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1.
J Mol Evol ; 51(6): 520-31, 2000 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-11116326

RESUMO

Pathogenic bacteria have evolved a wide variety of toxins to invade and attack host organisms. In particular, strains of the bacteria Staphylococcus aureus and Streptococcus pyogenes produce a family of pyrogenic toxin superantigens (PTSAgs) that can cause illness, e.g., toxic shock syndrome, or synergize with a number of other immune system disorders. The PTSAgs are all similar in size and have a conserved two-domain tertiary fold despite minimal amino acid sequence identity. The tertiary structure of PTSAg domain 1 is similar to the immunoglobulin binding motif of streptococcal proteins G and L. PTSAg domain 2 resembles members of the oligosaccharide/oligonucleotide binding fold family that includes the B subunits of the AB(5) heat-labile enterotoxins, cholera toxin, pertussis toxin, and verotoxin. The strong structural homology between the pyrogenic toxins and other bacterial proteins suggests that the PTSAgs evolved through the recombination of two smaller beta-strand motifs.


Assuntos
Toxinas Bacterianas/genética , Evolução Molecular , Staphylococcus aureus/genética , Streptococcus pyogenes/genética , Superantígenos/genética , Motivos de Aminoácidos , Sequência de Aminoácidos , Toxinas Bacterianas/química , Enterotoxinas/química , Enterotoxinas/genética , Dados de Sequência Molecular , Conformação Proteica , Homologia de Sequência de Aminoácidos , Staphylococcus aureus/imunologia , Streptococcus pyogenes/imunologia , Superantígenos/química
2.
Protein Sci ; 9(9): 1847-51, 2000 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-11045630

RESUMO

The streptococcal pyrogenic toxins A, B, and C (SPEA, SPEB, and SPEC) are responsible for the fever, rash, and other toxicities associated with scarlet fever and streptococcal toxic shock syndrome. This role, together with the ubiquity of diseases caused by Streptococcus pyogenes, have prompted structural analyses of SPEA by several groups. Papageorgiou et al. (1999) have recently reported the structure of SPEA crystallized in the absence of zinc. Zinc has been shown to be important in the ability of some staphylococcal and streptococcal toxins to stimulate proliferation of CD4+ T-cells. Since cadmium is more electron dense than zinc and typically binds interchangeably, we grew crystals in the presence of 10 mM CdCl2. Crystals have been obtained in three space groups, and the structure in the P2(1)2(1)2(1) crystal form has been refined to 1.9 A resolution. The structural analysis revealed an identical tetramer as well as a novel tetrahedral cluster of cadmium in all three crystal forms on a disulfide loop encompassing residues 87-98. No cadmium was bound at the site homologous to the zinc site in staphylococcal enterotoxins C (SECs) despite the high structural homology between SPEA and SECs. Subsequent soaking of crystals grown in the presence of cadmium in 10 mM ZnCl2 showed that zinc binds in this site (indicating it can discriminate between zinc and cadmium ions) using the three ligands (Asp77, His106, and His110) homologous to the SECs plus a fourth ligand (Glu33).


Assuntos
Proteínas de Bactérias , Exotoxinas/química , Proteínas de Membrana , Metais/química , Modelos Moleculares , Conformação Proteica
3.
Acta Crystallogr D Biol Crystallogr ; 56(Pt 9): 1187-90, 2000 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-10957642

RESUMO

The head-tail connector of bacteriophage phi29, an oligomer of gene product 10 (gp10), was crystallized into various forms. The most useful of these were an orthorhombic P22(1)2(1) form (unit-cell parameters a = 143.0, b = 157.0, c = 245.2 A), a monoclinic C2 form (a = 160.7, b = 143.6, c = 221.0 A, beta = 97.8 degrees ) and another monoclinic C2 form (a = 177.0, b = 169.1, c = 185.2 A, beta = 114.1 degrees ). Frozen crystals diffracted to about 3.2 A resolution. There is one connector per crystallographic asymmetric unit in each case. Rotation functions show the connector to be a dodecamer. Translation functions readily determined the position of the 12-fold axis in each unit cell. The structure is being determined by 12-fold electron-density averaging within each crystal and by averaging between the various crystal forms.


Assuntos
Proteínas do Capsídeo , Capsídeo/química , Fagos Bacilares/genética , Capsídeo/genética , Capsídeo/isolamento & purificação , Cristalização , Cristalografia por Raios X , Escherichia coli/química , Escherichia coli/genética , Escherichia coli/metabolismo , Estrutura Molecular , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação
4.
Infect Immun ; 68(9): 5011-7, 2000 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-10948118

RESUMO

Streptococcal pyrogenic exotoxins (SPEs) are superantigens that have been implicated in causing streptococcal toxic shock syndrome (STSS). Most notably, SPE serotype A is made by nearly all M-protein serotype 1 and 3 streptococci, the M types most associated with the illness (these strains contain one or more other SPEs, and those proteins are likely also to contribute to disease). We have prepared double-, triple-, and hexa-amino-acid mutants of SPE A by PCR and other mutagenesis procedures. The sites chosen for mutation were solvent-exposed residues thought to be important for T-cell receptor (TCR) or major histocompatibility complex (MHC) class II interaction. These mutants were nonsuperantigenic for human peripheral blood mononuclear cells and rabbit and mouse splenocytes and were nonlethal in two rabbit models of STSS. In addition, these mutants stimulated protective antibody responses. Interestingly, mutants that altered toxin binding to MHC class II were more immunogenic than mutants altering TCR binding. Collectively, these studies indicate that multiple-site mutants of SPE A are toxoids that may have use in protecting against the toxin's effects in STSS.


