Molecular insights into ligand recognition and signaling of OXGR1

Domain: neuroscience
Published: 2025-12-18
Editorial outcome: major_revision

Abstract
GPR99/OXGR1 is a G protein-coupled receptor (GPCR) with two endogenous agonists, the tricarboxylic acid cycle derivative 2-oxoglutarate (α-ketoglutarate) and the inflammatory mediator cysteinyl leukotriene E4 (LTE4), hence also termed CysLT3 receptor. How GPR99/OXGR1 recognizes two distinct ligands is a biologically important question. Here we present cryo-EM structures of GPR99/OXGR1-Gq complexed with oxoglutarate and LTE4, respectively. The oxoglutarate-bound structure shows a binding pocket surrounded by the transmembrane domains (TM), with a primary site and an accessory site for simultaneous binding of two oxoglutarate molecules for full activation of the receptor. The TM binding pocket, however, is too small to accommodate the cysteinyl leukotriene LTE4. Alanine substitution of key residues for oxoglutarate binding had little impact on LTE4-induced signaling. A distinct site in between TM3/4/5 just above intracellular loop 2 was identified in the solved structure with LTE4, but the densities were less well-defined. Alanine substitution of amino acids potentially involved in LTE4 interaction at this site abrogated LTE4-induced receptor activation without affecting oxoglutarate-induced signaling. Both ligands activated GPR99/OXGR1 primarily through the Gq pathway, but LTE4 also induced Gi signaling. These findings illustrate the structural basis for GPR99/OXGR1 to interact with structurally distinct oxoglutarate and LTE4.

Introduction
GPR99 was initially identified as a homologue of P2Y1, a purinergic GPCR that binds nucleotide ligands1. Further studies found that GPR99 does not bind nucleotide ligands2 but recognize endogenous dicarboxylates such as oxoglutarate3. Oxoglutarate is mainly derived from the tricarboxylic acid (TCA) cycle and serves as an essential myometabokine for autocrine and paracrine signaling, as well as an immunometabolite4. GPR99 recognizes oxoglutarate with an EC50 of approximately 200 μM and was subsequently named OXGR15. GPR99/OXGR1 is expressed in various human tissues including the kidney, respiratory epithelium, placenta, muscle, fetal brain and immune cells1,4,6,7. As a receptor of oxoglutarate, GPR99/OXGR1 is with its low affinity and low potency for this ligand, which is present in various tissues at concentrations in the micromolar range. In renal tubules, high concentrations of oxoglutarate promote Gq-mediated IP1 accumulation that contributes to the regulation of acid-base homeostasis4. Variations in renal pH may alter the polarity of oxoglutarate, thereby influencing Gq signaling through the activation of GPR99/OXGR14,8. These context-dependent features highlight how physiological environment may shape GPR99/OXGR1 signaling.

In addition to oxoglutarate, leukotriene E4 (LTE4) was identified as a ligand of GPR99/OXGR19. LTE4 is a cysteinyl leukotriene with a branched structure and established functions in allergy and asthma10–12. Elevated concentrations of LTE4 are observed in severe asthma, acute exacerbation of asthma, and aspirin-exacerbated respiratory disease (AERD)13–15, where it mediates sustained tracheobronchial constriction. An enrichment of GPR99/OXGR1 expression on the respiratory epithelial cells further exacerbates the inflammatory condition. Compared to LTC4 and LTD4 that are cysteinyl leukotrienes binding to different G protein-coupled receptors, LTE4 is more stable12. This feature of LTE4 contributes to asthma development due to the chronic effect of receptor/ligand-related hyperresponsiveness16. Other experimental evidence also supports the presence of the third cysteinyl leukotriene GPCR with high affinity for LTE417, whereas the LTE4 concentrations required to activate CysLTR1 and CysLTR2 are much higher than its physiological levels18. Thus, GPR99/OXGR1 is recognized as the physiologically relevant LTE4 receptor (CysLTR3), in addition to CysLTR1 and CysLTR2 that primarily bind to LTC4 and LTD45,9,18. CysLTR3 (GPR99/OXGR1) is markedly different from CysLTR1 and CysLTR2 in primary sequence with only 36% of amino acid identity9, and the binding affinity of LTE4 to GPR99/OXGR1 (Kd ~ 2.5 nM)9 is higher than that of LTC4 and LTD4 to CysLTR1 and CysLTR2, respectively7,9. Extensive studies have associated LTE4-CysLTR3 (GPR99/OXGR1) signaling with potent pro-inflammatory effects, such as enhanced bronchoconstriction3,19 and eosinophil influx into the airway6,7,15. The high affinity of LTE4 for GPR99/OXGR1 is in sharp contrast to the low affinity of oxoglutarate (Kd in hundreds of micromolar) for the same receptor, suggesting that the two structurally different ligands may interact with GPR99/OXGR1 in different manners. However, the structural basis for GPR99/OXGR1 to bind oxoglutarate and LTE4 is currently lacking despite progress in structural characterization of CysLTR1 and CysLTR220–22.

Here we report a cryo-EM structure of GPR99/OXGR1 bound to oxoglutarate. Structural and functional analyses identify a primary and an accessory binding site for oxoglutarate, featuring simultaneous binding of two oxoglutarate molecules for full activation of the receptor. Furthermore, we found that the transmembrane binding pocket in GPR99/OXGR1 is too small to accommodate the branched LTE4, and further cryo-EM analysis of a GPR99/OXGR1-Gq complex formed in the presence of LTE4 identified a potential LTE4 binding site outside the transmembrane binding pocket. Through functional analysis, we confirmed the alternative binding mode of LTE4 to GPR99/OXGR1, which likely serves as a promising target for future drug candidates. We found that LTE4 primarily activates the Gq-signaling pathways and the Gi pathway to a lesser extent. Although the poor EM density at this site prevented us to definitively locate the LTE4 binding site, our study provides a rational framework of the structural basis for and mechanistic insights into ligand recognition by GPR99/OXGR1.

