Head-to-Tail Interaction between Amphotericin B and Ergosterol Occurs in Hydrated Phospholipid Membrane

Abstract

Amphotericin B (AmB) is thought to exert its antifungal activity by forming an ion-channel assembly in the presence of ergosterol. In the present study we aimed to elucidate the mode of molecular interactions between AmB and ergosterol in hydrated phospholipid bilayers using the rotational echo double resonance (REDOR) spectra. We first performed (13)C{(19)F}REDOR experiments with C14-(19)F-labeled AmB and biosynthetically (13)C-labeled ergosterol and implied that both "head-to-head" and "head-to-tail" orientations occur for AmB-ergosterol interaction in the bilayers. To further confirm the "head-to-tail" pairing, (13)C-labeled ergosterol at the dimethyl terminus (C26/C27) was synthesized and subjected to the REDOR measurements. The spectra unambiguously demonstrated the presence of a "head-to-tail" orientation for AmB-ergosterol pairing. In order to obtain information on the position of the dimethyl terminus of ergosterol in membrane, (13)C{(31)P}REDOR were carried out using the labeled ergosterol and the phosphorus atom of a POPC headgroup. Significant REDOR dephasing was observed at the C26/C27 signal of ergosterol in the presence of AmB, but not in the absence of AmB, clearly indicating that the side-chain terminus of ergosterol in the AmB complex comes close to the bilayer surface.

Amphotericin forms an extramembranous and fungicidal sterol sponge

“…1b). 4–23 An extensive series of structural and biophysical studies, including those employing planar lipid bilayers, 4–10 liposome permeability, 9–13,17 Corey-Pauling-Kulton (CPK) modeling, 7–9 UV/Vis spectroscopy, 9–11,13,21 circular dichroism, 10,11,13,21 fluorescence spectroscopy, 9,11 Raman spectroscopy, 10 differential scanning calorimetry, 9,10,21 chemical modifications, 11–14,17 atomic force microscopy, 21 transmission electron microscopy, 20 computer modeling, 11,15 electron paramagnetic resonance, 10 surface plasmon resonance, 22 solution NMR spectroscopy, 11 and solid-state NMR (SSNMR) 16–19 spectroscopy have been interpreted through the lens of this ion channel model. Importantly, this model suggests that the path to an improved therapeutic index requires selective formation of ion channels in yeast versus human cells, 10–20 that the search for other resistance-refractory antimicrobials should focus on membrane-permeabilizing compounds, 24 and that the ion channel-forming and cytotoxic activities of AmB cannot be separated.…”

“…1b). 7–9,11,12,15–19,22,23 (ii) In an alternative surface adsorption model, AmB is primarily positioned in the intermediate/headgroup region, oriented parallel to the plane of the membrane, sequestering Erg to the membrane surface (Fig. 1c).…”

The influence of amphotericin B on the molecular organization and structural properties of DPPC lipid membranes modified by sterols

“…The layer of the DPPC-ergosterol-AmB phase is thicker than the one with cholesterol in multibilayers, which can be caused by the terminal dimethyl group of ergosterol which is close to the surface of the membrane in the complex [14].…”

“…There is some experimental research supporting the possibility of formation of AmB-sterol (head-to-head) complexes in the lipid bilayer and stoichiometries ranging from 1:1, 1:4 to 1:0.7 have been reported [16,24]. Another study shows that AmB and ergosterol can form head to tail association in the lipid environment [14]. A linear dichroism technique using Fourier Transform Infrared (FTIR) spectroscopy of lipid monolayers containing dipalmitoylphosphatidylcholine (DPPC) and sterols indicates that AmB molecules are located mainly on the membrane surface [8] of molecules in the polar heads of the lipids can disrupt the hydrophilic part of the membrane [8].…”

“…Until now, a lot of theoretical and experimental work has been carried out to understand the mode of AmB biological action [2,[5][6][7][8][9][10] and interaction with membranes [11][12][13][14]. The membrane activity of AmB is based on the fact that it has greater tendency to interact with ergosterol-enriched membranes of fungal cells than with mammalian membranes containing cholesterol, which was demonstrated in many studies especially regarding monolayers spread at the air-water interface [15][16][17][18][19].…”