Assuntos
Proteínas de Bactérias , Exotoxinas/imunologia , Proteínas de Membrana , Choque Séptico/prevenção & controle , Infecções Estreptocócicas/prevenção & controle , Streptococcus pyogenes/imunologia , Superantígenos/imunologia , Toxoides/imunologia , Animais , Antígenos de Histocompatibilidade Classe II/fisiologia , Humanos , Dose Letal Mediana , Camundongos , Camundongos Endogâmicos BALB C , Mutação , Coelhos , Receptores de Antígenos de Linfócitos T/fisiologia
5.
J Immunol ; 165(4): 2306-12, 2000 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-10925320

RESUMO

Streptococcal pyrogenic exotoxin C (SPE C) is a superantigen produced by many strains of Streptococcus pyogenes that (along with streptococcal pyrogenic exotoxin A) is highly associated with streptococcal toxic shock syndrome (STSS) and other invasive streptococcal diseases. Based on the three-dimensional structure of SPE C, solvent-exposed residues predicted to be important for binding to the TCR or the MHC class II molecule, or important for dimerization, were generated. Based on decreased mitogenic activity of various single-site mutants, the double-site mutant Y15A/N38D and the triple-site mutant Y15A/H35A/N38D were constructed and analyzed for superantigenicity, toxicity (lethality), immunogenicity, and the ability to protect against wild-type SPE C-induced STSS. The Y15A/N38D and Y15A/H35A/N38D mutants were nonmitogenic for rabbit splenocytes and human PBMCs and nonlethal in two rabbit models of STSS, yet both mutants were highly immunogenic. Animals vaccinated with the Y15A/N38D or Y15A/H35A/N38D toxoids were protected from challenge with wild-type SPE C. Collectively, these data indicate that the Y15A/N38D and Y15A/H35A/N38D mutants may be useful as toxoid vaccine candidates.


Assuntos
Proteínas de Bactérias , Vacinas Bacterianas/imunologia , Exotoxinas/imunologia , Proteínas de Membrana , Pirogênios/imunologia , Choque Séptico/imunologia , Choque Séptico/prevenção & controle , Streptococcus pyogenes/imunologia , Toxoides/imunologia , Animais , Vacinas Bacterianas/administração & dosagem , Vacinas Bacterianas/síntese química , Vacinas Bacterianas/genética , Células Cultivadas , Dimerização , Modelos Animais de Doenças , Exotoxinas/administração & dosagem , Exotoxinas/síntese química , Exotoxinas/genética , Humanos , Bombas de Infusão Implantáveis , Ativação Linfocitária , Modelos Moleculares , Mutagênese Sítio-Dirigida , Pirogênios/administração & dosagem , Pirogênios/síntese química , Pirogênios/genética , Coelhos , Streptococcus pyogenes/genética , Relação Estrutura-Atividade , Toxoides/administração & dosagem , Toxoides/síntese química , Toxoides/genética , Vacinas Sintéticas/química , Vacinas Sintéticas/genética , Vacinas Sintéticas/imunologia
6.
Biochemistry ; 39(27): 7943-55, 2000 Jul 11.
Artigo em Inglês | MEDLINE | ID: mdl-10891075

RESUMO

The crystal structures of protocatechuate 3,4-dioxygenase from the soil bacteria Acinetobacterstrain ADP1 (Ac 3,4-PCD) have been determined in space group I23 at pH 8.5 and 5.75. In addition, the structures of Ac 3,4-PCD complexed with its substrate 3, 4-dihydroxybenzoic acid (PCA), the inhibitor 4-nitrocatechol (4-NC), or cyanide (CN(-)) have been solved using native phases. The overall tertiary and quaternary structures of Ac 3,4-PCD are similar to those of the same enzyme from Pseudomonas putida[Ohlendorf et al. (1994) J. Mol. Biol. 244, 586-608]. At pH 8.5, the catalytic non-heme Fe(3+) is coordinated by two axial ligands, Tyr447(OH) (147beta) and His460(N)(epsilon)(2) (160beta), and three equatorial ligands, Tyr408(OH) (108beta), His462(N)(epsilon)(2) (162beta), and a hydroxide ion (d(Fe-OH) = 1.91 A) in a distorted bipyramidal geometry. At pH 5.75, difference maps suggest a sulfate binds to the Fe(3+) in an equatorial position and the hydroxide is shifted [d(Fe-OH) = 2.3 A] yielding octahedral geometry for the active site Fe(3+). This change in ligation geometry is concomitant with a shift in the optical absorbance spectrum of the enzyme from lambda(max) = 450 nm to lambda(max) = 520 nm. Binding of substrate or 4-NC to the Fe(3+) is bidentate with the axial ligand Tyr447(OH) (147beta) dissociating. The structure of the 4-NC complex supports the view that resonance delocalization of the positive character of the nitrogen prevents substrate activation. The cyanide complex confirms previous work that protocatechuate 3,4-dioxygenases have three coordination sites available for binding by exogenous substrates. A significant conformational change extending away from the active site is seen in all structures when compared to the native enzyme at pH 8.5. This conformational change is discussed in its relevance to enhancing catalysis in protocatechuate 3,4-dioxygenases.


