Mechanism of allosteric regulation of β2-adrenergic receptor by cholesterol
- Tampere University of Technology, Finland
- University of Helsinki, Finland
- ETH Zürich, Switzerland
- University of Southern Denmark, Denmark
Figures
Figure 1 with 1 supplement
Conformational dynamics of β2AR.
(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, …
Figure 1—figure supplement 1
Conformational distributions of β2AR in lipid bilayers with various cholesterol (Chol) concentrations.
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…
Figure 2 with 10 supplements
Cholesterol interaction sites on β2AR.
(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 …
Figure 2—figure supplement 1
Residues of β2AR involved in cholesterol binding, and cholesterol interaction sites on β2AR.
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 …
Figure 2—figure supplement 2
Sequence alignment of β2AR orthologues around the cholesterol-binding site IC1.
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 …
Figure 2—figure supplement 3
Sequence alignment of β2AR orthologues around the cholesterol-binding site IC2.
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 …
Figure 2—figure supplement 4
Sequence alignment of β2AR orthologues around the cholesterol-binding site EC1.
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 …
Figure 2—figure supplement 5
Cholesterol density around the receptor at low cholesterol concentrations.
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 …
Figure 2—figure supplement 6
Structure of cholesterol analogues and properties of sterol-containing bilayers.
(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 …
Figure 2—figure supplement 7
Interactions of cholesterol and cholesterol-like molecules with β2AR.
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 …
Figure 2—figure supplement 8
Densities of sterols around β2AR.
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 …
Figure 2—figure supplement 9
Conformational distributions of β2AR in lipid bilayers with different cholesterol analogues.
(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 …
Figure 2—figure supplement 10
IC1 interaction site.
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. …
Figure 3 with 1 supplement
Effect of cholesterol on the active conformation of β2AR.
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 …
Figure 3—figure supplement 1
Conformational distribution of β2AR starting from the active state.
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 …
Figure 4 with 1 supplement
Impact of membrane-mediated effects on the β2AR conformation.
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 …
Figure 4—figure supplement 1
Properties of thick and/or ordered cholesterol-free bilayers.
(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 …
Figure 5 with 1 supplement
Binding time of cholesterol.
(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 …
Figure 5—figure supplement 1
Interaction of cholesterol with β2AR.
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 …
Author response image 1
Specific cholesterol binding site in β2AR with CCM displayed with side chain positions of conserved amino acid residues, as found in (A) the crystal structure (1) and (B) during our simulation.
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).
Author response image 2
For the time-dependent distance betweenH4 and its average position, as the H4 helix fluctuates around its average location, shown here are results for the standard deviation of the distance fluctuations.
Data are given for cases, where IC1 is occupied (blue) or unoccupied (orange) by cholesterol.
Author response image 3
Distributions of LL and LG distances from individual trajectories (shown in different colors) for various cholesterol concentrations.
https://doi.org/10.7554/eLife.18432.028
Author response image 4
Area in the 2D histogram visited by the receptor conformations.
The bin edge length was set to 0.1 Å in both dimensions.
Author response image 5
Cytosolic view of β2AR (A) in the beginning of 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 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 …
Videos
Video 1
Spontaneous binding/unbinding of cholesterol at the three main cholesterol interaction sites of β2AR during a 2.5-μs simulation with 10 mol% of cholesterol.
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 …
Video 2
Spontaneous binding/unbinding of cholesterol at the three main cholesterol interaction sites of β2AR during a 2.5-μs simulation with 40 mol% of cholesterol.
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 …
Tables
Table 1
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 |
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*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.
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†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.
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‡Listed are the simulation times of production simulations; the equilibration time of the systems (100 ns) is not included.
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§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.
Table 2
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 |
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* 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).
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† Calculations are based on systems having ≥10 mol% cholesterol. Shown here are the average values over different trajectories.
