Membrane Damage by an α-Helical Pore-forming Protein, Equinatoxin II, Proceeds through a Succession of Ordered Steps

Abstract

Background: Actinoporins are pore-forming toxins that damage cellular membranes by ␣-helices. Results: An engineered mutant of actinoporin equinatoxin II reveals sequential steps during pore formation. Conclusion: Pore formation is composed of a succession of ordered steps: fast membrane binding followed by the N-terminal region association with the membrane and oligomerization. Significance: Equinatoxin II pore formation does not require stable prepore intermediate as is often found in other poreforming toxins.

A Pore-Forming Toxin Requires a Specific Residue for Its Activity in Membranes with Particular Physicochemical Properties

“…Their globular structure presents a common fold based on a ␤-sandwich core flanked by two ␣-helices (17,18). The best characterized actinoporins are equinatoxin II (19), sticholysin II (20), and FraC (16). Actinoporins have been studied as potential immunotoxins (21) and serve as models to study protein-membrane interactions, a process of great relevance in neurodegenerative diseases (22,23).…”

“…In particular, sphingomyelin (SM) is a lipid that facilitates binding and pore formation (24), although this lipid is not strictly necessary to permeabilize model membranes (25). Once bound to the membrane, actinoporins oligomerize and transfer their N-terminal helical region toward the hydrophobic core of the membrane, although the exact sequence of events is still under debate (19,26,27). During the insertion step, the N-terminal ␣-helix spans the entire thickness of the membrane and lines the wall of the octameric pore as recently shown by x-ray crystallography (28).…”

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

“…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.…”

“…Understanding the assembly mechanism of actinoporins has dramatically increased after determination of the crystal structure of FraC, which complemented a body of biochemical and biophysical data previously obtained by different groups (Tejuca et al 1996;Alvarez-Valcarcel et al 2001;Hong et al 2002;Alvarez et al 2003;Anderluh et al 2003;Rojko et al 2013;Antonini et al 2014;Subburaj et al 2015). However, the exact sequence of events that takes place during the assembly of actinoporins and the identification of the functionally relevant intermediates in the membrane remain under debate.…”

“…However, the exact sequence of events that takes place during the assembly of actinoporins and the identification of the functionally relevant intermediates in the membrane remain under debate. It is now generally accepted that actinoporins bind to membranes mainly as monomers (Tejuca et al 1996;Barlic et al 2004;Bakrac et al 2008;Pedrera et al 2014;Rojko et al 2014) and then undergo a conformational change that involves only the N-terminal segment (Rojko et al 2013(Rojko et al , 2015Tanaka et al 2015). 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).…”