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

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.

Disrupting a key hydrophobic pair in the oligomerization interface of the actinoporins impairs their pore‐forming activity

“…55 The dimer interface obtained by Tanaka et al 8 is been described as the basis for the octameric pore observed in the FraC structure and, more recently, for an octameric prepore structure of this toxin. 25 However, from our results and previous published data, 16 we cannot rule out the possibility that this dimeric interface can nucleate up to 8 units by the sequential addition of single or dimeric units to complete a final ring-like assembly. EqtII was described to be organized in the cell surface as a mixture of oligomeric species mostly including those formed by even number of units.…”

“…Moreover, it would be possible that different intermediate oligomeric states could form arc‐like toroidal pores with one edge filled by protein complexes and the other with bend lipids, similar to those observed in the MACPF/CDC protein superfamily or for the Bcl2 protein Bax . Further studies should confirm this hypothesis since current high quality data obtained by electron microscopy or atomic force microscopy at high protein concentration do not support the formation of arc‐like structures in the mechanism of action of actinoporins …”

“…The exact sequence of events that take place during the assembly of actinoporins and functionally relevant intermediates in the membrane remains under debate. Some studies suggest the relevance of protein–protein interactions while others assume that there is no need for such protein interfaces to stabilize oligomeric intermediates for the final pore assembly . However, evidence supports the structural and functional importance of dimeric structures in the transition of the monomer structure from solution to the pore state …”

“…4,23 There are mainly two hypothetical models under debate which describe the architecture of the pore formed by these toxins: the classical toroidal pore [11][12][13]24 and the hybrid protein-lipid pore. 8,25 These models differ in the stoichiometry of the pore and in the relevance of the protein-protein interactions for pore stability. The toroidal pore model was initially proposed and is mainly based on functional studies with sticholysins and EqtII.…”

Biophysical and biochemical strategies to understand membrane binding and pore formation by sticholysins, pore-forming proteins from a sea anemone

“…The monomeric units then oligomerize to form pores in which the N-terminal α-helix lines the channel walls in conjunction with lipids (Tanaka et al 2015). Some studies suggest the relevance of protein-protein interactions (Mechaly et al 2011;Tanaka et al 2015;Morante et al 2016), while others assume that there is no need for such protein interfaces to stabilize oligomeric intermediates for the final pore assembly (Mancheno et al 2003(Mancheno et al , 2006). …”