Imaging the Lipid-Phase-Dependent Pore Formation of Equinatoxin II in Droplet Interface Bilayers

Abstract

Using phase-separated droplet interface bilayers, we observe membrane binding and pore formation of a eukaryotic cytolysin, Equinatoxin II (EqtII). EqtII activity is known to depend on the presence of sphingomyelin in the target membrane and is enhanced by lipid phase separation. By imaging the ionic flux through individual pores in vitro, we observe that EqtII pores form predominantly within the liquid-disordered phase. We observe preferential binding of labeled EqtII at liquid-ordered/liquid-disordered domain boundaries before it accumulates in the liquid-disordered phase.

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

“…The amino acids Glu2, Phe15, Arg52, Pro80 and Trp111 replaced by Cys in the site-directed mutants are indicated in the figure. Images were produced with the UCSF Chimera program for Windows (Pettersen et al 2004) According to this model, the transition of the N-terminus to the bilayer occurs in a non-concerted manner, prior to or concurrently with the oligomerization process, as has been proposed for StII (Antonini et al 2014) or EqtII (Rojko et al 2014). Thus, the pore is constructed from the successive addition of N-termini of various protomers and lipid molecules (Antonini et al 2014).…”

“…Although the joint presence of SM and Chol in membranes significantly increases the binding and permeabilizing activity of actinoporins, this enhancement has been related to the coexistence of lipid phases (Barlic et al 2004;Martinez et al 2007;Schon et al 2008;Rojko et al 2014;GarciaLinares et al 2015). In model membrane systems, the liquid ordered (Lo) domains occur usually when Chol associates with saturated glycerophospholipids or sphingophospholipids to render phospholipid-Chol complexes.…”

“…In particular, SM has emerged as the major partner of Chol for Lo phase formation, and ternary mixtures of dioleoylphosphatidylcholine (DOPC), SM and Chol are the most widely studied systems used to characterize the Lo/liquid-disordered (Ld) phase coexistence (de Almeida et al 2003). In this regard, the influence of Lo/Ld phase coexistence in bilayers and their equivalent Lo and liquid-expanded phases in monolayers have been the most studied systems in the actinoporin family (Barlic et al 2004;Martinez et al 2007;Schon et al 2008;Rojko et al 2014;Garcia-Linares et al 2015). For example, lateral defects related to the presence of Lo domains have been considered the preferential binding sites for EqtII (de Almeida et al 2003;Barlic et al 2004).…”

“…For example, lateral defects related to the presence of Lo domains have been considered the preferential binding sites for EqtII (de Almeida et al 2003;Barlic et al 2004). However, more recently it has been proposed for EqtII that the lateral interphases between the Lo and Ld phases act as primary binding sites before the toxin accumulates in the Ld phase, where the pore formation takes place predominantly (Rojko et al 2014).…”

“…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).…”