Photobleaching Reveals Heterogeneous Stoichiometry for Equinatoxin II Oligomers

Abstract

Equinatoxin II (EqtII), a sea anemone cytolysin, is known to oligomerize to form pores that spontaneously insert into membranes. Crystallographic and cryo-EM studies of structurally similar cytolysins offer contradictory evidence for pore stoichiometry. Here we used single-molecule photobleaching of fluorescently labeled EqtII to determine the stoichiometry of EqtII oligomers in supported lipid bilayers. A frequency analysis of photobleaching steps revealed a log-normal distribution of stoichiometries with a mean of 3.4±2.3 standard deviations. Comparison of our experimental data with simulations of fixed stoichiometries supports our observation of a heterogeneous distribution of EqtII oligomerization. These data are consistent with a model of EqtII stoichiometry where pores are on average tetrameric, but with large variation in the number of subunits in individual pores.

Toxicity of an α-Pore-forming Toxin Depends on the Assembly Mechanism on the Target Membrane as Revealed by Single Molecule Imaging

“…Even though from our experimental data we cannot estimate what oligomeric state(s) would potentially be a functional unit for pore formation, mathematical modeling also suggests that tetramers are the most stable form of EqtII in the membrane, in agreement with recent determination of the stoichiometry of EqtII oligomers in supported bilayers (33), the structure of Sticholysin II in monolayers (13), and contrary to the models based on higher oligomeric species proposed for Fragaceatoxin C (15). However, one essential difference with structural studies reported so far is the detection of multiple coexisting oligomeric forms, which underscores the power of our single molecule approach when studying dynamic processes.…”

“…Under these conditions, the number of particles per area was comparable for both mutants. Moreover, differences in concentration between both mutants are not expected to have significant effects on the kinetics of oligomerization or the formation of complexes as suggested by Baker et al (33).…”

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

“…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).…”

“…Intriguingly, membranes made of a single type of lipid are not susceptible to the lytic activity of the toxin (25,26,30). In contrast, mixtures of lipids containing SM and other lipids such as phosphatidylcholine (PC) or cholesterol often trigger pore formation.…”

Disrupting a key hydrophobic pair in the oligomerization interface of the actinoporins impairs their pore‐forming activity

“…The toroidal pore model was initially proposed and is mainly based on functional studies with sticholysins and EqtII . Kinetic and electrophysiological studies and more recently single molecule imaging have revealed that predominantly 3‐4 monomers of these proteins form functional pores . Moreover, crystalografic data of a prepore like structure of StII in lipid monolayers shows that the units that form the assembly do not establish relevant protein–protein interactions .…”