Dynamics of the Apo µ-Opioid Receptor in Complex with Gi Protein

Abstract

Opioid receptors, particularly the µ-opioid receptor (μOR), play a pivotal role in mediating the analgesic and addictive effects of opioid drugs. G protein signaling is an important pathway of μOR function, usually associated with painkilling effects. However, the molecular mechanisms underlying the interaction between the μOR and G protein remain poorly understood. In this study, we employed classical all-atom molecular dynamics simulations to investigate the structural changes occurring with the μOR-G protein complex under two different conditions: with the G protein in the apo form (open) and with the GDP bound G protein (closed, holo form). The receptor was in the apo form and active conformation in both cases, and the simulation time comprised 1µs for each system. In order to assess the effect of the G protein coupling on the receptor activation state, three parameters were monitored: the correlation of the distance between TM3 and TM6 and the RMSD of the NPxxYA motif; the universal activation index (A100); and the χ² dihedral distribution of residue W293^6.48. When complexed with the open G protein, receptor conformations with intermediate activation state prevailed throughout the molecular dynamics, whereas in the condition with the closed G protein, mostly inactive conformations of the receptor were observed. The major effect of the G protein in the receptor conformation comes from a steric hindrance involving an intracellular loop of the receptor and a β-sheet region of the G protein. This suggests that G-protein precoupling is essential for receptor activation, but this fact is not sufficient for complete receptor activation.

Keywords: G protein signaling; Guanosine Di-phosphate (GDP); molecular dynamics.

Figure 1

Distances between the Cα of residue D147^3.32 of the µOR binding site and residue L348^Ga5 of the Gα monomer for the µOR-Gi apo (a) and µOR-Gi-GDP system (b). The blue line represents the adjacent averaging of 50 points during 1000 ns. The dashed lines mark the minimum (*) and maximum (**) values measured on the available structures (listed in Supplementary Table S1).

Figure 2

Distances between the Cα of residue R165^3.50 of the µOR and residue F354^Gα5 of the Gα monomer for the µOR-Gi apo (a) and µOR-Gi-GDP system (b). The blue line represents the adjacent averaging of 50 points during 1000 ns. The dashed lines mark the minimum (*) and maximum (**) values measured on the available structures (listed in Supplementary Table S1).

Figure 3

Contacts observed during MD for F354^Gαi on the µOR-Gi complex, where the receptor is cyan and the Gαi is green. The red dashed lines represent lost contacts and yellow dashed lines represent new contacts. TM6 was omitted, and H8 is transparent for the better visualization of residues.

Figure 4

RMSD values from the Cα of the µOR and the Gα protein for selected portions/systems: (a) α-helices µOR/µOR-Gi apo; (b) α-helices Gα/µOR-Gi apo; (c) β-sheets Gα/µOR-Gi apo; (d) α-helices µOR/µOR-Gi-GDP; (e) α-helices Gα/µOR-Gi-GDP; (f) β-sheets Gα/µOR-Gi-GDP.

Figure 5

Distances between the Cα of residues R165^3.50 and T279^6.34 and the RMSD of the NPxxYA motif relative to the inactive µOR (PDB id 4DKL) (Å) for µOR-Gi apo (a) and µOR-Gi-GDP (b).

Figure 6

Graph (a) shows the A¹⁰⁰ values for the µOR-Gi apo (black) and µOR-Gi-GDP (red) systems. The blue lines represent the moving averages of 50 points, while the orange ones represent the activation state classification threshold (A¹⁰⁰ < 0 is inactive, 0 < A¹⁰⁰ < 55 is intermediate, and A¹⁰⁰ > 55 is active). Graph (b) shows the Frequency Count (%) of the A¹⁰⁰ values obtained for both systems.

Figure 7

Structures of the µOR-Gi apo (a) and µOR-Gi-GDP (b) systems with highlighted portions, ICL3 (red), S6 (yellow), receptor (cyan), and Gα monomer (green).

Figure 8

χ₂ of W293^6.48 values obtained from the 1µs MD simulation for (a) µOR-Gi apo, (c) µOR-Gi-GDP, and the respective frequency counts (%) (b) and (d), respectively.

Figure 9

Normalized distances between the Center of Mass (COM) of the Gα N-terminal (a,b), Switch I (c,d), and Switch II (e,f) and the COM of Gβ. The blue line corresponds to the adjacent averaging of 50 points.

Figure 10

Salt bridge distances between D177^ICL3 and R32^Gαi and between K98^ICL1 and D332^Gβ for system µOR-Gi apo (a,b) and µOR-Gi-GDP (c,d). The blue line corresponds to the adjacent averaging of 50 points.

Figure 11

Salt bridge distances in the µOR-Gi apo between residues R182^4.40 (blue) and E38^Gαi (red) (a) and D26^Gαi (red) and K74^Gβ (blue) (b).

Figure 12

Values of the salt bridge distances obtained from the µOR-Gi apo and µOR-Gi-GDP between residues R182^4.40 and E28^Gαi (a,b) and D26^Gαi and K73^Gβ (c,d). Blue lines correspond to the adjacent averaging of 50 points.

Figure 13

Distances between the Centers of Mass (COM) of the AH domain and the Ras-like domain to assess the possible GDP binding site opening. The blue line shows the adjacent averaging of 50 points.

Figure 14

Model of the ligand-free µOR-Gi complex used in the simulations. Proteins are shown as ribbons: µOR in cyan, Gαi in green, Gβ in purple, and Gγ in orange. Ions are shown as spheres: Na⁺ in dark blue, and Cl⁻ in cyan. Lipids are shown as sticks; hydrogens not shown. Water molecules are omitted for clarity. In panel (a), the view from the side, and in panel (b), the view from the top.

Figure 15

Residues D177^ICL3–R32^Gαi (a), R182^4.40–E28^Gαi (b), D26^Gαi–K73^Gβ (c), and K98^ICL1–D332^Gβ (d), which may form salt bridges between the receptor and Gα and the receptor and Gβ, where µOR is in cyan, Gαi is in green, Gβ is purple, and the residues’ side chains are shown as sticks.

Figure 16

Parameters used to assess complex stability. Gα5 insertion into the receptor from the distance between Cα of D147^3.32 (blue sphere) and Cα of L348^Gαi (yellow sphere) (a), Cα of R165^3.50 (blue sphere) and Cα of F354^Gαi (yellow sphere) (b), where TM6 was omitted for better visualization, where µOR is in cyan, Gαi is in green, Gβ is purple, and the residues’ side chains are shown as sticks.

Figure 17

Gαi (green) model showing the Ras-like portion is in red, the alpha helical domain (AH) in blue, and the GDP (as surface, in cyan). Yellow spheres show the center of mass (COM) and the yellow dashed line indicates the distance between the COMs of the Ras-like and AH portions.

Figure 18

Gαi, Gβ, and Gγ from the µOR-Gi-GDP system, in green, purple, and orange, respectively. The N-terminal (N-term), Switch I (SwI), and Switch II (SwII) regions of Gαi are represented in red, black, and blue, respectively. The yellow spheres represent the centers of mass (COM) of the regions in which they are inserted, while the dashed yellow lines represent the distances between the COM of the areas in Gαi and the COM of Gβ. Residues R67^Gαi, T265^Gαi, and L355^Gβ were omitted for better visualization of the COM spheres.