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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.


Model for pore formation by FraC. A toxin monomer binds the membrane. The membrane promotes protein-protein interactions between monomers to produce a dimer (15) leading to prepore upon successive addition of monomer and/or dimers to the growing oligomer. In the prepore, the N-terminal α-helices are partially embedded in the membrane with their N termini exposed to the aqueous phase. The conversion to the transmembrane pore would be achieved by the concerted penetration and elongation of the helices across the lipid bilayer. The structures of the monomer, dimer, and pore were retrieved from the Protein Data Bank codes 3VWI, 4TSL, and 4TSY, respectively. The prepore illustrates a tentative model consistent with the presented in this study.
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Figure 8: Model for pore formation by FraC. A toxin monomer binds the membrane. The membrane promotes protein-protein interactions between monomers to produce a dimer (15) leading to prepore upon successive addition of monomer and/or dimers to the growing oligomer. In the prepore, the N-terminal α-helices are partially embedded in the membrane with their N termini exposed to the aqueous phase. The conversion to the transmembrane pore would be achieved by the concerted penetration and elongation of the helices across the lipid bilayer. The structures of the monomer, dimer, and pore were retrieved from the Protein Data Bank codes 3VWI, 4TSL, and 4TSY, respectively. The prepore illustrates a tentative model consistent with the presented in this study.

Mentions: Our results suggest a model where the N-terminal α-helices penetrate the bilayer in a concerted manner (Fig. 8), an alternative mechanism to that in which helix penetration occurs before protein oligomerization (21). Although pore formation by the successive insertion of single α-helices cannot be completely ruled out in membranes made of SM/DOPC (1:1), simple thermodynamic considerations suggest that would not be the case. The penetration of individual α-helices containing a large number of charged residues (FraC displays three Asp and one Glu in this region) in the hydrophobic core of biological membranes would be strongly disfavored (49–51).


Identification of a Membrane-bound Prepore Species Clarifies the Lytic Mechanism of Actinoporins * ♦
Model for pore formation by FraC. A toxin monomer binds the membrane. The membrane promotes protein-protein interactions between monomers to produce a dimer (15) leading to prepore upon successive addition of monomer and/or dimers to the growing oligomer. In the prepore, the N-terminal α-helices are partially embedded in the membrane with their N termini exposed to the aqueous phase. The conversion to the transmembrane pore would be achieved by the concerted penetration and elongation of the helices across the lipid bilayer. The structures of the monomer, dimer, and pore were retrieved from the Protein Data Bank codes 3VWI, 4TSL, and 4TSY, respectively. The prepore illustrates a tentative model consistent with the presented in this study.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 8: Model for pore formation by FraC. A toxin monomer binds the membrane. The membrane promotes protein-protein interactions between monomers to produce a dimer (15) leading to prepore upon successive addition of monomer and/or dimers to the growing oligomer. In the prepore, the N-terminal α-helices are partially embedded in the membrane with their N termini exposed to the aqueous phase. The conversion to the transmembrane pore would be achieved by the concerted penetration and elongation of the helices across the lipid bilayer. The structures of the monomer, dimer, and pore were retrieved from the Protein Data Bank codes 3VWI, 4TSL, and 4TSY, respectively. The prepore illustrates a tentative model consistent with the presented in this study.
Mentions: Our results suggest a model where the N-terminal α-helices penetrate the bilayer in a concerted manner (Fig. 8), an alternative mechanism to that in which helix penetration occurs before protein oligomerization (21). Although pore formation by the successive insertion of single α-helices cannot be completely ruled out in membranes made of SM/DOPC (1:1), simple thermodynamic considerations suggest that would not be the case. The penetration of individual α-helices containing a large number of charged residues (FraC displays three Asp and one Glu in this region) in the hydrophobic core of biological membranes would be strongly disfavored (49–51).

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.