The Structural Basis of Cholesterol Accessibility in Membranes

Abstract

Although the majority of free cellular cholesterol is present in the plasma membrane, cholesterol homeostasis is principally regulated through sterol-sensing proteins that reside in the cholesterol-poor endoplasmic reticulum (ER). In response to acute cholesterol loading or depletion, there is rapid equilibration between the ER and plasma membrane cholesterol pools, suggesting a biophysical model in which the availability of plasma membrane cholesterol for trafficking to internal membranes modulates ER membrane behavior. Previous studies have predominantly examined cholesterol availability in terms of binding to extramembrane acceptors, but have provided limited insight into the structural changes underlying cholesterol activation. In this study, we use both molecular dynamics simulations and experimental membrane systems to examine the behavior of cholesterol in membrane bilayers. We find that cholesterol depth within the bilayer provides a reasonable structural metric for cholesterol availability and that this is correlated with cholesterol-acceptor binding. Further, the distribution of cholesterol availability in our simulations is continuous rather than divided into distinct available and unavailable pools. This data provide support for a revised cholesterol activation model in which activation is driven not by saturation of membrane-cholesterol interactions but rather by bulk membrane remodeling that reduces membrane-cholesterol affinity.

Side-Chain Oxysterols Modulate Cholesterol Accessibility through Membrane Remodeling

“…18 As a measure of cholesterol activation, we measured the mean SASA of cholesterol molecules in each simulation (Figure 2A). At all concentrations of 25-HC, we find lower cholesterol accessibility at low cholesterol concentrations and large increases in accessibility at higher cholesterol concentrations.…”

“…18 In this setting, the phospholipids and the water interface move inward while the cholesterol remains fixed in position, leaving it more exposed to solvent and extramembrane acceptors. This membrane thinning observed at high cholesterol concentrations is consistent with both other simulated models and nuclear magnetic resonance and X-ray studies.…”

“…10,18,19 Production simulations were run for 400 ns of total simulation time. On the basis of examination of the membrane-projected area and system energy drift, we chose to discard the first 200 ns of each simulation as equilibration time, leaving 200 ns of steady-state simulation time for analysis of each membrane composition.…”

“…18 In this revised model, there are no distinct pools of accessible and inaccessible cholesterol; rather, changes in membrane structure at high cholesterol concentrations lead to decreased membrane thickness and an increase in the availability of all cholesterol molecules in the membrane. As additional cholesterol is added to a membrane, all cholesterol within the membrane becomes increasingly more accessible and, at some threshold concentration, becomes available to external cholesterol sensors.…”

Perfringolysin O Structure and Mechanism of Pore Formation as a Paradigm for Cholesterol-Dependent Cytolysins

“…Based on the requirement of high cholesterol levels, targeting of PFO to cholesterol rich domains or “lipid rafts” has been suggested (Ohno-Iwashita et al 2004). However, it has become clear that exposure of cholesterol at the membrane surface is a key factor to trigger PFO binding, and “lipid rafts” may not be necessary for toxin binding (Heuck et al 2007; Nelson et al 2008; Flanagan et al 2009; Moe and Heuck 2010; Sokolov and Radhakrishnan 2010; Olsen et al 2013). Moreover, the localization of PFO oligomers on the membrane surface may change from the original binding site after insertion of the β-barrel (Nelson et al 2010; Lin and London 2013).…”