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1.
Nat Commun ; 10(1): 2641, 2019 06 14.
Artigo em Inglês | MEDLINE | ID: mdl-31201325

RESUMO

Epsilon toxin (Etx), a potent pore forming toxin (PFT) produced by Clostridium perfringens, is responsible for the pathogenesis of enterotoxaemia of ruminants and has been suggested to play a role in multiple sclerosis in humans. Etx is a member of the aerolysin family of ß-PFTs (aß-PFTs). While the Etx soluble monomer structure was solved in 2004, Etx pore structure has remained elusive due to the difficulty of isolating the pore complex. Here we show the cryo-electron microscopy structure of Etx pore assembled on the membrane of susceptible cells. The pore structure explains important mutant phenotypes and suggests that the double ß-barrel, a common feature of the aß-PFTs, may be an important structural element in driving efficient pore formation. These insights provide the framework for the development of novel therapeutics to prevent human and animal infections, and are relevant for nano-biotechnology applications.


Assuntos
Toxinas Bacterianas/química , Clostridium perfringens/ultraestrutura , Animais , Toxinas Bacterianas/genética , Toxinas Bacterianas/isolamento & purificação , Toxinas Bacterianas/metabolismo , Biotecnologia/métodos , Linhagem Celular , Infecções por Clostridium/microbiologia , Infecções por Clostridium/prevenção & controle , Clostridium perfringens/genética , Clostridium perfringens/metabolismo , Clostridium perfringens/patogenicidade , Microscopia Crioeletrônica , Cães , Enterotoxemia/microbiologia , Enterotoxemia/prevenção & controle , Modelos Moleculares , Mutagênese Sítio-Dirigida , Nanotecnologia/métodos , Conformação Proteica em Folha beta/genética , Multimerização Proteica/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo
2.
Chem Cent J ; 11(1): 73, 2017 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-29086855

RESUMO

The crystal structure of a commercially available form of human recombinant (HR) insulin, Insugen (I), used in the treatment of diabetes has been determined to 0.92 Å resolution using low temperature, 100 K, synchrotron X-ray data collected at 16,000 keV (λ = 0.77 Å). Refinement carried out with anisotropic displacement parameters, removal of main-chain stereochemical restraints, inclusion of H atoms in calculated positions, and 220 water molecules, converged to a final value of R = 0.1112 and Rfree = 0.1466. The structure includes what is thought to be an ordered propanol molecule (POL) only in chain D(4) and a solvated acetate molecule (ACT) coordinated to the Zn atom only in chain B(2). Possible origins and consequences of the propanol and acetate molecules are discussed. Three types of amino acid representation in the electron density are examined in detail: (i) sharp with very clearly resolved features; (ii) well resolved but clearly divided into two conformations which are well behaved in the refinement, both having high quality geometry; (iii) poor density and difficult or impossible to model. An example of type (ii) is observed for the intra-chain disulphide bridge in chain C(3) between Sγ6-Sγ11 which has two clear conformations with relative refined occupancies of 0.8 and 0.2, respectively. In contrast the corresponding S-S bridge in chain A(1) shows one clearly defined conformation. A molecular dynamics study has provided a rational explanation of this difference between chains A and C. More generally, differences in the electron density features between corresponding residues in chains A and C and chains B and D is a common observation in the Insugen (I) structure and these effects are discussed in detail. The crystal structure, also at 0.92 Å and 100 K, of a second commercially available form of human recombinant insulin, Intergen (II), deposited in the Protein Data Bank as 3W7Y which remains otherwise unpublished is compared here with the Insugen (I) structure. In the Intergen (II) structure there is no solvated propanol or acetate molecule. The electron density of Intergen (II), however, does also exhibit the three types of amino acid representations as in Insugen (I). These effects do not necessarily correspond between chains A and C or chains B and D in Intergen (II), or between corresponding residues in Insugen (I). The results of this comparison are reported. Graphical abstract Conformations of PheB25 and PheD25 in three insulin structures: implications for biological activity? Insulin residues PheB25 and PheD25 are considered to be important for insulin receptor binding and changes in biological activity occur when these residues are modified. In porcine insulin and Intergen (II) PheB25 adopts conformation B and PheD25 conformation D. However, unexpectedly PheB25 in Insugen (I) human recombinant insulin adopts two distinct conformations corresponding to B and D, Figure 1 and PheD25 adopts a single conformation corresponding to B not D, Figure 2. Conformations of this residue in the ultra-high resolution structure of Insugen (I) are therefore unique within this set. Figures were produced with Biovia, Discovery Studio 2016.

