Concluding model of the predicted impact of Arg132^3.50 on dopamine-dependent activation of D2R.

<p>At the inactive-state system (left row), an equilibrium between a formed and a broken ionic lock was observed, whereas the presence of dopamine was found to reduce the stability of the ionic lock. At active-state D2R (right row), dopamine increased receptor-G protein interactions via the formation of an ionic interaction between Arg132^3.50 of D2R and Asp350 of Gα_i. For clarity, TM3 and TM6 are colored in light-blue and dark-blue, respectively.</p

Total occurrences of distances larger than 9.5 Å between Arg132^3.50 and Glu368^6.30 at the simulation systems A and B.

<p>The fractions of simulation time within the systems A and B, in which the distances between the Cα-atoms of Arg132^3.50 and Glu368^6.30 were found to be larger than 9.5 Å. The values above the bars represent mean ± standard error of the mean of the simulation systems A and B and indicate a higher frequency of distances larger than 9.5 Å in the presence of dopamine (unpaired t-test, two-tailed P value = 0.0960).</p

Conformational classification of the simulation systems A-D.

<p>(A, B) Intracellular view on cytoplasmic receptor domains of the simulations systems A-D indicating unchanged global conformational states throughout the MD simulations: An overlay of average structures of representative simulation systems (each derived from the final 25ns simulation times) is shown for (A) inactive-state systems A (light-grey) and B (light-orange) and (B) the active-state systems C (dark-grey) and D (brown). For comparison, the X-ray structures of inactive-state D3R (blue) and active-state β2AR (green), which were used for homology modeling, are depicted. (C, D) The distances (and occurrences of these distances) between the intracellular ends of TM3 and TM6, measured as the distances between the Cα-atoms of Arg132^3.50 and Glu368^6.30, are shown for (C) the inactive-state systems A and B and (D) the active-state systems C and D. For comparison, these distances at crystal structures of D3R and β2AR are highlighted with dashed lines. Colors are used as described above.</p

Distances of ionic lock residues at the inactive-state systems A and B.

<p>(A) Close view on representative conformations of an intact (= closed, grey) and a broken (= open, orange) ionic lock between residues Arg132^3.50 of TM3 (light-blue) and Glu368^6.30 of TM6 (dark-blue). In addition, Arg132^3.50 is stabilized by Asp131^3.49 of TM3. (B, C) Distances between the side chains of residues Arg132^3.50 (Cζ) and Glu368^6.30 (Cδ) in the course of the simulations A and B are shown. Cumulative occurrences of certain distances for system A (B) predominantly show distances, which are consistent with an intact ionic lock (green boxes). In contrast, the latter distances are less frequently populated at the dopamine-bound system B (C), when higher occurrences were observed for larger distances, consistent with an open ionic lock.</p

Schematic overview of the main simulation systems and their simulation times.

<p>To visually help distinguish the apo- (in which dopamine is absent, A and C) from the dopamine-bound complexes (dopamine in orange, B and D), TM 1, 2 and 7 are colored in light-grey and dark-grey, respectively. For clarity, TM3 and TM6 are colored in light-blue and dark-blue, respectively. The active-state systems (C and D) are represented by the characteristic outward movement of TM6 and the presence of Gα_i (in green). The asterisk refers to a previously published simulation. The simulation times of each system are given in bold.</p

Dihedral angle of His393^6.55 in the D2R-Gα_i-complexes.

<p>On the left side of the figure, the dihedral angle of residue His393^6.55 (atoms: C-CA-CB-CG) is depicted as green and red lines for the D2^DownR-Gα_i- and the D2^UpR-Gα_i-simulations, respectively. The right column shows representative snapshots taken from the D2R-Gα_i-simulations and visualizes the interactions of residue His393^6.55 with amino acids S193^5.43 and Y408^7.35 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 His^6.55 taken from the crystal structure of the dopaminergic D₃ receptor, as grey sticks.</p

Characterization of the ligand binding pockets within the-simulation systems.

<p>(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 D2^Down/UpR-models. Helices TM3, TM4 and TM5 are shown as ribbons (green: D2^DownR; red: D2^UpR), 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 D2^DownR- and D2^UpR-simulations are depicted. (B, D) Schematic representation of interactions between the ligands BI167107 (B) and dopamine (D) and residues from β2AR and D2^Down/UpR, respectively.</p

Summary of selectivity determining amino acids within theβ2AR-Gα_s- and the D2R-Gα_i-complexes and representative values of the alanine scanning mutagenesis.

<p>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-Gα_s complex and the D2^DownR-Gα_i and the D2^UpR-Gα_i-complexes, respectively. The orange and red rectangles besides the amino acids correspond to hydrophobic or polar interactions to other residues, respectively.</p

Crucial interactions between receptors and G-proteins.

<p>Residues in the receptor-G-protein interfaces of the simulation systems are shown as sticks. The receptors (dark-blue: β2AR, dark-green: D2^DownR, dark-red: D2^UpR) and the G-proteins (light-blue: Gα_s, light-green: Gα_i^Down, light-red: Gα_i^Up) are represented as ribbons. Overview structures of the β2AR- Gα_s-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 Gα_i with residues from D2^DownR are shown. (B) Ionic interactions between positively charged amino acids from IL3 of D2^DownR and negatively charged amino acids of α5 and α4-β6 are depicted. (C); Crucial interactions of amino acids from the C-terminal part of Gα_s with residues from β2AR are shown. (D) The salt bridge between R132 of D2^UpR and D350 of Gα_i is visualized. (E) Q229 of β2AR is a crucial amino acid within a hydrogen bond network formed between β2AR and Gα_s. (F) F139 of β2AR shows pronounced hydrophobic interactions with residues from Gα_s. (G, H) Interacting amino acids of IL2 from D2^DownR (G) and D2^UpR (H) with multiple domains of Gα_i are depicted. Residue M140 is differently stabilized within the D2^DownR and D2^UpR simulations.</p

Alignment of contacts areas to G-proteins of aminergic GPCRs.

<p>Amino acids of the receptors supposed to determine selective coupling between β2AR-Gα_s and D2R-Gα_i 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-Gα_s and D2R-Gα_i, but are not supposed to determine selective coupling, are colored in yellow.</p