Interaction between the Marine Sponge Cyclic Peptide Theonellamide A and Sterols in Lipid Bilayers As Viewed by Surface Plasmon Resonance and Solid-State 2H Nuclear Magnetic Resonance

Theonellamides (TNMs) are members of a distinctive family of antifungal and cytotoxic bicyclic dodecapeptides isolated from the marine sponge Theonella sp. Recently, it has been shown that TNMs recognize 3 beta-hydroxysterol-containing membranes, induce glucan overproduction, and damage cellular membranes. However, to date, the detailed mode of sterol binding at a molecular level has not been determined. In this study, to gain insight into the mechanism of sterol recognition of TNM in lipid bilayers, surface plasmon resonance (SPR) experiments and solid-state deuterium nuclear magnetic resonance (H-2 NMR) measurements were performed on theonellamide A (TNM-A). SPR results revealed that the incorporation of 10 mol % cholesterol or ergosterol into 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) membranes significantly enhances the affinity of the peptide for the membrane, particularly in the initial binding to the membrane surface. These findings, together with the fact that binding of TNM-A to epicholesterol (3 alpha-cholesterol)-containing liposomes and pure POPC liposomes was comparably weak, confirmed the preference of the peptide for the 3 beta-hydroxysterol-containing membranes. To further establish the formation of the complex of TNM-A with 3 beta-hydroxysterols in lipid bilayers, solid-state H-2 NMR measurements were conducted using deuterium-labeled cholesterol, ergosterol, or epicholesterol. The H-2 NMR spectra showed that TNM-A significantly inhibits the fast rotational motion of cholesterol and ergosterol, but not epicholesterol, therefore verifying the direct complexation between TNM-A and 3 beta-hydroxysterols in lipid bilayers. This study demonstrates that TNM-A directly recognizes the 3 beta-OH moiety of sterols, which greatly facilitates its binding to bilayer membranes.