Structures of the Human PGD2 Receptor CRTH2 Reveal Novel Mechanisms for Ligand Recognition

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Graphical Abstract Authors Lei Wang, Dandan Yao, R.N.V. Krishna Deepak, ..., Weimin Gong, Zhiyi Wei, Cheng Zhang

Correspondence [email protected] (Z.W.), [email protected] (C.Z.)

In Brief Wang et al. reported crystal structures of antagonist-bound human CRTH2 as a new asthma drug target. Chemically diverse antagonists occupy a similar semi-occluded pocket with distinct binding modes. Structural analysis suggests a potential ligand entry port and an opposite charge attraction-facilitated binding process for the endogenous

CRTH2 ligand prostaglandin D2.

Highlights d Crystal structures of antagonist-bound human CRTH2 are solved d A well-structured N terminus covers ligand binding pocket d Conserved and divergent binding features of CRTH2 antagonists are revealed d A multiple-step binding process of prostaglandin D2 is proposed

Wang et al., 2018, Molecular Cell 72, 48–59 October 4, 2018 ª 2018 Elsevier Inc. https://doi.org/10.1016/j.molcel.2018.08.009 Molecular Cell Article

Structures of the Human PGD2 Receptor CRTH2 Reveal Novel Mechanisms for Ligand Recognition

Lei Wang,1,7 Dandan Yao,2,3,7 R.N.V. Krishna Deepak,4 Heng Liu,1 Qingpin Xiao,1,5 Hao Fan,4 Weimin Gong,2,6 Zhiyi Wei,5,* and Cheng Zhang1,8,* 1Department of Pharmacology and Chemical Biology, School of Medicine, University of Pittsburgh, Pittsburgh, PA 15261, USA 2Key Laboratory of RNA Biology, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China 3University of Chinese Academy of Sciences, Chinese Academy of Sciences, Beijing 100049, China 4Bioinformatics Institute (BII), Agency for Science, Technology and Research (A*STAR), Singapore 138671, Singapore 5Department of Biology, Southern University of Science and Technology, Shenzhen, Guangdong 518055, China 6Hefei National Research Center for Physical Sciences at the Microscale, School of Life Sciences, University of Science and Technology of China, Hefei, Anhui 230027, China 7These authors contributed equally 8Lead Contact *Correspondence: [email protected] (Z.W.), [email protected] (C.Z.) https://doi.org/10.1016/j.molcel.2018.08.009

SUMMARY chemokine chemoattractant GPCRs, which also includes the receptors for anaphylatoxin C3a and C5a, formylpeptides, leu- The signaling of prostaglandin D2 (PGD2) through kotrienes, and some other eicosanoids (Fredriksson et al., G-protein-coupled receptor (GPCR) CRTH2 is a ma- 2003; Nagata and Hirai, 2003; Serhan, 2014)(Figure S1A). These jor pathway in type 2 inflammation. Compelling evi- non-chemokine chemoattractant receptors share a relatively dence suggests the therapeutic benefits of blocking high sequence similarity and the same preference for Gi protein, CRTH2 signaling in many inflammatory disorders. but they recognize diverse ligands, including lipids, peptides, Currently, a number of CRTH2 antagonists are under and large proteins. Despite much evidence linking this group of receptors to a number of inflammatory diseases, no drugs that clinical investigation, and one compound, fevipi- specifically target this group of GPCRs are currently commer- prant, has advanced to phase 3 clinical trials for cially available. asthma. Here, we present the crystal structures of CRTH2 is highly expressed in type 2 helper T cells (Th2), innate human CRTH2 with two antagonists, fevipiprant lymphoid cells (ILCs), eosinophils, and basophils (Cosmi et al., and CAY10471. The structures, together with dock- 2000; Hirai et al., 2001; Mjo¨ sberg et al., 2011; Nagata et al., ing and ligand-binding data, reveal a semi-occluded 1999). PGD2-CRTH2 signaling is a major pathway in type 2 pocket covered by a well-structured amino terminus inflammation, leading to the activation of immune cells and the and different binding modes of chemically diverse production of type 2 cytokines (Monneret et al., 2001; Xue CRTH2 antagonists. Structural analysis suggests et al., 2005). Thus, CRTH2 has emerged as a promising new a ligand entry port and a binding process that is target in treating type 2 inflammation-driven diseases, such facilitated by opposite charge attraction for PGD , as asthma and allergic rhinitis, which has spurred intensive 2 research efforts in developing CRTH2 antagonists for clinical which differs significantly from the binding pose investigation (Kupczyk and Kuna, 2017; Pettipher et al., 2007; and binding environment of lysophospholipids and Pettipher and Whittaker, 2012; Schuligoi et al., 2010). The first endocannabinoids, revealing a new mechanism for nonlipid CRTH2 antagonist, ramatroban, was discovered by lipid recognition by GPCRs. serendipity (Hirai et al., 2002; Sugimoto et al., 2003). Ramatro- ban was initially developed as a thromboxane receptor antagonist drug used in Japan for treating allergic diseases; it was INTRODUCTION then proven to also be a CRTH2 antagonist. Modification of ramatroban led to the discovery of the first potent and selective

Eicosanoid lipid prostaglandin D2 (PGD2) is the major prosta- CRTH2 antagonist, CAY10471 (also named TM30089), which glandin produced by activated mast cells (Lewis and Austen, exhibits insurmountable action, in contrast to the reversible ac-

1981). The physiological function of PGD2 is mainly mediated tion of ramatroban in some assays (Mathiesen et al., 2006; Ulven by two G protein-coupled receptors (GPCRs), PGD2 receptor 1 and Kostenis, 2005). Such early studies have inspired a number and 2 (DP1 and DP2), which share modest sequence similarity of companies to develop numerous CRTH2 antagonists with and couple to different G proteins (Monneret et al., 2001; Nagata diverse chemical scaffolds and pharmacological properties in et al., 1999). DP2 is more commonly called the chemoattrac- the past decade (Kupczyk and Kuna, 2017; Pettipher and Whit- tant receptor-homologous molecule expressed on Th2 cells taker, 2012; Santus and Radovanovic, 2016). Several of these

(CRTH2). While DP1 is closely related to other prostaglandin antagonists have been tested in asthma patients, but the results receptors, CRTH2 is more akin to a group of leukocyte non- were mixed (Barnes et al., 2012; Busse et al., 2013; Erpenbeck

