Identification of a membrane-bound prepore species clarifies the lytic mechanism of actinoporins

TL;DR

This study identifies and characterizes a membrane-bound prepore intermediate in actinoporins, providing new insights into the pore formation mechanism of alpha-pore-forming toxins using cryo-EM and AFM.

Contribution

First identification and structural characterization of a membrane-bound prepore in actinoporins, clarifying the pore assembly process.

Findings

The prepore size matches the pore except for the transmembrane region.

The N-terminus is exposed in the prepore, not inserted into the membrane.

Structural intermediates are crucial for pore formation in alpha-PFTs.

Abstract

Pore-forming toxins (PFT) 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 $\beta$-PFT. However, in the class of $\alpha$-PFT 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 (AFM) to identify, for the first time, a prepore species of the actinoporin fragaceatoxin C (FraC) bound to lipid vesicles. The size of the prepore coincides 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 exposed to the aqueous solution. Our study reveals the structure of the prepore complex in actinoporins, and highlights the role of structural intermediates for the formation of cytolytic pores by an $\alpha$-PFT.