The signaling of prostaglandin D 2 (PGD 2 ) through G-protein-coupled receptor (GPCR) CRTH2 is a major pathway in type 2 inflammation. Compelling evidence suggests the therapeutic benefits of blocking CRTH2 signaling in many inflammatory disorders. Currently, a number of CRTH2 antagonists are under clinical investigation, and one compound, fevipiprant, has advanced to phase 3 clinical trials for asthma. Here, we present the crystal structures of human CRTH2 with two antagonists, fevipiprant and CAY10471. The structures, together with docking and ligand-binding data, reveal a semi-occluded pocket covered by a well-structured amino terminus and different binding modes of chemically diverse CRTH2 antagonists. Structural analysis suggests a ligand entry port and a binding process that is facilitated by opposite charge attraction for PGD 2 , which differs significantly from the binding pose and binding environment of lysophospholipids and endocannabinoids, revealing a new mechanism for lipid recognition by GPCRs.

Similar to PGD, nearly all of the CRTH2 antagonists are carboxylic acid derivatives with a carboxylate moiety, which is believed to be a critical pharmacophore that interacts with the receptor () ( Figure 1 A). 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 PGD. Structural comparison analysis suggests a distinct binding pose of PGDcompared to the lysophospholipids and endocannabinoids.

(B) Competition radioactive ligand binding assays with HEK293T cell membranes expressing wtCRTH2 and CRTH2-mT4L. For each experiment, 2 nM [ 3 H] PGD 2 was used and various concentrations of PGD 2 (top), CAY10471 (middle), and fevipirant (bottom) were added as competing ligands. Data points are presented as the mean values ± SEM, n = 3.

CRTH2 is highly expressed in type 2 helper T cells (Th2), innate lymphoid cells (ILCs), eosinophils, and basophils (). PGD-CRTH2 signaling is a major pathway in type 2 inflammation, leading to the activation of immune cells and the production of type 2 cytokines (). Thus, CRTH2 has emerged as a promising new target in treating type 2 inflammation-driven diseases, such as asthma and allergic rhinitis, which has spurred intensive research efforts in developing CRTH2 antagonists for clinical investigation (). The first nonlipid CRTH2 antagonist, ramatroban, was discovered by serendipity (). Ramatroban was initially developed as a thromboxane receptor antagonist drug used in Japan for treating allergic diseases; it was then proven to also be a CRTH2 antagonist. Modification of ramatroban led to the discovery of the first potent and selective CRTH2 antagonist, CAY10471 (also named TM30089), which exhibits insurmountable action, in contrast to the reversible action of ramatroban in some assays (). Such early studies have inspired a number of companies to develop numerous CRTH2 antagonists with diverse chemical scaffolds and pharmacological properties in the past decade (). Several of these antagonists have been tested in asthma patients, but the results were mixed (). It has been suggested that a subpopulation of asthmatic patients whose airway inflammation is largely driven by Th2-type inflammation would benefit most from CRTH2 antagonists (). Recently, a potent CRTH2 antagonist, fevipiprant, showed promising clinical efficacy in patients with uncontrolled asthma in a few clinical trials (). Thus, CRTH2 antagonists hold the promise of being a new class of asthma drugs, and the development of new CRTH2 antagonists remains highly competitive, as evidenced by the continuing clinical investigation initiated by many companies with their own compounds ().

Eicosanoid lipid prostaglandin D(PGD) is the major prostaglandin produced by activated mast cells (). The physiological function of PGDis mainly mediated by two G protein-coupled receptors (GPCRs), PGDreceptor 1 and 2 (DPand DP), which share modest sequence similarity and couple to different G proteins (). DPis more commonly called the chemoattractant receptor-homologous molecule expressed on Th2 cells (CRTH2). While DPis closely related to other prostaglandin receptors, CRTH2 is more akin to a group of leukocyte non-chemokine chemoattractant GPCRs, which also includes the receptors for anaphylatoxin C3a and C5a, formylpeptides, leukotrienes, and some other eicosanoids () ( Figure S1 A). These non-chemokine chemoattractant receptors share a relatively high sequence similarity and the same preference for Gi protein, but they recognize diverse ligands, including lipids, peptides, and large proteins. Despite much evidence linking this group of receptors to a number of inflammatory diseases, no drugs that specifically target this group of GPCRs are currently commercially available.