Results
Overall structure of the oxoglutarate-GPR99/OXGR1-Gq signaling complex
The human GPR99/OXGR1 and Gq proteins were co-expressed in Sf9 insect cells and purified. The endogenous ligand oxoglutarate was introduced to facilitate the formation of the GPR99/OXGR1-Gq signaling complex, which was purified and subjected to cryo-EM analysis (Supplementary Fig. 1a, b). The structure of the signaling complex was then determined at a global resolution of 3.16 Å (Supplementary Fig. 1c–f, Table 1). The structure models were confidently built, and most residues were assigned based on the well-defined density map (Fig. 1a and Supplementary Fig. 2). Two separate ligand densities were observed in the orthosteric binding pocket, which fit well with two ligand molecules of oxoglutarate (Fig. 1b). GPR99/OXGR1 holds the canonical seven-transmembrane architecture and G protein coupling manner of GPCR (Fig. 1c). There are two disulfide bridges in the extracellular domains: C24N-term-C2747.25, and C1063.25-C183ECL2 (using GPCR Ballesteros-Weinstein numbering as superscripts23). The disulfide bonds are conserved among most Class A GPCRs and contribute to stabilization of the extracellular domains (Supplementary Fig. 3). The GPR99/OXGR1 in our structure adopts a conformation in the active state, with an intracellular pocket accommodating the α5 helix in Gq (Fig. 1b).

Molecular basis for oxoglutarate recognition by GPR99/OXGR1
In the oxoglutarate-GPR99/OXGR1-Gq complex, the transmembrane domains TM1-3, TM6-7, extracellular loop (ECL) 1 and ECL2 of GPR99/OXGR1 encircle a hydrophilic and positively charged orthosteric pocket suited for the binding of negatively charged oxoglutarate molecule (Supplementary Fig. 4). The oxoglutarates adopt a horizontal orientation (Supplementary Fig. 4). One oxoglutarate molecule (OG1 in Fig. 1b) is embedded at the bottom of the pocket with the carboxyl group pointing downwards, and the other oxoglutarate molecule (OG2) fits well in the solvent-accessible subpocket at the top of the TM pocket with the carboxyl group pointing upwards (Fig. 1b and Supplementary Fig. 4). OG1 is directly clamped by salt bridges with four positively charged amino acids: R1103.29 and R2887.39 for one carboxyl, and H1143.33 and R2616.55 for the other (Fig. 1d, e). There are sufficient interactions between OG1 and these amino acid residues that form a primary binding site for the ligand. In comparison, OG2 is salt-bridged with K321.31 and H2817.32 at one carboxyl, and a hydrogen bond with Y922.63 through the other carboxyl (Fig. 1d, e). The surrounding amino acids form an accessory site near ECL2, which is well exposed to the solvent environment and therefore less stable for OG2 binding. Notably, two gating arginine residues (R1103.29 and R2887.39), together with Y932.64 and D185ECL2, spatially separate OG1 from OG2 (Supplementary Fig. 5a). The interaction network of arginines3 stabilize the extracellular domain of GPR99/OXGR1 and separates OG1 from the extracellular solvent environment (Supplementary Fig. 5b).

Structural basis of selectivity for GPCR binding of dicarboxylates
Our findings reveal a distinctive binding mode for dicarboxylates in GPR99/OXGR1 compared to GPR91, suggesting a molecular mechanism underlying dicarboxylate selectivity in these GPCRs. The essential residues K321.31, H2817.32 and Y922.63 for OG2 binding in GPR99/OXGR1 are not found in GPR91 (E22, N274 and S82 in GPR91; Fig. 2 and Supplementary Fig. 6). Positively charged arginine residues are critical for the recognition and binding of negatively charged carboxyl groups, which are present in oxoglutarate (from oxoglutaric acid) and succinate (from succinic acid) in physiological pH. Most arginine residues in the orthosteric pocket are conserved between GPR91 and GPR99/OXGR1, namely R1103.29, R2616.55, R2646.58, R268ECL3 and R2887.39, but R177ECL2 and R180ECL2 in GPR99/OXGR1 are absent in GPR91 (Fig. 2 and Supplementary Fig. 6). In GPR91, only R993.29 and R2817.39 are known to participate in direct interactions with identified agonists and antagonists, including succinate, epoxysuccinate, maleic acid, compound 31, and NF-56-EJ4024,25.

We found that, in GPR99/OXGR1, R1103.29, R2616.55 and R2887.39 contributed to the binding of OG1 and were involved in receptor activation. The other four arginine residues, R177ECL2, R180ECL2, R2646.58 and R268ECL3, were positioned above the OG1 and at the top of the orthosteric pocket, with their positively charged guanidinium groups oriented down towards the orthosteric pocket (Supplementary Fig. 5b). This cluster of arginines likely acts as a gatekeeper of the orthosteric pocket, facilitating the recruitment of oxoglutarate. In particular, the flexibility of R177ECL2 and R180ECL2 at the tip of ECL2 in GPR99/OXGR1 may be crucial for capturing negatively charged molecules, allowing only appropriate ligands like oxoglutarate (here OG2) to engage with R2646.58 and R268ECL3 (Supplementary Fig. 5b). Interestingly, the positively charged R177ECL2 and R180ECL2 in GPR99/OXGR1 are replaced by the negatively charged D167ECL2 and N168ECL2 in GPR91. Additionally, the tip of ECL2 in GPR91 is positioned farther from the orthosteric pocket compared to GPR99/OXGR1. These observations suggest differences in ligand recognition and recruitment between GPR99/OXGR1 and GPR91, thus contributing to ligand selectivity in these two dicarboxylate receptors. In this structure, the closely positioned arginine residues at the orifice and surface of the orthosteric binding pocket form an “arginine network” that mediates the recruitment and binding of oxoglutarate to the receptor.

