Figure 1.
Initial conformation of dopamine in the D2R-Gαi-complexes.
The backbone of D2R is shown as green ribbon, with important amino acids (indicated as green sticks) that stabilize the ligand dopamine in its initial conformation. Dopamine is represented as orange sticks and stabilized by ionic interactions to D1143.32 and hydrogen bonds to S1935.42 and S1975.46. The second conformation of residue H3936.55 is shown as red sticks.
Figure 2.
Characterization of the ligand binding pockets within the-simulation systems.
(A) Extracellular view into the binding pocket of β2AR (blue ribbons). Residues involved in ligand binding are shown as blue sticks, whereas the ligand BI167107 is represented as orange sticks. (C) Side view into the binding pockets of the D2Down/UpR-models. Helices TM3, TM4 and TM5 are shown as ribbons (green: D2DownR; red: D2UpR), the other parts of the receptors are removed for clarity. Residues that stabilize dopamine in its binding pocket are represented as sticks. The different conformations of dopamine (green and red sticks) within the D2DownR- and D2UpR-simulations are depicted. (B, D) Schematic representation of interactions between the ligands BI167107 (B) and dopamine (D) and residues from β2AR and D2Down/UpR, respectively.
Figure 3.
Dihedral angle of His3936.55 in the D2R-Gαi-complexes.
On the left side of the figure, the dihedral angle of residue His3936.55 (atoms: C-CA-CB-CG) is depicted as green and red lines for the D2DownR-Gαi- and the D2UpR-Gαi-simulations, respectively. The right column shows representative snapshots taken from the D2R-Gαi-simulations and visualizes the interactions of residue His3936.55 with amino acids S1935.43 and Y4087.35 depending on its dihedral angle (orange: state 1; purple: state 2; dark-cyan: state 3). Helices 5, 6 and 7 are shown as ribbons, whereas the amino acids are represented as sticks. Additionally, state 2 shows the conformation of residue His6.55 taken from the crystal structure of the dopaminergic D3 receptor, as grey sticks.
Figure 4.
Alignment of the amino-acid contacts between receptors and G-proteins.
Individual alignments for the receptors and the G-proteins are shown. A colored background indicates that the residue forms contacts to other amino acids (yellow: 1 or 2 contacts; green: 3 or 4 contacts; blue: at least 5 contacts). Red letters indicate residues involved in ionic interactions, whereas dotted underlines indicate contacts present in the crystal structure of β2AR-Gαs.
Figure 5.
Summary of selectivity determining amino acids within theβ2AR-Gαs- and the D2R-Gαi-complexes and representative values of the alanine scanning mutagenesis.
The grey columns in the middle refer to the regions within GPCRs and G-proteins, to which the mentioned amino acids belong. Amino acids in italic letters have not been mutated in the computational alanine scanning (n.d.). Blue, green and red bars show the binding free energy differences of the alanine scanning mutagenesis for the β2AR-Gαs complex and the D2DownR-Gαi and the D2UpR-Gαi-complexes, respectively. The orange and red rectangles besides the amino acids correspond to hydrophobic or polar interactions to other residues, respectively.
Figure 6.
Crucial interactions between receptors and G-proteins.
Residues in the receptor-G-protein interfaces of the simulation systems are shown as sticks. The receptors (dark-blue: β2AR, dark-green: D2DownR, dark-red: D2UpR) and the G-proteins (light-blue: Gαs, light-green: GαiDown, light-red: GαiUp) are represented as ribbons. Overview structures of the β2AR- Gαs-complex are indicated as grey tubes. The yellow rectangles point to the areas of the complexes, from which snapshots from MD simulations are visualized. (A) Specific interactions of amino acids from the C-terminus of Gαi with residues from D2DownR are shown. (B) Ionic interactions between positively charged amino acids from IL3 of D2DownR and negatively charged amino acids of α5 and α4-β6 are depicted. (C); Crucial interactions of amino acids from the C-terminal part of Gαs with residues from β2AR are shown. (D) The salt bridge between R132 of D2UpR and D350 of Gαi is visualized. (E) Q229 of β2AR is a crucial amino acid within a hydrogen bond network formed between β2AR and Gαs. (F) F139 of β2AR shows pronounced hydrophobic interactions with residues from Gαs. (G, H) Interacting amino acids of IL2 from D2DownR (G) and D2UpR (H) with multiple domains of Gαi are depicted. Residue M140 is differently stabilized within the D2DownR and D2UpR simulations.
Figure 7.
Alignment of contacts areas to G-proteins of aminergic GPCRs.
Amino acids of the receptors supposed to determine selective coupling between β2AR-Gαs and D2R-Gαi are highlighted in dark-blue and dark-green, respectively. A brighter color, light-blue or light-green, is attributed to amino acids, which show an identical sequence compared to β2AR and D2R, or, in the case of arginine and lysine residues, a similar sequence, whereas a grey color points to sequence differences. Amino acids, which appear in the interface of β2AR-Gαs and D2R-Gαi, but are not supposed to determine selective coupling, are colored in yellow.
Figure 8.
Comparison of the C-terminal parts of Gαs and Gαi.
The different conformations of the C-termini of Gαs (light-blue ribbons) and Gαi (light-green ribbons) within their pockets in β2AR (dark-blue ribbons) and D2DownR (dark-green ribbons), respectively, are shown. Important residues are represented as sticks.