In addition to the large structural divergence of the extracellular regions, the ligand binding pocket in CRTH2 also exhibits different characteristics compared to that of S1P, LPA, and CB1. The most striking difference is the polar environment of the distal end of the ligand binding pocket in CRTH2, whereas the corresponding regions in S1P, LPA, and CB1 are largely hydrophobic ( Figure S6 ). Consequently, the carboxylate head group of PGDis buried deeply inside the distal end of the pocket, while the hydrocarbon chain possibly extends toward the ligand entry port. In contrast, the fatty-acyl chains of the endogenous ligands for S1P, LPA, and CB1 are buried deep inside the binding pocket, while the polar head groups are close to the extracellular surface (). Additionally, the electrostatic charge distributions of the ligand binding pockets in these lipid GPCRs are largely different ( Figure 6 B). In the structures of antagonist-bound S1Pand LPAand agonist-bound CB1, the ligand access port is positively charged, while the rest of the binding pockets are highly negatively charged, contrasting with the highly positively charged ligand binding pocket in CRTH2. Such a charge distribution may help to position the phosphate head groups of lysophospholipids or the hydroxyl groups of endocannabinoids at the ligand access port through both electrostatic attraction and repulsion to ensure that their acyl chains are buried in the binding pocket ( Figure 6 C). Therefore, it is likely that for those receptors the acyl chains of the lysophospholipids and the endocannabinoids go into the ligand-binding pockets without a large orientation change of the lipid molecules, which is different from the proposed multistep process for the recognition of PGDby CRTH2 ( Figure 5 E).

The feature of a structured N-terminal region as a lid domain covering the ligand-binding pocket has also been observed in a few other GPCRs for diffusible lipid ligands, such as sphingosine-1-phosphate (S1P) (), lysophosphatidic acid (LPA) (), and endocannabinoids (CB1) (), and has been proposed to be a conserved feature for many lipid GPCRs. However, CRTH2 differs significantly from these lipid GPCRs in the extracellular region. First, the overall conformation of the N-terminal region is largely different in CRTH2 than in other lipid GPCRs. The N termini of S1Pand LPAform a helical structure on top of the extracellular surface that packs against ECL1 and ECL2, while the N terminus of CB1 forms a loop structure followed by a very short helix, which is buried in the helical bundle and packs against ECL1, ECL2, and TM7 ( Figure 6 A). In CRTH2, the N-helix is nearly parallel to the β-hairpin of ECL2, forming the lid domain together with the long N-loop. Second, the ECL2s in the S1P, LPA, and CB1 receptors lack a β-hairpin motif and project toward the inside of the helical bundle ( Figure 6 A). As a result, the conserved extracellular disulfide bond linking ECL2 to TM3 in almost all class A GPCRs, including CRTH2, is missing in those receptors. Third, compared to those in S1P, LPA, and CB1, the long N-loop in CRTH2 results in a wider ligand entry port on the side of the helical bundle ( Figure S6 ). This may provide enough space for the orientation change of PGDat the ligand entry port to prepare the ligand for entering the ligand binding pocket ( Figure 5 E).

(C) Cartoon diagrams of the ligand-binding modes of four lipid GPCRs. From the left to the right: CRTH2 with prostaglandin D 2 (PGD 2 ), S1P 1 with sphingosine-1-phosphate (S1P), LPA 1 with lysophosphatidic acid (LPA), and CB1 with N-arachidonoylethanolamine (AEA). The lipid bilayer is shown as the pink background. For all ligands, the polar head groups are colored in green, while the rest hydrophobic moieties are colored in yellow.

(A) Extracellular regions in the structures of CRTH2 (blue), S1P(magenta, PDB ID 3V2W ), LPA(pink, PDB ID 4Z35 ), CB1 with an inverse agonist (green, PDB ID 5U09 ), and CB1 with an agonist (cyan, PDB ID 5XRA ). Fevipiprant (orange) and ligands in other GPCRs (light blue) are shown as spheres. For S1Pand LPA, only antagonist-bound structures are available. See also Figure S6 A.

