A lipid pathway for ligand binding is necessary for a cannabinoid G protein-coupled receptor

Abstract

Recent isothiocyanate covalent labeling studies have suggested that a classical cannabinoid, (-)-7'-isothiocyanato-11-hydroxy-1',1'dimethylheptyl-hexahydrocannabinol (AM841), enters the cannabinoid CB2 receptor via the lipid bilayer (Pei, Y., Mercier, R. W., Anday, J. K., Thakur, G. A., Zvonok, A. M., Hurst, D., Reggio, P. H., Janero, D. R., and Makriyannis, A. (2008) Chem. Biol. 15, 1207-1219). However, the sequence of steps involved in such a lipid pathway entry has not yet been elucidated. Here, we test the hypothesis that the endogenous cannabinoid sn-2-arachidonoylglycerol (2-AG) attains access to the CB2 receptor via the lipid bilayer. To this end, we have employed microsecond time scale all-atom molecular dynamics (MD) simulations of the interaction of 2-AG with CB2 via a palmitoyl-oleoyl-phosphatidylcholine lipid bilayer. Results suggest the following: 1) 2-AG first partitions out of bulk lipid at the transmembrane alpha-helix (TMH) 6/7 interface; 2) 2-AG then enters the CB2 receptor binding pocket by passing between TMH6 and TMH7; 3) the entrance of the 2-AG headgroup into the CB2 binding pocket is sufficient to trigger breaking of the intracellular TMH3/6 ionic lock and the movement of the TMH6 intracellular end away from TMH3; and 4) subsequent to protonation at D3.49/D6.30, further 2-AG entry into the ligand binding pocket results in both a W6.48 toggle switch change and a large influx of water. To our knowledge, this is the first demonstration via unbiased molecular dynamics that a ligand can access the binding pocket of a class A G protein-coupled receptor via the lipid bilayer and the first demonstration via molecular dynamics of G protein-coupled receptor activation triggered by a ligand binding event.

FIGURE 1.

Flowchart depicting the sequence of trajectories is given here.

FIGURE 2.

This plot of root mean square deviation (RMSD) versus time for trajectory A (see Fig. 1) shows that the CB2 inactive state model stabilized within the first 50 ns.

FIGURE 3.

Relationship between the intracellular ends of TMH3 and TMH6 at designated points along the simulation is illustrated here. The CB2 TMH bundle is shown in ribbon format with phosphorus atoms of the bilayer highlighted in gold. TMHs shown in color include TMH3 (blue), TMH5 (green with red intracellular extension), and TMH6 (magenta). Top and lower left, although we began trajectory A with a salt bridge between R3.50(131) and D6.30(240) at the IC ends of TMH3/6, the receptor rearranged to form a salt bridge between R3.55(136) and D6.30(240), with Y3.51(132) hydrogen bonding to the exposed backbone carbonyl of L6.29(239). In addition, the IC-3 loop assumed two additional helical turns (shown here in red). Lower right, this figure shows the broken R3.55(136)/D6.30(240) salt bridge and the broken Y3.51(132)/L6.29(239) carbonyl interaction at 184 ns into trajectory E (compare with lower left).

FIGURE 4.

Top, at 13 ns into trajectory E, 2-AG (yellow) is poised to enter CB2 between TMH6 (magenta) and TMH7 (cyan) (shown in surface view) via an opening above W6.48(258) at the level of the 3₁₀ helical region of TMH7, which formed in trajectory C. Middle, in the control trajectory (trajectory B) for which no 2-AG partitioning out of bulk lipid occurred, the distances between the centers of mass on TMH6 and TMH7 at three different levels show no major changes. Bottom, in contrast in trajectory C, an increase in distance between TMH6/7 occurs only extracellular to W6.48.

FIGURE 5.

This figure illustrates the progress of 2-AG into the binding pocket. The color scale represents the percentage of the trajectory in which any portion of 2-AG is within 4 Å of residues on CB2 (defined here as within contact distance). Residues within contact distance are listed on the right and are color-coded according to this scale. A, in trajectory C (40–275 ns), the 2-AG has partitioned out of bulk lipid and contacts residues in or near the TMH6/7 interface. Highest contact is with F7.35(281) and C7.38(284). B, in trajectory E (0–52 ns; up to the point of headgroup insertion), 2-AG interaction with residues in the TMH6/7 interface increases with greater than 80% contact occurring with F7.35(281), S7.39(285), and C6.47(257). C, in trajectory E, after 2-AG entry into CB2 (53–127 ns), 2-AG begins to contact binding pocket residues on TMH3 (V3.32(113)), TMH6 (W6.48(258)), TMH7 (C7.42(288)), and the EC-3 loop (D(275)). D, subsequent to protonation, in trajectory G (from 913 to 1668 ns), 2-AG contacts multiple residues on TMH3/6/7 and the EC-3 loop with formation of hydrogen bonds with D(275) in the EC-3 loop and to a lesser extent with S7.39(285) (see supplemental Table S1).

