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.

Figure 1

Various metrics of lipid condensation in POPC (blue) or DOPC (green) phospholipid membranes of varying cholesterol concentrations. Error bars show standard deviations of the mean, calculated using a bootstrap method. (Gray-shaded zone) Estimated division between condensing and relaxing regimes, at 25 mol % cholesterol. (A) Mean solvent-accessible surface area of single phospholipids. (B) Mean membrane thickness. (C) Mean tail-order parameters of the sn-1 (solid) and sn-2 (dotted) phospholipid tails. (D) Phospholipid interdigitation, measured as percentage overlap between phospholipid mass densities in each leaflet.

Figure 2

Cholesterol activation as measured in experimental and simulated membrane systems. (A) Binding of perfringolysin O to liposomes composed of POPC (blue) or DOPC (green) membranes of varying cholesterol concentrations. Error bars are derived from four replica experiments. (Solid lines) Sigmoidal fits to the binding data. (Shaded regions) Region of 2.5–25% of maximal binding, as estimated from the fit. (B) Mean cholesterol hydroxyl depth for cholesterol in simulated POPC (blue) or DOPC (green) membranes of varying cholesterol concentrations. Error bars show standard deviations of the mean. POPC (shaded blue) and DOPC (green) regions are calculated from the experimental data shown in panel A. (Gray region) Estimated mean depth of 0.33 nm at which PFO binding occurs for both lipid species.

Figure 3

(A) Mean positions of cholesterol hydroxyl oxygens (solid) and the bilayer/water interface (dotted), measured relative to the bilayer center, in POPC (blue) or DOPC (green) membranes at varying cholesterol concentrations. Error bars show standard deviations of the mean. (B) Distributions of cholesterol depth in POPC bilayers with 6 (black), 30 (blue), 41 (red), and 52 (green) mol % cholesterol. (Dashed vertical lines) Medians of each distribution.

Figure 4

(A) Percentage of POPC (blue) or DOPC (green) phospholipids that are neighbors of (solid) or distant from (dotted) cholesterol molecules in each bilayer leaflet. (B) Mean tail-order parameters for the sn-1 palmitoyl (black) and sn-2 oleoyl (red) chains of POPC, shown as the mean over all phospholipids (solid) or only those phospholipids that are neighbors of (dashed) or distant from (dotted) cholesterol molecules in each bilayer leaflet. (C) Percentage of cholesterol molecules in POPC (blue) or DOPC (green) membranes that have >4 (dashed), or ≤4 (dotted) phospholipid neighbors. (D) Depth of cholesterol hydroxyl oxygens in POPC (blue) or DOPC (green) membranes, shown as the mean over all cholesterols (solid), over only cholesterols with ≤4 PC neighbors (dotted), or over only cholesterols with >4 PC neighbors (dashed).

Figure 5

The proposed relation between underlying changes in cholesterol accessibility and experimentally measured binding to available cholesterol. (Four concentrations of cholesterol are marked in blue, cyan, magenta, and red in all four panels for comparison.) (A) Mean cholesterol accessibility is modeled as constant below a saturation threshold C_sat and increasing linearly above it. (B) The number of cholesterols of each accessibility at any particular concentration (thick lines) are modeled as normal distributions centered on the mean value (thin lines) and with a common variance. Those cholesterols above some high threshold A_binding are presumed to be available for binding to external acceptors. (C) The concentration of available cholesterol changes nonlinearly with total cholesterol concentration, with significant amounts of available cholesterol occurring only above the binding threshold C_binding. (D) Binding of an external acceptor is dependent only on the concentration of available cholesterol, and thus occurs only above C_binding before saturating once all acceptors have bound cholesterol.

Figure 6

The proposed model of cholesterol activation. (A) With no cholesterol present, phospholipids pack loosely with their tails in relatively disordered states. (B) As cholesterol is added to the membrane, it locally orders neighboring phospholipids, causing the bilayer to thicken and condense. (C) At very high cholesterol concentrations, length mismatch between cholesterol and phospholipids drives interdigitation of the acyl chains, resulting in membrane thinning and cholesterol exposure to solvent.