(A) The distances between the Cα atoms of D1133.32–S2075.46 (distance defined as LL) and R1313.50–E2686.30 (LG) pairs used to measure the fluctuations at the ligand and G-protein binding sites, …
In panels (A–F) the distributions are plotted as a function of LL (distance between the Cα atoms of D1133.32 and S2075.46) at the ligand binding site and LG (distance between the Cα atoms of R1313.50…
(A–B) 2D number densities of cholesterol (Chol) around β2AR. The data are averaged over all independent trajectories for a given cholesterol concentration (Table 1) and normalized with respect to …
Panels (A–B) (top): Cholesterol occupancy time per residue of β2AR described in terms of the normalized time fraction, where a value of one stands for a contact throughout the simulation trajectory …
The residues that play a major role (contact fraction ≥ 0.4, where one stands for maximum contact and zero for no contact) in cholesterol binding are highlighted. Here for IC1, the residues in the …
The residues that play a major role (contact fraction ≥ 0.4, where one stands for maximum contact and zero for no contact) in cholesterol binding are highlighted. Following sequence alignment, shown …
The residues that play a major role (contact fraction ≥ 0.4, where one stands for maximum contact and zero for no contact) in cholesterol binding are highlighted. Following sequence alignment, shown …
Two-dimensional (2D) averaged and normalized number densities of cholesterol around β2AR shown at low cholesterol concentrations (2 and 5 mol%). The intracellular and extracellular leaflets are …
(A) The different cholesterol analogues used in the current study. (B–D) Average lipid chain order parameter SCD of DOPC bilayers with different concentrations of cholesterol or …
The average interaction energies for van der Waals (vdW) and electrostatic interactions are determined separately. Error bars are in the range of 0.1–1 kJ/mol. The lower panel represents the …
Normalized 2D average number densities around β2AR: (A–B) CHSA (the deprotonated form of cholesteryl hemisuccinate (CHS)); (C–F) CHS. Densities of sterols in mixed sterol-containing bilayers with …
(A–B) Oxysterol-containing systems having 4 mol% of oxysterol (27-OH-Chol or 4β-OH-Chol) and 21% cholesterol. (C–D) DOPC bilayer with 10 mol% and 40 mol% of CHS. Conformational distributions are …
Specific cholesterol binding site in β2AR with the cholesterol consensus motif displayed with side chain positions of the conserved amino acid residues, as found in (A) the crystal structure (ref. …
Cytosolic view of β2AR (A) in the beginning of a simulation (active state) as well as in representative simulation snapshots in (B) a DOPC bilayer and (C) in the presence of 40 mol% cholesterol. The …
The conformational distributions of β2AR in (left) a DOPC bilayer and (right) a DOPC bilayer with 40 mol% cholesterol (Chol) as a function of LL and LG. The gray dotted lines represent the …
The conformational distribution of β2AR in bilayers composed of (A) long-chain PC-20:0/22:1 c13 lipids and (C) DOPC with 20 mol% pyrene (Pyrene20). (B) 3D-distribution of bilayer thickness in the …
(A–B) Long-chain PC bilayer properties compared to those of cholesterol-rich and DOPC systems. (A) The average bilayer thickness in several different bilayer systems (see Table 1). (B) The average …
(A–C) Time-correlation function of cholesterol (Chol) at the three major interaction sites (IC1, IC2, EC1) on the β2AR surface. Initially cholesterol is bound to the site (distance ≤ 0.5 nm) and the …
Time development for the distances of cholesterol molecules from the β2AR surface, where these cholesterol molecules were initially bound at the eight binding sites identified in this study …
In the simulation snapshot, residues are colored according to their strength of interaction with cholesterol (red represents the weakest and blue represents the strongest interaction).
Data are given for cases, where IC1 is occupied (blue) or unoccupied (orange) by cholesterol.
The bin edge length was set to 0.1 Å in both dimensions.
The dotted line represents the distance between the Cα atoms of R1313.50–E2686.30 (defined as LG) used to measure the fluctuation at the G protein binding site. (D) Simulation snapshot (in the …
Cholesterols interacting at the cholesterol-binding sites are highlighted (yellow at IC1; green at IC2; and blue and red at EC1). Other cholesterols are shown in gray. For clarity, other lipids in a …
Cholesterols interacting at the cholesterol-binding interaction sites are highlighted (yellow and green at IC1; red, blue and orange at IC2; and pink, purple and cyan at EC1). Other cholesterols are …
Descriptions of systems simulated: β2AR in bilayers with varying lipid compositions. ‘Chol’ stands for cholesterol.