48 Molecular Cell 72, 48–59, October 4, 2018 ª 2018 Elsevier Inc. Figure 1. CRTH2 Ligands and Overall Structures

(A) Chemical structures of PGD2, fevipiprant, CAY10471 and ramatroban and the conserved carboxylate group. 3 (B) Competition radioactive ligand binding assays with HEK293T cell membranes expressing wtCRTH2 and CRTH2-mT4L. For each experiment, 2 nM [ H] PGD2 was used and various concentrations of PGD2 (top), CAY10471 (middle), and fevipirant (bottom) were added as competing ligands. Data points are presented as the mean values ± SEM, n = 3. (C) Overall structures of fevipiprant-bound and CAY10471-bound CRTH2 are colored in blue and slate, respectively. Fevipiprant and CAY10471 are shown as orange and yellow spheres. et al., 2016; Kuna et al., 2016; Miller et al., 2017; Pettipher et al., Interesting characteristics of the ligand binding pocket, including 2014). It has been suggested that a subpopulation of asthmatic a widely open end as the potential ligand entry port and a patients whose airway inflammation is largely driven by Th2- gradually increased positive charge distribution, allow us to pro- type inflammation would benefit most from CRTH2 antagonists pose a novel mechanism for the binding of PGD2. Structural (Kupczyk and Kuna, 2017). Recently, a potent CRTH2 antago- comparison analysis suggests a distinct binding pose of PGD2 nist, fevipiprant, showed promising clinical efficacy in patients compared to the lysophospholipids and endocannabinoids. with uncontrolled asthma in a few clinical trials (White et al., 2018). Thus, CRTH2 antagonists hold the promise of being RESULTS a new class of asthma drugs, and the development of new CRTH2 antagonists remains highly competitive, as evidenced Crystallization of CRTH2 and Overall Structures by the continuing clinical investigation initiated by many com- To crystallize human CRTH2, a construct of human CRTH2 was panies with their own compounds (Kupczyk and Kuna, 2017; generated by inserting an engineered T4 lysozyme (mT4L) Pettipher and Whittaker, 2012). (Thorsen et al., 2014) with an additional N-terminal 8-amino

Similar to PGD2, nearly all of the CRTH2 antagonists are car- acid linker into the intracellular loop 3 (ICL3) for crystallization boxylic acid derivatives with a carboxylate moiety, which is (Figure S1B). The 8-amino acid linker greatly improved crystal believed to be a critical pharmacophore that interacts with the quality, which was achieved unintentionally. To further facilitate receptor (Pettipher and Whittaker, 2012)(Figure 1A). To under- crystallogenesis, the flexible C-terminal region from R340 to stand the molecular mechanisms for the action of CRTH2 li- S395 was removed before crystallization, and the potential gands, we solved the crystal structures of human CRTH2 bound glycosylation site N25 was mutated to alanine. No other muta- to two antagonists, fevipiprant and CAY10471. The structures, tions were introduced. Ligand competition binding assays together with the results from computational docking studies showed that the sequence modifications did not significantly and ligand binding assays, reveal conserved and divergent affect the ligand-binding properties of CRTH2 (Figure 1B). Using structural features for the binding of diverse CRTH2 antagonists, this construct, we solved the crystal structures of human CRTH2 which occupy a semi-occluded ligand-binding pocket covered in complex with two antagonists, fevipiprant and CAY10471, at by a well-structured N-terminal region with a novel conformation. 2.80 A˚ and 2.74 A˚ resolution, respectively (Figure 1C; Table 1).

Molecular Cell 72, 48–59, October 4, 2018 49 Table 1. Data Collection and Refinement Statistics the second extracellular loop (ECL2) with a conserved b-hairpin structure, is similar to the structures of two other non-chemokine CRTH2-fevipiprant CRTH2-CAY10471 chemoattractant GPCRs that are close phylogenetic neighbors, PDB ID 6D26 6D27 namely, the LTB4 receptor (BLT1) and the C5a receptor (C5aR) Data collection (Hori et al., 2018; Liu et al., 2018; Robertson et al., 2018; Fig- Space group P212121 P212121 ure S2A). Interestingly, even though C5aR recognizes peptide li- Cell dimensions gands, which are chemically distinct from the lipid mediators a, b, c (A˚ ) 50.457, 61.710, 52.175, 62.636, recognized by CRTH2, if the structures of C5aR and CRTH2 266.517 272.215 are superimposed on each other, the antagonists of CRTH2 a, b, g () 90, 90, 90 90, 90, 90 nearly overlap with part of the peptide antagonist PMX53 for C5aR (Figure S2B). However, unlike BLT1 and C5aR, CRTH2 Resolution (A˚ ) 50–2.8 (2.85–2.8) 50–2.7 (2.75–2.7) contains a well-folded N-terminal structure with a central alpha R (%)a 18.3 (77.4) 16.6 (62.8) merge helix (N-helix) connected to the transmembrane helix 1 (TM1) I/s(I) 8.9 (1.2) 11.6 (1.3) by a long loop (N-loop) (Figures 2A and S3A). The N-helix and b CC1/2 (0.71) (0.87) N-loop pack tightly against the second extracellular loop Completeness (%) 91.9 (76.5) 92.6 (74.1) (ECL2), forming a lid between ECL1 and ECL3 to largely cover Redundancy 4.9 (3.9) 5.5 (3.6) the ligand binding pocket (Figure 2A). Besides the disulfide 3.52 Refinement bond between C182 in ECL2 and C104 (Ballesteros-Wein- Resolution (A˚ ) 50–2.8 (2.95–2.8) 50–2.74 (2.86–2.74) stein numbering) in TM3 that is conserved in most rhodopsin- like GPCRs, an additional disulfide bond was found between No. reflections 19,525 (2,293) 25,865 (1,211) 5.31 c C11 and C199 , linking the N-terminal helix to TM5 and Rcryst/Rfree (%) 25.0 (35.9)/28.0 24.2 (33.8)/28.0 thereby fixing the position of the N-terminal region (Figure 2A). (44.4) (42.3) The N-terminal region of TM1 in CRTH2 tilts toward TM2 No. atoms compared to those in BLT1 and C5aR and thereby creates a Protein 3,498 3,447 gap between TM1 and TM7 as the only open end of the ligand- Ligand 149 163 binding pocket (Figures 2B and S2C). Because the extracellular Water 3 8 ligand access is restricted to the pocket by the lid domain, B factors (A˚ 2) this gap in the lateral side of CRTH2 is likely to be the ligand entry port. Protein 84.7 94.7 The C-terminal region in CRTH2, D310–L327, immediately af- Ligand 93.3 107.2 ter TM7 forms an unusually long helical structure, namely, helix 8. Water 55.4 70.0 Helix 8 in CRTH2 exhibits an interesting amphipathic nature RMSD characterized by four leucine residues and one valine residue lin- Bond lengths (A˚ ) 0.002 0.002 ing the membrane-facing side, suggesting a strong membrane Bond angles () 0.6 0.6 association, and positively charged residues lining the cyto- Ramachandran plot plasmic side (Figure S3B). A previous study showed that the C-terminal tail of CRTH2 negatively regulated receptor signaling Favored (%) 96.4 96.1 and that a truncation of the C terminus after R317 could enhance Allowed (%) 3.6 3.9 Gi signaling (Schro¨ der et al., 2009), indicating an important role Outline (%) 0 0 of the long helix 8 in CRTH2 signaling. In addition, the entire cyto- MolProbity score 1.47 1.24 plasmic surface of CRTH2 is highly positively charged with a All atom crash score 4.53 2.07 number of sulfate ions modeled in the region (Figure S3C). Values inP parenthesesP are for highest-resolution shell. Although speculative, such characteristics may suggest a poten- a Rmerge = jIi Imj/ Ii, where Ii is the intensity of the measured reflection tial regulation of receptor signaling by negatively charged phos- and Im is the mean intensity of all symmetry related reflections. pholipids (Huynh et al., 2009). b CC1/2 is the correlation coefficient of the half datasets. c jj jj jj j j Rcryst = S Fobs Fcalc /S Fobs , where Fobs and Fcalc are observed and Structural Basis for the Binding of CAY10471 and jj jj jj j j calculated structure factors. Rfree = ST Fobs Fcalc /ST Fobs , where T is Fevipiprant a test dataset of 5% of the total reflections randomly chosen and set The high-quality electron density maps allowed unambiguous aside prior to refinement. modeling of fevipiprant and CAY10471 as two slow-dissociating CRTH2 antagonists in the structures (Mathiesen et al., 2006; The high-quality electron density maps allowed us to model all Royer et al., 2007; Sykes et al., 2016)(Figures S4A and S4B). Fe- residues of CRTH2 from A5 to L327, except for G237, which was vipiprant and CAY10471 bind to a semi-occluded ligand-binding replaced by mT4L, in the structure of CRTH2 with fevipiprant. pocket with a widely open end surrounded by the N-helix, the The structure of CRTH2 with CAY10471 is nearly identical to N-loop and the extracellular parts of TM1 and TM7, and an the structure with fevipiprant, except for a few loop residues occluded distal end surrounded by TMs 3, 5, and 6 (Figures near the ligand binding pocket and the disordered S22–A25 re- 3A, S4A, and S4B). A majority of residues in the ligand binding gion at the N terminus. The overall structure of CRTH2, especially pocket are aromatic residues, with a few highly charged residues