Assuntos
Acinetobacter/enzimologia , Protocatecoate-3,4-Dioxigenase/química , Cristalização , Modelos Moleculares , Conformação Proteica , Protocatecoate-3,4-Dioxigenase/metabolismo
7.
Infect Immun ; 68(6): 3630-4, 2000 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-10816521

RESUMO

Staphylococcus aureus and Streptococcus pyogenes express pyrogenic toxin superantigens (PTSAgs) that are associated with toxic shock syndrome (TSS) and staphylococcal food poisoning (SFP). Most PTSAgs cause TSS in deep-tissue infections, whereas only TSS toxin 1 (TSST-1) is associated with menstrual, vaginal TSS. In contrast, SFP has been linked only with staphylococcal enterotoxins (SEs). Because of the differential abilities of PTSAgs to cause systemic or localized symptoms in a site-dependent manner, the present study was undertaken to assess the toxins' abilities to cross mucosal barriers. The activity of three PTSAgs when delivered orally, vaginally, or intravenously to rabbits and orally to monkeys was investigated. TSST-1 induced shock via all three routes in rabbits. Although active when administered intravenously, SEC1 and streptococcal pyrogenic exotoxin A (SPEA) did not cause symptoms when administered orally or vaginally. Only SEC1 induced emesis in the monkey feeding assay. TSST-1, albeit less stable than SEC1 and SPEA to pepsin, induced diarrhea in monkeys. Our results may explain the unique association of TSST-1 with menstrual TSS and why SPEA is only rarely associated with TSS after pharyngitis, despite being highly associated with TSS after subcutaneous infections. Finally, our studies indicate that enterotoxicity in SFP is not the result of superantigenicity.


Assuntos
Proteínas de Bactérias , Toxinas Bacterianas/toxicidade , Proteínas de Membrana , Pirogênios/toxicidade , Choque Séptico/etiologia , Intoxicação Alimentar Estafilocócica/etiologia , Infecções Estreptocócicas/etiologia , Superantígenos/toxicidade , Sequência de Aminoácidos , Animais , Enterotoxinas/toxicidade , Exotoxinas/toxicidade , Macaca nemestrina , Modelos Moleculares , Dados de Sequência Molecular , Coelhos , Homologia de Sequência de Aminoácidos
8.
Structure ; 8(4): 429-40, 2000 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-10801478

RESUMO

BACKGROUND: Intradiol dioxygenases catalyze the critical ring-cleavage step in the conversion of catecholate derivatives to citric acid cycle intermediates. Catechol 1,2-dioxygenases (1, 2-CTDs) have a rudimentary design structure - a homodimer with one catalytic non-heme ferric ion per monomer, that is (alphaFe(3+))(2). This is in contrast to the archetypical intradiol dioxygenase protocatechuate 3,4-dioxygenase (3,4-PCD), which forms more diverse oligomers, such as (alphabetaFe(3+))(2-12). RESULTS: The crystal structure of 1,2-CTD from Acinetobacter sp. ADP1 (Ac 1,2-CTD) was solved by single isomorphous replacement and refined to 2.0 A resolution. The structures of the enzyme complexed with catechol and 4-methylcatechol were also determined at resolutions of 1.9 A and 1.8 A, respectively. While the characteristics of the iron ligands are similar, Ac 1,2-CTD differs from 3,4-PCDs in that only one subunit is used to fashion each active-site cavity. In addition, a novel 'helical zipper', consisting of five N-terminal helices from each subunit, forms the molecular dimer axis. Two phospholipids were unexpectedly found to bind within an 8 x 35 A hydrophobic tunnel along this axis. CONCLUSIONS: The helical zipper domain of Ac 1, 2-CTD has no equivalent in other proteins of known structure. Sequence analysis suggests the domain is a common motif in all members of the 1,2-CTD family. Complexes with catechol and 4-methylcatechol are the highest resolution complex structures to date of an intradiol dioxygenase. Furthermore, they confirm several observations seen in 3,4-PCDs, including ligand displacement upon binding exogenous ligands. The structures presented here are the first of a new family of intradiol dioxygenases.


