Structural analysis of a 949-residue fragment of the [D130A,S512A] mutant of SCP from group B Streptococcus (S. agalactiae, SCPB) revealed SCPB is composed of five distinct domains. The N-terminal subtilisin-like protease domain has a 134-residue protease-associated domain inserted into a loop between two beta-strands. This domain also contains one of two Arg-Gly-Asp (RGD) sequences found in SCPB. At the C terminus are three fibronectin type III (Fn) domains. The second RGD sequence is located between Fn1 and Fn2. Our analysis suggests that SCP binding to integrins by the RGD motifs may stabilize conformational changes required for substrate binding.

Asunto(s)

Adhesinas Bacterianas/química , Pared Celular/enzimología , Endopeptidasas/química , Streptococcus agalactiae/enzimología , Adhesinas Bacterianas/metabolismo , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Cristalografía por Rayos X , Endopeptidasas/metabolismo , Modelos Moleculares , Unión Proteica , Estructura Cuaternaria de Proteína , Estructura Terciaria de Proteína

RESUMEN

Catechol 1,2-dioxygenase was first studied by Hayaishi and colleagues in 1950. In 1967, catechol 1,2-dioxygenase from Pseudomonas arvilla C-1 (PaCTD) was chosen as a model system for the catecholic intradiol dioxygenases due to its activity, stability and expression level. Here we report the 2.65 A structure of the betabeta isozyme of PaCTD. The structure supports the hypothesis first made by Vetting and Ohlendorf [The 1.8A crystal structure of catechol 1,2-dioxygenase reveals a novel hydrophobic helical zipper as a subunit linker, Struct. Fold. Des. 8 (2000) 429-440.] that the catechol 1,2-dioxygenases are lipid binding proteins. The 5 amino-terminal helices involved in dimerization and forming the lipid binding site are shown to be plastic in their positions and orientations. The sequence differences between the alpha and beta polypeptides are located at the part of the monomers distant from dimerization surface and thus permit the formation of the 3 isozymes (alphaalpha, alphabeta, and betabeta) of PaCTD. The reported inactivation by sulfhydryl-modifying reagents is explained by the structure. The 10-residue Helix F (residues 203-212) is proposed to be central in communicating between the lipid binding site and the active site.

Asunto(s)

Catecol 1,2-Dioxigenasa/química , Pseudomonas/enzimología , Secuencia de Aminoácidos , Sitios de Unión , Dominio Catalítico , Catecol 1,2-Dioxigenasa/genética , Cristalografía por Rayos X , Dimerización , Metabolismo de los Lípidos , Datos de Secuencia Molecular , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Pseudomonas/genética

RESUMEN

The active site Fe(III) of protocatechuate 3,4-dioxygenase (3,4-PCD) from Pseudomonas putida is ligated axially by Tyr447 and His462 and equatorially by Tyr408, His460, and OH(-). Tyr447 and OH(-) are displaced as protocatechuate (3,4-dihydroxybenzoate, PCA) chelates the iron and appear to serve as in situ bases to promote this process. The role(s) of Tyr408 is (are) explored here using mutant enzymes that exhibit less than 0.1% wild-type activity. The X-ray crystal structures of the mutants and their PCA complexes show that the new shorter residues in the 408 position cannot ligate the iron and instead interact with the iron through solvents. Moreover, PCA binds as a monodentate rather than a bidentate ligand, and Tyr447 fails to dissociate. Although the new residues at position 408 do not directly bind to the iron, large changes in the spectroscopic and catalytic properties are noted among the mutant enzymes. Resonance Raman features show that the Fe-O bond of the monodentate 4-hydroxybenzoate (4HB) inhibitor complex is significantly stronger in the mutants than in wild-type 3,4-PCD. Transient kinetic studies show that PCA and 4HB bind to 3,4-PCD in a fast, reversible step followed by a step in which coordination to the metal occurs; the latter process is at least 50-fold slower in the mutant enzymes. It is proposed that, in wild-type 3,4-PCD, the Lewis base strength of Tyr408 lowers the Lewis acidity of the iron to foster the rapid exchange of anionic ligands during the catalytic cycle. Accordingly, the increase in Lewis acidity of the iron caused by substitution of this residue by solvent tends to make the iron substitution inert. Tyr447 cannot be released to allow formation of the usual dianionic PCA chelate complex with the active site iron, and the rate of electrophilic attack by O(2) becomes rate limiting overall. The structures of the PCA complexes of these mutant enzymes show that hydrogen-bonding interactions between the new solvent ligand and the new second-sphere residue in position 408 allow this residue to significantly influence the spectroscopic and kinetic properties of the enzymes.