50 Molecular Cell 72, 48–59, October 4, 2018 Figure 2. N-Terminal Region and Ligand Entry Port in CRTH2 (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. (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. clustered at the occluded distal end (Figure 3B). The side chains tral tetracarcazole group of CAY10471, such as substitution with of two charged residues, R1704.64 and K2105.42, and the side an unsaturated carcazole group with a flat plane and ring open- chains of two tyrosine residues, Y184 in ECL2 and Y2626.51, ing, would change the position of the sulfonyl fluorophenyl tail point toward the carboxylate head groups of the two antagonists group relative to the surrounding aromatic residues and cause to form a strong polar interaction network, creating a highly steric clash, thus resulting in lower affinities (Pettipher and Whit- charged environment to hold the carboxylate group at the distal taker, 2012). end of the ligand binding pocket. Four phenylalanine residues, F872.60, F1113.32, F1123.33, and F2947.43, form the bottom of A New Binding Mode of Ramatroban Revealed by the ligand binding pocket. These residues, together with Docking F902.63, H1073.28, Y183, Y184, Y2626.51, and L2867.35, engage CAY10471 shares a high structural similarity with its parent com- in extensive aromatic and hydrophobic interactions with the pound ramatroban (Figure 1A). The major difference is that, central aromatic groups in the antagonists: the methyl azaindole instead of an acetate group, ramatroban has a longer propionate group in fevipirant and the tetrahydrocarbazole group in group attached to the central tetracarbazole group. Previous CAY10471. Both central aromatic groups also engage in studies have shown that CAY10471 is an insurmountable antag- cation-p interactions with the side chain of R1704.64. onist with slow dissociation, while ramatroban is a highly revers- Despite the conserved features, the tail groups of the two an- ible antagonist (Mathiesen et al., 2006). In addition, ramatroban tagonists show distinct binding modes and engage in different is less effective in stabilizing the receptor compared to fevipi- additional interactions with the receptor (Figures 3A and 3B). prant and CAY10471 in our thermostability assays (Figure 4A), For fevipiprant, the methylsulfonyl phenyl group extends toward indicating that ramatroban potentially engages in different inter- ECL2, with the substituted trifluoromethyl group facing a cleft actions with the receptor. We simulated the binding of ramatro- between TM1 and TM7. Additional aromatic interactions form ban to CRTH2 by computational docking. We used the structure between the phenyl group and aromatic residues F902.63, H95, of CRTH2 with CAY10471 as template because of the high struc- Y183, and W2837.32, and hydrogen bonds form between one ox- tural similarity between CAY10471 and ramatroban. We vali- ygen atom of the methylsulfonyl moiety and the main chain dated our docking methods by reproducing the same binding amine group of C182. Such a binding mode of fevipiprant, pose of CAY10471 as that in the crystal structure (Figure S4C). together with the details of the binding pocket, well explains Interestingly, the top-ranked docking poses of ramatroban differ the results of the structure and function relationship (SAR) significantly from those of CAY10471 in the crystal structure (Fig- studies for developing fevipiprant (Sykes et al., 2016). For ure 4B). The sulfonyl fluorophenyl tail group of ramatroban oc- CAY10471, the sulfonyl group also extends toward ECL2, while cupies a region similar to that occupied by the same moiety in the fluorophenyl group, as the tail group, extends toward TM7, CAY10471. However, compared to CAY10471, the tetracarba- resulting in a swing of the indole ring of W2837.32 compared to zole and propionate moieties of ramatroban, although still buried this residue in the structure with fevipiprant. Such a conformation in the aromatic pocket, adopt a flipped conformation to accom- of W2837.32 further causes the movement of L20 at the N termi- modate the longer propionate head group. This binding mode nus, potentially leading to a disordered region of S22–S24 in the positions the carboxylate group in ramatroban away from the structure of CRTH2 with CAY10471 (Figure 3C). The fluorophenyl spatially constrained Y184-K2105.42-Y2626.51 cluster, thereby group of CAY10471 participates in the aromatic interactions with disrupting the polar interaction network associated with residues Y183, W2837.32, and P2877.36. Modifications of the cen- CAY10471. Additionally, the tetracarbazole group of ramatroban

Molecular Cell 72, 48–59, October 4, 2018 51 Figure 3. Binding of CAY10471 and Fevipiprant (A) Ligand binding pocket with the open end and the distal end and binding poses of both ligands. (B) Residues involved in the binding of fevipiprant and CAY10471. Hydrogen bonds are shown as black dashed lines. Disulﬁde bonds are shown as yellow sticks. (C) Different conformations of W2837.32 and L20 in the structures of CRTH2 with two ligands. The disordered region between L20 and A25 in CRTH2-CAY10471 is shown as a slate dashed line.

resides in an unfavorable polar environment, further compro- two carbon-carbon double bonds in PGD2 to further stabilize mising ramatroban binding (Figures 4B and S4C). Taken the ligand. The hydrophobic environment of the ligand binding together, our findings well explain the weaker binding of ramatro- pocket is shielded from the extracellular aqueous milieu by the ban compared to that of CAY10471. More importantly, they sug- lid domain formed by the N terminus and ECL2. The C11A muta- gest that even small changes in chemical structures during drug tion, which presumably disrupts the disulfide bond linking the design may lead to significant changes in the binding affinities of N-terminal region to TM5 to destabilize the N-terminal region,

CRTH2 antagonists. could signiﬁcantly compromise PGD2 binding in our assays, suggesting the important role of the N-terminal region in PGD2 Insights into PGD2 Binding binding (Figure 5B). The conservation of the carboxylate group in PGD2 and most The well-structured N-terminal region results in a gap between CRTH2 antagonists suggests that the carboxylate in PGD2 the N-loop and TM7 as the only open end of the ligand binding occupies a similar site with a highly polar environment formed pocket, which could serve as a ligand entry port for lipid agonists by residues R1704.64, Y183, Y184, K2105.42, Y2626.51, and and antagonists (Figure 2). Three positively charged residues, E2696.58 (Figure 5A). Consistently, previous mutagenesis studies H95, R175, and R179 from ECL1 and ECL2, which do not directly have demonstrated the important roles of K2105.42 and E2696.58 interact with the antagonists, project their side chains into this in PGD2 binding (Hata et al., 2005). The rest of the hydrocarbon entry port (Figure 5C). Interestingly, strong electron density chain of PGD2 with a central cyclopentyl ring likely occupies the was observed around R175 and R179 in our structures. We hydrophobic space largely constituted by the aromatic residues modeled a succinate or a propylene glycol molecule from the (Figure 5A), which can potentially form p-p interactions with the crystallization conditions in the two structures to ﬁt the electron

