title: Structure and activity of the N-terminal region of the eukaryotic cytolysin equinatoxin II.
abstract: The actinoporins are a family of proteins from sea anemones that lyse cells by forming pores in cell membranes. Sphingomyelin plays an important role in their lytic activity, with membranes lacking this lipid being resistant to these toxins. Pore formation by the actinoporin equinatoxin II (EqTII) proceeds by membrane binding via a surface rich in aromatic residues, followed by translocation of the N-terminal region to the membrane and, finally, across the bilayer to form a functional pore. A key feature of this mechanism is the ability of the N-terminal region to form a stable, bilayer-spanning helix in the membrane, which in turn requires dissociation of the N-terminus from the bulk of the protein and significant extension of the N-terminal helix of native EqTII. In this study the structures of three peptides corresponding to residues 11-29, 11-32, and 1-32, respectively, of EqTII have been investigated by high-resolution nuclear magnetic resonance and Fourier transform infrared spectroscopy. The 32-residue peptide lacks ordered secondary structure in water, but residues 6-28 form a helix in dodecylphosphocholine micelles. Although this helix is long enough to span a bilayer membrane, this peptide and the shorter analogues display limited permeabilizing activity in large unilamellar vesicles and very weak hemolytic activity in human red blood cells. Thus, while the N-terminal region has the structural features required for this unusual mechanism of pore formation, the lack of activity of the isolated N-terminus shows that the bulk of the protein is essential for efficient pore formation by facilitating initial membrane binding, interacting with sphingomyelin, or stabilizing the oligomeric pore.
authors.affiliation: The Walter & Eliza Hall Institute of Medical Research, 1G Royal Parade, Parkville, Victoria 3050, Australia.
authors.forename: Alison
authors.initials: A
authors.lastname: Drechsler
authors.forename: Cristina
authors.initials: C
authors.lastname: Potrich
authors.forename: Jennifer K
authors.initials: JK
authors.lastname: Sabo
authors.forename: Mattia
authors.initials: M
authors.lastname: Frisanco
authors.forename: Graziano
authors.initials: G
authors.lastname: Guella
authors.forename: Mauro
authors.initials: M
authors.lastname: Dalla Serra
authors.forename: Gregor
authors.initials: G
authors.lastname: Anderluh
authors.forename: Frances
authors.initials: F
authors.lastname: Separovic
authors.forename: Raymond S
authors.initials: RS
authors.lastname: Norton
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002462:Cell Membrane / Q000378:metabolism; D003064:Cnidarian Venoms / Q000737:chemistry* / Q000378:metabolism*; D005057:Eukaryotic Cells / Q000378:metabolism*; D006461:Hemolysis; D006801:Humans; D008051:Lipid Bilayers / Q000378:metabolism; D008562:Membrane Glycoproteins / Q000378:metabolism*; D008823:Micelles; D008958:Models, Molecular; D008969:Molecular Sequence Data; D019906:Nuclear Magnetic Resonance, Biomolecular; D054353:Perforin; D010767:Phosphorylcholine / Q000031:analogs & derivatives / Q000737:chemistry; D052899:Pore Forming Cytotoxic Proteins; D017434:Protein Structure, Tertiary; D012615:Sea Anemones / Q000737:chemistry; D017550:Spectroscopy, Fourier Transform Infrared; D013109:Sphingomyelins / Q000378:metabolism
chemical_list: D003064:Cnidarian Venoms; D008051:Lipid Bilayers; D008562:Membrane Glycoproteins; D008823:Micelles; D052899:Pore Forming Cytotoxic Proteins; D013109:Sphingomyelins; D010767:Phosphorylcholine; D054353:Perforin; C028810:dodecylphosphocholine; C012307:equinatoxin