Cornelio Kimberly, Espiritu Rafael Atillo, Todokoro Yasuto, Hanashima Shinya, Kinoshita Masanao, Matsumori Nobuaki, Murata Michio, Nishimura Shinichi, Kakeya Hideaki, Yoshida Minoru, Matsunaga Shigeki
Department of Chemistry, Graduate School of Science, Osaka University, Osaka 563-0043, Japan; Lipid Active Structure Project, ERATO, Japan Science and Technology Agency, Osaka 560-0043, Japan.
Department of Chemistry, Graduate School of Science, Osaka University, Osaka 563-0043, Japan.
Bioorg Med Chem. 2016 Nov 1;24(21):5235-5242. doi: 10.1016/j.bmc.2016.08.043. Epub 2016 Aug 24.
Theonellamide A (TNM-A) is an antifungal bicyclic dodecapeptide isolated from a marine sponge Theonella sp. Previous studies have shown that TNM-A preferentially binds to 3β-hydroxysterol-containing membranes and disrupts membrane integrity. In this study, several H NMR-based experiments were performed to investigate the interaction mode of TNM-A with model membranes. First, the aggregation propensities of TNM-A were examined using diffusion ordered spectroscopy; the results indicate that TNM-A tends to form oligomeric aggregates of 2-9 molecules (depending on peptide concentration) in an aqueous environment, and this aggregation potentially influences the membrane-disrupting activity of the peptide. Subsequently, we measured the H NMR spectra of TNM-A with sodium dodecyl sulfate-d (SDS-d) micelles and small dimyristoylphosphatidylcholine (DMPC)-d/dihexanoylphosphatidylcholine (DHPC)-d bicelles in the presence of a paramagnetic quencher Mn. These spectra indicate that TNM-A poorly binds to these membrane mimics without sterol and mostly remains in the aqueous media. In contrast, broader H signals of TNM-A were observed in 10mol% cholesterol-containing bicelles, indicating that the peptide efficiently binds to sterol-containing bilayers. The addition of Mn to these bicelles also led to a decrease in the relative intensity and further line-broadening of TNM-A signals, indicating that the peptide stays near the surface of the bilayers. A comparison of the relative signal intensities with those of phospholipids showed that TNM-A resides in the lipid-water interface (close to the C2' portion of the phospholipid acyl chain). This shallow penetration of TNM-A to lipid bilayers induces an uneven membrane curvature and eventually disrupts membrane integrity. These results shed light on the atomistic mechanism accounting for the membrane-disrupting activity of TNM-A and the important role of cholesterol in its mechanism of action.