Assuntos
Dioxigenases , Oxigenases/química , Oxigenases/metabolismo , Acinetobacter/enzimologia , Sítios de Ligação , Domínio Catalítico , Catecol 1,2-Dioxigenase , Cristalografia por Raios X , Inibidores Enzimáticos/farmacologia , Cloreto de Mercúrio/farmacologia , Modelos Moleculares , Oxigenases/antagonistas & inibidores , Fosfolipídeos/metabolismo , Conformação Proteica , Protocatecoate-3,4-Dioxigenase/química
9.
Infect Immun ; 68(4): 2366-8, 2000 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-10722646

RESUMO

The staphylococcal exfoliative toxins (ETs) A and B (ETA and ETB) are 27-kDa exotoxins produced by certain strains of Staphylococcus aureus and are the causative agents of staphylococcal scalded-skin syndrome. The crystal structures of the ETs strongly indicate that the proteins are members of the serine protease family of enzymes, although protease activity until now has not yet been conclusively demonstrated. Here, we show that the peptide beta-melanocyte-stimulating hormone (beta-MSH) is cleaved by ETA and that both ETA and ETB are capable of cleaving alpha-MSH. Both toxins exhibit cleavage at specific glutamic acid residues in MSH peptides. Moreover, biologically inactive mutants of ETA were incapable of cleaving beta-MSH.


Assuntos
Enterotoxinas/metabolismo , alfa-MSH/metabolismo , beta-MSH/metabolismo , Sequência de Aminoácidos , Animais , Bovinos , Eletroforese em Gel de Poliacrilamida , Ácido Glutâmico/metabolismo , Coração/microbiologia , Concentração de Íons de Hidrogênio , Dados de Sequência Molecular , Mutação , Serina Endopeptidases/metabolismo , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Síndrome da Pele Escaldada Estafilocócica/metabolismo , Staphylococcus/imunologia , Temperatura , alfa-MSH/genética , beta-MSH/genética
10.
J Immunol ; 164(4): 2207-13, 2000 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-10657676

RESUMO

Exfoliative toxin A (ETA) is known to be a causative agent of staphylococcal scalded skin syndrome (SSSS). Although relatively little is known about exactly how the exfoliative toxins (ETs) cause SSSS, much has been discovered recently that may help elucidate the mechanism(s) by which ETA exhibits activities such as lymphocyte mitogenicity and epidermolytic activity. Here, we have shown that highly purified ETA does have T lymphocyte mitogenic activity in that wild-type ETA induced T cell proliferation whereas several single amino acid mutants lacked significant activity. Neither wild-type ETA nor any single amino acid mutants were proteolytic for a casein substrate, yet esterase activity was detected in wild-type ETA and several mutants, but eliminated in other mutants. A mutation in aa 164 (Asp to Ala) showed a 9-fold increase in esterase activity as well. Finally, we correlated esterase activity with epidermolytic activity. All mutants that lost esterase activity also lost epidermolytic activity. Conversely, mutants that retained esterase activity also retained exfoliative activity, implicating serine protease or serine protease-like activity in the causation of SSSS. Moreover, the mutants that displayed markedly reduced T cell superantigenic activity retained their epidermolytic activity (although some of these mutants required higher doses of toxin to cause disease), which suggests an ancillary role for this activity in SSSS causation.


Assuntos
Exfoliatinas/genética , Superantígenos/genética , Substituição de Aminoácidos/genética , Animais , Sítios de Ligação/genética , Sítios de Ligação/imunologia , Análise Mutacional de DNA , Esterases/genética , Esterases/imunologia , Esterases/metabolismo , Exfoliatinas/química , Exfoliatinas/metabolismo , Ativação Linfocitária , Mitógenos/imunologia , Mutagênese Sítio-Dirigida , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/imunologia , Fragmentos de Peptídeos/metabolismo , Estrutura Secundária de Proteína/genética , Coelhos , Síndrome da Pele Escaldada Estafilocócica/imunologia , Síndrome da Pele Escaldada Estafilocócica/patologia , Especificidade por Substrato/genética , Especificidade por Substrato/imunologia , Superantígenos/química , Superantígenos/metabolismo
11.
J Bacteriol ; 181(20): 6478-87, 1999 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-10515940

RESUMO

Protocatechuate 3,4-dioxygenase is a member of a family of bacterial enzymes that cleave the aromatic rings of their substrates between two adjacent hydroxyl groups, a key reaction in microbial metabolism of varied environmental chemicals. In an appropriate genetic background, it is possible to select for Acinetobacter strains containing spontaneous mutations blocking expression of pcaH or -G, genes encoding the alpha and beta subunits of protocatechuate 3, 4-dioxygenase. The crystal structure of the Acinetobacter oxygenase has been determined, and this knowledge affords us the opportunity to understand how mutations alter function in the enzyme. An earlier investigation had shown that a large fraction of spontaneous mutations inactivating Acinetobacter protocatechuate oxygenase are either insertions or large deletions. Therefore, the prior procedure of mutant selection was modified to isolate Acinetobacter strains in which mutations within pcaH or -G cause a heat-sensitive phenotype. These mutations affected residues distributed throughout the linear amino acid sequences of PcaH and PcaG and impaired the dioxygenase to various degrees. Four of 16 mutants had insertions or deletions in the enzyme ranging in size from 1 to 10 amino acid residues, highlighting areas of the protein where large structural changes can be tolerated. To further understand how protein structure influences function, we isolated strains in which the phenotypes of three different deletion mutations in pcaH or -G were suppressed either by a spontaneous mutation or by a PCR-generated random mutation introduced into the Acinetobacter chromosome by natural transformation. The latter procedure was also used to identify a single amino acid substitution in PcaG that conferred activity towards catechol sufficient for growth with benzoate in a strain in which catechol 1,2-dioxygenase was inactivated.