52 Molecular Cell 72, 48–59, October 4, 2018 Figure 4. Ramatroban Docking Results

(A) Thermostability of unliganded CRTH2 and CRTH2 bound to ramatroban, fevipiprant and CAY10471. Apparent melting temperatures, Tms, were calculated and are shown in the brackets. Data points are presented as the mean values ± SEM, n = 2. (B) Binding pose of ramatroban, shown as dark purple sticks, from docking. CAY10471 is shown as thin yellow sticks for comparison. The carboxylate group in ramatroban is circled. See also Figure S4C. density (Figures 5C and S5). Both compounds contain polar the N-terminal region of BLT1 is completely disordered in the groups that are the same as or similar to the carboxylate group structure of guinea pig BLT1 bound to an atypical antagonist in PGD2, forming salt bridges or hydrogen bonds with R175 BIIL260 (Hori et al., 2018), leaving the ligand binding pocket and R179. These observations suggest that the carboxylate open to the extracellular milieu. This is likely an inherent feature group of PGD2 may form similar interactions with those two res- of BLT1 since its short N-terminal sequence does not favor idues at the ligand entry port during the early stage of ligand a structured motif and no cysteine residue is present at the recognition. Moreover, the entire ligand binding pocket of N terminus to form an additional disulﬁde bond. The ligand bind- CRTH2 exhibits a gradually increased positive charge distribu- ing port between TM1 and TM7 in CRTH2 is also absent in BLT1 tion from the entry port to the distal end (Figure 5D). We propose because of a difference TM1 conformation (Figure 2B). Such a that such a feature plays an important role in guiding PGD2 to ac- large structural divergence of the ligand binding pockets in cess the pocket by attracting its carboxylate group to reach the BLT1 and CRTH2 suggests that those two receptors adopt distal end. Collectively, as shown in Figure 5E, the process of different mechanisms for the lipid recognition, even though their

PGD2 binding suggested by our results includes the anchoring ligands are both eicosanoids with a high chemical similarity of the carboxylate group of PGD2 to the ligand entry port and (Figure S2A). the following access to the ligand-binding pocket facilitated by The feature of a structured N-terminal region as a lid domain the positive charge gradient. The nonuniform charge distribution covering the ligand-binding pocket has also been observed in may also help PGD2 to change its orientation in the lipid bilayer to a few other GPCRs for diffusible lipid ligands, such as sphingo- enter the ligand-binding pocket. Considering the negative sine-1-phosphate (S1P1)(Hanson et al., 2012), lysophosphatidic charge property of the carboxylate group, the change in the acid (LPA1)(Chrencik et al., 2015), and endocannabinoids (CB1) orientation of PGD2 likely does not occur spontaneously in the (Hua et al., 2016, 2017; Shao et al., 2016), and has been pro- lipid bilayer. Supporting this mechanism for PGD2 recognition, posed to be a conserved feature for many lipid GPCRs. How- previous studies have shown that mutations of R179 could ever, CRTH2 differs signiﬁcantly from these lipid GPCRs in reduce the afﬁnity of PGD2 by 5- to 10-fold (Hata et al., 2005), the extracellular region. First, the overall conformation of the and we also showed that mutating the positively charged residue N-terminal region is largely different in CRTH2 than in other lipid 4.64 R170 at the distal end of the ligand binding pocket nearly GPCRs. The N termini of S1P1 and LPA1 form a helical structure abolished PGD2 binding (Figure 5B). on top of the extracellular surface that packs against ECL1 and ECL2, while the N terminus of CB1 forms a loop structure fol- Structural Comparison with Other Lipid GPCRs lowed by a very short helix, which is buried in the helical bundle

BLT1, as the receptor for the lipid LTB4, is closely related to and packs against ECL1, ECL2, and TM7 (Figure 6A). In CRTH2, CRTH2, and their endogenous ligands LTB4 and PGD2 are the N-helix is nearly parallel to the b-hairpin of ECL2, forming the both eicosanoids with a common precursor (Smith, 1989). To lid domain together with the long N-loop. Second, the ECL2s in the best of our knowledge, BLT1 and CRTH2 are the only two the S1P1, LPA1, and CB1 receptors lack a b-hairpin motif and eicosanoid GPCRs with solved structures. Although their struc- project toward the inside of the helical bundle (Figure 6A). As a tures share a high similarity (Figure S2A), compared to CRTH2, result, the conserved extracellular disulﬁde bond linking ECL2

Molecular Cell 72, 48–59, October 4, 2018 53 Figure 5. PGD2 Binding

(A) Potential binding pocket for PGD2 as shown by the transparent gray cavity. The polar residues that may be involved in the interactions with the carboxylate group in PGD2 are labeled in purple. 3 (B) Cell surface expression levels of wild-type CRTH2 (wtCRTH2) and three mutants (left) and their speciﬁc saturation binding of H-PGD2 (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

(legend continued on next page)

54 Molecular Cell 72, 48–59, October 4, 2018 Figure 6. Structural Comparison of CRTH2 to Lipid GPCRs S1P1, LPA1, and CB1

(A) Extracellular regions in the structures of CRTH2 (blue), S1P1 (magenta, PDB ID 3V2W), LPA1 (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 S1P1 and LPA1, only antagonist-bound structures are available. See also Figure S6A. (B) Charge distribution of the ligand-binding pockets pointed by arrows of these GPCRs shown in (A). The potential ligand access ports are marked with black dashed circles.

(C) Cartoon diagrams of the ligand-binding modes of four lipid GPCRs. From the left to the right: CRTH2 with prostaglandin D2 (PGD2), S1P1 with sphingosine-1- phosphate (S1P), LPA1 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. to TM3 in almost all class A GPCRs, including CRTH2, is missing In addition to the large structural divergence of the extracel- in those receptors. Third, compared to those in S1P1, LPA1, and lular regions, the ligand binding pocket in CRTH2 also exhibits CB1, the long N-loop in CRTH2 results in a wider ligand entry different characteristics compared to that of S1P1, LPA1, and port on the side of the helical bundle (Figure S6). This may pro- CB1. The most striking difference is the polar environment of vide enough space for the orientation change of PGD2 at the the distal end of the ligand binding pocket in CRTH2, whereas ligand entry port to prepare the ligand for entering the ligand the corresponding regions in S1P1, LPA1, and CB1 are largely binding pocket (Figure 5E). hydrophobic (Figure S6). Consequently, the carboxylate head

N terminus of the receptor through flow cytometry. The specific saturation binding assays were performed using cell membranes. The W283A mutant is shownas a positive control, in which the binding of PGD2 was not significantly altered. Data points are presented as the mean values ± SEM, n = 3. (C) Succinate (pink) and propylene glycol (brown) molecules modeled in the structures of fevipiprant-bound CRTH2 (blue) and CAY10471-bound CRTH2 (slate). The hydrogen bonds are shown as black dashed lines. (D) Charge distribution of the ligand-binding pocket of CRTH2 (open-book view). Fevipiprant is shown as orange sticks.

(E) Cartoon diagram of the proposed PGD2 binding process. The positive charge potential is indicated as ‘‘+.’’ The lipid bilayer is shown as the pink background.

