The Structural Basis of Cholesterol Activation in Membranes

Regulation of cholesterol homeostasis is essential to mammalian cell function, and multiple response pathways are responsible for detecting and maintaining cholesterol levels in different membranes. Recent work has suggested that these pathways selectively respond to an active form of cholesterol that is observed only above a threshold concentration. However, the differences between active and inactive forms of cholesterol are still poorly understood. To identify structural changes in cholesterol-membrane interactions associated with activation, we use molecular dynamics simulations of phospholipid/cholesterol membrane bilayers to obtain atomic-level detail of how the dynamic structure of membranes is perturbed as the cholesterol concentration increases, which are analyzed for changes consistent with activation. We find that at high cholesterol concentrations, cholesterol becomes significantly more exposed to solvent and partially shifts out of the membrane. This effect is seen in membranes composed of either saturated or unsaturated lipids, and shifts in experimentally measured activation thresholds with membrane composition are consistent with changes in increased cholesterol exposure in simulations. We have identified several structural changes in cholesterol within membranes coincident with cholesterol activation as measured in experimental systems. These results demonstrate that the activation of cholesterol believed to be responsible for regulating cholesterol trafficking and homeostasis has a common structural basis in its interactions with the membrane. Future studies will focus on validation of these simulated results with biophysical and spectroscopic experiments and exploration of the effects of small molecules on cholesterol activation.