Assuntos
Acinetobacter/genética , Catecóis/metabolismo , Dioxigenases , Hidroxibenzoatos/metabolismo , Oxigenases/genética , Protocatecoate-3,4-Dioxigenase/genética , Acinetobacter/enzimologia , Sequência de Aminoácidos , Catecol 1,2-Dioxigenase , Reparo do DNA , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese , Oxigenases/metabolismo , Mutação Puntual , Protocatecoate-3,4-Dioxigenase/metabolismo , Análise de Sequência de DNA , Especificidade por Substrato , Supressão Genética , Transformação Bacteriana
12.
Biochemistry ; 38(32): 10239-46, 1999 Aug 10.
Artigo em Inglês | MEDLINE | ID: mdl-10441117

RESUMO

The exfoliative toxins (ETs) cause staphylococcal scalded skin syndrome, a disease characterized by specific separation of layers of the skin. Evidence suggests that the toxins act as serine proteases, though the specific substrate and mode of action are not known for certain. The crystal structure of exfoliative toxin A (ETA) was reported earlier and shown to be similar to that of the chymotrypsin-like serine proteases. Here, we report the 2.4 A resolution crystal structure of the other exfoliative toxin, ETB, which is 40% identical to ETA. The overall structures of ETA and ETB are similar including the positions of key residues within the active site. The structure of ETB supports the previous findings that the ETs are serine proteases that cleave substrates after glutamic acid residues. In this study we also discuss a number of structural differences including a large 14 residue loop insertion which may be a key feature involved in the differing biological properties of the ETs, particularly the pyrogenic and lethal activities of ETB not shared by ETA.


Assuntos
Exfoliatinas/química , Exfoliatinas/metabolismo , Staphylococcus aureus/enzimologia , Superantígenos/química , Superantígenos/metabolismo , Sequência de Aminoácidos , Ânions , Sítios de Ligação , Domínio Catalítico , Cristalografia por Raios X , Exfoliatinas/isolamento & purificação , Glicina/química , Glicina/metabolismo , Antígenos de Histocompatibilidade Classe II/química , Antígenos de Histocompatibilidade Classe II/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Receptores de Antígenos de Linfócitos T/química , Receptores de Antígenos de Linfócitos T/metabolismo , Superantígenos/isolamento & purificação
13.
J Immunol ; 162(8): 4550-9, 1999 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-10201994

RESUMO

Certain strains of Staphylococcus aureus express one or both of two related, but immunologically distinct, exfoliative toxins (ETA and ETB). These toxins induce the symptoms associated with staphylococcal scalded skin syndrome. Both ETs have been shown to stimulate T cell proliferation. Recently, it was reported that ETA is a superantigen that stimulates T cells bearing human Vbeta2 or several murine Vbetas. However, other investigators have proposed that the superantigenicity reported for ETA resulted from contaminants in commercial preparations. This present study addresses those conflicting reports by assessing the biological and immunologic activities of highly purified rETs. ETA and ETB required APCs to induce selective polyclonal expansion of several human Vbetas (huVbetas), although, neither toxin expanded huVbeta2. ETB induced expansion of murine T cells bearing Vbetas 7 and 8, those that have the highest homology to the huVbetas expanded by ETA and ETB. Although flow cytometry of ETB-stimulated T cells matched PCR results, stimulation by ETA reduced percentages of T cells positive for several huVbetas that had been shown to have increased levels of mRNA transcripts. ETA and ETB induced contrasting reactions in vivo. In rabbits, ETB was moderately pyrogenic and enhanced susceptibility to lethal shock, while ETA lacked both activities. Predictions based on comparisons with other superantigens suggest molecular regions potentially involved in receptor binding in the ETA crystal structure and a modeled ETB three-dimensional structure. These results show that ETs are superantigens with unique properties that could account for the discrepancies reported.


Assuntos
Exfoliatinas/imunologia , Superantígenos/imunologia , Animais , Células Cultivadas , Células Clonais , Epitopos de Linfócito T/imunologia , Exfoliatinas/química , Exfoliatinas/toxicidade , Regulação da Expressão Gênica/imunologia , Genes Codificadores da Cadeia beta de Receptores de Linfócitos T/imunologia , Humanos , Imunofenotipagem , Injeções Intravenosas , Ativação Linfocitária , Contagem de Linfócitos , Camundongos , Camundongos Endogâmicos C3H , Modelos Moleculares , Coelhos , Superantígenos/química , Superantígenos/toxicidade , Linfócitos T/imunologia , Linfócitos T/metabolismo
14.
J Mol Biol ; 280(1): 129-36, 1998 Jul 03.
Artigo em Inglês | MEDLINE | ID: mdl-9653036

