Membrane insertion of the N-terminal alpha-helix of equinatoxin II, a sea anemone cytolytic toxin.
Equinatoxin II (Eqt-II) is a member of the actinoporins, a unique family of cytotoxins comprising 20 kDa pore-forming proteins isolated from sea anemones. Actinoporins bind preferentially to lipid membranes containing sphingomyelin, and create cation-selective pores by oligomerization of three to four monomers. Previous studies have shown that regions of Eqt-II crucial for its cytolytic mechanism are an exposed aromatic cluster and the N-terminal region containing an amphipathic alpha-helix. In the present study, we have investigated the transfer of the N-terminal alpha-helix into the lipid membrane by the use of three mutants containing an additional tryptophan residue in different positions within the amphipathic alpha-helix (Ile18-->Trp, Val22-->Trp and Ala25-->Trp). The interaction of the mutants with different model systems, such as lipid monolayers, erythrocytes and ghost membranes, was extensively characterized. Intrinsic fluorescence measurements and the use of vesicles containing brominated phospholipids indicated a deep localization of the N-terminal amphipathic helix in the lipid bilayer, except for the case of Val22-->Trp. This mutant is stabilized in a state immediately prior to final pore formation. The introduction of additional tryptophan residues in the sequence of Eqt-II has proved to be a suitable approach to monitor the new environments that surround defined regions of the molecule upon membrane interaction.
Unidad de Biofísica (CSIC-UPV/EHU) and Departamento de Bioquímica y Biología Molecular, Universidad del País Vasco, Apartado 644, 48080 Bilbao, Spain. ion.gutierrez@bf.uni-lj.si
Ion
I
Gutiérrez-Aguirre
Ariana
A
Barlic
Zdravko
Z
Podlesek
Peter
P
Macek
Gregor
G
Anderluh
Juan M
JM
González-Mañas
D000818:Animals; D001923:Brain Chemistry; D002417:Cattle; D003064:Cnidarian Venoms / Q000737:chemistry / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D004530:Egg Yolk / Q000737:chemistry; D004912:Erythrocytes / Q000187:drug effects; D006461:Hemolysis / Q000187:drug effects; D006736:Horses / Q000097:blood; D008567:Membranes, Artificial; D009154:Mutation / Q000235:genetics; D010455:Peptides / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010713:Phosphatidylcholines / Q000378:metabolism; D017434:Protein Structure, Tertiary; D012615:Sea Anemones / Q000737:chemistry*; D013050:Spectrometry, Fluorescence / Q000379:methods; D013109:Sphingomyelins / Q000378:metabolism
D003064:Cnidarian Venoms; D008567:Membranes, Artificial; D010455:Peptides; D010713:Phosphatidylcholines; D013109:Sphingomyelins; C012307:equinatoxin