Transmembrane helical domain of the cannabinoid CB1 receptor

Abstract

Brain cannabinoid (CB(1)) receptors are G-protein coupled receptors and belong to the rhodopsin-like subfamily. A homology model of the inactive state of the CB(1) receptor was constructed using the x-ray structure of beta(2)-adrenergic receptor (beta(2)AR) as the template. We used 105 ns duration molecular-dynamics simulations of the CB(1) receptor embedded in a 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) bilayer to gain some insight into the structure and function of the CB(1) receptor. As judged from the root mean-square deviations combined with the detailed structural analyses, the helical bundle of the CB(1) receptor appears to be fully converged in 50 ns of the simulation. The results reveal that the helical bundle structure of the CB(1) receptor maintains a topology quite similar to the x-ray structures of G-protein coupled receptors overall. It is also revealed that the CB(1) receptor is stabilized by the formation of extensive, water-mediated H-bond networks, aromatic stacking interactions, and receptor-lipid interactions within the helical core region. It is likely that these interactions, which are often specific to functional motifs, including the S(N)LAxAD, D(E)RY, CWxP, and NPxxY motifs, are the molecular constraints imposed on the inactive state of the CB(1) receptor. It appears that disruption of these specific interactions is necessary to release the molecular constraints to achieve a conformational change of the receptor suitable for G-protein activation.

Figure 1

(A) Simulation system consisting of the CB₁ receptor, 140 POPC molecules, 14,625 water molecules, 62 Na⁺, and 77 Cl⁻. The PAL moiety that is covalently bonded to C⁴¹⁵ is represented by space-filling. Lipid heads, water molecules, and ions are represented by space-filling, and lipid tails by sticks. Lipid hydrogen atoms are omitted for clarity. The system at 105 ns of the simulation is shown. Color coding: C, cyan; O, red; N, blue; P, orange; Na⁺, cyan; Cl⁻, yellow; and H, white. (B) The RMSDs, calculated by RMS fitting to the initial coordinates with respect to the backbone heavy atoms of the residues as defined in the helical boundaries, of the CB₁ receptor: TM helices (black), extracellular loops (blue), intracellular loops (green), and H8 (red). (C) Superposition of the CB₁ receptor (green) to the x-ray structures of opsin (17) (orange), rhodopsin (18) (blue), β₁AR (19) (purple), and β₂AR (20) (red), with respect to the Cα atoms of the TM helical domain.

Figure 2

(A) H-bond networks, viewed from the intracellular side, of the helical bundle of the CB₁ receptor at the intracellular side. (B) H-bond networks, viewed from the extracellular side, of the helical bundle of the CB₁ receptor at the extracellular side. The H-bonds are represented by broken lines (white), whereas the water-mediated, clustered H-bond networks are circled by continuous lines (red). All water molecules (red) are represented in the space-filling mode. Hydrogen atoms are omitted for clarity. The structure was taken at 105 ns of the simulation. (C) Energy stabilization by water-mediated H-bond networks is estimated by nonbonding interaction energy (in kcal/mol) (in green), which is a summation of the electrostatic (black) and van der Waals (red) components. As the H-bonding partner, see Table 2. Color coding for the TM helices (ribbons): red, H1; orange, H2; yellow, H3; green, H4; cyan, H5; blue, H6; purple, H7; and gray, H8.

Figure 3

(A) Snapshots of the conserved water contact site in the middle of the helical core of H1/H2/H7 near the S(N)LAxAD motif at 0.0 ns, 20.0 ns, 75.0 ns, and 100.0 ns of the simulation are represented. (B) WAT1 coordination to both N1.50¹³⁴ and D2.50¹⁶³ during the simulation.

Figure 4

(A) Development of the aromatic cluster near the D(E)RY motif by F3.44²⁰⁸, Y3.51²¹⁵, F5.53²⁸⁹, Y5.56²⁹², and Y5.60²⁹⁶ is shown by a comparison of the snapshots taken at the time of 0.0 ns and 100.0 ns of the simulation. D3.49²¹³ (red) and R3.50²¹⁴ (blue) are also represented. (B) Energy stabilization by the aromatic stacking interactions between the individual aromatic stacking pairs. Only the side chain was considered for nonbonding interaction energy.

Figure 5

(A) The hydrophobic pore-forming residues, including F3.36²⁰⁰, V3.40²⁰⁴, W5.43²⁷⁹, L5.52²⁸⁸, V5.55²⁹¹, L6.44³⁵², I6.45³⁵³, and L6.52³⁶⁰, at the H5/H6 kinked region in the middle of the helical bundle are represented by white space-filling. The kink-causing P6.50³⁵⁸ and part of the residues (L6.51³⁵⁹and C7.38³⁸²) that form H-bonds to WAT6 are represented by sticks. The coordinated water WAT6 is represented by space-filling. The lipid, whose tails are inserted into the hydrophobic pore, is represented by pink sticks. The nonbonding interactions between these hydrophobic pore-forming residues and the lipid tails are traced along the simulation at: 0.0 ns, 20.0 ns, 100.0 ns (side view), and 100.0 ns (extracellular view). For the snapshot of 100.0 ns (extracellular view), the intracellular half of the lipid bilayer and L6.52³⁶⁰ are omitted for clarity. Color coding for the TM helices (ribbons) is the same as in Fig. 2A. (B) Nonbonding interaction energy (in kcal/mol) between the hydrophobic pore-forming residues, including F3.36²⁰⁰, V3.40²⁰⁴, W5.43²⁷⁹, L5.52²⁸⁸, V5.55²⁹¹, L6.44³⁵², I6.45³⁵³, and L6.52³⁶⁰, and a lipid that is found to be in close contact. Color coding for the TM helices (ribbons) is the same as in Fig. 2A.

Figure 6

(A) The hydrophobic pore-forming residues, including L1.52¹³⁶, V1.53¹³⁷, V1.56¹⁴⁰, L¹⁴⁷, P7.50³⁹⁴, I7.51³⁹⁵, A7.54³⁹⁸, L⁴⁰⁴, and A⁴⁰⁷, at the H7/H8 junction are represented by white space-filling. The two lipids, whose tails are inserted into the hydrophobic pore, are represented by orange and red sticks. The helical bundle is represented by the green cartoon. The PAL moiety that is covalently bonded to C⁴¹⁵ is represented by sticks. The nonbonding interactions between these hydrophobic pore-forming residues and the lipid tails are traced along the simulation at 0.0 ns, 10.0 ns, 30.0 ns, and 100.0 ns. Color coding for the palmitoylated C⁴¹⁵: C, cyan; O, red; N, blue; P, orange; and S, yellow. (B) Nonbonding interaction energy (in kcal/mol) between the hydrophobic pore-forming residues, including L1.52¹³⁶, V1.53¹³⁷, V1.56¹⁴⁰, L¹⁴⁷, P7.50³⁹⁴, I7.51³⁹⁵, A7.54³⁹⁸, L⁴⁰⁴, and A⁴⁰⁷, and the two lipids in close contact.