1.C.12.1.1
Perfringolysin O, PFO. In the formation of the pore forming toxin, the elongated toxin monomer binds stably to the membrane in an "end-on" orientation, with its long axis approximately perpendicular to the plane of the membrane bilayer (Ramachandran et al. 2005). This orientation is largely retained, even after monomers associate to form an oligomeric prepore complex. The domain 3 (D3) polypeptide segments that ultimately form transmembrane beta-hairpins remain far above the membrane surface in both the membrane-bound monomer and prepore oligomer. Upon pore formation, these segments enter the bilayer, whereas D1 moves to a position that is substantially closer to the membrane. Therefore, the extended D2 beta-structure that connects D1 to membrane-bound D4 appears to bend or otherwise reconfigure during the prepore-to-pore transition of the perfringolysin O oligomer (Ramachandran et al. 2005). The prepore to pore transition has been visualized by electron microscopy (Dang et al. 2005). Phosphatidylcholine in the outer leaflet increases the cholesterol concentration required to induce PFO binding while phosphatidylethanolamine and phosphatidylserine in the inner leaflet of asymmetric vesicles stabilized the formation of a deeply inserted conformation that does not form pores, even though it contains transmembrane segments (Lin and London 2014). This conformation may represent an important intermediate stage in PFO pore formation.  Cholesterol recognition, oligomerization, and the conformational changes involved in pore formation have been reviewed (Johnson and Heuck 2014), and the involvement of the D1 domain in structural transitions leading to pore formation has been studied (Kacprzyk-Stokowiec et al. 2014). Interaction of PFO with cholesterol is sufficient to initiate an irreversible sequence of coupled conformational changes that extend throughout the toxin molecule and induce pore formation (Heuck et al. 2007).  Once this transmembrane beta-barrel protein is inserted, PFO assembles into pore-forming oligomers containing 30-50 PFO monomers. These form a pore of up to 300 Å, far exceeding the size of most other proteinaceous pores.  Decreasing the length of the β-strands causes the pore to shrink (Lin et al. 2015). Site-directed mutagenesis data combined with binding studies performed with different sterols, and molecular modeling are beginning to shed light on the interaction with cholesterol (Savinov and Heuck 2017). Fine-tuning of the stability of beta-strands by Y181 in perfringolysin O directs the prepore to pore transition (Kulma et al. 2019)c. Cholesterol-specific binding motifs in perfringolysin O have been identified (Šakanović et al. 2024). Loading fluoroquinolones into Escherichia coli outer membrane vesicles provides an  antibiotic delivery platform (Wu et al. 2024).