Atomic Resolution Studies of Sterol Interactions by Solid-State NMR Spectroscopy

Abstract

Sterols, vital and abundant components of plasma membranes, are involved in numerous cellular biochemical processes such as regulation of membrane properties and signaling events. However, these sterol interactions are not completely elucidated at the atomic level; this impedes further investigations into disorders related to these biological activities. Combining solid-state NMR (SSNMR), high yield biosynthesis of isotopically labeled sterols, and molecular dynamics, we aim to understand the key interactions that determine the sterol-specificity to amphotericin B (AmB), a powerful but toxic antifungal drug used to treat life-threatening fungal infections. AmB forms large aggregates that are neither crystalline nor soluble, making structural information difficult or impossible to obtain via x-ray crystallography or solution-state NMR. We have recently determined that AmB kills yeast cells primarily by binding to ergosterol1 (Erg) and forming a large extramembranous sterol sponge.2 These findings were enabled by the biosynthesis of fractionally 13C labeled Erg and state-of-the-art SSNMR techniques. The sterol sponge model hypothesizes that interactions of AmB with Erg determine its ability to kill yeast, whereas binding to cholesterol is responsible for determining its toxicity in human cells. SSNMR spectroscopy is uniquely able to detect and quantify the binding of sterols to AmB in the sterol sponge in atomistic detail. These studies provide a roadmap towards an improved therapeutic index3 for this drug while simultaneously elucidating additional details about the molecular mechanism of AmB-sterol interactions.(1)“Amphotericin primarily kills yeast by simply binding ergosterol”, Gray et al. Proc. Natl. Acad. Sci. 2011, 109, 2234-2239.(2)“Amphotericin forms an extramembranous sterol sponge”, Anderson et al. Nat. Chem. Bio. 2014, 10, 400-406.(3)“C2’-OH of amphotericin B plays an important role in binding the primary sterol of human cells but not yeast cells”, Wilcock et al. J. Am. Chem. Soc. 2013, 135, 8488-8491.