RESUMO

The structure of the Cro repressor protein from phage lambda has been refined to a crystallographic R-value of 19.3% at 2.3 A resolution. The re fined model supports the structure as originally described in 1981 and provides a basis for comparison with the Cro-operator complex described in the accompanying paper. Changes in structure seen in different crystal forms and modifications of Cro suggest that the individual subunits are somewhat plastic in nature. In addition, the dimer of Cro suggests a high degree of flexibility, which may be important in forming the Cro-DNA complex. The structure of the Cro subunit as determined by NMR agrees reasonably well with that in the crystals (root-mean-square discrepancy of about 2 A for all atoms). There are, however, only a limited number of intersubunit distance constraints and, presumably for this reason, the different NMR models for the dimer vary substantially among themselves (discrepancies of 1.3 to 5.5 A). Because of this variation it is not possible to say whether the range of discrepancies between the X-ray and NMR Cro dimers (2.9 to 7.5 A) represent a significant difference between the X-ray and solution structures. It has previously been proposed that substitutions of Tyr26 in Cro increase thermal stability by the "reverse hydrophobic effect", i.e. by exposing 40% more hydrophobic surface to solvent in the folded form than in the unfolded state. The refined structure, however, suggests that Tyr26 is equally solvent exposed in the folded and unfolded states. The most stabilizing substitution is Tyr26-->Asp and in this case it appears that interaction with an alpha-helix dipole is at least partly responsible for the enhanced stability.


Assuntos
Bacteriófago lambda/química , Proteínas de Ligação a DNA , Estrutura Secundária de Proteína , Proteínas Repressoras/química , Proteínas Repressoras/genética , Cristalografia por Raios X , Calefação , Modelos Moleculares , Mutagênese Sítio-Dirigida , Solventes , Proteínas Virais , Proteínas Virais Reguladoras e Acessórias
15.
Biochemistry ; 37(20): 7194-202, 1998 May 19.
Artigo em Inglês | MEDLINE | ID: mdl-9585531

RESUMO

The three-dimensional structures of five mutants of toxic shock syndrome toxin-1 (TSST-1) have been determined. These mutations are in the long central alpha helix and are useful in mapping portions of TSST-1 involved in superantigenicity and lethality. The T128A, H135A, Q139K, and I140T mutations appear to reduce superantigenicity by altering the properties of the T-cell receptor interaction surface. The Q136A mutation is at a largely buried site and causes a dramatic change in the conformation of the beta7-beta9 loop which covers the back of the central alpha helix. As this mutation has the unique ability to reduce the toxin's lethality in rabbits while retaining its superantigenicity, it raises the possibility that this rear loop mediates the ability of TSST-1 to induce lethality and suggests a route for producing nonlethal toxins for therapeutic development.


Assuntos
Toxinas Bacterianas , Enterotoxinas/química , Enterotoxinas/genética , Mutação , Staphylococcus aureus/imunologia , Superantígenos/toxicidade , Alanina/genética , Animais , Cristalografia por Raios X , Enterotoxinas/toxicidade , Glutamina/genética , Histidina/genética , Humanos , Lisina/genética , Camundongos , Modelos Moleculares , Coelhos , Staphylococcus aureus/química , Staphylococcus aureus/genética , Superantígenos/química , Superantígenos/genética , Treonina/genética
16.
Biochemistry ; 37(8): 2131-44, 1998 Feb 24.
Artigo em Inglês | MEDLINE | ID: mdl-9485360

RESUMO

The essential active site Fe3+ of protocatechuate 3,4-dioxygenase [3, 4-PCD, subunit structure (alphabetaFe3+)12] is bound by axial ligands, Tyr447 (147beta) and His462 (162beta), and equatorial ligands, Tyr408 (108beta), His460 (160beta), and a solvent OH- (Wat827). Recent X-ray crystallographic studies have shown that Tyr447 is dissociated from the Fe3+ in the anaerobic 3,4-PCD complex with protocatechuate (PCA) [Orville, A. M., Lipscomb, J. D., and Ohlendorf, D. H. (1997) Biochemistry 36, 10052-10066]. The importance of Tyr447 to catalysis is investigated here by site-directed mutation of this residue to His (Y447H), the first such mutation reported for an aromatic ring cleavage dioxygenase containing Fe3+. The crystal structure of Y447H (2.1 A resolution, R-factor of 0.181) is essentially unchanged from that of the native enzyme outside of the active site region. The side chain position of His447 is stabilized by a His447(N)delta1-Pro448(O) hydrogen bond, placing the Nepsilon2 atom of His447 out of bonding distance of the iron ( approximately 4.3 A). Wat827 appears to be replaced by a CO32-, thereby retaining the overall charge neutrality and coordination number of the Fe3+ center. Quantitative metal and amino acid analysis shows that Y447H binds Fe3+ in approximately 10 of the 12 active sites of 3,4-PCD, but its kcat is nearly 600-fold lower than that of the native enzyme. Single-turnover kinetic analysis of the Y447H-catalyzed reaction reveals that slow substrate binding accounts for the decreased kcat. Three new kinetically competent intermediates in this process are revealed. Similarly, the product dissociation from Y447H is slow and occurs in two resolved steps, including a previously unreported intermediate. The final E.PCA complex (ES4) and the putative E.product complex (ESO2*) are found to have optical spectra that are indistinguishable from those of the analogous intermediates of the wild-type enzyme cycle, while all of the other observed intermediates have novel spectra. Once the E.S complex is formed, reaction with O2 is fast. These results suggest that dissociation of Tyr447 occurs during turnover of 3,4-PCD and is important in the substrate binding and product release processes. Once Tyr447 is removed from the Fe3+ in the final E.PCA complex by either dissociation or mutagenesis, the O2 attack and insertion steps proceed efficiently, suggesting that Tyr447 does not have a large role in this phase of the reaction. This study demonstrates a novel role for Tyr in a biological system and allows evaluation and refinement of the proposed Fe3+ dioxygenase mechanism.