Structural insights into LTE4 binding to GPR99/OXGR1
Unlike oxoglutarate, LTE4 has a relatively large, hydrophobic tail and two charged carboxyl groups at the head (Fig. 3a). Like other CysLTs, the “Y” shaped carboxyl terminus of LTE4 differs from the straight chain of hydroxyacids found in LTB410,26. To learn how LTE4 binds GPR99/OXGR1, we compared our solved structure with available structures of CysLTR1 and CysLTR220,21, bound to Pranlukast (Fig. 3b) and ONO-2570366 (Fig. 3c), respectively. Amino acid residues known to interact with these ligands were identified with the superimposed receptor structures shown in the background. The binding pockets showed little resemblance at these critical residues, an observation consistent with the grouping of GPR99/OXGR1 in the P2Y nucleotide receptor subfamily and the two CysLT receptors in a different subfamily of GPCRs1,27. We also compared the receptor structures sliced at the TM binding pocket in the same plane. As shown in Fig. 3d–f, the binding pockets of CysLTR1 and CysLTR2 are similar in shape and volume (Fig. 3d, e), snugly holding their respective ligands. In comparison, the binding pocket of GPR99/OXGR1 differs drastically from these two receptors in shape and volume, suitable only for the much smaller oxoglutarate molecules. Other notable features include the absence of positively charged arginine network, which is important for binding of the bicarboxylic ligands such as oxoglutarate and succinate3,25,28, in the CysLT receptors. Moreover, the large access cleft from the lipid membrane between TM4 and TM5 in the CysLT receptors22,27, which allow lateral entry of the bulky tails of the lipid ligands, is missing in GPR99/OXGR1. While revising the manuscript, we compared the structure of GPR99/OXGR1 with the LTD4-bound CysLT2R structure that became available only recently22, and found little resemblance between the two at the orthosteric binding pockets (Supplementary Fig. 7). These results strongly suggest that LTE4 uses a different binding site in GPR99/OXGR1.

To illustrate how LTE4 binds to GPR99/OXGR1, an LTE4-GPR99/OXGR1-Gq complex was obtained with an excessive amount of LTE4 (~1.00 μM), and then subjected to cryo-EM analysis (Supplementary Fig. 8a, b). The resulting structure of the complex was resolved at an overall resolution of 2.84 Å (Fig. 4a, Supplementary Fig. 8c–g and 9, Table 1). An analysis of the structure found no extra EM density for LTE4 in the orthosteric pocket. Instead, a binding site was identified above the intracellular loop 2 (ICL2) and surrounded by TM3-5. Although the EM density could not definitively indicate binding of LTE4 at this site, the shape fits well with an LTE4 molecule (Fig. 4b, c).

Several approaches were taken to further explore the potential site for LTE4. A structural complex model was built based on the EM density, and potential interactions between LTE4 and GPR99/OXGR1 were identified. Specifically, L1223.41, T1253.44, I1293.48, F1303.49, V1524.44, A1534.45, V1574.49, I1604.52, T2055.45, L2095.49 could form extensive hydrophobic interactions with the aliphatic chain of LTE4 (Supplementary Fig. 10a). The negatively charged head of LTE4 could be attached to a positively charged subpocket formed by ICL2 and TM3-4 (Fig. 4d and Supplementary Fig. 10b). In detail, R1484.40 could form a salt bridge with one carboxyl group, C141ICL2 could possibly form a hydrogen bond with the other carboxyl group, and C1494.41 could form a hydrogen bond with the hydroxyl group as well as another hydrogen bond with a carboxyl group (Fig. 4d, e). The presence of these interactions was validated with alanine substitutions and the effects on receptor signaling by LTE4 as well as oxoglutarate were compared (see below).

Computational approaches were taken to further evaluate possible binding of LTE4 to this alternative site. Molecular docking was applied to dock LTE4 to the TM pocket and the alternative site, respectively. The corresponding binding energies were calculated, showing an energetic preference for LTE4 at the alternative binding site (Supplementary Fig. 11a). Furthermore, three independent 1-µs MD simulations captured a stable binding pose of LTE4 at this alternative site (Supplementary Fig. 11b, c).

Validation of GPR99/OXGR1-ligand interactions by site-directed mutagenesis
To further elucidate ligand interaction with GPR99/OXGR1, site-directed mutagenesis and functional assays were performed. Amino acid residues exhibiting polar interactions with oxoglutarate and LTE4 were substituted by alanine, and all mutants were positive for cell surface expression (Supplementary Fig. 12a). The accumulation of IP1, downstream of the Gq-phospholipase C (PLC) pathway, was measured for both WT and mutants of GPR99/OXGR1 (Fig. 5). Alanine substitution of residues interacting with OG1 (R110A, H114A, R261A, R288A) resulted in a significant reduction in both the potency and efficacy of IP1 accumulation following oxoglutarate stimulation (Fig. 5a). In comparison, mutations in OG2-interacting residues (K32A and Y92A) led to a more than 100-fold increase in EC50, but the maximum response (Emax) remained unchanged (Fig. 5a). These findings suggest that, while OG2 is essential for maintaining ligand occupation, it plays a less significant role in receptor activation compared to OG1.

