Abstract
Actinoporin equinatoxin II (EqtII) is an archetypal example of α-helical pore-forming toxins that porate cellular membranes by the use of α-helices. Previous studies proposed several steps in the pore formation: binding of monomeric protein onto the membrane, followed by oligomerization and insertion of the N-terminal α-helix into the lipid bilayer. We studied these separate steps with an EqtII triple cysteine mutant. The mutant was engineered to monitor the insertion of the N terminus into the lipid bilayer by labeling Cys-18 with a fluorescence probe and at the same time to control the flexibility of the N-terminal region by the disulfide bond formed between cysteines introduced at positions 8 and 69. The insertion of the N terminus into the membrane proceeded shortly after the toxin binding and was followed by oligomerization. The oxidized, non-lytic, form of the mutant was still able to bind to membranes and oligomerize at the same level as the wild-type or the reduced form. However, the kinetics of the N-terminal helix insertion, the release of calcein from erythrocyte ghosts, and hemolysis of erythrocytes was much slower when membrane-bound oxidized mutant was reduced by the addition of the reductant. Results show that the N-terminal region needs to be inserted in the lipid membrane before the oligomerization into the final pore and imply that there is no need for a stable prepore formation. This is different from β-pore-forming toxins that often form β-barrel pores via a stable prepore complex.
Highlights
Actinoporins are pore-forming toxins that damage cellular membranes by ␣-helices
We have previously shown that labeling of the EqtIII18C was sufficiently good and that this residue was transferred to the lipid bilayer during the pore formation [25]
The C terminus is located on the side opposite that occupied by regions of the molecule that participate in membrane binding [18, 19, 36] or formation of the final pore [23, 25], and any labeling does not interfere with various steps in pore formation
Summary
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. Previous studies proposed several steps in the pore formation: binding of monomeric protein onto the membrane, followed by oligomerization and insertion of the N-terminal ␣-helix into the lipid bilayer. The pore was proposed to contain lipid molecules from the membrane in a toroidal arrangement [27, 28] This model of the final pore was recently challenged by the crystal structure of fragaceatoxin (FraC), an actinoporin from the sea anemone Actinia fragacea [12]. The inactive mutant, which has a membrane-penetrating region attached to the core of the molecule by an introduced disulfide bond, still binds to the lipid membrane and is able to oligomerize on the membrane surface When such a membrane-bound molecule was treated with reductant dithiothreitol (DTT), the kinetics of the N-terminal insertion and pore forming ability was much slower as compared with the prereduced form. Results imply that stable prepores, observed in almost all cases of -PFT, are not needed in the pore formation of actinoporins
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