BLT1, as the receptor for the lipid LTB, is closely related to CRTH2, and their endogenous ligands LTBand PGDare both eicosanoids with a common precursor (). To the best of our knowledge, BLT1 and CRTH2 are the only two eicosanoid GPCRs with solved structures. Although their structures share a high similarity ( Figure S2 A), compared to CRTH2, the N-terminal region of BLT1 is completely disordered in the structure of guinea pig BLT1 bound to an atypical antagonist BIIL260 (), leaving the ligand binding pocket open to the extracellular milieu. This is likely an inherent feature of BLT1 since its short N-terminal sequence does not favor a structured motif and no cysteine residue is present at the N terminus to form an additional disulfide bond. The ligand binding port between TM1 and TM7 in CRTH2 is also absent in BLT1 because of a difference TM1 conformation ( Figure 2 B). Such a large structural divergence of the ligand binding pockets in BLT1 and CRTH2 suggests that those two receptors adopt different mechanisms for the lipid recognition, even though their ligands are both eicosanoids with a high chemical similarity ( Figure S2 A).

The well-structured N-terminal region results in a gap between the N-loop and TM7 as the only open end of the ligand binding pocket, which could serve as a ligand entry port for lipid agonists and antagonists ( Figure 2 ). Three positively charged residues, H95, R175, and R179 from ECL1 and ECL2, which do not directly interact with the antagonists, project their side chains into this entry port ( Figure 5 C). Interestingly, strong electron density was observed around R175 and R179 in our structures. We modeled a succinate or a propylene glycol molecule from the crystallization conditions in the two structures to fit the electron density ( Figures 5 C and S5 ). Both compounds contain polar groups that are the same as or similar to the carboxylate group in PGD, forming salt bridges or hydrogen bonds with R175 and R179. These observations suggest that the carboxylate group of PGDmay form similar interactions with those two residues at the ligand entry port during the early stage of ligand recognition. Moreover, the entire ligand binding pocket of CRTH2 exhibits a gradually increased positive charge distribution from the entry port to the distal end ( Figure 5 D). We propose that such a feature plays an important role in guiding PGDto access the pocket by attracting its carboxylate group to reach the distal end. Collectively, as shown in Figure 5 E, the process of PGDbinding suggested by our results includes the anchoring of the carboxylate group of PGDto the ligand entry port and the following access to the ligand-binding pocket facilitated by the positive charge gradient. The nonuniform charge distribution may also help PGDto change its orientation in the lipid bilayer to enter the ligand-binding pocket. Considering the negative charge property of the carboxylate group, the change in the orientation of PGDlikely does not occur spontaneously in the lipid bilayer. Supporting this mechanism for PGDrecognition, previous studies have shown that mutations of R179 could reduce the affinity of PGDby 5- to 10-fold (), and we also showed that mutating the positively charged residue R170at the distal end of the ligand binding pocket nearly abolished PGDbinding ( Figure 5 B).

The conservation of the carboxylate group in PGDand most CRTH2 antagonists suggests that the carboxylate in PGDoccupies a similar site with a highly polar environment formed by residues R170, Y183, Y184, K210, Y262, and E269 Figure 5 A). Consistently, previous mutagenesis studies have demonstrated the important roles of K210and E269in PGDbinding (). The rest of the hydrocarbon chain of PGDwith a central cyclopentyl ring likely occupies the hydrophobic space largely constituted by the aromatic residues ( Figure 5 A), which can potentially form π-π interactions with the two carbon-carbon double bonds in PGDto further stabilize the ligand. The hydrophobic environment of the ligand binding pocket is shielded from the extracellular aqueous milieu by the lid domain formed by the N terminus and ECL2. The C11A mutation, which presumably disrupts the disulfide bond linking the N-terminal region to TM5 to destabilize the N-terminal region, could significantly compromise PGDbinding in our assays, suggesting the important role of the N-terminal region in PGDbinding ( Figure 5 B).

