The equinatoxin N‐terminus is transferred across planar lipid membranes and helps to stabilize the transmembrane pore

Abstract

Equinatoxin II is a cytolytic protein isolated from the sea anemone Actinia equina. It is a member of the actinoporins, a family of eukaryotic pore‐forming toxins with a unique mechanism of pore formation. Equinatoxin II is a 20 kDa cysteineless protein, with sphingomyelin‐dependent activity. Recent studies showed that the N‐terminal region of the molecule requires conformational flexibility during pore formation. An understanding of the N‐terminal position in the final pore and its role in membrane insertion and pore stability is essential to define the precise molecular mechanism of pore formation. The formation of pores and their electrophysiologic characteristics were studied with planar lipid membranes. We show that amino acids at positions 1 and 3 of equinatoxin II are exposed to the lumen of the pore. Moreover, sulfhydryl reagents and a hexa‐histidine tag attached to the N‐terminus revealed that the N‐terminus of the toxin extends through the pore to the other (trans) side of the membrane and that negatively charged residues inside the pore are crucial to define the electrophysiologic characteristics of the channel. Finally, we detected a new, less stable, state with a lower conductance by using a deletion mutant in which the first five N‐terminal amino acids were removed. We propose that the first five amino acids help to anchor the amphipathic helix on the trans side of the membrane and consequently stabilize the final transmembrane pore.

A Toxin-based Probe Reveals Cytoplasmic Exposure of Golgi Sphingomyelin

“…His-tagged EqtII-GFP is less hemolytically active than the wild-type EqtII, but similar to EqtII that is simply His-tagged (supplemental Fig. S1B), in agreement with the published data (21). Surface plasmon resonance (SPR) experiments showed much greater binding of EqtII-GFP to membranes composed of porcine brain SM and 1,2-dioleoyl-sn-glyc-ero-3-phosphocholine (DOPC) as compared with membranes containing only DOPC (Fig.…”

“…All other chemicals were from Sigma unless stated otherwise. Recombinant EqtII, mutant EqtII 8 -69, and histidine-tagged variant were prepared in Escherichia coli as described previously (20,21). The fusion protein composed of EqtII and GFP (EqtII-GFP) was expressed as a His 6 fusion protein in an E. coli Rosetta (DE3) pLysS strain as detailed in supplemental data.…”

Molecular Determinants of Sphingomyelin Specificity of a Eukaryotic Pore-forming Toxin

“…All other chemicals were from Sigma unless stated otherwise. Recombinant EqtII, mutants and histidine-tagged variants were prepared in Escherichia coli as described previously (19,32). The tryptophan fluorescence and far UV circular dichroism spectra showed that mutations did not cause large structural changes.…”

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

“…The N-terminal region, residues 1-28, was shown to be the only part of the molecule that undergoes a conformational change, and its flexibility is essential for the formation of the final pore (19,(22)(23)(24). After membrane binding, the N terminus is detached from the protein core and translocated into the lipidwater interface (22,25) and finally across the lipid membrane, where it participates in formation of the final pore (25,26). The molecular details about the final transmembrane pore of actinoporins are still lacking.…”