3.
Nat Commun ; 7: 11293, 2016 Apr 06.
Artigo em Inglês | MEDLINE | ID: mdl-27048994

RESUMO

Lysenin from the coelomic fluid of the earthworm Eisenia fetida belongs to the aerolysin family of small ß-pore-forming toxins (ß-PFTs), some members of which are pathogenic to humans and animals. Despite efforts, a high-resolution structure of a channel for this family of proteins has been elusive and therefore the mechanism of activation and membrane insertion remains unclear. Here we determine the pore structure of lysenin by single particle cryo-EM, to 3.1 Å resolution. The nonameric assembly reveals a long ß-barrel channel spanning the length of the complex that, unexpectedly, includes the two pre-insertion strands flanking the hypothetical membrane-insertion loop. Examination of other members of the aerolysin family reveals high structural preservation in this region, indicating that the membrane-insertion pathway in this family is conserved. For some toxins, proteolytic activation and pro-peptide removal will facilitate unfolding of the pre-insertion strands, allowing them to form the ß-barrel of the channel.


Assuntos
Toxinas Bacterianas/metabolismo , Membrana Celular/metabolismo , Microscopia Crioeletrônica , Proteínas Citotóxicas Formadoras de Poros/metabolismo , Toxinas Biológicas/química , Humanos , Lipídeos/química , Modelos Moleculares , Estrutura Secundária de Proteína , Solubilidade , Água/química
4.
J Mol Biol ; 426(18): 3134-3147, 2014 Sep 09.
Artigo em Inglês | MEDLINE | ID: mdl-25020226

RESUMO

CPE (Clostridium perfringens enterotoxin) is the major virulence determinant for C. perfringens type-A food poisoning, the second most common bacterial food-borne illness in the UK and USA. After binding to its receptors, which include particular human claudins, the toxin forms pores in the cell membrane. The mature pore apparently contains a hexamer of CPE, claudin and, possibly, occludin. The combination of high binding specificity with cytotoxicity has resulted in CPE being investigated, with some success, as a targeted cytotoxic agent for oncotherapy. In this paper, we present the X-ray crystallographic structure of CPE in complex with a peptide derived from extracellular loop 2 of a modified, CPE-binding Claudin-2, together with high-resolution native and pore-formation mutant structures. Our structure provides the first atomic-resolution data on any part of a claudin molecule and reveals that claudin's CPE-binding fingerprint (NPLVP) is in a tight turn conformation and binds, as expected, in CPE's C-terminal claudin-binding groove. The leucine and valine residues insert into the binding groove while the first residue, asparagine, tethers the peptide via an interaction with CPE's aspartate 225 and the two prolines are required to maintain the tight turn conformation. Understanding the structural basis of the contribution these residues make to binding will aid in engineering CPE to target tumor cells.


Assuntos
Claudina-2/química , Clostridium perfringens/química , Enterotoxinas/química , Modelos Moleculares , Substituição de Aminoácidos , Claudina-2/metabolismo , Clostridium perfringens/genética , Clostridium perfringens/isolamento & purificação , Clostridium perfringens/metabolismo , Cristalografia por Raios X , Enterotoxinas/genética , Enterotoxinas/isolamento & purificação , Enterotoxinas/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Mutação , Peptídeos/genética , Peptídeos/metabolismo , Ligação Proteica , Conformação Proteica , Multimerização Proteica
5.
Vaccine ; 32(23): 2682-7, 2014 May 13.
Artigo em Inglês | MEDLINE | ID: mdl-24709588