Asunto(s)

Proteínas Bacterianas/química , Compuestos Férricos/química , Hierro/química , Protocatecuato-3,4-Dioxigenasa/química , Pseudomonas putida/enzimología , Tirosina/química , Sustitución de Aminoácidos/genética , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Sitios de Unión/fisiología , Catálisis , Cristalografía por Rayos X , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/metabolismo , Compuestos Férricos/metabolismo , Histidina/química , Histidina/metabolismo , Hidróxidos/química , Hidróxidos/metabolismo , Hierro/metabolismo , Quelantes del Hierro/química , Quelantes del Hierro/metabolismo , Oxígeno/química , Oxígeno/metabolismo , Parabenos/química , Parabenos/metabolismo , Unión Proteica/fisiología , Estructura Terciaria de Proteína , Protocatecuato-3,4-Dioxigenasa/genética , Protocatecuato-3,4-Dioxigenasa/metabolismo , Pseudomonas putida/genética , Tirosina/genética , Tirosina/metabolismo

RESUMEN

The catechol dioxygenases allow a wide variety of bacteria to use aromatic compounds as carbon sources by catalyzing the key ring-opening step. These enzymes use specifically either catechol or protocatechuate (2,3-dihydroxybenozate) as their substrates; they use a bare metal ion as the sole cofactor. To learn how this family of metalloenzymes functions, a structural analysis of designed and selected mutants of these enzymes has been undertaken. Here we review the results of this analysis on the nonheme ferric iron intradiol dioxygenase protocatechuate 3,4-dioxygenase.

Asunto(s)

Catecoles/metabolismo , Protocatecuato-3,4-Dioxigenasa/química , Acinetobacter/enzimología , Secuencia de Aminoácidos , Cristalografía por Rayos X , Modelos Moleculares , Datos de Secuencia Molecular , Mutación Puntual , Conformación Proteica , Protocatecuato-3,4-Dioxigenasa/genética , Protocatecuato-3,4-Dioxigenasa/metabolismo , Pseudomonas/enzimología , Alineación de Secuencia

RESUMEN

The X-ray crystal structures of homoprotocatechuate 2,3-dioxygenases isolated from Arthrobacter globiformis and Brevibacterium fuscum have been determined to high resolution. These enzymes exhibit 83% sequence identity, yet their activities depend on different transition metals, Mn2+ and Fe2+, respectively. The structures allow the origins of metal ion selectivity and aspects of the molecular mechanism to be examined in detail. The homotetrameric enzymes belong to the type I family of extradiol dioxygenases (vicinal oxygen chelate superfamily); each monomer has four betaalphabetabetabeta modules forming two structurally homologous N-terminal and C-terminal barrel-shaped domains. The active-site metal is located in the C-terminal barrel and is ligated by two equatorial ligands, H214NE1 and E267OE1; one axial ligand, H155NE1; and two to three water molecules. The first and second coordination spheres of these enzymes are virtually identical (root mean square difference over all atoms, 0.19 A), suggesting that the metal selectivity must be due to changes at a significant distance from the metal and/or changes that occur during folding. The substrate (2,3-dihydroxyphenylacetate [HPCA]) chelates the metal asymmetrically at sites trans to the two imidazole ligands and interacts with a unique, mobile C-terminal loop. The loop closes over the bound substrate, presumably to seal the active site as the oxygen activation process commences. An "open" coordination site trans to E267 is the likely binding site for O2. The geometry of the enzyme-substrate complexes suggests that if a transiently formed metal-superoxide complex attacks the substrate without dissociation from the metal, it must do so at the C-3 position. Second-sphere active-site residues that are positioned to interact with the HPCA and/or bound O2 during catalysis are identified and discussed in the context of current mechanistic hypotheses.

