Structural Bases for the Involvement of Phosphatidylinositol-4,5-bisphosphate in the Internalization of the Cell-Penetrating Peptide Penetratin

Cell-penetrating peptides cross cell membranes through various parallel internalization pathways. Herein, we analyze the role of the negatively charged lipid phosphatidylinositol-4,S-bisphosphate (PI(4,5)P-2) in the internalization of Penetratin. Contributions of both inner leaflet and outer leaflet pools of PI(4,5)P-2 were revealed by quantifying the internalization of Penetratin in cells treated with PI(4,5)P-2 binders. Studies on model systems showed that Penetratin has a strong affinity for PI(4,S)P-2 and interacts selectively with this lipid, even in the presence of other negatively charged lipids, as demonstrated by affinity photo-crosslinking experiments. Differential scanning calorimetry experiments showed that Penetratin induces lateral segregation in PI(4,5)P-2-containing liposomes, which was confirmed by coarse-grained molecular dynamics simulations. NMR experiments indicated that Penetratin adopts a stabilized helical conformation in the presence of PI(4,5)P-2-containing membranes, with an orientation parallel to the bilayer plane, which was also confirmed by all-atom simulations. NMR and photo-crosslinking experiments also suggest a rather shallow insertion of the peptide in the membrane. Put together, our findings suggest that PI(4,S)P-2 is a privileged interaction partner for Penetratin and that it plays an important role in Penetratin internalization.