Assuntos
Protocatecoate-3,4-Dioxigenase/química , Protocatecoate-3,4-Dioxigenase/metabolismo , Sequência de Bases , Sítios de Ligação/genética , Clonagem Molecular , Eletroquímica , Ferro/química , Cinética , Ligantes , Modelos Químicos , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Oligodesoxirribonucleotídeos/genética , Mutação Puntual , Conformação Proteica , Proteus mirabilis/genética , Protocatecoate-3,4-Dioxigenase/genética , Pseudomonas fluorescens/enzimologia , Pseudomonas fluorescens/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Especificidade por Substrato , Tirosina/química
17.
Biochemistry ; 36(38): 11504-13, 1997 Sep 23.
Artigo em Inglês | MEDLINE | ID: mdl-9298971

RESUMO

The crystal structure of the anaerobic complex of Pseudomonas putida protocatechuate 3,4-dioxygenase (3,4-PCD) bound with the alternative substrate, 3,4-dihydroxyphenylacetate (HPCA), is reported at 2.4 A resolution and refined to an R factor of 0.17. Formation of the active site Fe(III).HPCA chelated complex causes the endogenous axial tyrosinate, Tyr447 (147beta), to dissociate from the iron and rotate into an alternative orientation analogous to that previously observed in the anaerobic 3,4-PCD.3,4-dihydroxybenzoate complex (3, 4-PCD.PCA) [Orville, A. M., Lipscomb, J. D., & Ohlendorf, D. H. (1997) Biochemistry 36, 10052-10066]. Two orientations of the aromatic ring of HPCA related by an approximate 180 degrees rotation within the active site are consistent with the electron density. Resonance Raman (rR) spectroscopic data from Brevibacteriumfuscum 3,4-PCD.HPCA complex in solution reveals low frequency rR vibrational bands between 500 and 650 cm-1 as well as a band at approximately 1320 cm-1 which are diagnostic of a HPCA. Fe(III) chelate complex. 18O labeling of HPCA at either the C4 or C3 hydroxyl group unambiguously establishes the vibrational coupling modes associated with the five-membered chelate ring system. Analysis of these data suggests that the Fe(III)-HPCAO4 bond is shorter than the Fe(III)-HPCAO3 bond. This consequently favors the model for the crystal structure in which the C3 phenolic function occupies the Fe3+ ligand site opposite the endogenous ligand Tyr408(Oeta) (108beta). This is essentially the same binding orientation as proposed for PCA in the crystal structure of the anaerobic 3,4-PCD.PCA complex based solely on direct modeling of the 2Fo - Fc electron density and suggests that this is the conformation required for catalysis.


Assuntos
Ácido 3,4-Di-Hidroxifenilacético/química , Compostos Férricos/química , Protocatecoate-3,4-Dioxigenase/química , Anaerobiose , Brevibacterium/enzimologia , Cristalografia por Raios X , Modelos Moleculares , Dados de Sequência Molecular , Pseudomonas putida/enzimologia , Especificidade da Espécie , Análise Espectral Raman
18.
Biochemistry ; 36(33): 10039-51, 1997 Aug 19.
Artigo em Inglês | MEDLINE | ID: mdl-9254599

RESUMO

Protocatechuate 3,4-dioxygenase (3,4-PCD) catalyzes the oxidative ring cleavage of 3,4-dihydroxybenzoate to produce beta-carboxy-cis, cis-muconate. Crystal structures of Pseudomonas putida3,4-PCD [quaternary structure of (alphabetaFe3+)12] complexed with seven competitive inhibitors [3-hydroxyphenylacetate (MHP), 4-hydroxyphenylacetate (PHP), 3-hydroxybenzoate (MHB), 4-hydroxybenzoate (PHB), 3-fluoro-4-hydroxybenzoate (FHB), 3-chloro-4-hydroxybenzoate (CHB), and 3-iodo-4-hydroxybenzoate (IHB)] are reported at 2.0-2.2 A resolution with R-factors of 0. 0.159-0.179. The inhibitors bind in a narrow active site crevasse lined with residues that provide a microenvironment that closely matches the chemical characteristics of the inhibitors. This results in as little as 20% solvent-exposed surface area for the higher-affinity inhibitors (PHB, CHB, and FHB). In uncomplexed 3,4-PCD, the active site Fe3+ is bound at the bottom of the active site crevasse by four endogenous ligands and a solvent molecule (Wat827). The orientations of the endogenous ligands are relatively unperturbed in each inhibitor complex, but the inhibitors themselves bind to or near the iron in a range of positions, all of which perturb the position of Wat827. The three lowest-affinity inhibitors (MHP, PHP, and IHB) yield distorted trigonal bipyramidal iron coordination geometry in which the inhibitor C4-phenolate group displaces the solvent ligand. MHB binds within the active site, but neither its C3-OH group nor the solvent molecule binds to the iron. The C4-phenolate group of the three highest-affinity inhibitors (PHB, CHB, and FHB) coordinates the Fe3+ adjacent to Wat827, resulting in a shift in its position to yield a six-coordinate distorted octahedral geometry. The range of inhibitor orientations may mimic the mechanistically significant stages of substrate binding to 3, 4-PCD. The structure of the final substrate complex is reported in the following paper [Orville, A. M., Lipscomb, J. D., & Ohlendorf, D. H. (1997) Biochemistry 36, 10052-10066].


