Molecular basis for ligand recognition and receptor activation of the prostaglandin D2 receptor DP1

Abstract

The prostaglandin D2 receptor 1 (DP1), a rhodopsin-like Class A GPCR, orchestrates critical physiological and pathological processes, ranging from sleep regulation to inflammatory responses and cardiovascular function. Despite its therapeutic significance, structural insights into DP1 activation mechanisms have remained elusive. Here, using cryoelectron microscopy (cryo-EM), we determined high-resolution structures of human DP1 in both inactive and active states, with the latter captured in complex with its endogenous agonist PGD2 or the synthetic agonist BW245C, bound to the stimulatory G protein, Gs. Our structures, coupled with functional and mutagenesis studies, unveiled unique structural features of DP1, including an alternative activation mechanism, ligand-selectivity determinants, and G protein coupling characteristics. These molecular insights provide a rational framework for designing selective DP1-targeted therapeutics, both agonists and antagonists, with enhanced specificity and reduced off-target effects, opening broad avenues for treating DP1-associated disorders.

Keywords: drug design; prostaglandin D2 receptor 1; receptor activation.

Fig. 1.

Structure determination of DP1 -G_s complexes. (A) Cryo-EM density and cartoon representation of the model of the PGD₂-DP1-Gs complex. DP1 is shown in Indian red, Gα_s in light salmon, Gβ in medium purple, Gγ in forest green, Nb35 in light gray, and PGD₂ in gold. (B) Cryo-EM density and cartoon representation of the model of the BW245C-DP1-G_s complex. DP1 is shown in light sea green, G_s in light salmon, Gβ in medium purple, Gγ in forest green, Nb35 in light gray, and BW245C in orchid. (C) Cryo-EM density map and cartoon representation of the inactive DP1 receptor model fused with BRIL in the third intracellular loop (ICL3). The DP1 receptor is rendered in burly wood coloration, while the BRIL fusion protein is displayed in slate gray. (D) Comparison of the ligand-bound DP1-G_s complex with the EP2-G_s complex (PDB ID: 7CX2), EP3-G_i structure (PDB ID: 8GDC), EP4-G_s structure (PDB ID: 8GDB), and FP-G_q structure (PDB ID: 8IUK) at their receptor parts. DP1 is shown in light sea green, EP2 in violet, EP3 in medium purple, EP4 in pink, and FP in yellow. (E) Detailed diagram comparing the ligand-bound DP1-G_s complex with the EP2 structure (PDB ID: 7CX2), EP3 structure (PDB ID: 8GDC), EP4 structure (PDB ID: 8GDB), and FP-G_q structure (PDB ID: 8IUK) at their receptor parts. DP1 is shown in light sea green, EP2 in violet, EP3 in medium purple, EP4 in pink, and FP in yellow.

Fig. 2.

The PGD₂ binding pocket of DP1. (A) Vertical cross-section of the PGD₂ binding pocket in the DP1 receptor. (B) Corresponding interactions that contribute to PGD₂ binding in the DP1 receptor. (C) Region division of PGD₂ and corresponding interactions that contribute to the PGD₂ binding with the DP1 receptor. Hydrogen bonds are depicted as red dashed lines. (D) cAMP activation assay of key mutants in the DP1 receptor that bind to PGD₂. ΔpEC₅₀ = pEC₅₀ of PGD₂ for the specific mutant - pEC₅₀ of PGD₂ for the wild-type (WT) receptor. Data are presented as mean values ± SEM; n = 3 independent samples; significance was determined using One-way ANOVA; n.s. = not significant; *P < 0.05; **P < 0.01; ***P < 0.001.

Fig. 3.

The BW245C binding pocket of the DP1 receptor and the selective mechanism of BW245C. (A) Vertical cross-section of the BW245C binding pocket in the DP1 receptor. (B) Comparison of the binding patterns of PGD₂ and BW245C. (C) Corresponding interactions that contribute to BW245C binding in the DP1 receptor. (D) Region division of BW245C and corresponding interactions that contribute to the BW245C binding with the DP1 receptor. Hydrogen bonds are depicted as red dashed lines. (E) cAMP activation assay of key mutants in the DP1 receptor that bind to BW245C. ΔpEC₅₀ = pEC₅₀ of BW245C for the specific mutant - pEC₅₀ of BW245C for the WT receptor. Data are presented as mean values ± SEM; n = 3 independent samples; significance was determined using One-way ANOVA; n.s. = not significant; *P < 0.05; **P < 0.01; ***P < 0.001. (F) Distribution of minimal distance of BW245C’s carboxyl group to sidechain oxygen of T181 and Y87. Red and blue lines represent WT and R310A systems, respectively. (G) Sequence alignment of prostanoid receptors. Hydrophobic residues are shown in yellow, polar charged residues in blue, and polar uncharged residues in green.

Fig. 4.

Active structure of DP1. (A) Comparison of transmembrane helices TM5, TM6, and TM7 of the active DP1 receptor, β₂ adrenergic receptor (β₂AR), and inactive DP1 receptor. (B) Distance display between the ligands and the residue at position 6.48. (C) The steric clashes that exists between TM6 and TM7. (D) Distance display between the ligands and the residue at position 7.47. (E and F) The rotation and the map density of K76 in active and inactive DP1. (G) A steric clash exists between I317 in the inactive DP1 receptor and the backbone atoms of TM1 in the active DP1 conformation. (H) Distance display between the ligands and the residue K76^2.54. (I) Distribution of sidechain heavy atom RMSD for K76^2.54. (J and K) cAMP accumulation assay of WT and K76A, I317A mutants in DP1 with PGD₂ and BW245C. (L) Comparison of the D/ERY motif between the active DP1, inactive DP1 and EP4 receptors.

Fig. 5.

DP1-G_s coupling. (A) Comparison of the structures of the Gα_s-coupled DP1 receptor, β₂ adrenergic receptor (β₂AR; PDB ID: 3SN6), and EP2 receptor (PDB ID: 7CX2). The DP1 receptor is shown in light sea green, β₂AR in light purple, and EP2 in pink. (B) The residues in the DP1 receptor, β₂AR, and EP2 receptor that contact the Gα_s subunit. (C) Detailed interactions of the intracellular loop 1 (ICL1) and intracellular loop 2 (ICL2) with the Gα_s subunit. Residues are shown as sticks, with the corresponding cryo-EM density represented as a mesh. (D) Detailed interactions of the “ECW” motif with the Gα_s subunit.