br Results br Discussion GPCRs recognize a
Discussion GPCRs recognize a broad range of molecules with a vast chemical influenza a virus through different mechanisms. Our understanding of the recognition of lipid mediators by GPCRs primarily comes from the structural studies of receptors for lysophospholipids and endocannabinoids including S1P1, LPA1, LPA6, and CB1, which have revealed two different types of extracellular ligand recognition domains (Taniguchi et al., 2017). In S1P1, LPA1, and CB1, the N-terminal region folds on top of the ligand binding pocket and the ECL2 projects toward the inside of the 7-TM bundle to interact with the ligands, while in LPA6, the ligand binding pocket is open to the extracellular environment, with the ECL2 extending away from the 7-TM bundle, similar to BLT1. Our structures of CRTH2 reveal a new conformation of the extracellular region that, to the best of our knowledge, has not been observed in other GPCR structures. In the structures, the well-folded N-terminal region packs tightly against the ECL2, resulting in a widely open end of the ligand binding pocket as the ligand entry port. The structural analysis allows us to propose a novel mechanism for the binding of the lipid molecule PGD2 to CRTH2, in which the carboxylate group of PGD2 first binds to the ligand entry port through interactions with positively charged residues and then extends deeply into the ligand-binding pocket following the positive charge gradient, while the rest of the hydrocarbon chain is stabilized by many aromatic residues in the ligand binding pocket (Figure 5E). Our studies thus offer new insights into how GPCRs recognize chemically diverse endogenous lipid mediators. Additionally, despite the structural divergence of the extracellular domains in CRTH2, S1P1, LPA1, and CB1, these receptors share a similar feature characterized by a gap between the N-terminal segments of TM1 and TM7 (Figure S6), which also extends to the photoreceptor rhodopsin (Palczewski et al., 2000, Park et al., 2008). Such a feature may be highly conserved in a majority of lipid-activated GPCRs, providing a common structural basis for the uptake and release of lipophilic ligands. 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 S1A and S2A). 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, Y6.51, 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 F6.44XXCW6.48XP6.50 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). We propose that for the group of non-chemokine chemoattractant GPCRs, the three conserved residues, F6.44, 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. 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, LTB4, and PGD2, respectively, are both eicosanoid lipid mediators with a high chemical similarity (Figure S2A). 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 LTB4 and PGD2. 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). 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.