Dissecting the mechanism of action of actinoporins. Role of the N-terminal amphipathic α-helix in membrane binding and pore activity of sticholysins I and II

Abstract

Actinoporins sticholysin I and sticholysin II (St I, St II) are proposed to lyse model and biomembranes via toroidal pore formation by their N-terminal domain. Based on the hypothesis that peptide fragments can reproduce the structure and function of this domain, the behavior of peptides containing St I residues 12-31 (StI12-31), St II residues 11-30 (StII11-30), and its TOAC-labeled analogue (N-TOAC-StII11-30) was examined. Molecular modeling showed a good match with experimental structures, indicating amphipathic α-helices in the same regions as in the toxins. CD spectra revealed that the peptides were essentially unstructured in aqueous solution, acquiring α-helical conformation upon interaction with micelles and large unilamellar vesicles (LUV) of variable lipid composition. Fluorescence quenching studies with NBD-containing lipids indicated that N-TOAC-StII11-30's nitroxide moiety is located in the membranes polar head group region. Pyrene-labeled phospholipid inter-leaflet redistribution suggested that the peptides form toroidal pores, according to the mechanism of action proposed for the toxins. Binding occurred only to negatively charged LUV, indicating the importance of electrostatic interactions; in contrast the peptides bound to both negatively charged and zwitterionic micelles, pointing to a lesser influence of these interactions. In addition, differences between bilayers and micelles in head group packing and in curvature led to differences in peptide-membrane interaction. We propose that the peptides topography in micelles resembles that of the toxins in the toroidal pore. The peptides mimicked the toxins permeabilizing activity, St II peptides being more effective than StI12-31. To our knowledge, this is the first demonstration that differences in the toxins N-terminal amphipathic α-helix play a role in the difference between St I and St II activities.

Fig 1. Peptides modeled structures match toxins high resolution structures.

Superposition of the rainbow-colored structure of St I (A) [5] and St II (B) [4], with the black-colored structure of StI_12-31 and StII_11-30 obtained by the PEP-FOLD program, respectively. Each toxin is shown at two orientations. Peptide and toxin sequence alignment and bars of RMSD values (between 0 and 1 Å) of the superimposed structures. RMSD > 1 not shown.

Fig 2. Atomic representation of the models of peptides StI_12-31 and StII_11-30 obtained by the PEP-FOLD program.

StI_12-31 (A) and StII_11-30 (B) structures. Colors of residues types: Non-polar (black), polar uncharged (green), negatively charged (red) and positively charged (blue).

Fig 3. CD spectra of the peptides and binding isotherms.

CD spectra of 12 μM StI_12-31 (A), StII_11-30 (B) and N-TOAC-StII_11-30 (C) in solution and in the presence of 10 mM LPC micelles or 0.4 mM POPC or POPC:POPA (90:10) LUV, pH 7.0. (D) Peptide-membrane binding isotherms of StII_11-30 obtained from normalized [θ] values as a function of [Lipid]/[Peptide] ratio.

Fig 4. NDB fluorescence emission quenching by TOAC residue of N-TOAC-StII_11-30.

(A) Fluorescence emission spectra of 0.5 μM DPPE-NBD incorporated in 50 μM DPPC:DMPA (90:10) LUV in the presence of increasing concentrations of N-TOAC-StII_11-30. (B) Normalized maximum fluorescence emission as a function of peptide concentration.

Fig 5. Lipid redistribution triggered by the peptides.

Normalized redistribution of 5 μM DOPE-Pyr between bilayer leaflets of 100 μM POPC:POPA:DOPE-Pyr (85:10:5) in the presence of increasing concentrations of StI_12-31 (A), StII_11-30 (B) and N-TOAC-StII_11-30 (C).

Fig 6. CF leakage from LUV triggered by peptide-membrane interaction.

(A) Kinetics of CF release from 20 μM POPC:POPA (90:10) LUV triggered by increasing StII_11-30 concentration. Total CF release after 50 minutes from LUV of variable lipid composition as function of peptide concentration. StI_12-31 (B), StII_11-30 (C) and, N-TOAC-StII_11-30 (D). The lines represent fittings of Hill’s equation to the experimental data.

Fig 7. Hemolytic activity of the peptides.

(A) Released hemoglobin from red blood cells by StI_12-31, StII_1-30, StII_11-30 and N-TOAC-StII_11-30. The last column displays the results for negative and positive controls. (B) Total percentage of hemoglobin released from red blood cells by the peptides.