FIGURE 6.

Effect of 2-AG entry into CB2 is illustrated here. The CB2 TMH bundle is shown in ribbon format. TMHs shown in color include TMH3 (blue), TMH5 (green), and TMH6 (magenta). The IC-3 loop is shown in red. a, receptor before 2-AG entry at 1 ns into trajectory E; b, 130 ns after 2-AG entry; and c, 131.14 ns after 2-AG entry. The C-α distances between Y3.51(132) and D6.30(240) (yellow balls) are 7.56 Å (a), 12.05 Å (b), and 13.86 Å (c). The hinge points for the movement of the IC3 loop, G5.53(204), and G6.38(248) are shown as red balls.

FIGURE 7.

Top, change in the distance between TMH3/6 in trajectory E is illustrated that occurs near the IC side of the receptor (A3.47(128) to A6.34(244) distance (blue) and Y3.51(132) to D6.30(240) distance (black)) starting at 59 ns into trajectory E, which is 5 ns after 2-AG enters CB2 (ligand entry time point marked by purple arrowhead). The distance between residues more EC do not change substantially (V3.40(121) to A6.42(252) distance (red) or L3.44(125) to G6.38(248) distance (green)). In contrast, bottom shows that in the control trajectory (trajectory D), there are no significant changes in the distance between TMH3/6. The orange arrowhead in top marks the time point frame used for protonation studies.

FIGURE 8.

This figure illustrates a comparison of the SASAs of R3.50(131), R3.55(136), and R(229), the latter being a CB2 IC-3 loop residue. Top, SASAs of these three arginines are shown for trajectory C + D in which no ligand binding event occurred. Bottom, these SASAs are shown for trajectory C + E in which ligand entry occurred at 54 ns into trajectory E (marked by purple arrowhead).

FIGURE 9.

This figure provides a plot of the N(R3.55(136))-O(D6.30(240)) distance versus time for the sequence trajectory C → E (up to protonation point) → trajectory F → G. The purple arrowhead marks the point of 2-AG entry, and the orange arrowhead marks the point at which CB2 was protonated. The ionic lock salt bridge between R3.55(136) and D6.30(240) was considered broken if the N(R3.55(136))-O(D6.30(240)) distance was 4 Å or greater. Distances greater than or equal to 4 Å were plotted at 4 Å.

FIGURE 10.

This figure illustrates that I5.47(198)/F3.36(117)/W6.48(258) may constitute the toggle switch for the CB2 receptor. A, at the start of trajectory G, I5.47(198) has a g+ χ_1. This places a methyl group (dashed circle) adjacent to W6.48(258) and fills the space needed for a W6.48(258) χ₁ g+ → trans change. B, at 912 ns into trajectory G, I5.47(198) undergoes a χ₁ g+ → g− transition that creates an open space (dashed circle) adjacent to W6.48(258). C, at 913 ns into trajectory G, W6.48(258) undergoes the χ₁ g+ → trans change and F3.36(117) undergoes a χ₁ trans → g+ change concomitantly. This change in F3.36(117) effectively prevents the movement of the W6.48 back to its original position in the bundle, even when D at 988 ns into trajectory G, the χ₁ of W6.48(258) returns to g+ and its χ₂ moves to trans.

FIGURE 11.

A, water location (shown in green surface display) before 2-AG entry 53 ns into trajectory E. B, water location (shown in orange surface display) after 2-AG entry (913–1668 ns, trajectory G). Here, the TMHs have been assigned the following colors: TMH1 (pink), TMH2 (gray), TMH3 (dark blue), TMH4 (gold), TMH5 (green), TMH6 (magenta), TMH7 (cyan), and Hx8 (brown). Insets illustrate the position of the phosphorus atoms (colored gold) of the lipid bilayer relative to the TMH bundle in both A and B.