Molecular basis for lipid recognition by the prostaglandin D2 receptor CRTH2

Abstract

Prostaglandin D₂ (PGD₂) signals through the G protein-coupled receptor (GPCR) CRTH2 to mediate various inflammatory responses. CRTH2 is the only member of the prostanoid receptor family that is phylogenetically distant from others, implying a nonconserved mechanism of lipid action on CRTH2. Here, we report a crystal structure of human CRTH2 bound to a PGD₂ derivative, 15R-methyl-PGD₂ (15mPGD₂), by serial femtosecond crystallography. The structure revealed a "polar group in"-binding mode of 15mPGD₂ contrasting the "polar group out"-binding mode of PGE₂ in its receptor EP3. Structural comparison analysis suggested that these two lipid-binding modes, associated with distinct charge distributions of ligand-binding pockets, may apply to other lipid GPCRs. Molecular dynamics simulations together with mutagenesis studies also identified charged residues at the ligand entry port that function to capture lipid ligands of CRTH2 from the lipid bilayer. Together, our studies suggest critical roles of charge environment in lipid recognition by GPCRs.

Keywords: CRTH2 (DP2); MD simulations; crystal structure; lipid binding; prostaglandin D2.

Fig. 1.

Overall structure of CRTH2 bound to 15mPGD₂ and its ligand-binding pocket. (A) Chemical structures of 15mPGD₂, PGD₂, and PGE₂. C-9, C-11, and C-15 positions are indicated with italic numbers. (B) Overall structure of CRTH2 bound to 15mPGD₂ and structure alignment with CRTH2 bound to fevipiprant. (C) Overlapping, binding positions of 15mPGD₂ and fevipiprant. (D) Potential lipid entry port. (E) Binding pocket of 15mPGD₂. (F) Docking of PGE₂ (pink) into the 15mPGD₂- (yellow) binding pocket. Hydrogen bonds are shown as dashed lines.

Fig. 2.

Structural comparison analysis. (A) CRTH2 with 15mPGD₂. The N-terminal region preceding TM1 is colored in light pink. (B–D) EP2, EP3, and EP4 with PGE₂ (Protein Data Bank [PDB] IDs: 7CX2, 6AK3, and 7D7M, respectively). (E) TP with ramatroban (PDB ID: 6IIU). The N-terminal region preceding TM1 is colored in salmon. ECL2 in each receptor is colored in magenta. 15mPGD₂, PGE₂, and ramatroban are colored in yellow, green, and purple, respectively. The carboxyl group in each ligand is circled. The charge distributions of ligand-binding pockets of these prostanoid receptors are shown in the lower panels. (F) Sequence alignment of all prostanoid receptors. The alignment of ECL2 sequences was based on the alignments of two highly conserved residues, P^4.50 and the cysteine residue, in ECL2, forming the conserved extracellular disulfide bond. The alignment of TM5 sequences was based on the alignment of 5.50 residues in the receptors. (G) Two lipid-binding modes.

Fig. 3.

PGE₂ and PGD₂ interact with different sets of residues to activate their receptors. (A) EP3 (brown) with PGE₂ (green) (PDB ID: 6AK3). (B) EP2 (light brown) with PGE₂ (green) (PDB ID: 7CX2). The ω-chain of PGE₂ extends below the 6.51 residue to interact with W295^6.48 in EP3 or M124^3.40 in EP2. (C) CRTH2 (cyan) with 15mPGD₂ (yellow). PGD₂ binds to a superficial binding pocket in CRTH2 so that the ω-chain of PGD₂ is above the residue Y^6.51. The hydrogen bond is shown as a black dashed line.

Fig. 4.

Conformational dynamics of 15mPGD₂ in CRTH2 holo-1 and holo-2 systems. The CRTH2 structures at t = 0 ns (translucent gray ribbon) are superposed with structures at t = 1 µs in holo-1 (A) (translucent green ribbon) and t = 1 µs in holo-2 (B) (translucent purple ribbon). In both panels, conformations of bound 15mPGD₂ at t = 0 ns (translucent gray) and t = 1 µs (gold) are shown in stick and ball-and-stick representation, respectively. Salt–bridge interactions formed by the carboxyl group of 15mPGD₂ with R170^ECL2, R175^ECL2, and K210^5.42 are shown as black dotted lines in A and B. Evolution of the salt bridges during the 1-µs simulation in holo-1 (C) and holo-2 (D) systems are presented as blue, red, and yellow lines (50-point moving average) corresponding to 15mPGD₂-R170^ECL2, 15mPGD₂-R175^ECL2, and 15mPGD₂-K210^5.42 interactions, respectively, with the dotted black line indicating the cutoff distance of 4.0 Å.

Fig. 5.

CRTH2–POPC interaction in holo-1 simulation and mutagenesis data. (A) Interaction of a bilayer POPC molecule, with the residues lining the entry port in holo-1 simulation. The structures of the receptor at t = 0 ns (translucent gray) and t = 1 μs (green) are shown in ribbon representation. The entry port residues (pink), bound 15mPGD₂ (yellow), POPC (gray), and binding site residues R170^ECL2 and K210^5.42 (purple) are shown in sphere representation. (B) Close up of the CRTH2 entry port–POPC interaction, with the bound POPC (gray) and entry port residues (pink) shown in ball-and-stick and stick representation, respectively. The interactions between the POPC and the protein residues are shown as black dotted lines. (C) Evolution of CRTH2–POPC intermolecular salt bridges during the 1-µs holo-1 simulation, with the dotted black line indicating the cutoff distance of 4.0 Å. (D) Saturation-binding assays on different CRTH2 constructs using ³H-PGD₂. All CRTH2 constructs were transiently expressed in human embryonic kidney 293 cells, and cell membranes were prepared and used in the ligand-binding assays. Expression of each construct was confirmed by cell surface staining. Nonspecific binding measured in the presence of access amount of PGD₂ was subtracted. The dissociation equilibrium constants (K_ds) of PGD₂ for the wtCRTH2 and the R284A mutant are listed in the table shown on the right of panel D. For other CRTH2 mutants, no saturable ³H-PGD₂ binding was observed, indicating mostly nonspecific binding. Each data point in the left panel is shown as mean ± SEM and n = 3.

Fig. 6.

Conformational dynamics of the ligand-binding pocket in MD simulations. (A) Evolution of radius of gyration (50-point moving average) of the binding site residues in 1-µs trajectories. Evolution of S-π–type W283^7.31–M17^N-helix (B) and Y183^ECL2–M17^N-helix (C) interactions in 1-µs trajectories. Scatter plot showing distribution of W283^7.31 (ring centroid)–M17^N-helix (S-atom) distances and W283^7.31 χ1 rotamer angles in W283^7.31 apo-1 (D), apo-2 (E), holo-1 (F), and holo-2 (G) trajectories. Structures with W283^7.31 χ1 rotamer angle equal to the average W283^7.31 χ1 rotamer angle in the last 500 ns of the simulation are selected as representatives from each trajectory. The distance and χ1 rotamer angle corresponding to the crystal structure and representative structure from each simulation are shown using a black star and square in the scatter plots. Organization of the W283^7.31–M17^N-helix–Y183^ECL2 bridge motif in representative structures, along with their W283^7.31 χ1 rotamer angle, from apo-1 (H) (yellow), apo-2 (I) (pink), holo-1 (J) (green), and holo-2 (K) (purple) trajectories. The bridge motif from the crystal structure (gray) is also shown superposed to the representative structure.