Consistent with the predicted structure for LTE4 binding, alanine substitution of the OG1 and OG2 sites had little effect on LTE4-induced receptor signaling (Fig. 5b). Substitution of the projected LTE4-binding residues (C141ICL2, R1484.40 and C1494.41) markedly reduced LTE4-induced IP1 accumulation (Fig. 5c), but had little effect on oxoglutarate-induced receptor activation (Fig. 5d). This finding suggests that LTE4 binding to GPR99/OXGR1 depends on hydrogen bonding with key residues C141ICL2, R1484.40 and C1494.41 in the binding site, but this site was not utilized by oxoglutarate. Altogether, results obtained from mutagenesis experiments provide functional clues that support the binding of LTE4 to a site distinct from the TM site for oxoglutarate. We have also noticed that some mutants (C141ICL2A, C1494.41A) exhibited a reduced Emax albeit having comparable cell surface expression with the WT GPR99/OXGR1. As C141ICL2 and C1494.41 are proximal to the ICL2 region where the receptor extensively interacts with G proteins, we suspect that mutations at these sites may alter the overall stability of ICL2 and consequently the recruitment and activation of G proteins, thus affecting the efficiency of Gq response. These functional data distinguish the TM binding mode of oxoglutarate from the distinct binding mode of LTE4 at GPR99/OXGR1.

Ligand-induced signaling through GPR99/OXGR1
Given the structural difference between oxoglutarate binding and LTE4 binding at GPR99/OXGR1, it is suspected that their downstream signaling properties might vary. We therefore examined the signaling pathways triggered by oxoglutarate and LTE4. Notably, oxoglutarate preferentially activated the PLC pathway downstream of Gq signaling, leading to increased IP1 accumulation; however, oxoglutarate failed to stimulate cAMP reduction downstream of Gi coupling (Fig. 5e). In contrast, LTE4 treatment caused reduction in cAMP accumulation (Fig. 5e), suggesting activation of Gαi. This interesting finding was further verified by treatment of the cells with pertussis toxin (PTX), which ADP-ribosylates Gαi and breaks the Gi activation cycle29. PTX abrogated the cAMP response elicited by LTE4 (Fig. 5f) without affecting IP1 accumulation (Fig. 5g). These findings indicate that LTE4 binding to GPR99/OXGR1 can engage Gi-mediated signaling in addition to its Gq-coupled activity.

To investigate the basal activity of GPR99/OXGR1, we used a NanoBiT-based G protein dissociation assay, with SmBiT fused to Gγ and LgBiT fused to Gα30. Gα dissociation from Gβγ, that accompanies G protein activation, reduces the NanoBiT signal. HEK293T cells were co-transfected with equal amounts of plasmids encoding tagged G proteins and varying amounts of plasmids encoding GPR99/OXGR1 or the constitutively active KSHV-GPCR as a control31. Basal G protein dissociation of GPR99/OXGR1 was compared to that of KSHV-GPCR. Increasing KSHV-GPCR plasmid levels resulted in a dose-dependent decrease in NanoBiT signal, indicating agonist-independent G protein dissociation, whereas GPR99/OXGR1 showed no signal change, suggesting negligible basal activity of GPR99/OXGR1 (Supplementary Fig. 12b). These results indicate that GPR99/OXGR1 is not a constitutively active GPCR that can complex with G proteins without agonist binding.

The G protein coupling interface of GPR99/OXGR1
The G protein interfaces are highly similar between oxoglutarate-bound and LTE4-bound GPR99/OXGR1 signaling complex. However, subtle differences are observed for side chain orientations of the α5 helix of Gq protein (Fig. 6a). As a result, the G protein interaction profiles are slightly different between oxoglutarate-bound and LTE4-bound GPR99/OXGR1 structures. In the oxoglutarate-GPR99/OXGR1-Gq complex, more extensive polar interactions between Gq and GPR99/OXGR1 were identified (Fig. 6b). Specifically, Y351 of the α5 helix of Gq protein forms polar contacts with R1313.50 and S682.39 of GPR99/OXGR1. E350 forms a hydrogen bond with H145ICL2, and K349 interacts with D3078.48. Q345 forms polar interactions with Q230ICL3, and D341 has a polar contact with K2366.30 (Fig. 6b). In the LTE4-GPR99/OXGR1-Gq complex, four pairs of polar contacts are found between GPR99/OXGR1 and the Gq protein: R1313.50-Y351, D3078.48-K349, V1343.53-N347 and K2366.30-D341 (Fig. 6c). These subtle structural differences may help to explain the distinct biological outcomes induced by oxoglutarate versus LTE4.

Ligand-induced activation mechanism of GPR99/OXGR1
To elucidate the activation mechanism induced by orthosteric coupling of oxoglutarate, the recently reported structures of GPR91 in the active state25 (PDB: 8WOG) and inactive state24 (PDB: 6RNK) were used as references for comparison. The outward movement of TM5-6 and inward movement of TM7, which accommodated the α5 helix of Gq, illustrated a typical conformational change associated with receptor activation (Fig. 7a, b). Additionally, the structural motifs responsible for GPCR activation, specifically DRY, CWXP, NPXXY and PIF (corresponding to FRY, CFLP, NLLLY and PIF in GPR99), were compared between GPR99/OXGR1 and GPR91 (PDB: 8WOG). The oxoglutarate-GPR99/OXGR1-Gq complex represented a molecular mechanism for receptor activation triggered from the orthosteric pocket. Specifically, the salt bridge between OG1 and R2616.55 gave rise to an inward turning of TM6, which was transduced through the CFLP motif (Fig. 7c). The turning of TM6, along with the bridge between OG1 and H1143.33, caused a rearrangement of the hydrophobic core of the PIF motif (Fig. 7d). Besides, the salt bridge between OG1 and H1143.33, and the hydrogen bond between OG1 and R2887.39, triggered a conformational change that propagates through TM7. The salt bridge between oxoglutarate and R1103.29 initiated conformational changes that were transduced through TM3. Furthermore, OG2 played an accessory role, alongside OG1, in initiating receptor signaling. In a synergistic manner, the extracellular signal induced a rearrangement of the NLLLY (NPXXY) motif (Fig. 7e) and the FRY (DRY) motif (Fig. 7f) in the intracellular domain, opening the intracellular pocket to accommodate α5 helix of the Gq protein (Fig. 7e, f).