(E) Cartoon diagram of the proposed PGD 2 binding process. The positive charge potential is indicated as “+.” The lipid bilayer is shown as the pink background. The carboxylate group in PGD 2 is colored in green, while the rest hydrophobic moiety of PGD 2 is colored in yellow.

(B) Cell surface expression levels of wild-type CRTH2 (wtCRTH2) and three mutants (left) and their specific saturation binding of 3 H-PGD 2 (right). The cell surface expression of each construct in HEK293T cells was assessed by measuring the binding of fluorescent anti-FLAG antibodies to the FLAG epitope displayed at the N terminus of the receptor through flow cytometry. The specific saturation binding assays were performed using cell membranes. The W283A mutant is shown as a positive control, in which the binding of PGD 2 was not significantly altered. Data points are presented as the mean values ± SEM, n = 3.

CAY10471 shares a high structural similarity with its parent compound ramatroban ( Figure 1 A). The major difference is that, instead of an acetate group, ramatroban has a longer propionate group attached to the central tetracarbazole group. Previous studies have shown that CAY10471 is an insurmountable antagonist with slow dissociation, while ramatroban is a highly reversible antagonist (). In addition, ramatroban is less effective in stabilizing the receptor compared to fevipiprant and CAY10471 in our thermostability assays ( Figure 4 A), indicating that ramatroban potentially engages in different interactions with the receptor. We simulated the binding of ramatroban to CRTH2 by computational docking. We used the structure of CRTH2 with CAY10471 as template because of the high structural similarity between CAY10471 and ramatroban. We validated our docking methods by reproducing the same binding pose of CAY10471 as that in the crystal structure ( Figure S4 C). Interestingly, the top-ranked docking poses of ramatroban differ significantly from those of CAY10471 in the crystal structure ( Figure 4 B). The sulfonyl fluorophenyl tail group of ramatroban occupies a region similar to that occupied by the same moiety in CAY10471. However, compared to CAY10471, the tetracarbazole and propionate moieties of ramatroban, although still buried in the aromatic pocket, adopt a flipped conformation to accommodate the longer propionate head group. This binding mode positions the carboxylate group in ramatroban away from the spatially constrained Y184-K210-Y262cluster, thereby disrupting the polar interaction network associated with CAY10471. Additionally, the tetracarbazole group of ramatroban resides in an unfavorable polar environment, further compromising ramatroban binding ( Figures 4 B and S4 C). Taken together, our findings well explain the weaker binding of ramatroban compared to that of CAY10471. More importantly, they suggest that even small changes in chemical structures during drug design may lead to significant changes in the binding affinities of CRTH2 antagonists.

Despite the conserved features, the tail groups of the two antagonists show distinct binding modes and engage in different additional interactions with the receptor ( Figures 3 A and 3B). For fevipiprant, the methylsulfonyl phenyl group extends toward ECL2, with the substituted trifluoromethyl group facing a cleft between TM1 and TM7. Additional aromatic interactions form between the phenyl group and aromatic residues F90, H95, Y183, and W283, and hydrogen bonds form between one oxygen atom of the methylsulfonyl moiety and the main chain amine group of C182. Such a binding mode of fevipiprant, together with the details of the binding pocket, well explains the results of the structure and function relationship (SAR) studies for developing fevipiprant (). For CAY10471, the sulfonyl group also extends toward ECL2, while the fluorophenyl group, as the tail group, extends toward TM7, resulting in a swing of the indole ring of W283compared to this residue in the structure with fevipiprant. Such a conformation of W283further causes the movement of L20 at the N terminus, potentially leading to a disordered region of S22–S24 in the structure of CRTH2 with CAY10471 ( Figure 3 C). The fluorophenyl group of CAY10471 participates in the aromatic interactions with residues Y183, W283, and P287. Modifications of the central tetracarcazole group of CAY10471, such as substitution with an unsaturated carcazole group with a flat plane and ring opening, would change the position of the sulfonyl fluorophenyl tail group relative to the surrounding aromatic residues and cause steric clash, thus resulting in lower affinities ().

