Membrane Sterols Modulate the Binding Mode of Amphotericin B without Affecting Its Affinity for a Lipid Bilayer

Membrane-active antibiotics are known
to selectively target certain
pathogens based on cell membrane properties, such as fluidity, lipid
ordering, and phase behavior. These are in turn modulated by the composition
of a lipid bilayer and in particular by the presence and type of membrane
sterols. Amphotericin B (AmB), the golden standard of antifungal treatment,
exhibits higher activity toward ergosterol-rich fungal membranes,
which permits its use against systemic mycoses; however, the selectivity
for fungal membranes is far from satisfactory leading to severe side
effects. Despite decades of research, no consensus has emerged on
the origin of AmB specificity for fungal cells and its actual mode
of action at the molecular level. Previously, it has been proposed
that the specific action of AmB is related to differences in its affinity
for membranes of different composition. In this work, we investigate
this relationship by employing molecular dynamics simulations to compare
the free energy of insertion of AmB into three types of membranes:
a pure DMPC bilayer and DMPC bilayers containing 30% of cholesterol
or ergosterol. We analyze the orientation of AmB molecules within
the bilayer in order to unambiguously establish their membrane binding
mode and relate the orientational freedom to the sterol-dependent
tightness of lipid packing. Our results strongly indicate that the
membrane insertion of AmB proceeds virtually to completion independent
of membrane type, and hence the higher toxicity against fungal membranes
may rather result from differences in subsequent oligomerization in
the membrane and assembly of monomers into functional transmembrane
pores. In particular, the latter could be facilitated by sterol-induced
ordering of AmB molecules along the membrane normal, revealed by our
free energy profiles. Moreoverin contrast to certain claimswe
find no stable binding mode corresponding to the horizontal adsorption
of AmB on the membrane surface.