RESUMO

Epsilon toxin (Etx) is a ß-pore-forming toxin produced by Clostridium perfringens toxinotypes B and D and plays a key role in the pathogenesis of enterotoxemia, a severe, often fatal disease of ruminants that causes significant economic losses to the farming industry worldwide. This study aimed to determine the potential of a site-directed mutant of Etx (Y30A-Y196A) to be exploited as a recombinant vaccine against enterotoxemia. Replacement of Y30 and Y196 with alanine generated a stable variant of Etx with significantly reduced cell binding and cytotoxic activities in MDCK.2 cells relative to wild type toxin (>430-fold increase in CT50) and Y30A-Y196A was inactive in mice after intraperitoneal administration of trypsin activated toxin at 1000× the expected LD50 dose of trypsin activated wild type toxin. Moreover, polyclonal antibody raised in rabbits against Y30A-Y196A provided protection against wild type toxin in an in vitro neutralisation assay. These data suggest that Y30A-Y196A mutant could form the basis of an improved recombinant vaccine against enterotoxemia.


Assuntos
Toxinas Bacterianas/imunologia , Vacinas Bacterianas/imunologia , Enterotoxemia/prevenção & controle , Animais , Cães , Feminino , Células Madin Darby de Rim Canino , Camundongos , Camundongos Endogâmicos BALB C , Mutagênese Sítio-Dirigida , Testes de Neutralização , Estrutura Terciária de Proteína , Coelhos , Proteínas Recombinantes/imunologia , Vacinas Sintéticas/imunologia
6.
Toxins (Basel) ; 6(3): 1049-61, 2014 Mar 12.
Artigo em Inglês | MEDLINE | ID: mdl-24625763

RESUMO

Necrotic enteritis toxin B (NetB) is a ß-pore-forming toxin produced by Clostridium perfringens and has been identified as a key virulence factor in the pathogenesis of avian necrotic enteritis, a disease causing significant economic damage to the poultry industry worldwide. In this study, site-directed mutagenesis was used to identify amino acids that play a role in NetB oligomerisation and pore-formation. NetB K41H showed significantly reduced toxicity towards LMH cells and human red blood cells relative to wild type toxin. NetB K41H was unable to oligomerise and form pores in liposomes. These findings suggest that NetB K41H could be developed as a genetic toxoid vaccine to protect against necrotic enteritis.


Assuntos
Toxinas Bacterianas/química , Enterotoxinas/química , Proteínas Citotóxicas Formadoras de Poros/química , Aminoácidos/química , Animais , Toxinas Bacterianas/genética , Toxinas Bacterianas/metabolismo , Linhagem Celular Tumoral , Células Cultivadas , Galinhas , Enterotoxinas/genética , Enterotoxinas/metabolismo , Eritrócitos/metabolismo , Fluoresceínas/metabolismo , Hemólise , Humanos , Proteínas Citotóxicas Formadoras de Poros/genética , Proteínas Citotóxicas Formadoras de Poros/metabolismo , Estrutura Secundária de Proteína
7.
PLoS One ; 8(6): e66673, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23805259

RESUMO

Clostridium perfringens Delta toxin is one of the three hemolysin-like proteins produced by C. perfringens type C and possibly type B strains. One of the others, NetB, has been shown to be the major cause of Avian Nectrotic Enteritis, which following the reduction in use of antibiotics as growth promoters, has become an emerging disease of industrial poultry. Delta toxin itself is cytotoxic to the wide range of human and animal macrophages and platelets that present GM2 ganglioside on their membranes. It has sequence similarity with Staphylococcus aureus ß-pore forming toxins and is expected to heptamerize and form pores in the lipid bilayer of host cell membranes. Nevertheless, its exact mode of action remains undetermined. Here we report the 2.4 Å crystal structure of monomeric Delta toxin. The superposition of this structure with the structure of the phospholipid-bound F component of S. aureus leucocidin (LukF) revealed that the glycerol molecules bound to Delta toxin and the phospholipids in LukF are accommodated in the same hydrophobic clefts, corresponding to where the toxin is expected to latch onto the membrane, though the binding sites show significant differences. From structure-based sequence alignment with the known structure of staphylococcal α-hemolysin, a model of the Delta toxin pore form has been built. Using electron microscopy, we have validated our model and characterized the Delta toxin pore on liposomes. These results highlight both similarities and differences in the mechanism of Delta toxin (and by extension NetB) cytotoxicity from that of the staphylococcal pore-forming toxins.


