Structural Insights into the Oligomerization and Architecture of Eukaryotic Membrane Pore-Forming Toxins

Abstract

Pore-forming toxins (PFTs) are proteins that are secreted as soluble molecules and are inserted into membranes to form oligomeric transmembrane pores. In this paper, we report the crystal structure of Fragaceatoxin C (FraC), a PFT isolated from the sea anemone Actinia fragacea, at 1.8 Å resolution. It consists of a crown-shaped nonamer with an external diameter of about 11.0 nm and an internal diameter of approximately 5.0 nm. Cryoelectron microscopy studies of FraC in lipid bilayers reveal the pore structure that traverses the membrane. The shape and dimensions of the crystallographic oligomer are fully consistent with the membrane pore. The FraC structure provides insight into the interactions governing the assembly process and suggests the structural changes that allow for membrane insertion. We propose a nonameric pore model that spans the membrane by forming a lipid-free α-helical bundle pore.

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

“…The conical pore is depicted as being funnelshaped and characterized by an upper diameter of 5 nm and a lower one of 1.5 nm, with the latter diameter similar to that previously calculated for EqtII and Sts (Belmonte et al 1993;Tejuca et al 2001). Initially, the nonameric structure of FraC in the presence of detergent was considered to be a prepore state because it did not exhibit the N-terminal helices detached from the protein body (Mechaly et al 2011); however, this model was later reconsidered by its own authors, taking into account that it constitutes an oligomer with a low probability to evolve to a competent lytic structure due to steric problems (Tanaka et al 2015).…”

“…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.…”

“…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 conical pore model was deduced from the structural determinations of the crystals of a FraC nonameric structure in the presence of detergent and the cryo-electron microscopy studies of FraC in the presence of large, unilamellar vesicles (LUV) composed of an equimolar mixture of dioleoylphosphatidylcholine (DOPC) and SM (Mechaly et al 2011). From these studies, it was proposed that the pores of actinoporins are composed of the α-helices from the Nterminus of nine monomers, without the involvement of membrane lipids.…”