The carboxylate group in PGD2 is colored in green, while the rest hydrophobic moiety of PGD2 is colored in yellow.

Molecular Cell 72, 48–59, October 4, 2018 55 group of PGD2 is buried deeply inside the distal end of the in a majority of lipid-activated GPCRs, providing a common pocket, while the hydrocarbon chain possibly extends toward structural basis for the uptake and release of lipophilic ligands. the ligand entry port. In contrast, the fatty-acyl chains of the CRTH2 belongs to a group of non-chemokine chemoattrac- endogenous ligands for S1P1, LPA1, and CB1 are buried deep in- tant GPCRs that are phylogenetically close to each other but side the binding pocket, while the polar head groups are close to recognize very diverse ligands from lipids to peptides to large the extracellular surface (Chrencik et al., 2015; Hanson et al., proteins (Figures S1A and S2A). The structures of CRTH2 re- 2012; Hua et al., 2017). Additionally, the electrostatic charge dis- ported here, together with the previously reported structures of tributions of the ligand binding pockets in these lipid GPCRs are BLT1 and C5aR, show a large structural divergence of the extra- largely different (Figure 6B). In the structures of antagonist- cellular region in those receptors, likely accounting for the recog- bound S1P1 and LPA1 and agonist-bound CB1, the ligand ac- nition of diverse ligands by those GPCRs. On the other hand, the cess port is positively charged, while the rest of the binding structures also reveal a conserved structural feature in these pockets are highly negatively charged, contrasting with the high- receptors. One residue in TM6, Y6.51, which is conserved as a ly positively charged ligand binding pocket in CRTH2. Such a Y or F in other non-chemokine chemoattractant GPCRs, directly charge distribution may help to position the phosphate head interacts with the ligands of all three receptors (Figure S7). This groups of lysophospholipids or the hydroxyl groups of endocan- residue sits on top of a structural motif F6.44XXCW6.48XP6.50 nabinoids at the ligand access port through both electrostatic that is highly conserved in rhodopsin-like GPCRs and interacts attraction and repulsion to ensure that their acyl chains are with W6.48, which has been suggested to function as a toggle buried in the binding pocket (Figure 6C). Therefore, it is likely switch in the activation of some GPCRs (Smit et al., 2007). We that for those receptors the acyl chains of the lysophospholipids propose that for the group of non-chemokine chemoattractant and the endocannabinoids go into the ligand-binding pockets GPCRs, the three conserved residues, F6.44,W6.48, and Y/F6.51, without a large orientation change of the lipid molecules, which line up in TM6 to constitute a critical structural motif that is different from the proposed multistep process for the recogni- mediates the propagation of signal from the extracellular ligand tion of PGD2 by CRTH2 (Figure 5E). binding pocket to the cytoplasmic region that interacts with intracellular signaling molecules in receptor activation. DISCUSSION The two receptors, CRTH2 and BLT1, apparently adopt different mechanisms for lipid recognition, with distinct ligand GPCRs recognize a broad range of molecules with a vast chem- binding pockets, even though the endogenous ligands for ical diversity through different mechanisms. Our understanding BLT1 and CRTH2, LTB4, and PGD2, respectively, are both eicos- of the recognition of lipid mediators by GPCRs primarily comes anoid lipid mediators with a high chemical similarity (Figure S2A). from the structural studies of receptors for lysophospholipids Some other members of this group of GPCRs, including FPR2/ and endocannabinoids including S1P1, LPA1, LPA6, and CB1, ALX, ChemR23 (CMKLR1), and GPR32, recognize a special which have revealed two different types of extracellular ligand group of eicosanoid lipids called specialized pro-resolving lipid recognition domains (Taniguchi et al., 2017). In S1P1, LPA1, and mediators (SPMs). SPMs can promote the resolution of inflam- CB1, the N-terminal region folds on top of the ligand binding mation, in contrast to the primary pro-inflammatory function of pocket and the ECL2 projects toward the inside of the 7-TM most eicosanoid lipids, including LTB4 and PGD2. Whether the bundle to interact with the ligands, while in LPA6, the ligand bind- recognition of SPMs by their receptors is similar to the lipid ing pocket is open to the extracellular environment, with the ECL2 recognition by BLT1 or by CRTH2 needs further investigation. extending away from the 7-TM bundle, similar to BLT1. Our struc- This is important considering the increasing research interests tures of CRTH2 reveal a new conformation of the extracellular re- in developing new pro-resolving mediators as a novel therapy gion that, to the best of our knowledge, has not been observed in for treating inflammatory diseases (Dalli and Serhan, 2018). other GPCR structures. In the structures, the well-folded N-termi- In addition, one member of this group, FPR2/ALX, can sense nal region packs tightly against the ECL2, resulting in a widely both formyl peptides and SPMs. The molecular mechanism for open end of the ligand binding pocket as the ligand entry port. such promiscuous ligand recognition remains elusive. The structural analysis allows us to propose a novel mechanism Our structures also provide new insights into CRTH2 drug for the binding of the lipid molecule PGD2 to CRTH2, in which development. The ligand binding pocket revealed by our struc- the carboxylate group of PGD2 first binds to the ligand entry tures comprises many aromatic residues and a few polar resi- port through interactions with positively charged residues and dues at the distal end. Correspondingly, most CRTH2 antago- then extends deeply into the ligand-binding pocket following nists share a similar structural feature characterized by an the positive charge gradient, while the rest of the hydrocarbon acetate polar group attached to a central aromatic group to fit chain is stabilized by many aromatic residues in the ligand bind- the ligand binding pocket (Pettipher and Whittaker, 2012). The ing pocket (Figure 5E). Our studies thus offer new insights into different binding poses of the tail groups of CAY10471 and fevi- how GPCRs recognize chemically diverse endogenous lipid piprant associated with the different conformations of W2837.32 mediators. Additionally, despite the structural divergence of the and the N-loop indicate that the open end of the ligand binding extracellular domains in CRTH2, S1P1, LPA1, and CB1, these re- pocket, which we propose to be the ligand entry port, exhibits ceptors share a similar feature characterized by a gap between certain structural flexibility. Additional structures of CRTH2 the N-terminal segments of TM1 and TM7 (Figure S6), which with other antagonists that have distinct tail groups are needed also extends to the photoreceptor rhodopsin (Palczewski et al., to further investigate the conformational diversity of residues 2000; Park et al., 2008). Such a feature may be highly conserved in this region, as it may significantly affect the results of

