Structure of the dopamine D3 receptor bound to a bitopic agonist reveals a new specificity site in an expanded allosteric pocket

Abstract

Although aminergic GPCRs are the target for ~25% of approved drugs, developing subtype selective drugs is a major challenge due to the high sequence conservation at their orthosteric binding site. Bitopic ligands are covalently joined orthosteric and allosteric pharmacophores with the potential to boost receptor selectivity, driven by the binding of the secondary pharmacophore to non-conserved regions of the receptor. Although bitopic ligands have great potential to improve current medications by reducing off-target side effects, the lack of structural information on their binding mode impedes rational design. Here we determine the cryo-EM structure of the hD₃R coupled to a G_O heterotrimer and bound to the D₃R selective bitopic agonist FOB02-04A. Structural, functional and computational analyses provide new insights into its binding mode and point to a new TM2-ECL1-TM1 region, which requires the N-terminal ordering of TM1, as a major determinant of subtype selectivity in aminergic GPCRs. This region is underexploited in drug development, expands the established secondary binding pocket in aminergic GPCRs and could potentially be used to design novel and subtype selective drugs.

Keywords: G protein-coupled receptors; GPCR; bitopic drugs; cryo-EM; dopamine D3 receptor; drug selectivity.

Figure 1.. Overall cryo-EM reconstruction of the D₃R-G_O:FOB02–04A complex.

Cryo-EM maps for the D₃R-G_O:FC)B02–04A complex in Conformation A (A) and B (B) are shown with an inset into the ligand binding site from the top view. Cryo-EM density is colored according to subunit with the bitopic ligand colored in red (Conformation A) and green (Conformation B). Coordinates for Conformation A (C) and B (D) for both complexes are shown as cartoons and colored by subunit with the bitopic ligand colored in red (Conformation A) and green (Conformation B).

Figure 2.. Coupling of the D₃R to a G_O heterotrimer.

Zooms into the D₃R (yellow) interface with the C-terminal α5 of G_O (turquoise) or G_i (purple) shown as cartoons with relevant residues as sticks. (A) Interaction details of the D₃R:G_O interface when bound to FOB02–04A. The cryo-EM density of the C-terminal α5 of G_O is shown as mesh. (B) Comparison of the D₃R coupling interface to G_i (PDB 7CMU, green) and G_O (D₃R-G_O:FC)B02–04A). (C) C-terminal α5 interactions of G_O vs G_i (PDB 7CMU) coupling. (D) Interactions between ICL2 and intracellular section of TM4 with the αN of the G_i (PDB 7CMU, green) or G_O protein.

Figure 3.. Binding of the bitopic FOB02–04A to the D₃R receptor.

(A) Schematic of dopamine, pramipexole, rotigotine and the bitopic FOB02–04A ligand shown as sticks and colored by component. (B) Binding of the secondary pharmacophore (SP) (sticks, dark red) to a groove-shaped pocket at the D₃R (yellow, surface representation) formed by ECL1 and TM1. (C) Two views of a comparison of FOB02–04A (dark red carbon, sticks), pramipexole (green carbon, sticks) and rotigotine (cyan carbon, sticks) binding into the D₃R pocket (yellow, surface representation). Dashed circles indicate OBS, established SBP and the new SBP_2-ECL1–1 site. (D) Overall binding mode of the bitopic molecule to the D₃R and ordering of TM1 upon bitopic binding. FOB02–04A (dark red, sticks) is displayed on superposed structures of D3R bound to eticlopride (cartoon, cyan) and FOB02–04A (cartoon, yellow) (E) Schematic of the FOB02–04A binding into the D₃R ligand binding pocket. Residues at the OBS, SBP and SBP_2-ECL1–1 are indicated. (F) Binding details of FOB02–04A (dark red, sticks) at the D₃R (yellow sticks) with cryo-EM density as grey mesh. (G) Binding details of FOB02–04A (dark red, sticks) at the D₃R (yellow cartoons) with residues at the ligand binding pocket colored by functional effect when mutated to alanine: decreased efficacy – green carbons, decreased potency – blue carbon and non-detectable binding – red carbon. (H) pEC50 values for alanine mutation of the residues at the ligand binding site in response to activation by FOB02–04A using the TRUPATH assay. All data are means ± SEM of three independent experiments performed in technical triplicate. *P< 0.05 (one-way ANOVA with Dunnett post hoc analysis), nd, non-detectable. (I) Dose response curves of D₃R WT (blue curves) compared to dose response curves of relevant alanine mutants (orange curves) upon activation by FOB02–04A (shown as net BRET). Data are presented as means ± SEM of three independent experiments performed in technical triplicate.

Figure 4.. Sequence and structural diversity of the SBP_2-ECL1–1 in aminergic GPCRs.

(A) Comparison of the D₃R (yellow cartoons with relevant residues as sticks) and D₂R (light blue cartoons with relevant residues as sticks) TM2-ECL1 and TM1 regions within reach of FOB02–04A (dark red, sticks), (B) Sequence alignment of TMl and TM2-ECL1 regions in aminergic GPCRs with residues around the SBP_2-ECL1–1 embedded in a box. Sequence conservation is color-coded above each residue position (gradient from dark red, conserved, to dark blue, non-conserved). Structural differences at the SBP_2-ECL1–1 site among closely related adrenergic receptors (C, D) and serotonin receptors (E, F). Receptor are shown as cartoons colored by receptor with relevant residues shown as sticks.

Figure 5.. Conformation A and B within the D₃R-G_O:FC)B02–04A complex.

Coordinates of the D₃R (yellow cartoon, with relevant residues as sticks) are shown with FOB02–04A in Conformation A (dark red, sticks) superposed to Conformation B (green, sticks), Cryo-EM density is shown as grey mesh for Conformation A (A) and Conformation B (B) with both superposed FOB02–04A conformations. (C) Predicted binding poses of bitopic FOB02–04A with D₃R showing Conformation A and Conformation B with intramolecular interactions shown as black dashes lines. Black arrows indicate distances for assessing bitopic FOB02–04A binding pose distribution between Conformations A and B with specified closest distances (E90^2.65 carboxyl group in D₃R to FOB02–04A indole atom N5 and from Y365^7.35 4-hydroxyphenyl moiety in D₃R to the phenyl ring of FOB02–04A lFI-indole-2-carboxamide SP). A semi-transparent skin reveals the receptor molecular surface, which is colored by the residue properties (red (acidic), blue (basic), green (hydrophobic)). (D) Interaction dynamics between D₃R E90^2.65 and FOB02–04A SP (depicted in brown pallet) compared with proximity distance between D3R Y365^7,35 and the SP of FOB02–04A (shown in green palette) suggest that FOB02–04A predominantly adopts Conformation A over B. Data from five independent simulations of D₃R-Gαoβγ heterotrimer crystal complex are shown, spanning 0.6 μs of cumulative time per system, with the sampling rate of 10 frames per ns, solid lines and same-color shadows representing moving average values and one standard deviation respectively from 50 frames in all cases.