Assuntos
Toxinas Bacterianas/metabolismo , Clostridium perfringens/metabolismo , Toxinas Bacterianas/química , Toxinas Bacterianas/genética , Cristalografia por Raios X , Células HeLa , Humanos , Microscopia Eletrônica , Imagem Óptica , Ligação Proteica , Estrutura Terciária de Proteína , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/metabolismo
8.
Vaccine ; 31(37): 4003-8, 2013 Aug 20.
Artigo em Inglês | MEDLINE | ID: mdl-23727000

RESUMO

NetB (necrotic enteritis toxin B) is a recently identified ß-pore-forming toxin produced by Clostridium perfringens. This toxin has been shown to play a major role in avian necrotic enteritis. In recent years, a dramatic increase in necrotic enteritis has been observed, especially in countries where the use of antimicrobial growth promoters in animal feedstuffs has been banned. The aim of this work was to determine whether immunisation with a NetB toxoid would provide protection against necrotic enteritis. The immunisation of poultry with a formaldehyde NetB toxoid or with a NetB genetic toxoid (W262A) resulted in the induction of antibody responses against NetB and provided partial protection against disease.


Assuntos
Toxinas Bacterianas/imunologia , Infecções por Clostridium/veterinária , Clostridium perfringens/genética , Enterite/veterinária , Toxoides/farmacologia , Animais , Anticorpos Antibacterianos/análise , Toxinas Bacterianas/genética , Galinhas/imunologia , Galinhas/microbiologia , Infecções por Clostridium/microbiologia , Infecções por Clostridium/prevenção & controle , Eletroforese em Gel de Poliacrilamida , Enterite/imunologia , Enterite/prevenção & controle , Ensaio de Imunoadsorção Enzimática , Formaldeído/imunologia , Imunização/métodos , Mutação , Doenças das Aves Domésticas/microbiologia , Toxoides/imunologia
9.
Protein Sci ; 22(5): 650-9, 2013 May.
Artigo em Inglês | MEDLINE | ID: mdl-23504825

RESUMO

Clostridium perfringens epsilon toxin (Etx) is a pore-forming toxin responsible for a severe and rapidly fatal enterotoxemia of ruminants. The toxin is classified as a category B bioterrorism agent by the U.S. Government Centres for Disease Control and Prevention (CDC), making work with recombinant toxin difficult. To reduce the hazard posed by work with recombinant Etx, we have used a variant of Etx that contains a H149A mutation (Etx-H149A), previously reported to have reduced, but not abolished, toxicity. The three-dimensional structure of H149A prototoxin shows that the H149A mutation in domain III does not affect organisation of the putative receptor binding loops in domain I of the toxin. Surface exposed tyrosine residues in domain I of Etx-H149A (Y16, Y20, Y29, Y30, Y36 and Y196) were mutated to alanine and mutants Y30A and Y196A showed significantly reduced binding to MDCK.2 cells relative to Etx-H149A that correlated with their reduced cytotoxic activity. Thus, our study confirms the role of surface exposed tyrosine residues in domain I of Etx in binding to MDCK cells and the suitability of Etx-H149A for further receptor binding studies. In contrast, binding of all of the tyrosine mutants to ACHN cells was similar to that of Etx-H149A, suggesting that Etx can recognise different cell surface receptors. In support of this, the crystal structure of Etx-H149A identified a glycan (ß-octyl-glucoside) binding site in domain III of Etx-H149A, which may be a second receptor binding site. These findings have important implications for developing strategies designed to neutralise toxin activity.


Assuntos
Toxinas Bacterianas/genética , Toxinas Bacterianas/metabolismo , Infecções por Clostridium/microbiologia , Clostridium perfringens/fisiologia , Interações Hospedeiro-Patógeno , Receptores de Superfície Celular/metabolismo , Animais , Toxinas Bacterianas/química , Sítios de Ligação , Linhagem Celular , Sobrevivência Celular , Clostridium perfringens/química , Clostridium perfringens/genética , Cães , Células Madin Darby de Rim Canino , Modelos Moleculares , Mutação Puntual , Ligação Proteica , Conformação Proteica
10.
J Biol Chem ; 288(5): 3512-22, 2013 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-23239883