56 Molecular Cell 72, 48–59, October 4, 2018 structure-based virtual screening for developing novel CRTH2 Writing – Original Draft, H.F., Z.W., and C.Z.; Writing – Review & Editing, antagonists. Furthermore, the unexpected small molecules H.F., Z.W., and C.Z.; Visualization, Z.W. and C.Z.; Funding Acquisition, H.F., modeled in this region suggest that the ligand entry port may Z.W., and C.Z.; Supervision, W.G., Z.W., and C.Z. offer an additional site for designing new synthetic CRTH2 antagonists, which compared to CAY10471 and fevipiprant would DECLARATION OF INTERESTS engage in additional interactions with the receptor to achieve The authors declare no competing interests. stronger binding and a longer duration of action. Collectively, our structures offer novel structural insights into Received: June 26, 2018 the action of diverse CRTH2 antagonists, which will facilitate Revised: July 25, 2018 CRTH2 drug development for a number of inflammatory dis- Accepted: August 6, 2018 Published: September 13, 2018 eases, including asthma. They also reveal interesting features of the ligand binding pocket and suggest a novel mechanism REFERENCES for the binding of the endogenous lipid molecule PGD2, thus shedding light on the structural and mechanistic diversity of Adams, P.D., Afonine, P.V., Bunko´ czi, G., Chen, V.B., Davis, I.W., Echols, N., GPCRs for the recognition of lipid mediators. Headd, J.J., Hung, L.W., Kapral, G.J., Grosse-Kunstleve, R.W., et al. (2010). PHENIX: a comprehensive Python-based system for macromolecular struc- STAR+METHODS ture solution. Acta Crystallogr. D Biol. Crystallogr. 66, 213–221. Baker, N.A., Sept, D., Joseph, S., Holst, M.J., and McCammon, J.A. (2001). Detailed methods are provided in the online version of this paper Electrostatics of nanosystems: application to microtubules and the ribosome. Proc. Natl. Acad. Sci. USA 98, 10037–10041. and include the following: Barnes, N., Pavord, I., Chuchalin, A., Bell, J., Hunter, M., Lewis, T., Parker, D., d KEY RESOURCES TABLE Payton, M., Collins, L.P., Pettipher, R., et al. (2012). A randomized, double- d CONTACT FOR REAGENT AND RESOURCE SHARING blind, placebo-controlled study of the CRTH2 antagonist OC000459 in moder- ate persistent asthma. Clin. Exp. Allergy 42, 38–48. d EXPERIMENTAL MODEL AND SUBJECT DETAILS B Cell lines Baxter, C.A., Murray, C.W., Clark, D.E., Westhead, D.R., and Eldridge, M.D. (1998). Flexible docking using Tabu search and an empirical estimate of bind- d METHOD DETAILS ing affinity. Proteins 33, 367–382. B Protein expression and purification Busse, W.W., Wenzel, S.E., Meltzer, E.O., Kerwin, E.M., Liu, M.C., Zhang, N., B Crystallization Chon, Y., Budelsky, A.L., Lin, J., and Lin, S.L. (2013). Safety and efficacy of B Data collection and structure determination the prostaglandin D2 receptor antagonist AMG 853 in asthmatic patients. B Protein thermostability assay J. Allergy Clin. Immunol. 131, 339–345. B HEK293T cell surface expression of CRTH2 constructs Caffrey, M. (2009). Crystallizing membrane proteins for structure determina- B Membrane preparation and radioactive ligand-binding tion: use of lipidic mesophases. Annu. Rev. Biophys. 38, 29–51. assays Chen, V.B., Arendall, W.B., 3rd, Headd, J.J., Keedy, D.A., Immormino, R.M., B Molecular docking Kapral, G.J., Murray, L.W., Richardson, J.S., and Richardson, D.C. (2010). d QUANTIFICATION AND STATISTICAL ANALYSIS MolProbity: all-atom structure validation for macromolecular crystallography. B Protein thermostability assay Acta Crystallogr. D Biol. Crystallogr. 66, 12–21. B Cell surface expression and ligand binding assay Chrencik, J.E., Roth, C.B., Terakado, M., Kurata, H., Omi, R., Kihara, Y., d DATA AND SOFTWARE AVAILABILITY Warshaviak, D., Nakade, S., Asmar-Rovira, G., Mileni, M., et al. (2015). Crystal structure of antagonist bound human lysophosphatidic acid receptor 1. Cell 161, 1633–1643. SUPPLEMENTAL INFORMATION Cosmi, L., Annunziato, F., Nagata, K., and Romagnani, S.; Galli MIG; Maggi RME (2000). CRTH2 is the most reliable marker for the detection of circulating Supplemental Information includes seven figures and can be found with this human type 2 Th and type 2 T cytotoxic cells in health and disease. Eur. J. article online at https://doi.org/10.1016/j.molcel.2018.08.009. Immunol. 30, 2972–2979.

ACKNOWLEDGMENTS Dalli, J., and Serhan, C.N. (2018). Identification and structure elucidation of the pro-resolving mediators provides novel leads for resolution pharmacology. We thank the staff at the GM/CA at APS of Argonne National Laboratory at Br. J. Pharmacol. Published online April 21, 2018. https://doi.org/10.1111/ Chicago for their assistance with X-ray diffraction data collection. We thank bph.14336. Dr. James C. Burnett and Dr. Peter Wipf for discussion. We acknowledge Eldridge, M.D., Murray, C.W., Auton, T.R., Paolini, G.V., and Mee, R.P. (1997). the financial support from the University of Pittsburgh, the NIH (Maximizing Empirical scoring functions: I. The development of a fast empirical scoring Investigators’ Research Award [MIRA] R351R35GM128641 to C.Z.), the function to estimate the binding affinity of ligands in receptor complexes. Biomedical Research Council (A*STAR to R.N.V.K.D. and H.F.), and the Na- J. Comput. Aided Mol. Des. 11, 425–445. tional Natural Science Foundation of China (31770791 and 315707410 to Emsley, P., and Cowtan, K. (2004). Coot: model-building tools for molecular Z.W.). Z.W. is also supported by the startup funds from Southern University graphics. Acta Crystallogr. D Biol. Crystallogr. 60, 2126–2132. of Science and Technology and the Recruitment Program of Global Youth Ex- Erpenbeck, V.J., Popov, T.A., Miller, D., Weinstein, S.F., Spector, S., perts of China. Magnusson, B., Osuntokun, W., Goldsmith, P., Weiss, M., and Beier, J. (2016). The oral CRTh2 antagonist QAW039 (fevipiprant): A phase II study in AUTHOR CONTRIBUTIONS uncontrolled allergic asthma. Pulm. Pharmacol. Ther. 39, 54–63. Conceptualization, L.W., Z.W., and C.Z.; Methodology, L.W., H.F., Z.W., and Fredriksson, R., Lagerstro¨ m, M.C., Lundin, L.G., and Schio¨ th, H.B. (2003). The C.Z.; Investigation, L.W., D.Y., H.L., Q.X., R.N.V.K.D., H.F., Z.W., and C.Z.; G-protein-coupled receptors in the human genome form five main families.

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Molecular Cell 72, 48–59, October 4, 2018 59 STAR+METHODS