The reported inactive state structures of CysLTR1 and CysLTR2 were used as references to illustrate the activation mechanism of GPR99/OXGR1 induced by LTE420,21 (Fig. 8). In this structure, polar interactions caused the ICL2 of GPR99/OXGR1 to move outward when LTE4 is present (Fig. 8a), thus inducing the opening of the G protein-coupling pocket (Fig. 8b). The hydrophobic interaction driven by the aliphatic chain of LTE4 directly triggered the rearrangement of the NLxxY motif (Fig. 8c) and the FRY motif (Fig. 8d). Consequently, the CFxP and PIF motifs rearranged synergistically, with TM5-6 moving outward and TM7 moving inward (Fig. 8e, f). Additionally, a salt bridge formed between R318 and D312, further stabilizing the activated conformation and resulting in an ordered helix 8, which was absent in the inactive structure due to its flexibility.

Methods
Design of constructs
The cDNA encoding human wild-type GPR99 was synthesized by General Biol (Chuzhou, China). The constructs were created by cloning the full-length coding sequence into a pFastbac1 vector. To facilitate protein expression and purification, a hemagglutinin (HA) signal peptide, a FLAG tag, a human rhinovirus 3C (HRV3C) protease cleavage site (LEVLFQGP) and a thermostabilized apocytochrome b562RIL (BRIL) were added to the N terminus, following our previously reported method25. The Gq protein was designed based on the Gi backbone by inserting the C terminus of Gq (residues 332 to 359) to DNGαi1 (dominant negative Gαi1 with G203A and A326S mutation) backbone (residues 1 to 326), which provided an additional site for the single-chain antibody variable fragment scFv16 to stabilize the complex. This strategy and the resulting chimeric Gq were widely used, as demonstrated in the reported structure determination of GPCR-Gq complexes50–52. Human Gβ1 and Gγ2 cDNAs with N-terminal 6×His tag were respectively cloned into a pFastBac-Dual vector. scFv16 was fused with a GP67 signal peptide at the N-terminal and an 8×His tag at the C-terminal. For the functional assay, the human GPR99 coding sequence was cloned into the pcDNA3.1 vector. Point mutations were introduced using PCR-mediated site-directed mutagenesis.

Purification of scFv16
Secreted scFv16 was purified from expression media of baculovirus-infected Sf9 insect cell (Invitrogen, Cat #: 11496015) culture using Ni-NTA. The supernatant from 2 L of culture was collected and loaded onto a gravity column packed with Ni-NTA resin. The resin was washed with 20 mM HEPES pH 7.5, 500 mM NaCl, and the protein was eluted with 20 mM HEPES pH 7.5, 150 mM NaCl and 250 mM imidazole. Eluted protein was concentrated and loaded onto Superdex 200 increase 10/300 size exclusion column (GE Healthcare Life Sciences, Sweden). The peak fractions were collected and concentrated, rapidly frozen in liquid nitrogen, and stored at −80 °C.

Expression and purification of the signaling complex
The bac-to-bac baculovirus expression system and Sf9 insect cells were used to express protein for structure determination. The baculoviruses were prepared according to the manual (Thermo Fisher Scientific). For protein expression, baculoviruses of GPR99, Gαq, and Gβ1γ2 were co-transfected into Sf9 cells when they reached a density of 2×106 cells/mL. The ratio of baculoviruses for transfection was 1:1:1, and then the proteins were expressed over a 48-hour period. The cell culture was harvested by centrifugation at 2000×g for 15 min and kept frozen at −80 °C.

To purify the complex, the ligand (oxoglutarate or LTE4) was added to induce GPR99-Gq complex formation. Cell pellets were resuspended in buffer containing 25 mM HEPES pH 7.4, 50 mM of NaCl, 5 mM of KCl, 5 mM of CaCl2, 5 mM of MgCl2, 5% glycerol, 25 mU/mL apyrase, 2.5 μg/ml leupeptin, 0.16 mg/ml benzamidine, 100 μM of oxoglutarate or 1 μM of LTE4. Cell membranes were collected by centrifugation after 30 minutes of incubation. The cell membranes were then solubilized in 20 mM HEPES pH 7.4, 100 mM NaCl, 0.8% LMNG, 0.1% CHS, 10% glycerol, 2.5 μg/ml leupeptin, 0.16 mg/ml benzamidine, 50 μM of oxoglutarate or 1 μM of LTE4. After 2 h incubation at 4 °C, the supernatant was cleared by centrifugation at 20,000×g for 30 min. A gravity-flow column with anti-FLAG affinity resin (GenScript Biotech) was used to capture the complex, followed by washing with 10 column volumes of wash buffer containing 20 mM of HEPES (pH 7.4), 100 mM of NaCl, 5% glycerol, 2 mM of CaCl2, 2 mM of MgCl2, 0.05% (w/v) LMNG, 0.05% (w/v) GDN (Anatrace), 0.003% (w/v) CHS, 20 μM of oxoglutarate or 0.4 μM of LTE4. The protein complex was eluted with buffer containing 20 mM of HEPES (pH 7.4), 100 mM of NaCl, 2 mM of CaCl2, 2 mM of MgCl2, 0.01% (w/v) LMNG, 0.001% (w/v) GDN (Anatrace), 0.001% (w/v) CHS, 0.2 mg/ml FLAG peptide (Sigma-Aldrich), 20 μM of oxoglutarate or 0.4 μM of LTE4. The eluate was concentrated by an Amicon Ultra-15 Centrifugal Filter Unit (Millipore) and separated on a Superose 6 10/300 size-exclusion chromatography column (Cytiva) pre-equilibrated with 20 mM HEPES (pH 7.4), 100 mM NaCl, 0.0015% LMNG, and 0.0005% GDN, 0.0003% CHS, 20 μM of oxoglutarate or 0.4 μM of LTE4. The relevant peak fractions of the protein complex were collected, analyzed on SDS-PAGE, analyzed on western-blotting, concentrated to approximately 10 mg/mL, and stored at -80 °C.