Assuntos
Inibidores Enzimáticos/química , Protocatecoate-3,4-Dioxigenase/antagonistas & inibidores , Sítios de Ligação , Ligação Competitiva , Simulação por Computador , Cristalografia por Raios X , Inibidores Enzimáticos/farmacologia , Cinética , Ligantes , Dados de Sequência Molecular , Estrutura Molecular , Protocatecoate-3,4-Dioxigenase/química , Pseudomonas putida/enzimologia
19.
Biochemistry ; 36(33): 10052-66, 1997 Aug 19.
Artigo em Inglês | MEDLINE | ID: mdl-9254600

RESUMO

Protocatechuate 3,4-dioxygenase (3,4-PCD) utilizes a ferric ion to catalyze the aromatic ring cleavage of 3,4-dihydroxybenzoate (PCA) by incorporation of both atoms of dioxygen to yield beta-carboxy-cis, cis-muconate. The crystal structures of the anaerobic 3,4-PCD.PCA complex, aerobic complexes with two heterocyclic PCA analogs, 2-hydroxyisonicotinic acid N-oxide (INO) and 6-hydroxynicotinic acid N-oxide (NNO), and ternary complexes of 3,4-PCD.INO.CN and 3,4-PCD. NNO.CN have been determined at 2.1-2.2 A resolution and refined to R-factors between 0.165 and 0.184. PCA, INO, and NNO form very similar, asymmetrically chelated complexes with the active site Fe3+ that result in dissociation of the endogenous axial tyrosinate Fe3+ ligand, Tyr447 (147beta). After its release from the iron, Tyr447 is stabilized by hydrogen bonding to Tyr16 (16alpha) and Asp413 (113beta) and forms the top of a small cavity adjacent to the C3-C4 bond of PCA. The equatorial Fe3+ coordination site within this cavity is unoccupied in the anaerobic 3,4-PCD.PCA complex but coordinates a solvent molecule in the 3,4-PCD.INO and 3,4-PCD.NNO complexes and CN- in the 3,4-PCD.INO.CN and 3,4-PCD.NNO.CN complexes. This shows that an O2 analog can occupy the cavity and suggests that electrophilic O2 attack on PCA is initiated from this site. Both the dissociation of the endogenous Tyr447 and the expansion of the iron coordination sphere are novel features of the 3,4-PCD. substrate complex which appear to play essential roles in the activation of substrate for O2 attack. Together, the structures presented here and in the preceding paper [Orville, A. M., Elango, N. , Lipscomb, J. D., & Ohlendorf, D. H. (1997) Biochemistry 36, 10039-10051] provide atomic models for several steps in the reaction cycle of 3,4-PCD and related Fe3+-containing dioxygenases.


Assuntos
Compostos Férricos/química , Protocatecoate-3,4-Dioxigenase/química , Cristalografia por Raios X , Ligantes , Estrutura Molecular , Protocatecoate-3,4-Dioxigenase/metabolismo , Especificidade por Substrato
20.
Protein Sci ; 6(6): 1220-7, 1997 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-9194182

RESUMO

The structure of toxic shock syndrome toxin-1 (TSST-1), the causative agent in toxic shock syndrome, has been determined in three crystal forms. The three structural models have been refined to R-factors of 0.154, 0.150, and 0.198 at resolutions of 2.05 A, 2.90 A, and 2.75 A, respectively. One crystal form of TSST-1 contains a zinc ion bound between two symmetry-related molecules. Although not required for biological activity, zinc dramatically potentiates the mitogenicity of TSST-1 at very low concentrations. In addition, the structure of the tetramutant TSST-1H [T69I, Y80W, E132K, I140T], which is nonmitogenic and does not amplify endotoxin shock, has been determined and refined in a fourth crystal form (R-factor = 0.173 to 1.9 A resolution).


Assuntos
Toxinas Bacterianas/química , Enterotoxinas/química , Superantígenos/química , Toxinas Bacterianas/genética , Toxinas Bacterianas/imunologia , Cristalografia por Raios X , Enterotoxinas/genética , Enterotoxinas/imunologia , Mitógenos/química , Mitógenos/genética , Mitógenos/imunologia , Modelos Moleculares , Mutação , Conformação Proteica , Staphylococcus aureus , Superantígenos/genética , Superantígenos/imunologia , Zinco/imunologia
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