Kinetics of Streptolysin O Self-Assembly

Palmer, Michaël; Valeva, Angela; Kehoe, Michael; Bhakdi, Sucharit

doi:10.1111/j.1432-1033.1995.tb20711.x

Cited by 47 publications

(44 citation statements)

References 20 publications

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“…All thiol-activated toxins are rendered inactive by chemical modification of a single cysteine residue that is located in a highly conserved region located close to the C terminus. The inactivated proteins no longer associate with membranes (9,10), which proves a role of this sequence element for membrane binding. Several point mutations within this conserved sequence have confirmed this conclusion (11).…”

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confidence: 97%

Membrane-penetrating Domain of Streptolysin O Identified by Cysteine Scanning Mutagenesis

Palmer¹,

Saweljew²,

Vulicevic³

et al. 1996

Journal of Biological Chemistry

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Streptolysin O (SLO), a polypeptide of 571 amino acids, belongs to a family of highly homologous toxins that bind to cell membranes containing cholesterol and then polymerize to form large transmembrane pores. A conserved region close to the C terminus contains the single cysteine residue of SLO and has been implicated in membrane binding, which has been the only clear assignment of function to a part of the sequence. We have used a cysteine-less active mutant of SLO to introduce single cysteine residues at 19 positions distributed throughout the sequence. The cysteines were derivatized with the polarity-sensitive fluorophore acrylodan, and the fluorescence emission of the label was examined at the different stages of SLO pore assembly. With several mutants, oligomerization on membranes was accompanied by emission blue-shifts, indicating movement of the label into a more hydrophobic environment. These effects were essentially confined to the range of amino acids 213-305. With oligomeric mutants L274C, S286C, and S305C, additional environmental alterations were induced when different nondenaturing detergents were used to dislodge the membrane lipids from the oligomers. The corresponding amino acid residues thus insert into the lipid bilayer during pore formation. Conversely, the spectra of oligomeric mutants A213C and T245C were not affected by detergents. Devoid of contact with the lipid bilayer, these amino acid residues probably participate in the interaction of SLO molecules within the oligomer.

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confidence: 97%

Membrane-penetrating Domain of Streptolysin O Identified by Cysteine Scanning Mutagenesis

Palmer¹,

Saweljew²,

Vulicevic³

et al. 1996

Journal of Biological Chemistry

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“…The mechanism of action of the CDCs involves a complex series of events that are known to include the binding and stable association with cholesterol-containing membranes by the toxin monomers (11,12), the lateral diffusion on the bilayer with concomitant formation of cytolysin oligomers (13)(14)(15)(16)(17), and ordered and coupled conformational changes that result in pore formation (9,(17)(18)(19).…”

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confidence: 99%

Assembly and Topography of the Prepore Complex in Cholesterol-dependent Cytolysins

Heuck

Tweten

Johnson

2003

Journal of Biological Chemistry

106

142

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Cholesterol-dependent cytolysins are a family of poreforming proteins that have been shown to be virulence factors for a large number of pathogenic bacteria. The mechanism of pore formation for these toxins involves a complex series of events that are known to include binding, oligomerization, and insertion of a transmembrane ␤-barrel. Several features of this mechanism remain poorly understood and controversial. Whereas a prepore mechanism has been proposed for perfringolysin O, a very different mechanism has been proposed for the homologous member of the family, streptolysin O. To distinguish between the two models, a novel approach that directly measures the dimension of transmembranes pores was used. Pore formation itself was examined for both cytolysins by encapsulating fluoresceinlabeled peptides and proteins of different sizes into liposomes. When these liposomes were re-suspended in a solution containing anti-fluorescein antibodies, toxinmediated pore formation was monitored directly by the quenching of fluorescein emission as the encapsulated molecules were released, and the dyes were bound by the antibodies. The analysis of pore formation determined using this approach reveals that only large pores are produced by perfringolysin O and streptolysin O during insertion (and not small pores that grow in size). These results are consistent only with the formation of a prepore complex intermediate prior to insertion of the transmembrane ␤-barrel into the bilayer. Fluorescence quenching experiments also revealed that PFO in the prepore complex contacts the membrane via domain 4, and that the individual transmembrane ␤-hairpins in domain 3 are not exposed to the nonpolar core of the bilayer at this intermediate stage.Cholesterol-dependent cytolysins (CDCs) 1 are produced by a variety of pathogenic Gram-positive bacteria (reviewed in Refs.1 and 2). The monomeric forms of the CDCs are highly water soluble, but the proteins bind to cholesterol-containing membranes and then spontaneously self-associate to form large aqueous pores in the bilayer. These oligomeric complexes vary in size and may contain up to 50 individual monomers (3-5).The only crystal structure of a water soluble, monomeric form of a CDC was solved by Rossjohn et al. (6) for perfringolysin O (PFO) from Clostridium perfringens, and their data revealed that PFO is comprised of four domains. The crystal structure of a membrane-inserted oligomer of a CDC is not presently available. However, several fluorescence-based studies have identified the regions of PFO that form a transmembrane ␤-barrel and have also provided other structural information about the membrane-inserted oligomer. Domain 3 of PFO contains two stretches of amino acids (190 -217 and 288 -311) that interact with the membrane during pore formation and create an amphipathic ␤-sheet that serves as an aqueouslipid interface after insertion into the membrane (7,8). Domain 4 (residues 391-500) is involved in membrane recognition and binding, and remains close to the membrane surface in the ...