Systems* | Initial lipid arrangement around β2AR | Lipids | Sterol mol % | No. of repeats† | Time (μs)‡ | ||
---|---|---|---|---|---|---|---|
DOPC | Random | DOPC | 0 | 3 | 3×2.5 | ||
DOPC-active | Random | DOPC | 0 | 3 | 3×2.5 | ||
C H O L | Chol2 | Random | DOPC + Chol | 2 | 3 | 3×2.5 | R A N D O M |
Chol5 | Random | DOPC + Chol | 5 | 3 | 3×2.5 | ||
Chol10 | Random | DOPC + Chol | 10 | 3 | 3×2.5 | ||
Chol25 | Random | DOPC + Chol | 25 | 2 | 2×2 | ||
Chol40 | Random | DOPC + Chol | 40 | 3 | 3×2.5 | ||
Chol40-active | Random | DOPC + Chol | 40 | 3 | 3×2.5 | ||
C H S | CHS10 | Random | DOPC + CHS | 10 | 2 | 2×2 | |
CHS40 | Random | DOPC + CHS | 40 | 2 | 2×2 | ||
CHSA10 [A for anionic] | Random | DOPC + CHSA | 10 | 1 | 2 | ||
CHSA40 | Random | DOPC + CHSA | 40 | 1 | 2 | ||
O X Y S T E R O L | 27-OH-Chol | Random [16 mol % Chol was randomly replaced by 27-OH-Chol] | DOPC + Chol + 27-OH-Chol | 25 (4 mol% 27-OH-Chol + 21 mol% Chol) | 3 | 2 + 1 + 1 | |
4β-Chol | Random [16 mol% Chol was randomly replaced by 4β-OH-Chol] | DOPC + Chol + 4β-OH-Chol | 25 (4 mol% 4β-OH-Chol + 21 mol% Chol) | 3 | 1 + 1 + 1 | ||
Chol-Bound§ | 8 cholesterols bound at sites predicted by simulations | DOPC + Chol | 1.9 | 3 | 3×2.5 | B O U N D | |
Chol-IC1 | 2 Chol bound at IC1 | DOPC + Chol | <1 | 2 | 2×2 | ||
CHS-IC1 | 2 CHS bound at IC1 | DOPC + CHS | <1 | 1 | 2 | ||
CHSA-IC1 | 2 CHSA bound at IC1 | DOPC + CHSA | <1 | 1 | 2 | ||
PC-20:0–22:1 c13 [Double bond at carbon 13] | Random | PC-20:0–22:1 c13 | 0 | 3 | 3×1.5 | ||
Pyrene20 | Random | DOPC + 20 mol% pyrene | 0 | 3 | 3×1.5 |
*In the DOPC-active and Chol40-active systems, we used the active-state conformation of the receptor as the starting structure; for all the other systems, we used the inactive conformation.
†For systems with no sterols initially bound to β2AR, i.e., the systems which started with a random distribution of lipids, a number of different repeat simulations for each lipid composition were performed with different initial lipid arrangements around the receptor. For systems with sterols initially bound to β2AR (seed and BOUND), different replicas were generated with different starting velocities.
‡Listed are the simulation times of production simulations; the equilibration time of the systems (100 ns) is not included.
§In the Chol-Bound system, eight cholesterol molecules were initially (at time zero of the simulation) bound at eight binding sites predicted by the present simulations, while the rest of the system had no cholesterol at all.
Interactions* of sterols at the three high-affinity cholesterol-binding sites.
Cholesterol/Cholesterol analogue | High-affinity cholesterol interaction sites | |||||
---|---|---|---|---|---|---|
IC1 | IC2 | EC1 | ||||
vdW interaction energy (kJ/mol) | No. of contacts | vdW interaction energy (kJ/mol) | No. of contacts | vdW interaction energy (kJ/mol) | No. of contacts | |
Cholesterol† | −138.04 ± 0.20 | 141.02 ± 0.22 | −95.06 ± 0.12 | 90.65 ± 0.16 | −129.51 ± 0.29 | 104.38 ± 0.28 |
CHS | −29.63 ± 0.14 | 28.78 ± 0.16 | −98.75 ± 0.11 | 96.30 ± 0.16 | - | - |
27-OH-Chol | −32.17 ± 0.30 | 34.95 ± 0.33 | −22.69 ± 0.23 | 28.41 ± 0.28 | −132.85 ± 0.27 | 120.20 ± 0.30 |
4β-OH-Chol | - | - | - | - | −41.80 ± 0.48 | 33.41 ± 0.42 |
* Shown are the total van der Waals (vdW) interaction energy and the number of contacts between cholesterol and β2AR, when cholesterol is in the IC1, IC2, or EC1 binding site (and similarly for the cholesterol analogues).
† Calculations are based on systems having ≥10 mol% cholesterol. Shown here are the average values over different trajectories.