Amphotericin B-Ergosterol Complex Spans a Lipid Bilayer as a Single-Length Assembly

Tomoya Yamamoto, Yuichi Umegawa, Hiroshi Tsuchikawa, Shinya Hanashima, Nobuaki Matsumori, Kosuke Funahashi, Sangjae Seo, Wataru Shinoda, and Michio Murata

Amphotericin B (AmB) is a polyene macrolide antibiotic clinically used as an antifungal drug. Its preferential complexation with ergosterol (Erg), the major sterol of fungal membranes, leads to the formation of barrel-stave-like ion channel across a lipid bilayer. To gain a better understanding of the mechanism of action, a mode of lipid-bilayer spanning provides essential information. However, because of the lack of methodologies to observe it directly, it has not been revealed for the Erg-containing channel assembly for a long time. In this study, we disclosed that the AmB-Erg complex spans a lipid bilayer with single-molecular length, using solid state NMR experiments. Paramagnetic relaxation enhancement by Mn2+ residing near the surface of lipid bi-layers induced the depth-dependent decay of 13C NMR signals for individual carbon atoms of AmB. We found that both terminal segments, 41-COOH group and C38/C39/C40 methyl groups, come close to the lipid bilayer surfaces, suggesting that the AmB-Erg complex spans a palmitoyloleoylphosphatidylcholine (POPC) bilayer with single-molecular length. MD simulation experiments further confirmed the stabilization of AmB-Erg as a single-length spanning complex. These results provide the experimental evidence on the single-length com-plex incorporated in membrane by thinning a POPC-Erg bilayer that mimics fungal membranes.