Figure 5. Localization of cholesterol around aquaporin-0 (AQP0) monomers from unbiased molecular dynamics (MD) simulations of individual AQP0 tetramers in sphingomyelin (SM) membranes with low and high cholesterol concentration.

Density maps representing the localization of cholesterol around AQP0 over time were computed from simulations starting from unbiased cholesterol positions in membranes at the indicated SM:Chol ratios. After combining the four maps calculated individually for the four subunits of the tetramer, cholesterol densities were projected (blue areas) onto the surface of a single AQP0 monomer (white surface). Projections are shown for the S1 and S2 monomer surfaces, as defined in the representations to the right. Lipids seen in the electron crystallographic structures obtained in membranes at the respective SM:Chol ratios are displayed as sticks and labeled according to the electron crystallographic structures. Densities are contoured at 10σ for the 2:1 SM:Chol membrane and at 9σ for the 1:2 SM:Chol membrane. The density hotspot indicated with an asterisk coincides with the Chol3 position seen at the 1:2 SM:Chol ratio. See also Figure 5—figure supplements 1–3.

Figure 5—figure supplement 1. Molecular dynamics simulations of aquaporin-0 (AQP0) in sphingomyelin (SM)/cholesterol (Chol) membrane systems.

(A–D) Top views (left panels) and side views (right panels) of the simulated systems. AQP0 tetramers are shown in white surface representation. Lipids in positions seen in the electron crystallographic AQP0_1SM:2Chol structure are shown in VDW representation; SM in green and Chol in red and blue, respectively. Bulk cholesterol molecules are shown in red stick representation, whereas only the phosphorous atoms of bulk SM lipids are shown as dark tan spheres. The membranes were solvated by explicit water molecules (not shown for clarity). (A) In the ‘No AQP0’ systems used as control, lipid-only bilayers consisting of molar SM:Chol mixtures of 1:0, 2:1, and 1:2 (shown) were simulated. In each system, one cholesterol molecule was inserted in the position of the deep cholesterol seen in the AQP0_1SM:2Chol structure. (B) In the ‘AQP0’ system, a single AQP0 tetramer by itself without the annular lipids was inserted into membranes consisting of molar SM:Chol mixtures of 2:1 and 1:2 (shown). (C) In the ‘AQP0+EC lipids’ system, a single AQP0 tetramer with the lipids observed in the AQP0_1SM:2Chol structure was inserted into membranes consisting of molar SM:Chol mixtures of 1:0, 2:1, and 1:2 (shown). Note the deep cholesterols at the center. (D) In the ‘2×AQP0’ system, a pair of AQP0 tetramers was inserted into a pure SM membrane. The interfacial lipids between the pair of tetramers were either pure SM (inset ‘SM only’) from the AQP0_2SM:1Chol structure or a hybrid interface that replaces the two central SM molecules with the EC deep cholesterol molecules seen in the AQP0_1SM:2Chol structure (shown, inset ‘with Chol’).

Figure 5—figure supplement 2. Deuterium order parameters for the membrane patches in equilibration.

Deuterium order parameters S_CD for the hydrophobic acyl chains for the sphingomyelin (SM) lipids in a pure SM bilayer (Pure SM, orange), and in SM:Chol membranes at molar mixing ratios of 2:1 (2:1 SM:Chol, cyan) and 1:2 (1:2 SM:Chol, purple). The continuous line shows the values for the SN1 acyl chains and the dashed line shows the values for the SN2 acyl chains. The values correspond to the last 50 ns of simulation, independent of the patch size and total equilibration time. The three boxes correspond to the pure lipid membrane patches, used for the No-AQP0, AQP0, and 2×AQP0 simulations, respectively, before the protein was inserted in the latter two cases. Membrane patch size indicated on top of each box.

Figure 5—figure supplement 3. Average area-per-lipid (APL) for the membrane patches in equilibration.

Average APL as a function of time (log scale) is shown for the warm-up NPT simulations after removal of position restraints. The average APL value for the last 50 ns of simulation for the pure sphingomyelin (SM) systems were 55.5±0.7 Ų, 54.2±0.5 Ų, and 53.7±0.2 Ų, for the system with 64, ~244, and 797 lipids/leaflet, respectively.