Asunto(s)

Dioxigenasas , Hierro/metabolismo , Manganeso/metabolismo , Oxigenasas/química , Secuencia de Aminoácidos , Cristalización , Cristalografía por Rayos X , Dimerización , Modelos Moleculares , Datos de Secuencia Molecular , Oxigenasas/genética , Oxigenasas/metabolismo , Especificidad por Sustrato

RESUMEN

Staphylococcus aureus is an important human pathogen, causing a variety of diseases. Major virulence factors of this organism include staphylococcal enterotoxins (SEs) that cause food poisoning and toxic shock syndrome. Our study identified a novel enterotoxin-like protein that is a member of the new subfamily (group V) of pyrogenic toxin superantigens (PTSAgs) and examined its biochemical and immunobiological properties. The gene encoding the SE-like protein is directly 5' of another recently identified PTSAg, SEK. The SE-like protein had a molecular weight of 26000 and an experimentally determined isoelectric point between 7.5 and 8.0. We demonstrated that the PTSAg had many of the biological activities associated with SEs, including superantigenicity, pyrogenicity, and ability to enhance endotoxin shock, but lacked both lethality in rabbits when administered in subcutaneous miniosmotic pumps and emetic activity in monkeys. Recombinant protein stimulated human CD4 and CD8 T cells in a T cell receptor variable region, beta chain (TCRVbeta) specific manner. T cells bearing TCRVbeta 2, 5.1, and 21.3 were significantly stimulated.

Asunto(s)

Toxinas Bacterianas/química , Pirógenos/química , Staphylococcus aureus/química , Superantígenos/química , Secuencia de Aminoácidos , Toxinas Bacterianas/genética , Toxinas Bacterianas/aislamiento & purificación , Secuencia de Bases , Clonación Molecular , Cartilla de ADN , Humanos , Datos de Secuencia Molecular , Filogenia , Pirógenos/genética , Pirógenos/aislamiento & purificación , Proteínas Recombinantes/química , Staphylococcus aureus/genética , Staphylococcus aureus/patogenicidad , Superantígenos/genética , Superantígenos/aislamiento & purificación , Virulencia

RESUMEN

The pathogenesis of acute rheumatic fever (ARF) is poorly understood. We identified two contiguous bacteriophage genes, designated speL and speM, encoding novel inferred superantigens in the genome sequence of an ARF strain of serotype M18 group A streptococcus (GAS). speL and speM were located at the same genomic site in 33 serotype M18 isolates, and no nucleotide sequence diversity was observed in the 33 strains analyzed. Furthermore, the genes were absent in 13 non-M18 strains tested. These data indicate a recent acquisition event by a distinct clone of serotype M18 GAS. speL and speM were transcribed in vitro and upregulated in the exponential phase of growth. Purified SpeL and SpeM were pyrogenic and mitogenic for rabbit splenocytes and human peripheral blood mononuclear cells in picogram amounts. SpeL preferentially expanded human T cells expressing T-cell receptors Vbeta1, Vbeta5.1, and Vbeta23, and SpeM had specificity for Vbeta1 and Vbeta23 subsets, indicating that both proteins had superantigen activity. SpeL was lethal in two animal models of streptococcal toxic shock, and SpeM was lethal in one model. Serologic studies indicated that ARF patients were exposed to serotype M18 GAS, SpeL, and SpeM. The data demonstrate that SpeL and SpeM are pyrogenic toxin superantigens and suggest that they may participate in the host-pathogen interactions in some ARF patients.

Asunto(s)

Proteínas Bacterianas/inmunología , Brotes de Enfermedades , Fiebre Reumática/epidemiología , Streptococcus pyogenes/inmunología , Superantígenos/inmunología , Enfermedad Aguda , Secuencia de Aminoácidos , Animales , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Humanos , Leucocitos Mononucleares/inmunología , Datos de Secuencia Molecular , Pirógenos/química , Pirógenos/genética , Pirógenos/inmunología , Pirógenos/metabolismo , Conejos , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/metabolismo , Fiebre Reumática/inmunología , Fiebre Reumática/microbiología , Análisis de Secuencia de ADN , Choque Séptico/inmunología , Choque Séptico/mortalidad , Choque Séptico/fisiopatología , Infecciones Estreptocócicas/epidemiología , Infecciones Estreptocócicas/inmunología , Infecciones Estreptocócicas/microbiología , Superantígenos/química , Superantígenos/genética , Superantígenos/metabolismo