The high-quality electron density maps allowed unambiguous modeling of fevipiprant and CAY10471 as two slow-dissociating CRTH2 antagonists in the structures () ( Figures S4 A and S4B). Fevipiprant and CAY10471 bind to a semi-occluded ligand-binding pocket with a widely open end surrounded by the N-helix, the N-loop and the extracellular parts of TM1 and TM7, and an occluded distal end surrounded by TMs 3, 5, and 6 ( Figures 3 A, S4 A, and S4B). A majority of residues in the ligand binding pocket are aromatic residues, with a few highly charged residues clustered at the occluded distal end ( Figure 3 B). The side chains of two charged residues, R170and K210, and the side chains of two tyrosine residues, Y184 in ECL2 and Y262, point toward the carboxylate head groups of the two antagonists to form a strong polar interaction network, creating a highly charged environment to hold the carboxylate group at the distal end of the ligand binding pocket. Four phenylalanine residues, F87, F111, F112, and F294, form the bottom of the ligand binding pocket. These residues, together with F90, H107, Y183, Y184, Y262, and L286, engage in extensive aromatic and hydrophobic interactions with the central aromatic groups in the antagonists: the methyl azaindole group in fevipirant and the tetrahydrocarbazole group in CAY10471. Both central aromatic groups also engage in cation-π interactions with the side chain of R170

The C-terminal region in CRTH2, D310–L327, immediately after TM7 forms an unusually long helical structure, namely, helix 8. Helix 8 in CRTH2 exhibits an interesting amphipathic nature characterized by four leucine residues and one valine residue lining the membrane-facing side, suggesting a strong membrane association, and positively charged residues lining the cytoplasmic side ( Figure S3 B). A previous study showed that the C-terminal tail of CRTH2 negatively regulated receptor signaling and that a truncation of the C terminus after R317 could enhance Gi signaling (), indicating an important role of the long helix 8 in CRTH2 signaling. In addition, the entire cytoplasmic surface of CRTH2 is highly positively charged with a number of sulfate ions modeled in the region ( Figure S3 C). Although speculative, such characteristics may suggest a potential regulation of receptor signaling by negatively charged phospholipids ().

The high-quality electron density maps allowed us to model all residues of CRTH2 from A5 to L327, except for G237, which was replaced by mT4L, in the structure of CRTH2 with fevipiprant. The structure of CRTH2 with CAY10471 is nearly identical to the structure with fevipiprant, except for a few loop residues near the ligand binding pocket and the disordered S22–A25 region at the N terminus. The overall structure of CRTH2, especially the second extracellular loop (ECL2) with a conserved β-hairpin structure, is similar to the structures of two other non-chemokine chemoattractant GPCRs that are close phylogenetic neighbors, namely, the LTBreceptor (BLT1) and the C5a receptor (C5aR) ( Figure S2 A). Interestingly, even though C5aR recognizes peptide ligands, which are chemically distinct from the lipid mediators recognized by CRTH2, if the structures of C5aR and CRTH2 are superimposed on each other, the antagonists of CRTH2 nearly overlap with part of the peptide antagonist PMX53 for C5aR ( Figure S2 B). However, unlike BLT1 and C5aR, CRTH2 contains a well-folded N-terminal structure with a central alpha helix (N-helix) connected to the transmembrane helix 1 (TM1) by a long loop (N-loop) ( Figures 2 A and S3 A). The N-helix and N-loop pack tightly against the second extracellular loop (ECL2), forming a lid between ECL1 and ECL3 to largely cover the ligand binding pocket ( Figure 2 A). Besides the disulfide bond between C182 in ECL2 and C104(Ballesteros-Weinstein numbering) in TM3 that is conserved in most rhodopsin-like GPCRs, an additional disulfide bond was found between C11 and C199, linking the N-terminal helix to TM5 and thereby fixing the position of the N-terminal region ( Figure 2 A). The N-terminal region of TM1 in CRTH2 tilts toward TM2 compared to those in BLT1 and C5aR and thereby creates a gap between TM1 and TM7 as the only open end of the ligand-binding pocket ( Figures 2 B and S2 C). Because the extracellular ligand access is restricted to the pocket by the lid domain, this gap in the lateral side of CRTH2 is likely to be the ligand entry port.