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“…The initial conformational change that is associated with membrane binding and which sets the stage for oligomerization (ref) involves conformational flexibility between domains 2 and 3. The complementation of PLO-DS oligomerization by wild type PLO supports a two-step model of oligomerization that so far had been based on kinetic modeling only (20). The fact that membrane insertion and pore formation can occur in oligomers that contain some insertion-deficient subunits indicates that the process of insertion is only partially cooperative.…”

Section: Discussionmentioning

confidence: 77%

“…Another possible explanation for the incorporation of PLO-DS into hybrid but not homogeneous oligomers may be related to the existence of different kinetic stages of oligomerization (20). In the rate-limiting initiation reaction, two or more monomers react with one another, and the kinetic handicaps of multiple mutant molecules would likely act additively at this stage.…”

Section: Discussionmentioning

confidence: 99%

Partial oligomerization of pyolysin induced by a disulfide-tethered mutant

Pokrajac

Baik

Harris

et al. 2012

Biochem. Cell Biol.

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These authors contributed equally to this work. Final version of this article is published by NRC Research Press and available at: http://dx.doi.org/10.1139/ O2012-029 SummaryThe bacterial toxin pyolysin (PLO) belongs to the family of Cholesterol-Dependent Cytolysins (CDCs), which form large, oligomeric pores in cholesterol-containing membranes. Monomeric CDC molecules have a structure of four domains, with domains 2 and 3 packed against each other. After binding to target membranes containing cholesterol, toxin monomers oligomerize into pre-pore complexes. Trans-membrane pores form when the pre-pores insert into the lipid bilayer. Membrane insertion requires each subunit in the pre-pore to undergo a significant change in conformation, including the separation of domains 2 and 3. We here describe a pyolysin mutant with an engineered disulfide bond between domains 2 and 3 that is unable to complete this conformational change and thus does not form pores. In contrast to a similar mutant previously described for the homologous toxin perfringolysin O, the disulfide-tethered mutant does not form pre-pore oligomers, indicating that it is already inhibited at the stage of oligomerization. When mixed with wild type PLO, the mutant partially inhibits oligomerization of the latter, so that the resulting hybrid oligomers are reduced in size and arc-shaped rather than ring-shaped. Osmotic protection experiments indicate that such oligomers can form functional pores of reduced size. These findings indicate that conformational flexibility between domains 2 and 3 is required for oligomerization. Moreover, membrane insertion can be triggered in oligomers that contain some insertion-deficient subunits and is therefore only partially cooperative.2

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Kinetics of Streptolysin O Self-Assembly

Cited by 47 publications

References 20 publications

Membrane-penetrating Domain of Streptolysin O Identified by Cysteine Scanning Mutagenesis

Membrane-penetrating Domain of Streptolysin O Identified by Cysteine Scanning Mutagenesis

Assembly and Topography of the Prepore Complex in Cholesterol-dependent Cytolysins

Partial oligomerization of pyolysin induced by a disulfide-tethered mutant

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