RESUMO

NetB is a pore-forming toxin produced by Clostridium perfringens and has been reported to play a major role in the pathogenesis of avian necrotic enteritis, a disease that has emerged due to the removal of antibiotics in animal feedstuffs. Here we present the crystal structure of the pore form of NetB solved to 3.9 Å. The heptameric assembly shares structural homology to the staphylococcal α-hemolysin. However, the rim domain, a region that is thought to interact with the target cell membrane, shows sequence and structural divergence leading to the alteration of a phosphocholine binding pocket found in the staphylococcal toxins. Consistent with the structure we show that NetB does not bind phosphocholine efficiently but instead interacts directly with cholesterol leading to enhanced oligomerization and pore formation. Finally we have identified conserved and non-conserved amino acid positions within the rim loops that significantly affect binding and toxicity of NetB. These findings present new insights into the mode of action of these pore-forming toxins, enabling the design of more effective control measures against necrotic enteritis and providing potential new tools to the field of bionanotechnology.


Assuntos
Toxinas Bacterianas/química , Toxinas Bacterianas/metabolismo , Clostridium perfringens/metabolismo , Animais , Toxinas Bacterianas/toxicidade , Linhagem Celular Tumoral , Forma Celular/efeitos dos fármacos , Galinhas , Colesterol/metabolismo , Cristalografia por Raios X , Modelos Moleculares , Proteínas Mutantes/metabolismo , Mutação/genética , Fosfolipídeos/metabolismo , Ligação Proteica , Multimerização Proteica/efeitos dos fármacos , Estrutura Terciária de Proteína , Solubilidade , Eletricidade Estática
11.
J Mol Biol ; 413(1): 138-49, 2011 Oct 14.
Artigo em Inglês | MEDLINE | ID: mdl-21839091

RESUMO

Clostridium perfringens enterotoxin (CPE) is a major cause of food poisoning and antibiotic-associated diarrhea. Upon its release from C. perfringens spores, CPE binds to its receptor, claudin, at the tight junctions between the epithelial cells of the gut wall and subsequently forms pores in the cell membranes. A number of different complexes between CPE and claudin have been observed, and the process of pore formation has not been fully elucidated. We have determined the three-dimensional structure of the soluble form of CPE in two crystal forms by X-ray crystallography, to a resolution of 2.7 and 4.0 Å, respectively, and found that the N-terminal domain shows structural homology with the aerolysin-like ß-pore-forming family of proteins. We show that CPE forms a trimer in both crystal forms and that this trimer is likely to be biologically relevant but is not the active pore form. We use these data to discuss models of pore formation.


Assuntos
Enterotoxinas/química , Toxinas Bacterianas/química , Clostridium perfringens/química , Cristalografia por Raios X , Modelos Moleculares , Proteínas Citotóxicas Formadoras de Poros/química , Conformação Proteica , Multimerização Proteica
12.
FEBS J ; 278(23): 4589-601, 2011 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-21518257

RESUMO

Clostridium perfringens ε-toxin is produced by toxinotypes B and D strains. The toxin is the aetiological agent of dysentery in newborn lambs but is also associated with enteritis and enterotoxaemia in goats, calves and foals. It is considered to be a potential biowarfare or bioterrorism agent by the US Government Centers for Disease Control and Prevention. The relatively inactive 32.9 kDa prototoxin is converted to active mature toxin by proteolytic cleavage, either by digestive proteases of the host, such as trypsin and chymotrypsin, or by C. perfringens λ-protease. In vivo, the toxin appears to target the brain and kidneys, but relatively few cell lines are susceptible to the toxin, and most work has been carried out using Madin-Darby canine kidney (MDCK) cells. The binding of ε-toxin to MDCK cells and rat synaptosomal membranes is associated with the formation of a stable, high molecular weight complex. The crystal structure of ε-toxin reveals similarity to aerolysin from Aeromonas hydrophila, parasporin-2 from Bacillus thuringiensis and a lectin from Laetiporus sulphureus. Like these toxins, ε-toxin appears to form heptameric pores in target cell membranes. The exquisite specificity of the toxin for specific cell types suggests that it binds to a receptor found only on these cells.