KEY RESOURCES TABLE

REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Mouse monoclonal Anit-FLAG M1 antibody This paper N/A Chemicals, Peptides, and Recombinant Proteins CAY10471 Cayman Chemical Cat# 10006735 Fevipiprant MedKoo Biosciences Cat# 319671 N-dodecyl-b-D-maltoside Anatrace Cat# D310S Cholesterol hemisuccinate Anatrace Cat# CH210 Lauryl maltose neopentyl glycol Anatrace Cat# NG310 PNGase F NEB Cat# P0704S CPM (7-diethylamino-3-(4-maleinidylphenyl)- Sigma Aldrich Cat# C1484 4-methylcoumarin) FuGENE Transfection Reagent Promega Cat# E2311 Neomycin Fisher Scientific Cat# 21810031 DyLight 488 Fisher Scientific Cat# 46402 [3H]PGD2 Perkin Elmer Cat# NET428025UC PGD2 Cayman Chemical Cat# 12010 Monoolein (1-Oleoyl-rac-glycerol) Sigma Aldrich Cat# M7765 Cholesterol Sigma Aldrich Cat# C8667 Salt Active Nuclease Arcticzymes Cat# 70920 DMEM Fisher Scientific Cat# MT10013CV FBS Gemini Bio-Products Cat# 900-108 Iodoacetamide Fisher Scientific Cat# AC122271000 Geneticin Fisher Scientific Cat# 10-131-035 FLAG peptide GL Biochem Custom synthesis Deposited Data Crystal Structure of CRTH2-CAY10471 This paper PDB: 6D27 Crystal Structure of CRTH2-fevipiprant This paper PDB: 6D26 Original data published as Mendeley dataset This paper https://data.mendeley.com/datasets/m57xsf7v5n/1 Experimental Models: Cell Lines Spodoptera frugiperda Sf9 cells Expression Systems Cat# 94-001F HEK293T cells ATCC Cat# CRL-3216 Recombinant DNA pFastBac-CRTH2-T4L This paper N/A pcDNA3.1+-wtCRTH2 This paper N/A pcDNA3.1+-CRTH2-C11A This paper N/A pcDNA3.1+-CRTH2-R170A This paper N/A pcDNA3.1+-CRTH2-W283A This paper N/A Software and Algorithms HKL2000 software Otwinowski and Minor, 1997 http://www.hkl-xray.com/ PHENIX Adams et al., 2010 https://www.phenix-online.org/ COOT Emsley and Cowtan, 2004 https://www2.mrc-lmb.cam.ac.uk/personal/ pemsley/coot/ PyMOL 2.0 The PyMOL Molecular Graphics System, https://pymol.org/2/ Schro¨ dinger, LLC. (Continued on next page)

e1 Molecular Cell 72, 48–59.e1–e4, October 4, 2018 Continued REAGENT or RESOURCE SOURCE IDENTIFIER Prism 7 GraphPad https://www.graphpad.com/scientific- software/prism/ GOLD 5.5 Jones et al., 1997 https://www.ccdc.cam.ac.uk/solutions/ csd-discovery/components/gold/ ChemScore fitness function Baxter et al., 1998; Eldridge et al., 1997 http://scbx.mssm.edu/mezeilab/molmod/ gold_docs/gold.1.80.html#260363 APBS Baker et al., 2001 http://www.poissonboltzmann.org/ Other Nickel Sepharose resin GE healthcare Cat#17526801 Superdex 200 Increase column GE healthcare Cat#28990944 Crystal Gryphon robot Art Robbins N/A Spectrophotometer SPECTRAMax N/A FACScan flow cytometer BD CellQuestTM Pro N/A LS6500 scintillation counter Beckman N/A

CONTACT FOR REAGENT AND RESOURCE SHARING

Further information and requests for resources and reagents should be directed to and will be fulﬁlled by the Lead Contact, Cheng Zhang ([email protected]). DNA constructs and other research reagents generated by the authors will be distributed upon request to other research investigators under a Material Transfer Agreement.

EXPERIMENTAL MODEL AND SUBJECT DETAILS

Cell lines Spodoptera frugiperda Sf9 cells were purchased from Expression Systems. Cells were cultured in ESF 921 medium (Expression Systems) at 27C and infected with baculovirus at a density of 4 3 106 cells per ml for large-scale protein expression. HEK293T cells were initially obtained form the American Type Culture Collection (ATCC). Cell were cultured in the DMEM medium with 4.5 g/L glucose, L-glutamine & sodium pyruvate plus 10% FBS at 37 C with 5% CO2. 300 mg/ml geneticin and neomycin were used to select the clones for constructing stable cell lines. Plasmocin (Fisher Scientiﬁc) was used to prevent mycoplasma contamination.

METHOD DETAILS

Protein expression and purification To facilitate crystallization, an engineered CRTH2 construct was designed by inserting a modified T4 lysozyme (eT4L) (Thorsen et al., 2014) into the third intracellular loop (ICL3) between residues R236 an R238, along with a linker (ADLGLQHR) at the N terminus of eT4L, which was introduced by accident, and introducing a mutation of glycosylation site N25A. A human rhinovirus (3C) protease cleavage site was inserted at residue S339 for C terminus removal. The construct gene was synthesized by gBlocks (Integrated DNA Technologies) and cloned into a modified pFastBac vector (Invitrogen), which contains a Flag-tag followed by a tobacco etch virus (TEV) protease cleavage site at the N terminus and an 8 3 His-tag at the C terminus. The engineered protein was expressed in Sf9 cells. Cells were infected by virus at a density of 4 3 106 cells per ml and cultured at 27C for 48 h with antagonist CAY10471 (Cayman Chemical) or fevipiprant (MedKoo Biosciences) at a final concentration of 100 nM in the medium. The transfected cells were collected by centrifugation and stored at 80C. To stabilize the receptor, 1 mM CAY10471 or fevipiprant was added during all purification steps. The frozen Sf9 cells were lysed by stirring in buffer containing 20 mM Tris-HCl, pH7.5, 0.2 mg/ml leupeptin, 100 mg/ml benzamidine and 2mg/ml iodoacetamide. After centrifugation, the pellet was resuspended and solubilized in buffer containing 20mM HEPES, pH7.5, 750mM NaCl, 1% (w/v) n-dodecyl-b-D-maltoside (DDM, Anatrace), 0.2% (w/v) sodium cholate (Sigma), 0.2% (w/v) cholesterol hemisuccinate (CHS, Anatrace), 20% (v/v) glycerol, 0.2 mg/ml leupeptin, 100 mg/ml benzamidine, 500 unit Salt Active Nuclease (Arcticzymes) and 2mg/ml iodoacetamide at 4C for 2h. The supernatant was isolated by centrifugation at 25,000 g for 30 min and incubated with nickel Sepharose resin (GE healthcare) plus 10 mM imidazole at 4C overnight. The resin was washed with buffer containing 20 mM HEPES, pH 7.5, 500 mM NaCl, 0.1% (w/v) DDM, 0.02% (w/v) CHS and 30 mM imidazole. The protein was eluted by 400 mM imidazole and directly loaded onto anti-Flag M1 antibody resin (homemade) after adding 2mM CaCl2. The detergent was slowly exchanged to 0.01% (w/v) lauryl maltose neopentyl glycol (MNG, Anatrace) on M1 antibody resin. The receptor was finally eluted with buffer containing 20 mM HEPES, pH 7.5, 100mM NaCl, 0.002% (w/v) MNG, 0.001% (w/v) CHS, 200 mg/ml Flag peptide and 5 mM EDTA. After

Molecular Cell 72, 48–59.e1–e4, October 4, 2018 e2 treatment with TEV protease, 3C protease and PNGase F (NEB) at 4C overnight, the N-terminal Flag-tag, the C-terminal His-tag and glycosylation were removed. The monodispersed receptor was collected after size-exclusion chromatography using a Superdex 200 Increase column (GE healthcare). The puriﬁed receptor was concentrated to 50-60 mg/ml for crystallization.