Cryo-grid preparation and EM data collection
Negative stain electron microscopy was performed on all the samples to confirm homogeneity and complex formation. For cryo-grid preparation, aliquots of 3 µL of purified protein complex were applied onto an Ultrafoil 300 mesh R1.2/1.3 holy gold (Au) grid, which had already been glow-discharged by Tergeo-EM plasma cleaner. Afterward, the grids were blotted for 3.5 s with a blot force of 1 in 100% humidity at 4 °C to remove excess samples, and then quickly plunged into liquid ethane using a Vitrobot Mark IV (Thermo Fisher Scientific). The prepared grids were stored in liquid nitrogen.

The grid sample screening and data collection were performed using SerialEM software (Mastronarde, 2005). The final datasets were collected by a Gatan K3 direct electron detector installed on a 300 kV Titan Krios G3 microscope. During data collection, a GIF Quantum energy filter (Gatan, USA) was used to exclude inelastically scattered electrons, with the energy slit width set to 20 eV. Movie stacks were acquired at a nominal magnification of 105,000, resulting in a calibrated pixel size of 0.85 Å. The total exposure time was 2.5 s, fractionated into 50 frames at a dose rate of 20.8 e/pixel/s. The defocus range for dataset collection was set from -1.2 to -2.5 μm.

Image processing and 3D reconstructions
The cryoSPARC version v4.2.153 and Relion version 4.054 were used to process the cryo-EM datasets. The pipeline was similar to that reported previously25. Motion correction and dose-weighting were applied to align the movie stacks. After contrast transfer function (CTF) estimation, micrographs were manually inspected, and obviously bad micrographs were discarded. Representative particles were then manually picked to generate initial two-dimensional (2D) templates for auto-picking.

For the oxoglutarate-GPR99-Gq dataset, a total of 2,676,089 particles were template-based picked and subjected to 2D classification. After three rounds of 2D classification, particles were selected from the 2D averages with clear secondary features. Ab initio reconstruction was performed by cryoSPARC to generate initial three-dimensional (3D) templates, followed by rounds of 3D classification. Finally, a dataset containing 167,941 particles was used for homogeneous refinement, non-uniform refinement, and local refinement. The global resolution was 3.16 Å, estimated by the ‘gold standard’ criterion (FSC = 0.143).

For the LTE4-GPR99-Gq dataset, template-based particle picking resulted in a dataset of 2,043,778 particles, which was subjected to three rounds of 2D classification. Good particles with clear secondary features in the 2D averages were selected. Then, after multiple rounds of 3D classification, a final dataset containing 100,083 particles was obtained, resulting in a final map with an estimated global resolution of 2.84 Å.

Model building and refinement
As no experimental GPR99 structures had been reported previously, we used the predicted structure from the AlphaFold Protein Structure Database (AF-Q96P68-F1) as an initial template for model building. The model building of the G protein heterotrimer and scFv16 was facilitated by our previously reported structure (PDB: 8WOG) as the starting template. The model was docked into the electron microscope density by UCSF chimera55 and manually rebuilt with Coot56, as well as iterative refinement with Phenix57. Final model validation and statistical analysis were performed using Molprobity58. The graphic structural figures were prepared using UCSF Chimera, ChimeraX and PyMOL.

IP-one accumulation assay
Human GPR99, both in its wild-type form and in mutants, were expressed in HeLa cells (ATCC, Cat #: CCL-2) 24 hours before harvesting (Invitrogen, L3000001). Gq downstream signaling, which leads to the accumulation of IP1, was measured using the IP-One Gq HTRF kit (Cisbio). Cells were resuspended in the stimulation buffer (Cisbio) and incubated with varying concentrations of oxoglutarate (A610290, Sangon Biotech, Shanghai, China) or leukotriene E4 (HY-113465, MedChemExpress, Monmouth Junction, NJ) diluted in the stimulation buffer for 30 minutes at 37 °C. IP1 accumulation was then assessed according to the manufacturer’s instructions. Fluorescence intensities were recorded using an Envision 2105 multimode plate reader (PerkinElmer). Intracellular IP1 levels were determined based on the fluorescence signals from the samples and IP1 standards.

cAMP inhibition assay
Human GPR99 (WT/mutants) were expressed in HeLa cells for 24 hrs (Invitrogen, L3000001), and pretreated with 250 ng/mL PTX or vehicle for 12 h at 37 °C. Gi downstream signaling, which results in the inhibition of cAMP accumulation was measured. Cells were harvested in HBSS supplemented with 5 mM HEPES, 0.1% (w/v) BSA, and 0.5 mM 3-isobutyl-1-methylxanthine. The plated cells were subsequently stimulated with varying concentrations of chemerin alongside 2.5 μM forskolin for 30 minutes. Intracellular cAMP levels were quantified using the LANCE Ultra cAMP kit (TRF0263, PerkinElmer) according to the manufacturer’s protocol, with readings taken on an EnVision 2105 multimode plate reader (PerkinElmer).

