Molecular basis for lipid recognition by the prostaglandin D2 receptor CRTH2

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.

Eicosanoids constitute a group of signaling lipid mediators that are derived from arachidonic acid or other polyunsaturated fatty acids (1, 2). As the name indicates, they all possess a long hydrocarbon chain with 20 carbon units, which is usually attached to a carboxylic acid head group. Prostanoids including prostaglandins D₂, E₂, and F₂ (PGD₂, PGE₂, and PGF₂); prostacyclin (PGI₂); thromboxane A₂ (TXA₂) and leukotrienes including leukotriene B₄, C₄, and D₄ (LTB₄, LTC₄, and LTD₄) are representative endogenous eicosanoids that can induce signaling through G protein–coupled receptors (GPCRs) to play critical roles in inflammation, immunity, hemostasis, and tissue repair (1, 3–5). In humans, nine prostanoid receptors have been identified: PGD₂ receptors 1 and 2 (DP1 and DP2), PGE₂ receptors 1 to 4 (EP1 to EP4), PGF₂ receptor (FP), PGI₂ receptor (IP), and TXA₂ receptor (TP) (5). All of these receptors, except for DP2, belong to the α-branch of Class A GPCRs as close phylogenetic neighbors of aminergic receptors (5, 6). DP2, chemoattractant receptor–homologous molecule expressed on type 2 helper T cells (Th2) (also named CRTH2), is more closely related to a group of chemotactic GPCRs in the γ-branch of Class A GPCRs, including chemokine receptors and receptors for LTB₄, formylpeptides, and complement peptides C3a and C5a (6, 7).Certain types of immune cells, including eosinophils, innate lymphoid cells, and Th2 cells, express high levels of CRTH2 (8–11). PGD₂ signaling through CRTH2 can induce chemotaxis of these immune cells, which is a major pathway that drives the onset of type 2 inflammation (11–15). Therefore, the roles of PGD₂–CRTH2 signaling axis in type 2 inflammation-related diseases such as asthma and allergic rhinitis have attracted outstanding research interest (14, 16, 17). CRTH2 antagonists hold the potential of being a new class of anti-inflammatory drugs (16, 18–24). Although clinical trials have generated mixed results on different CRTH2 antagonists for asthma (17–20, 25), it is likely that a certain subpopulation of patients, such as those with a high baseline of Th2 cells or eosinophils, may benefit most from anti-CRTH2 therapy (17, 24). In addition, it has been proposed that insurmountable CRTH2 antagonists with prolonged receptor residence time can provide better, therapeutic efficacy, compared to reversible and fast, dissociating CRTH2 antagonists, which needs further investigation (26).We have previously reported crystal structures of CRTH2 bound to two antagonists, CAY10471 and fevipiprant, which revealed a positively charged environment of the ligand-binding pocket and a potential ligand entry port (27). The structural analysis of antagonist-bound CRTH2 suggested that PGD₂ may enter the ligand-binding pocket through the ligand entry port by opposite charge attraction (27). Recently, crystal structures of several other prostanoid receptors, EP3, EP4, and TP, and cryogenic electron microscopy (cryo-EM) structures of the EP2 and EP4 signaling complexes with PGE₂ have also been reported (28–33). To further study the binding of lipid agonists to CRTH2 and investigate if CRTH2 differs from other prostanoid receptors in lipid recognition and receptor activation, we determined a 2.6-Å resolution, room temperature crystal structure of CRTH2 bound to a PGD₂ derivative by serial femtosecond crystallography (SFX) using an X-ray free electron laser (XFEL) source. We performed computational simulation studies using the CRTH2–15R-methyl-PGD₂ (15mPGD₂) structure, which helped identify the roles of specific residues surrounding the ligand entry port and provided molecular insights into the events that could facilitate ligand binding. Results from our mutational analysis of the identified residues helped us further strengthen the proposed model for ligand capture and entry in CRTH2.