Structural basis for self-assembly of a cytolytic pore lined by protein and lipid

Abstract

Pore-forming toxins (PFT) are water-soluble proteins that possess the remarkable ability to self-assemble on the membrane of target cells, where they form pores causing cell damage. Here, we elucidate the mechanism of action of the haemolytic protein fragaceatoxin C (FraC), a α-barrel PFT, by determining the crystal structures of FraC at four different stages of the lytic mechanism, namely the water-soluble state, the monomeric lipid-bound form, an assembly intermediate and the fully assembled transmembrane pore. The structure of the transmembrane pore exhibits a unique architecture composed of both protein and lipids, with some of the lipids lining the pore wall, acting as assembly cofactors. The pore also exhibits lateral fenestrations that expose the hydrophobic core of the membrane to the aqueous environment. The incorporation of lipids from the target membrane within the structure of the pore provides a membrane-specific trigger for the activation of a haemolytic toxin.

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

“…The three-dimensional (3D) solution structures of four actinoporins have been solved: StI (García-Linares et al 2013), StII (Mancheno et al 2003), equinatoxin II (EqtII) from Actinia equina (Athanasiadis et al 2001;Hinds et al 2002) and fragaceatoxin C (FraC) from Actinia fragacea (Mechaly et al 2011;Tanaka et al 2015). The comparison of these structures in solution show a similar 3D fold formed by a central rigid and compact core consisting of two β sheets.…”

“…These studies revealed the existence of multiple sites for lipid binding (Tanaka et al 2015); in fact two of these sites (L2 and L3) were considered to be initial binding sites similar to the POC binding site described for StII (Mancheno et al 2003), while L4 and L5 were hypothesized to be sites of low affinity for POC or perhaps highaffinity binding sites for lipids with headgroups other than POC (Tanaka et al 2015).…”

“…Thus, the pore is constructed from the successive addition of N-termini of various protomers and lipid molecules (Antonini et al 2014). In contrast, there is debate about the possible existence of prepore structures in the mechanism of actinoporins (Mancheno et al 2003(Mancheno et al , 2006Mechaly et al 2011;Tanaka et al 2015). However, there is no evidence showing that such prepore structures can evolve into a conducting channel (Rojko et al 2013(Rojko et al , 2015.…”

“…Three models have been proposed to describe the pore structure formed by actinoporins in membrane, the toroidal pore (Alvarez-Valcarcel et al 2001), the conical pore (Mechaly et al 2011) and the hybrid pore (Tanaka et al 2015). The toroidal pore model describes a channel formed by three or four protein monomers lined by non-bilayer forming lipids (Alvarez-Valcarcel et al 2001).…”

“…The toroidal pore model describes a channel formed by three or four protein monomers lined by non-bilayer forming lipids (Alvarez-Valcarcel et al 2001). In contrast, the conic pore hypothesis assumes that the pore is formed by nine toxin protomers, excluding the involvement of lipids in the structure, while the hybrid pore proposal assumes that the pore is composed of eight monomers with lipid participation (Tanaka et al 2015).…”