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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.


Related in: MedlinePlus

Two alternative routes for pore formation in actinoporins. The binding of the water-soluble monomer to the cell or model membranes leads to a lytic (active) pore by at least two alternative routes, as shown in the figure. Top, formation of a non-lytic oligomer (prepore) precedes insertion into the membrane (20). Bottom, insertion of the N-terminal region into the membrane occurs prior to oligomerization of the functional pore (21).
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Figure 1: Two alternative routes for pore formation in actinoporins. The binding of the water-soluble monomer to the cell or model membranes leads to a lytic (active) pore by at least two alternative routes, as shown in the figure. Top, formation of a non-lytic oligomer (prepore) precedes insertion into the membrane (20). Bottom, insertion of the N-terminal region into the membrane occurs prior to oligomerization of the functional pore (21).

Mentions: A variety of membrane-bound species has been proposed in the mechanism of actinoporins (17–19). However, the nature of some of the intermediate species and the order at which they appear during pore formation remain unclear. Some studies have proposed that the protein subunits first assemble into an oligomeric prepore, followed by the concerted insertion of the N-terminal region in the lipid bilayer that gives rise to the functional pore (20). Other studies, on the contrary, have suggested that the α-helix inserts deeply in the membrane prior to the oligomerization step, and therefore, this model does not contemplate the appearance of stable prepores (Fig. 1) (21).


Identification of a Membrane-bound Prepore Species Clarifies the Lytic Mechanism of Actinoporins * ♦
Two alternative routes for pore formation in actinoporins. The binding of the water-soluble monomer to the cell or model membranes leads to a lytic (active) pore by at least two alternative routes, as shown in the figure. Top, formation of a non-lytic oligomer (prepore) precedes insertion into the membrane (20). Bottom, insertion of the N-terminal region into the membrane occurs prior to oligomerization of the functional pore (21).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC5016661&req=5

Figure 1: Two alternative routes for pore formation in actinoporins. The binding of the water-soluble monomer to the cell or model membranes leads to a lytic (active) pore by at least two alternative routes, as shown in the figure. Top, formation of a non-lytic oligomer (prepore) precedes insertion into the membrane (20). Bottom, insertion of the N-terminal region into the membrane occurs prior to oligomerization of the functional pore (21).
Mentions: A variety of membrane-bound species has been proposed in the mechanism of actinoporins (17–19). However, the nature of some of the intermediate species and the order at which they appear during pore formation remain unclear. Some studies have proposed that the protein subunits first assemble into an oligomeric prepore, followed by the concerted insertion of the N-terminal region in the lipid bilayer that gives rise to the functional pore (20). Other studies, on the contrary, have suggested that the α-helix inserts deeply in the membrane prior to the oligomerization step, and therefore, this model does not contemplate the appearance of stable prepores (Fig. 1) (21).

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