(B) Structural comparison of N-terminal region and TM1 in CRTH2 (blue), BLT1 (cyan) and C5aR (gray). In both (A) and (B), the open end of the ligand binding pocket in CRTH2 as the potential ligand entry port is marked with a red dashed circle, which is occupied by the extracellular regions of TM1 in BLT1 and C5aR.

(A) Well-folded N-terminal region with an N-helix and N-loop in the structure of CRTH2 bound to fevipiprant (green). The disulfide bond connecting the N terminus and TM5 is indicated with an arrow. Fevipiprant is shown as orange spheres.

To crystallize human CRTH2, a construct of human CRTH2 was generated by inserting an engineered T4 lysozyme (mT4L) () with an additional N-terminal 8-amino acid linker into the intracellular loop 3 (ICL3) for crystallization ( Figure S1 B). The 8-amino acid linker greatly improved crystal quality, which was achieved unintentionally. To further facilitate crystallogenesis, the flexible C-terminal region from R340 to S395 was removed before crystallization, and the potential glycosylation site N25 was mutated to alanine. No other mutations were introduced. Ligand competition binding assays showed that the sequence modifications did not significantly affect the ligand-binding properties of CRTH2 ( Figure 1 B). Using this construct, we solved the crystal structures of human CRTH2 in complex with two antagonists, fevipiprant and CAY10471, at 2.80 Å and 2.74 Å resolution, respectively ( Figure 1 C; Table 1 ).

Discussion

1 , LPA 1 , LPA 6 , and CB1, which have revealed two different types of extracellular ligand recognition domains ( Taniguchi et al., 2017 Taniguchi R.

Inoue A.

Sayama M.

Uwamizu A.

Yamashita K.

Hirata K.

Yoshida M.

Tanaka Y.

Kato H.E.

Nakada-Nakura Y.

et al. Structural insights into ligand recognition by the lysophosphatidic acid receptor LPA 6 . 1 , LPA 1 , 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 LPA 6 , 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 PGD 2 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 ( 1 , LPA 1 , and CB1, these receptors share a similar feature characterized by a gap between the N-terminal segments of TM1 and TM7 ( Palczewski et al., 2000 Palczewski K.

Kumasaka T.

Hori T.

Behnke C.A.

Motoshima H.

Fox B.A.

Le Trong I.

Teller D.C.

Okada T.

Stenkamp R.E.

et al. Crystal structure of rhodopsin: A G protein-coupled receptor. Park et al., 2008 Park J.H.

Scheerer P.

Hofmann K.P.

Choe H.W.

Ernst O.P. Crystal structure of the ligand-free G-protein-coupled receptor opsin. 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 S1P, LPA, LPA, and CB1, which have revealed two different types of extracellular ligand recognition domains (). In S1P, LPA, 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 LPA, 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 PGDto 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 5 E). 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, S1P, LPA, 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 (). 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.

6.51, which is conserved as a Y or F in other non-chemokine chemoattractant GPCRs, directly interacts with the ligands of all three receptors (6.44XXCW6.48XP6.50 that is highly conserved in rhodopsin-like GPCRs and interacts with W6.48, which has been suggested to function as a toggle switch in the activation of some GPCRs ( Smit et al., 2007 Smit M.J.

Vischer H.F.

Bakker R.A.

Jongejan A.

Timmerman H.

Pardo L.

Leurs R. Pharmacogenomic and structural analysis of constitutive g protein-coupled receptor activity. 6.44, W6.48, and Y/F6.51, line up in TM6 to constitute a critical structural motif that mediates the propagation of signal from the extracellular ligand binding pocket to the cytoplasmic region that interacts with intracellular signaling molecules in receptor activation. CRTH2 belongs to a group of non-chemokine chemoattractant GPCRs that are phylogenetically close to each other but recognize very diverse ligands from lipids to peptides to large proteins ( Figures S1 A and S2 A). The structures of CRTH2 reported here, together with the previously reported structures of BLT1 and C5aR, show a large structural divergence of the extracellular region in those receptors, likely accounting for the recognition of diverse ligands by those GPCRs. On the other hand, the structures also reveal a conserved structural feature in these receptors. One residue in TM6, Y, which is conserved as a Y or F in other non-chemokine chemoattractant GPCRs, directly interacts with the ligands of all three receptors ( Figure S7 ). This residue sits on top of a structural motif FXXCWXPthat is highly conserved in rhodopsin-like GPCRs and interacts with W, which has been suggested to function as a toggle switch in the activation of some GPCRs (). We propose that for the group of non-chemokine chemoattractant GPCRs, the three conserved residues, F, W, and Y/F, line up in TM6 to constitute a critical structural motif that mediates the propagation of signal from the extracellular ligand binding pocket to the cytoplasmic region that interacts with intracellular signaling molecules in receptor activation.