Assuntos
Toxinas Bacterianas/química , Toxinas Bacterianas/metabolismo , Animais , Toxinas Bacterianas/toxicidade , Clostridium perfringens/metabolismo , Humanos , Modelos Moleculares , Conformação Proteica , Ratos , Sinaptossomos/metabolismo
13.
Artigo em Inglês | MEDLINE | ID: mdl-20606275

RESUMO

Clostridium perfringens is a Gram-positive anaerobic species of bacterium that is notable for its ability to produce a plethora of toxins, including membrane-active toxins (alpha-toxins), pore-forming toxins (-toxins) and binary toxins (iota-toxins). Here, the crystallization of the full-length wild-type C. perfringens enterotoxin is reported, which is the causative agent of the second most prevalent food-borne illness in the United States and has been implicated in many other gastrointestinal pathologies. Several crystal forms were obtained. However, only two of these optimized crystal forms (I and II) were useable for X-ray diffraction data collection. The form I crystals diffracted to d(min) = 2.7 A and belonged to space group C2, while the form II crystals diffracted to d(min) = 4 A and belonged to space group P2(1)3.


Assuntos
Clostridium perfringens/química , Enterotoxinas/química , Cristalização , Cristalografia por Raios X
14.
Nat Struct Mol Biol ; 11(8): 797-8, 2004 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-15258571

RESUMO

Epsilon-toxin from Clostridium perfringens is a lethal toxin. Recent studies suggest that the toxin acts via an unusually potent pore-forming mechanism. Here we report the crystal structure of epsilon-toxin, which reveals structural similarity to aerolysin from Aeromonas hydrophila. Pore-forming toxins can change conformation between soluble and transmembrane states. By comparing the two toxins, we have identified regions important for this transformation.


Assuntos
Toxinas Bacterianas/química , Aeromonas/metabolismo , Sequência de Aminoácidos , Bacillus/metabolismo , Membrana Celular/metabolismo , Cristalografia por Raios X , Modelos Moleculares , Dados de Sequência Molecular , Proteínas Citotóxicas Formadoras de Poros , Conformação Proteica , Estrutura Terciária de Proteína , Homologia de Sequência de Aminoácidos
15.
J Mol Biol ; 333(4): 759-69, 2003 Oct 31.
Artigo em Inglês | MEDLINE | ID: mdl-14568535

RESUMO

Clostridium absonum phospholipase C (Caa) is a 42.7 kDa protein, which shows 60% amino acid sequence identity with the Clostridium perfringens phospholipase C, or alpha-toxin (Cpa), and has been isolated from patients suffering from gas gangrene. We report the cloning and sequencing, purification, characterisation and crystal structure of the Caa enzyme. Caa had twice the phospholipid-hydrolysing (lecithinase) activity, 1.5 times the haemolytic activity and over seven times the activity towards phosphatidylcholine-based liposomes when compared with Cpa. However, the Caa enzyme had a lower activity than Cpa to the free (i.e. not in lipid bilayer) substrate para-nitrophenylphosphorylcholine, towards sphingomyelin-based liposomes and showed half the cytotoxicity. The lethal dose (LD(50)) of Caa in mice was approximately twice that of Cpa. The crystal structure of Caa shows that the 72-93 residue loop is in a conformation different from those of previously determined open-form alpha-toxin structures. This conformational change suggests a role for W84 in membrane binding and a possible route of entry into the active site along a hydrophobic channel created by the re-arrangement of this loop. Overall, the properties of Caa are compatible with a role as a virulence-determinant in gas gangrene caused by C.absonum.


Assuntos
Toxinas Bacterianas/química , Toxinas Bacterianas/metabolismo , Clostridium/enzimologia , Fosfolipases Tipo C/química , Fosfolipases Tipo C/metabolismo , Sequência de Aminoácidos , Animais , Toxinas Bacterianas/genética , Sítios de Ligação , Cristalografia por Raios X , Camundongos , Modelos Moleculares , Dados de Sequência Molecular , Ligação Proteica , Alinhamento de Sequência , Especificidade por Substrato , Fosfolipases Tipo C/genética
16.
J Mol Biol ; 319(2): 275-81, 2002 May 31.
Artigo em Inglês | MEDLINE | ID: mdl-12051905

