Table 1.
Description of the simulated systems and a summary of the main results as to binding processes.
Fig 1.
Snapshots of simulated system with a lipid composition of bmp:chol:sm:dopg:popc = 20:10:0:0:70 (System 5).
A) A typical starting configuration for npc2 in the water phase, above the membrane. B) Final configuration after a simulation of 400 ns in a case, where the cholesterol-binding pocket faces the membrane (Prone mode). C) Final configuration of a simulation, where the cholesterol-binding pocket faces the water phase (Supine mode). The residue G61 residing at the opening of the binding pocket is highlighted. Color code for lipids: bmp (violet), chol (green), popc (light brown). For clarity’s sake, water is not shown.
Fig 2.
The collective variables and the sampling approach.
npc2 structure overlaid with vectors showing the long (A) and the short (B) axes of the protein and the collective variables θ (A) and ϕ (B). The membrane normal defined as the vector between the com of P atoms of the upper and the lower leaflets of the membrane is represented with a solid arrow. A) The long axis is defined as a vector connecting the Cα com of residues 1–6, 19–38, 55–69, 93–109, 123–130 (pink), and that of residues 7–18, 39–54, 70–92, 110–122 (blue). B) The short axis is defined as a vector connecting the Cα com of residues 1–6, 13–45, 75–94, 124–130 (pink), and and that of residues 7–12, 46–74, 95–123 (blue). C) A schematic representation of the sampling approach. The numbers indicate the replica indices (i). Three separate wt-mtd simulations biasing |z| (i = 0, 3, …), θ (i = 1, 4, …), and ϕ (i = 2, 5, …) were performed within each window (w, shown in different colors) flanked by half harmonic restraints. Bias exchanges were attempted between simulations i and i + 1 in even (solid arrows) and odd pairings (dashed arrows). For a detailed discussion of the sampling procedure, see S1 Text.
Fig 3.
The free energy surfaces for npc2 binding to membranes.
A) Potential of mean force (pmf) profiles shown as a function of min zr, which stands for the minimum of the protein Cα z-coordinates for the residue r. The origin (z = 0) is set to comz of the upper leaflet P atoms. B) pmf surfaces shown as a function of min zr and ϕ for anionic membranes. Contours were plotted every 2 kJ/mol increments. The two binding orientations are marked with dashed lines. The thumbnail images of npc2 structure for the corresponding orientations are shown on the upper left corner for Prone mode and on the upper right corner for Supine mode. See S9 Fig for the local errors and S1 Fig for the pmf projected onto |z|. The labels indicate membrane content in molar fractions for a mixture of bmp:chol:sm:dopg:popc, and the corresponding system numbers (Table 1) are provided in parentheses. The local errors are given in S9 Fig.
Fig 4.
Normalized contact frequency analysis (ncf).
A) The black rectangles show the ncf averaged over all bmp mixtures (bmp:chol:sm:dopg:popc = 20:10:0:0:70 (System 5), 20:35:0:0:45 (System 6) and 20:10:20:0:50 (System 7)), and the gray bars show the standard deviation. B) Representative snapshots showing npc2 interacting with bmp in Prone mode (left) and Supine mode (right). The protein is colored based on the ncf and the residues with ncf > 0.4 are shown in licorice and labeled.
Fig 5.
Cholesterol-binding free energy change upon membrane binding.
A) Thermodynamic cycle for the estimation of cholesterol binding free energy change. Solid arrows represent the transformations performed in this work and dashed arrows represent the alternative transformations. B) The cholesterol binding free energy change as a function of min zr, calculated by ΔΔGchol-b.(min zr) = ΔGchol-b.(min zr) − ΔGapo(min zr). The labels indicate membrane content in molar fractions for a mixture of bmp:chol:sm:dopg:popc, and the corresponding system numbers (Table 1) are provided in parentheses.
Fig 6.
Schematic representation of the membrane binding of npc2.
The thumbnail images for npc2apo (green) and npc2chol-bound (red) are placed on the y-axis based on the pmf data (Fig 3A) of the bmp:chol:sm:dopg:popc = 20:10:0:0:70 (System 5) (low chol), bmp:chol:sm:dopg:popc = 20:35:0:0:45 (System 6) (high chol), and bmp:chol:sm:dopg:popc = 20:10:20:0:50 (System 7) (low chol with sm) systems. A putative cycle for transferring cholesterol from high chol to low chol membranes are indicated by thick arrows. The competing processes (the interconversion between the two binding modes and binding to sm-containing membranes) are indicated by dashed arrows.