4 , and PGD 2 , respectively, are both eicosanoid lipid mediators with a high chemical similarity ( 4 and PGD 2 . Whether the recognition of SPMs by their receptors is similar to the lipid recognition by BLT1 or by CRTH2 needs further investigation. This is important considering the increasing research interests in developing new pro-resolving mediators as a novel therapy for treating inflammatory diseases ( Dalli and Serhan, 2018 Dalli J.

Serhan C.N. Identification and structure elucidation of the pro-resolving mediators provides novel leads for resolution pharmacology. The two receptors, CRTH2 and BLT1, apparently adopt different mechanisms for lipid recognition, with distinct ligand binding pockets, even though the endogenous ligands for BLT1 and CRTH2, LTB, and PGD, respectively, are both eicosanoid lipid mediators with a high chemical similarity ( Figure S2 A). Some other members of this group of GPCRs, including FPR2/ALX, ChemR23 (CMKLR1), and GPR32, recognize a special group of eicosanoid lipids called specialized pro-resolving lipid mediators (SPMs). SPMs can promote the resolution of inflammation, in contrast to the primary pro-inflammatory function of most eicosanoid lipids, including LTBand PGD. Whether the recognition of SPMs by their receptors is similar to the lipid recognition by BLT1 or by CRTH2 needs further investigation. This is important considering the increasing research interests in developing new pro-resolving mediators as a novel therapy for treating inflammatory diseases (). In addition, one member of this group, FPR2/ALX, can sense both formyl peptides and SPMs. The molecular mechanism for such promiscuous ligand recognition remains elusive.

Pettipher and Whittaker, 2012 Pettipher R.

Whittaker M. Update on the development of antagonists of chemoattractant receptor-homologous molecule expressed on Th2 cells (CRTH2). From lead optimization to clinical proof-of-concept in asthma and allergic rhinitis. 7.32 and the N-loop indicate that the open end of the ligand binding pocket, which we propose to be the ligand entry port, exhibits certain structural flexibility. Additional structures of CRTH2 with other antagonists that have distinct tail groups are needed to further investigate the conformational diversity of residues in this region, as it may significantly affect the results of structure-based virtual screening for developing novel CRTH2 antagonists. Furthermore, the unexpected small molecules modeled in this region suggest that the ligand entry port may offer an additional site for designing new synthetic CRTH2 antagonists, which compared to CAY10471 and fevipiprant would engage in additional interactions with the receptor to achieve stronger binding and a longer duration of action. Our structures also provide new insights into CRTH2 drug development. The ligand binding pocket revealed by our structures comprises many aromatic residues and a few polar residues at the distal end. Correspondingly, most CRTH2 antagonists share a similar structural feature characterized by an acetate polar group attached to a central aromatic group to fit the ligand binding pocket (). The different binding poses of the tail groups of CAY10471 and fevipiprant associated with the different conformations of W283and the N-loop indicate that the open end of the ligand binding pocket, which we propose to be the ligand entry port, exhibits certain structural flexibility. Additional structures of CRTH2 with other antagonists that have distinct tail groups are needed to further investigate the conformational diversity of residues in this region, as it may significantly affect the results of structure-based virtual screening for developing novel CRTH2 antagonists. Furthermore, the unexpected small molecules modeled in this region suggest that the ligand entry port may offer an additional site for designing new synthetic CRTH2 antagonists, which compared to CAY10471 and fevipiprant would engage in additional interactions with the receptor to achieve stronger binding and a longer duration of action.