The Cholesterol-Dependent Cytolysin Signature Motif: A Critical Element in the Allosteric Pathway that Couples Membrane Binding to Pore Assembly

Department of Microbiology and Immunology, The University of Oklahoma Health Sciences Center, Oklahoma City, OK, USA.
PLoS Pathogens (Impact Factor: 7.56). 07/2012; 8(7):e1002787. DOI: 10.1371/journal.ppat.1002787
Source: PubMed


The cholesterol-dependent cytolysins (CDCs) constitute a family of pore-forming toxins that contribute to the pathogenesis of a large number of Gram-positive bacterial pathogens.The most highly conserved region in the primary structure of the CDCs is the signature undecapeptide sequence (ECTGLAWEWWR). The CDC pore forming mechanism is highly sensitive to changes in its structure, yet its contribution to the molecular mechanism of the CDCs has remained enigmatic. Using a combination of fluorescence spectroscopic methods we provide evidence that shows the undecapeptide motif of the archetype CDC, perfringolysin O (PFO), is a key structural element in the allosteric coupling of the cholesterol-mediated membrane binding in domain 4 (D4) to distal structural changes in domain 3 (D3) that are required for the formation of the oligomeric pore complex. Loss of the undecapeptide function prevents all measurable D3 structural transitions, the intermolecular interaction of membrane bound monomers and the assembly of the oligomeric pore complex. We further show that this pathway does not exist in intermedilysin (ILY), a CDC that exhibits a divergent undecapeptide and that has evolved to use human CD59 rather than cholesterol as its receptor. These studies show for the first time that the undecapeptide of the cholesterol-binding CDCs forms a critical element of the allosteric pathway that controls the assembly of the pore complex.

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    • "Circularization of the CDC oligomeric ring then triggers a dramatic conformational change in which helical segments from D3 convert to amphipathic hairpins and arrange into a transmembrane b-barrel pore (Tilley et al., 2005). For a subclass of CDCs, of which ILY is an archetypal member, control of regulating prepore transitions is transferred from the undecapeptide to CD59 binding (Dowd and Tweten, 2012). Our structural and functional analysis of the ILY-CD59 complex supports a model for these pores in which membraneanchored CD59 recruits soluble ILY monomers to the plasma membrane of human cells, orienting the CRM and undecapeptide for insertion into cholesterol-containing lipid bilayers. "
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    ABSTRACT: Pore-forming proteins containing the structurally conserved membrane attack complex/perforin fold play an important role in immunity and host-pathogen interactions. Intermedilysin (ILY) is an archetypal member of a cholesterol-dependent cytolysin subclass that hijacks the complement receptor CD59 to make cytotoxic pores in human cells. ILY directly competes for the membrane attack complex binding site on CD59, rendering cells susceptible to complement lysis. To understand how these bacterial pores form in lipid bilayers and the role CD59 plays in complement regulation, we determined the crystal structure of human CD59 bound to ILY. Here, we show the ILY-CD59 complex at 3.5 Å resolution and identify two interfaces mediating this host-pathogen interaction. An ILY-derived peptide based on the binding site inhibits pore formation in a CD59-containing liposome model system. These data provide insight into how CD59 coordinates ILY monomers, nucleating an early prepore state, and suggest a potential mechanism of inhibition for the complement terminal pathway.
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    • "LLO engages cholesterol as a native membrane receptor in dependence on the two amino acids threonine 515 and leucine 516, oligomerises to a prepore complex of up to 50 monomers, and forms a membrane pore in a concerted refolding step with each protomer contributing two beta-hairpins to the membrane-spanning β-barrel, which originates from five α-helices in the soluble state [16, 59–61]. The allosteric monomer assembly and prepore refolding process were recently shown to rely on an undecapeptide sequence (483-ECTGLAWEWWR-493), which was originally thought to be soley responsible for cholesterol binding [62]. The exact nature of the membrane pore remains controversial, as arciform pores, that is, membrane pores with a seemingly incomplete protein ring lining the aqueous membrane hole, are often observed in electron microscopic and atomic force microscopic imaging of various CDCs [22, 63–65]. "
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    ABSTRACT: Targeted disruption of the plasma membrane is a ubiquitous form of attack used in all three domains of life. Many bacteria secrete pore-forming proteins during infection with broad implications for pathogenesis. The cholesterol-dependent cytolysins (CDC) are a family of pore-forming toxins expressed predominately by Gram-positive bacterial pathogens. The structure and assembly of some of these oligomeric toxins on the host membrane have been described, but how the targeted cell responds to intoxication by the CDCs is not as clearly understood. Many CDCs induce lysis of their target cell and can activate apoptotic cascades to promote cell death. However, the extent to which intoxication causes cell death is both CDC- and host cell-dependent, and at lower concentrations of toxin, survival of intoxicated host cells is well documented. Additionally, the effect of CDCs can be seen beyond the plasma membrane, and it is becoming increasingly clear that these toxins are potent regulators of signaling and immunity, beyond their role in intoxication. In this review, we discuss the cellular response to CDC intoxication with emphasis on the effects of pore formation on the host cell plasma membrane and subcellular organelles and whether subsequent cellular responses contribute to the survival of the affected cell.
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