NanoBiT-based GPR99-G protein dissociation and Gα-Gβγ protein dissociation assays
HEK293T cells (ATCC, Cat #: CRL-3216) were seeded in 24-well plates and incubated for 24 hours. For GPR99-G protein dissociation assay, cells were co-transfected with plasmids encoding GPR99-SmBiT (333 ng/well) and Gαi-LgBiT (167 ng/well). For Gα-Gβγ protein dissociation, cells were co-transfected with plasmids encoding GPR99 (92 ng/well), Gαi-LgBiT (46 ng/well), Gβ (230 ng/well), and SmBiT-Gγ (230 ng/well). Following a 24-hour incubation at 37 °C, the transfected cells were harvested and seeded into white 384-well plates. For the luminescence assay, coelenterazine H (Yeasen Biotechnology, Shanghai, China) was added to a final concentration of 10 μM for 1-hour incubation at room temperature, and baseline luminescence signals were measured for 10 minutes using an Envision 2105 multimode plate reader (PerkinElmer). Ligands were then added, and luminescence detection continued for 1 hour.

Flow cytometry analysis
HEK293T cells were transfected with expression plasmids encoding FLAG-tagged wild-type (WT) or mutant GPR99 for 24 hours at 37 °C. The cells were then collected and washed with HBSS containing 0.5% BSA. After washing, the cells were incubated with a FITC-labeled anti-FLAG antibody (Sigma, Cat# F4049; diluted 1:50 in HBSS buffer) for 30 minutes on ice and washed again with HBSS. Flow cytometry (CytoFLEX, Beckman Coulter) was used to quantify the FITC fluorescence signals on cell surfaces. The fluorescence signals were then analyzed to determine the relative expression levels of GPR99 mutants.

Molecular docking analysis
Flexible docking followed by Binding Free Energy calculations were performed using the BIOVIA Discovery Studio (v2019) to evaluate the differential binding affinity of the ligand LTE4 at the transmembrane pocket and the distinct site. Flexible docking in DS is an induced fit protocol to simulate both protein and ligand flexibility. During flexible docking, there are three distinct phases: (i) protein side-chain conformation generation through CHARMM-based ChiFlex method, (ii) generation of ligand conformations and rigid ligand placement into each protein conformation through LibDock, and (iii) induced fit protein flexibility through ChiRotor-based side-chain reconstruction and final pose refinement through CDOCKER. Protein flexibility is introduced in the first and third phases.

Prior to docking, LTE4 was extracted from the cryo-EM structure and preprocessed using the “Prepare Ligands” and “Minimize Ligands” protocols in DS. This step ensured correction of valency errors, adjustment of protonation states, and geometry optimization under the CHARMM force field. The receptor extracted from the cryo-EM structure was prepared by adding hydrogen, defining the binding site, and specifying flexible side chains for induced fit modeling. Residues within 5 Å of the bound ligand in the cryo-EM structure were selected as flexible for ChiFlex/ChiRotor processing. Docking regions for the two binding sites were defined using a spherical selection with a radius of 20 Å centered on the geometric center of LTE4 and oxoglutarate. For each receptor, multiple low-energy conformations were generated by ChiFlex. Ligand conformations were generated using the BEST mode in CatConf. Rigid docking poses were generated using LibDock based on binding-site hotspots, which were clustered and filtered before a final refinement stage using CDOCKER.

After the flexible docking, poses were sorted by -CDOCKER_ENERGY, and the top-ranked poses were selected for binding free energy calculations. The “Calculate Binding Energies” section in Discovery Studio estimates the free energy of binding for a receptor-ligand complex by calculating the free energies of the complex, the receptor, and the ligand. Binding free energies were computed using CHARMM-based molecular mechanics and implicit solvation via the PBSA model. Ligand conformational entropy was included in the estimation by enabling the “Estimate Entropy” and “Ligand Conformational Entropy” options. The reported energy terms include van der Waals, electrostatic, ligand strain, receptor strain, and solvation contributions, providing an overall estimate of binding energy for each pose. The poses with the lowest binding energy were selected as the final poses for further analysis.

Molecular dynamics (MD) simulations and analysis
Molecular dynamics (MD) simulations started with the atomic coordinates of LTE4 and GPR99 extracted from the cryo-EM structure of LTE4-GPR99-Gq complex resolved in this study; the Gq protein heterotrimer complex was removed before the system setup. The protonation states of the ligand-receptor complex were assigned for a pH of 7.4 using the H++ web server. Next, the CHARMM-GUI membrane builder was used to embed the complex within a POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine) lipid bilayer59. The entire system was then solvated in a periodic water box (TIP3P model) containing 0.1 M NaCl. CHARMM36m force field was applied to model the system. Simulations were produced on GPUs using GROMACS (version 2024.2)60. The system was maintained at 310 K and 1.0 bar, and energy minimized in 10,000 steps. Then a 200-ns restrained MD simulations was performed to equilibrate and relax the system. After initial energy minimization and equilibration, three independent production simulations were performed, each lasting 1 μs. The production runs were conducted under an NPT ensemble, using the leap-frog algorithm. Bonds involving hydrogen atoms were constrained using the LINCS algorithm. Long-range electrostatic interactions were computed with the Particle Mesh Ewald (PME) method, while a 1.2 nm cutoff was used for short-range van der Waals and Coulombic interactions. The stability of the ligand-receptor complex was monitored throughout the simulations by calculating the root mean square deviation (RMSD). Trajectory frames were first aligned to the protein backbone Cα atoms of the initial structure, and the ligand’s RMSD was then computed relative to its starting pose. Analysis of these trajectories confirmed that LTE4 consistently adopted a stable conformation in the alternative pocket above ICL2 of GPR99. Initial coordinate and simulation input files and a coordinate file of the final output of molecular dynamics simulations are provided as Supplementary Data 1.

