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Identification of a Membrane-bound Prepore Species Clarifies the Lytic Mechanism of Actinoporins * ♦

View Article: PubMed Central - PubMed

ABSTRACT

Pore-forming toxins (PFTs) are cytolytic proteins belonging to the molecular warfare apparatus of living organisms. The assembly of the functional transmembrane pore requires several intermediate steps ranging from a water-soluble monomeric species to the multimeric ensemble inserted in the cell membrane. The non-lytic oligomeric intermediate known as prepore plays an essential role in the mechanism of insertion of the class of β-PFTs. However, in the class of α-PFTs, like the actinoporins produced by sea anemones, evidence of membrane-bound prepores is still lacking. We have employed single-particle cryo-electron microscopy (cryo-EM) and atomic force microscopy to identify, for the first time, a prepore species of the actinoporin fragaceatoxin C bound to lipid vesicles. The size of the prepore coincides with that of the functional pore, except for the transmembrane region, which is absent in the prepore. Biochemical assays indicated that, in the prepore species, the N terminus is not inserted in the bilayer but is exposed to the aqueous solution. Our study reveals the structure of the prepore in actinoporins and highlights the role of structural intermediates for the formation of cytolytic pores by an α-PFT.

No MeSH data available.


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Structure of the oligomeric prepore of FraC bound to DOPC vesicles.a, representative image of FraC in DOPC vesicles obtained by cryo-EM. Top and side views of the protein oligomers were selected (red squares) for subsequent classification analysis. The scale bar corresponds to 100 nm. b, density map projections (top row) and two-dimensional class-averaged particles (bottom row) employed to build a three-dimensional model of the protein oligomer (see below). c, set of particles obtained by maximum-likelihood (ML2D, top row) and hierarchical clustering (CL2D, bottom row) procedures. d, top view, and e, side view of the three-dimensional model of the prepore of FraC bound to vesicles of DOPC. The atomic model of FraC was built as an octamer using the coordinates of the protomer of FraC prior to pore formation (entry code 4TSL).
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Figure 3: Structure of the oligomeric prepore of FraC bound to DOPC vesicles.a, representative image of FraC in DOPC vesicles obtained by cryo-EM. Top and side views of the protein oligomers were selected (red squares) for subsequent classification analysis. The scale bar corresponds to 100 nm. b, density map projections (top row) and two-dimensional class-averaged particles (bottom row) employed to build a three-dimensional model of the protein oligomer (see below). c, set of particles obtained by maximum-likelihood (ML2D, top row) and hierarchical clustering (CL2D, bottom row) procedures. d, top view, and e, side view of the three-dimensional model of the prepore of FraC bound to vesicles of DOPC. The atomic model of FraC was built as an octamer using the coordinates of the protomer of FraC prior to pore formation (entry code 4TSL).

Mentions: To visualize the structure of membrane-bound FraC, vitrified samples of toxin-treated DOPC liposomes were imaged by cryo-EM. Ring-shaped particles covering the lipid vesicles were attributed to protein oligomers (Fig. 3A). A total of 1,562 top view and side view images were selected to build a three-dimensional model of the protein oligomer. The model was built by common-lines procedures, followed by iterative refinement using projection matching of the class-averaged images and the density map projections (Fig. 3B). A second classification method based on maximum likelihood (41) and hierarchical clustering approaches (Fig. 3C) (42) rendered images similar to those used to generate the final density map.


Identification of a Membrane-bound Prepore Species Clarifies the Lytic Mechanism of Actinoporins * ♦
Structure of the oligomeric prepore of FraC bound to DOPC vesicles.a, representative image of FraC in DOPC vesicles obtained by cryo-EM. Top and side views of the protein oligomers were selected (red squares) for subsequent classification analysis. The scale bar corresponds to 100 nm. b, density map projections (top row) and two-dimensional class-averaged particles (bottom row) employed to build a three-dimensional model of the protein oligomer (see below). c, set of particles obtained by maximum-likelihood (ML2D, top row) and hierarchical clustering (CL2D, bottom row) procedures. d, top view, and e, side view of the three-dimensional model of the prepore of FraC bound to vesicles of DOPC. The atomic model of FraC was built as an octamer using the coordinates of the protomer of FraC prior to pore formation (entry code 4TSL).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
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getmorefigures.php?uid=PMC5016661&req=5

Figure 3: Structure of the oligomeric prepore of FraC bound to DOPC vesicles.a, representative image of FraC in DOPC vesicles obtained by cryo-EM. Top and side views of the protein oligomers were selected (red squares) for subsequent classification analysis. The scale bar corresponds to 100 nm. b, density map projections (top row) and two-dimensional class-averaged particles (bottom row) employed to build a three-dimensional model of the protein oligomer (see below). c, set of particles obtained by maximum-likelihood (ML2D, top row) and hierarchical clustering (CL2D, bottom row) procedures. d, top view, and e, side view of the three-dimensional model of the prepore of FraC bound to vesicles of DOPC. The atomic model of FraC was built as an octamer using the coordinates of the protomer of FraC prior to pore formation (entry code 4TSL).
Mentions: To visualize the structure of membrane-bound FraC, vitrified samples of toxin-treated DOPC liposomes were imaged by cryo-EM. Ring-shaped particles covering the lipid vesicles were attributed to protein oligomers (Fig. 3A). A total of 1,562 top view and side view images were selected to build a three-dimensional model of the protein oligomer. The model was built by common-lines procedures, followed by iterative refinement using projection matching of the class-averaged images and the density map projections (Fig. 3B). A second classification method based on maximum likelihood (41) and hierarchical clustering approaches (Fig. 3C) (42) rendered images similar to those used to generate the final density map.

View Article: PubMed Central - PubMed

ABSTRACT

Pore-forming toxins (PFTs) are cytolytic proteins belonging to the molecular warfare apparatus of living organisms. The assembly of the functional transmembrane pore requires several intermediate steps ranging from a water-soluble monomeric species to the multimeric ensemble inserted in the cell membrane. The non-lytic oligomeric intermediate known as prepore plays an essential role in the mechanism of insertion of the class of β-PFTs. However, in the class of α-PFTs, like the actinoporins produced by sea anemones, evidence of membrane-bound prepores is still lacking. We have employed single-particle cryo-electron microscopy (cryo-EM) and atomic force microscopy to identify, for the first time, a prepore species of the actinoporin fragaceatoxin C bound to lipid vesicles. The size of the prepore coincides with that of the functional pore, except for the transmembrane region, which is absent in the prepore. Biochemical assays indicated that, in the prepore species, the N terminus is not inserted in the bilayer but is exposed to the aqueous solution. Our study reveals the structure of the prepore in actinoporins and highlights the role of structural intermediates for the formation of cytolytic pores by an α-PFT.

No MeSH data available.


Related in: MedlinePlus