Similar to PGD nearly all of the CRTH

Similar to PGD2, nearly all of the CRTH2 antagonists are carboxylic Kaempferol derivatives with a carboxylate moiety, which is believed to be a critical pharmacophore that interacts with the receptor (Pettipher and Whittaker, 2012) (Figure 1A). To understand the molecular mechanisms for the action of CRTH2 ligands, we solved the crystal structures of human CRTH2 bound to two antagonists, fevipiprant and CAY10471. The structures, together with the results from computational docking studies and ligand binding assays, reveal conserved and divergent structural features for the binding of diverse CRTH2 antagonists, which occupy a semi-occluded ligand-binding pocket covered by a well-structured N-terminal region with a novel conformation. Interesting characteristics of the ligand binding pocket, including a widely open end as the potential ligand entry port and a gradually increased positive charge distribution, allow us to propose a novel mechanism for the binding of PGD2. Structural comparison analysis suggests a distinct binding pose of PGD2 compared to the lysophospholipids and endocannabinoids.
Results
Discussion GPCRs recognize a broad range of molecules with a vast chemical diversity through different mechanisms. Our understanding of the recognition of lipid mediators by GPCRs primarily comes from the structural studies of receptors for lysophospholipids and endocannabinoids including S1P1, LPA1, LPA6, and CB1, which have revealed two different types of extracellular ligand recognition domains (Taniguchi et al., 2017). In S1P1, LPA1, and CB1, the N-terminal region folds on top of the ligand binding pocket and the ECL2 projects toward the inside of the 7-TM bundle to interact with the ligands, while in LPA6, the ligand binding pocket is open to the extracellular environment, with the ECL2 extending away from the 7-TM bundle, similar to BLT1. Our structures of CRTH2 reveal a new conformation of the extracellular region that, to the best of our knowledge, has not been observed in other GPCR structures. In the structures, the well-folded N-terminal region packs tightly against the ECL2, resulting in a widely open end of the ligand binding pocket as the ligand entry port. The structural analysis allows us to propose a novel mechanism for the binding of the lipid molecule PGD2 to CRTH2, in which the carboxylate group of PGD2 first binds to the ligand entry port through interactions with positively charged residues and then extends deeply into the ligand-binding pocket following the positive charge gradient, while the rest of the hydrocarbon chain is stabilized by many aromatic residues in the ligand binding pocket (Figure 5E). Our studies thus offer new insights into how GPCRs recognize chemically diverse endogenous lipid mediators. Additionally, despite the structural divergence of the extracellular domains in CRTH2, S1P1, LPA1, and CB1, these receptors share a similar feature characterized by a gap between the N-terminal segments of TM1 and TM7 (Figure S6), which also extends to the photoreceptor rhodopsin (Palczewski et al., 2000, Park et al., 2008). Such a feature may be highly conserved in a majority of lipid-activated GPCRs, providing a common structural basis for the uptake and release of lipophilic ligands.