RESUMO

Clostridium perfringens biotype A strains are the causative agents of gas-gangrene in man and are also implicated as etiological agents in sudden death syndrome in young domestic livestock. The main virulence factor produced by these strains is a zinc-dependent, phosphatidylcholine-preferring phospholipase C (alpha-toxin). The crystal structure of alpha-toxin, at pH 7.5, with the active site open and therefore accessible to substrate has previously been reported, as has calcium-binding to the C-terminal domain of the enzyme at pH 4.7. Here we focus on conformation changes in the N-terminal domain of alpha-toxin in crystals grown at acidic pH. These changes result in both the obscuring of the toxin active site and the loss of one of three zinc ions from it. Additionally, this "closed" form contains a small alpha helix, not present in the open structure, which hydrogen bonds to both the N and C-terminal domains. In conjunction with the previously reported findings that alpha-toxin can exist in active and inactive forms and that Thr74Ile and Phe69Cys substitutions markedly reduced the haemolytic activity of the enzyme, our work suggests that these loop conformations play a critical role in the activity of the toxin.


Assuntos
Clostridium perfringens/enzimologia , Fosfolipases Tipo C/química , Sítios de Ligação , Cálcio/metabolismo , Cristalografia por Raios X , Ligações de Hidrogênio , Concentração de Íons de Hidrogênio , Modelos Moleculares , Movimento , Maleabilidade , Estrutura Terciária de Proteína , Fosfolipases Tipo C/metabolismo , Zinco/metabolismo
17.
Biochemistry ; 41(20): 6253-62, 2002 May 21.
Artigo em Inglês | MEDLINE | ID: mdl-12009886

RESUMO

Clostridium perfringens alpha-toxin is a 370-residue, zinc-dependent, phospholipase C that is the key virulence determinant in gas gangrene. It is also implicated in the pathogenesis of sudden death syndrome in young animals and necrotic enteritis in chickens. Previously characterized alpha-toxins from different strains of C. perfringens are almost identical in sequence and biochemical properties. We describe the cloning, nucleotide sequencing, expression, characterization, and crystal structure of alpha-toxin from an avian strain, SWan C. perfringens (SWCP), which has a large degree of sequence variation and altered substrate specificity compared to these strains. The structure of alpha-toxin from strain CER89L43 has been previously reported in open (active site accessible to substrate) and closed (active site obscured by loop movements) conformations. The SWCP structure is in an open-form conformation, with three zinc ions in the active site. This is the first example of an open form of alpha-toxin crystallizing without the addition of divalent cations to the crystallization buffer, indicating that the protein can retain three zinc ions bound in the active site. The topology of the calcium binding site formed by residues 269, 271, 336, and 337, which is essential for membrane binding, is significantly altered in comparison with both the open and closed alpha-toxin structures. We are able to relate these structural changes to the different substrate specificity and membrane binding properties of this divergent alpha-toxin. This will provide essential information when developing an effective vaccine that will protect against C. perfringens infection in a wide range of domestic livestock.


Assuntos
Toxinas Bacterianas/química , Toxinas Bacterianas/metabolismo , Aves/microbiologia , Proteínas de Ligação ao Cálcio , Clostridium perfringens/química , Fosfolipases Tipo C/química , Fosfolipases Tipo C/metabolismo , Sequência de Aminoácidos , Animais , Toxinas Bacterianas/genética , Toxinas Bacterianas/toxicidade , Doenças das Aves/microbiologia , Cádmio/metabolismo , Bovinos , Clonagem Molecular , Clostridium perfringens/genética , Clostridium perfringens/isolamento & purificação , Cristalização , Cristalografia por Raios X , Enterocolite Pseudomembranosa/microbiologia , Enterocolite Pseudomembranosa/veterinária , Genes Bacterianos , Hemólise/efeitos dos fármacos , Cinética , Masculino , Lipídeos de Membrana/química , Lipídeos de Membrana/metabolismo , Camundongos , Dados de Sequência Molecular , Ligação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Homologia de Sequência de Aminoácidos , Fosfolipases Tipo C/genética , Fosfolipases Tipo C/toxicidade , Zinco/metabolismo
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