Sterol-dependent membrane association of the marine sponge-derived bicyclic peptide Theonellamide A as examined by1H NMR.
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, several1H 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 the1H NMR spectra of TNM-A with sodium dodecyl sulfate-d25(SDS-d25) micelles and small dimyristoylphosphatidylcholine (DMPC)-d54/dihexanoylphosphatidylcholine (DHPC)-d22bicelles in the presence of a paramagnetic quencher Mn2+. These spectra indicate that TNM-A poorly binds to these membrane mimics without sterol and mostly remains in the aqueous media. In contrast, broader1H signals of TNM-A were observed in 10mol% cholesterol-containing bicelles, indicating that the peptide efficiently binds to sterol-containing bilayers. The addition of Mn2+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.
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.
Kimberly
K
Cornelio
Department of Chemistry, Graduate School of Science, Osaka University, Osaka 563-0043, Japan.
Rafael Atillo
RA
Espiritu
Department of Chemistry, Graduate School of Science, Osaka University, Osaka 563-0043, Japan.
Yasuto
Y
Todokoro
Department of Chemistry, Graduate School of Science, Osaka University, Osaka 563-0043, Japan.
Shinya
S
Hanashima
Lipid Active Structure Project, ERATO, Japan Science and Technology Agency, Osaka 560-0043, Japan; Department of Chemistry, Graduate School of Sciences, Kyushu University, Fukuoka 819-0395, Japan.
Masanao
M
Kinoshita
Department of Chemistry, Graduate School of Science, Osaka University, Osaka 563-0043, Japan; Department of Chemistry, Graduate School of Sciences, Kyushu University, Fukuoka 819-0395, Japan.
Nobuaki
N
Matsumori
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.
Michio
M
Murata
Division of Bioinformatics and Chemical Genomics, Graduate School of Pharmaceutical Sciences, Kyoto University, Kyoto 606-8501, Japan.
Shinichi
S
Nishimura
Division of Bioinformatics and Chemical Genomics, Graduate School of Pharmaceutical Sciences, Kyoto University, Kyoto 606-8501, Japan.
Hideaki
H
Kakeya
Advanced Science Institute, RIKEN Center for Sustainable Resource Science, Saitama 351-0198, Japan.
Minoru
M
Yoshida
Graduate School of Agricultural and Life Sciences, The University of Tokyo, Tokyo 113-8657, Japan.
Shigeki
S
Matsunaga
D000818:Animals; D015394:Molecular Structure; D010456:Peptides, Cyclic / Q000737:chemistry*; D011161:Porifera / Q000737:chemistry*; D066244:Proton Magnetic Resonance Spectroscopy; D013261:Sterols / Q000737:chemistry*
D010456:Peptides, Cyclic; D013261:Sterols; C581283:theonellamide A
Cholesterol; Cyclic peptide; Marine sponge; Membrane curvature