Crystallization The CRTH2 receptor in complex with CAY10471 or fevipiprant was crystallized using the lipidic cubic phase (LCP) method (Caffrey, 2009). The protein sample was mixed with the monoolein/cholesterol lipid (10:1 w/w) at a weight ratio of 1: 1.5 (protein: lipid) using two glass syringes to form clear LCP. Then the LCP mixture was then dispensed onto glass plates in 20 nL drops and overlaid with 700 nL of precipitant solution using a Gryphon robot (Art Robbins). The crystallization condition of CRTH2-CAY10471 was 100 mM MES, pH 6.5, 100 mM ammonium sulfate, 30% (v/v) PEG400 and 2% (v/v) polypropylene glycol P400, while the same condition plus 1 mM succinate salt was used to crystallize CRTH2-fevipiprant. The crystallization plates were placed in a 15C incubator. Crystals appeared in 3 days and grew to full size in 2 weeks, which were then harvested from LCP using micro mounts (MiTeGen) and ﬂash frozen in liquid nitrogen.

Data collection and structure determination X-ray diffraction data was collected at the Chicago Advanced Photon Source (APS) beam line 23ID-B of GM/CA with a microbeam with a 10 mm diameter. Each crystal was exposed with a 10 mm 3 10 mm beam for 0.2 s and 0.2 degree oscillation per frame to collect 20-40 degrees of rotation data. Data from 21 crystals of CRTH2-CAY10471 and 25 crystals of CRTH2-fevipiprant were processed and merged by HKL2000 software (Otwinowski and Minor, 1997). The initial phase of CRTH2-fevipiprant complex was determined by molecular replacement in Phaser (McCoy et al., 2007) using the

C5aR receptor structure (PDB ID 6C1R) and T4L portion of ETBR (PDB ID 5XPR) as search models. The structure model was refined and rebuilt using PHENIX (Adams et al., 2010) and COOT (Emsley and Cowtan, 2004), respectively. The structure of CRTH2- CAY10471 was solved by molecular replacement using the CRTH2-fevipiprant model and refined using the same method. Finally, MolProbity (Chen et al., 2010; Williams et al., 2018) was used to check the quality of the two models. All the structure figures were produced with PyMOL (https://pymol.org/2/). The charge distribution was calculated by using APBS (Baker et al., 2001).

Protein thermostability assay The wild-type CRTH2 was used to determine the thermostability of the receptor with different ligands. The receptor was expressed and purified in a similar way as for the crystallization trials except that the ligand was finally removed by size exclusion chromatography using buffer without any ligand. The unliganded receptor was incubated with buffer only or with buffers containing 10 mM of the different ligands, ramatroban, fevipiprant and CAY10471, and then labeled with 7-diethylamino-3-(4-maleinidylphenyl)-4-methylcoumarin (CPM) dye (Sigma) at a concentration of 0.1 mg/ml. The CPM fluorescence intensity (excitation 387 nm, emission 463 nm) of each sample at different temperature points (from 30Cto75C) was measured by a spectrophotometer (SPECTRAMax Paradigm).

HEK293T cell surface expression of CRTH2 constructs All radioactive ligand-binding experiments were performed using the membranes of stably transfected HEK293T cells. To build the stable cell lines, constructs of the wild-type CRTH2 and CRTH2 mutants C11A, R170A and W283A were cloned into the vector pcDNA3.1+ (Invitrogen) with a FLAG-tag at the N terminus, and then transfected into HEK293T cell using FuGENE Transfection Reagent (Promega) for constructing stable cell lines. To determine the surface expression of each CRTH2 construct, flow cytometry experiments were performed. Cells were sus- pended in PBS, washed twice, and incubated with 1 mg/ml DyLight 488 (Thermo Fisher)-labeled anti-Flag M1 antibody (homemade) plus 2 mM CaCl2 in the dark for 15 min at room temperature. The cells were then analyzed for the DyLight488 fluorescence on a FACScan flow cytometer (BD CellQuestTM Pro). The protein expression levels were represented by the median fluorescence values of the sorted cells. All of the cells stably expressing wild-type CRTH2 and mutants showed comparable receptor expression levels.

Membrane preparation and radioactive ligand-binding assays To prepare cell membranes, HEK-239T cells stably expressing different CRTH2 constructs were rinsed and detached with PBS buffer. After centrifugation at 1000 x g for 10 min, the cell pellets were resuspended in buffer containing 20 mM Tris-HCl, pH 7.5, 1 mM EDTA, 0.2 mg/ml leupeptin and 100 mg/ml benzamidine. After homogenization, the samples were centrifuged first at 1000 x g for 10 min, then at 150,000 g for 1 h. The membrane pellet was resuspended in buffer containing 20 mM HEPES, pH7.5, 100 mM NaCl, 1 mM EDTA and homogenized, then frozen in liquid nitrogen and stored at 80C. 3 For the radioactive ligand-binding assays, 20 mg of membrane protein was incubated with [ H]PGD2 (Perkin Elmer) and nonradioactive ligands for 2 h at room temperature in 200 mL of binding buffer containing 20 mM HEPES, pH 7.5, 1 mM EDTA, 5 mM MgCl2, 3 5 mM MnCl2 and 0.1% (w/v) BSA. For the saturation binding, [ H]PGD2 was added in various concentrations from 0 to 20 nM. Total and nonspecific binding was measured in the absence and presence of 10 mM non-radioactive PGD2 (Cayman Chemical), respec- 3 tively. For the competition binding assays, the cell membranes were incubated with 2 nM [ H]PGD2 and various concentrations of competing ligands (nonradioactive PGD2, CAY10471 or fevipiprant) from 0.01 nM to 10 mM. After incubation, the reaction was terminated by adding 5 mL of cold binding buffer and rapidly filtering through glass giber prefilters (Millipore Sigma). The filters e3 Molecular Cell 72, 48–59.e1–e4, October 4, 2018 3 were washed three times with 5 mL cold binding buffer, and the retained receptor-bound [ H]PGD2 was incubated with 5 mL of CytoScint liquid scintillation cocktail (MP Biomedicals) and counted on a Beckman LS6500 scintillation counter.

Molecular docking We chose the CAY10471-bound CRTH2 structure as the receptor/host for docking ramatroban. Before docking ramatroban, we performed cognate docking of CAY10471 back to this CRTH2 structure to ascertain that the docking protocol could reproduce its crystal geometry. Following validation of the docking protocol, ramatroban (structure obtained from PubChem) (Kim et al., 2016) was docked to this CRTH2 structure. All docking runs were performed using GOLD 5.5 (Jones et al., 1997). Before docking, all crystal waters and ligands were deleted and hydrogen atoms were added to the receptor. The docking solutions were scored on the basis of the ChemScore ﬁtness function (Baxter et al., 1998; Eldridge et al., 1997).

QUANTIFICATION AND STATISTICAL ANALYSIS

Protein thermostability assay Results are represented as the mean ± SEM from 2 independent experiments. Data were analyzed with the Boltzmann sigmoidal equation in GraphPad Prism 7 (GraphPad Software) to calculate the melting temperature (Tm).

Cell surface expression and ligand binding assay Protein expression levels are represented by the median ﬂuorescence values of the sorted cells. Results are represented as the mean ± SEM from 3 independent measurements. Ligand binding data are represented as the mean ± SEM from 3 independent experiments. Data were analyzed with the one site competitive and saturation binding methods in GraphPad Prism 7 (GraphPad Software).

DATA AND SOFTWARE AVAILABILITY

The accession numbers for the coordinates and structure factors for CRTH2-fevipiprant and CRTH2-CAY10471 reported in this paper are PDB: 6D26 and 6D27, respectively. The original data for the protein thermostability assay and the ligand-binding assay have been published on Mendeley: https:// data.mendeley.com/datasets/m57xsf7v5n/1.

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