Statistical analysis
The data analysis was performed using Prism 9.5.0 (GraphPad, San Diego, CA). Dose-response curves for agonist analysis were generated using the log[agonist] vs. response equation (three parameters) within the software. For the IP1 and cAMP assay, data points were expressed as percentages (mean ± SEM) relative to the maximum response level for each sample, based on at least three independent experiments, as indicated in the figure legends. For NanoBiT assays, data points were expressed as fold changes (mean ± SEM) relative to negative control (cells treated without ligands) for each sample, based on at least three independent experiments, as indicated in the figure legends. EC50 values were derived from the dose-response curves. For cell surface expression, data points were shown as percentages (mean ± SEM) of the flow cytometry fluorescence signals of wild-type GPR99. Statistical comparisons were made using one-way analysis of variance (ANOVA). A p-value of 0.05 or less was considered statistically significant.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "Dr. Liu reported the cryo-EM structure of GPR99 (OXGR1) bound to oxoglutarate or LTE4, in complex with Gq protein. The\nauthors claim a simultaneous binding of two oxoglutarates in the orthosteric pocket and an allosteric binding of LTE4, which\nseems novel. The authors also reveal Gq coupling information and ligand induced activation mechanism of GPR99.\nHowever, the study was poorly executed and presented, especially the assertion that LTE4 acts as an allosteric modulator of\nGPR99 is questionable, as outlined below.\n1) The authors claim that an ambiguous density appearing above ECL2 and in the groove formed by the intracellular sides\nof TM3, TM4, and TM5 corresponds to LTE4. However, the same density is also present in the map of oxoglutarate-bound\nGPR99 (see Fig. 1 of reviewing), raising doubts about whether this is truly LTE4 or simply an endogenous lipid density. In\nfact, many GPCRs exhibit similar lipid densities in this region, such as GPR35 (8h8j), GPR174 (8izb), PTHR (6nbf), and\nCB2 (6pt0). Therefore, the authors need to provide solid evidence that the observed density is indeed from LTE4.\n2) The density of oxoglutarates is also very poor.\n3) The structure of oxoglutarate-bound GPR99 is almost identical to the LTE4-bound GPR99 with only 0.259 Å of r.m.s.d.,\nmaking it suspicious whether this is the self-activation conformation of GPR99. An assessment of the autonomous activity of\nGPR99 is needed.\n4) No lipids (including cholesterol) were built on clear density.\n5) Poor refinement (MP>2, clash>20).\n6) No expression level of mutants.\n7) A functional assay, such as a BRET assay, that directly links ligand binding to G-protein coupling is needed to assess the\ndose-response relationship between the ligand and G-protein activation.\nSee attachment.", "reviewer_2": "The study investigates the molecular mechanisms of GPR99 by examining high-resolution structures of the receptor bound\nto its endogenous ligands, oxaloacetate and LTE4. This analysis uncovers the basis for ligand recognition and the activation\nof specific signaling pathways, offering valuable structural and functional insights into GPR99 that will inform subsequent\nresearch. To further enhance the manuscript's quality, the following recommendations are proposed:\n1. It is recommended to include the structure of GPR99 coupled with Gαi protein to elucidate the molecular mechanisms\nunderlying the G protein selectivity preferences of the two ligands, oxaloacetate and LTE4. Additionally, discussing whether\nthese preferences are influenced by varying physiological environments, such as tissue and cell specificity, and their\nimplications for physiological significance and drug development, would be beneficial. An in-depth discussion is advised.\n2. The manuscript currently lacks data on other potential downstream signals, such as β-arrestin signaling or receptor\ninternalization, which may limit a comprehensive understanding of the receptor's functionality. It is suggested to include\ncomparative analyses of the effects of both ligands on these alternative signaling pathways.\n3. While the manuscript mentions allosteric sites as potential drug targets, it does not provide a detailed discussion on how\nstructural information can be utilized to design effective small molecules or antibody-based therapeutics. A comparative\nanalysis with other GPCRs possessing similar allosteric binding sites and mechanisms, such as S1PR3 and GPR88, would\nenrich the discussion.\n4. Some functional experiments lack clear information on the number of replicates and statistical significance, which may\naffect the credibility of the results. Additionally, the impact of site-directed mutations on receptor expression levels has not\nbeen addressed, and corresponding data are absent. Providing this information would strengthen the manuscript's validity."}, "author_response": "Point-by-Point Response to Reviewers’ Comments\nReviewer #1 (Remarks to the Author):\nDr. Liu reported the cryo-EM structure of GPR99 (OXGR1) bound to\noxoglutarate or LTE , in complex with G protein. The authors claim a\n4 q\nsimultaneous binding of two oxoglutarates in the orthosteric pocket and an\nallosteric binding of LTE , which seems novel. The authors also reveal G\n4 q\ncoupling information and ligand induced activation mechanism of GPR99.\nHowever, the study was poorly executed and presented, especially the\nassertion that LTE acts as an allosteric modulator of GPR99 is questionable,\n4\nas outlined below.\nResponse: We appreciate your recognition of our efforts in resolving the cryo-\nEM structures of GPR99 in complex with oxoglutarate, LTE , and Gq protein,\n4\nas well as our exploration of its activation mechanism. We acknowledge your\nconcerns regarding the assertion that LTE functions as an allosteric\n4\nmodulator. In response, we have carefully re-examined our data and\nconducted additional analyses to further substantiate our conclusions. Our\nnew evidence supports LTE binding at a distinct site as well as its influence\n4\non receptor conformation in a manner consistent with allosteric modulation.\nAdditionally, we have refined the presentation of our findings to improve clarity\nand ensure a more rigorous interpretation of the data. All modifications in the\nrevised manuscript are highlighted in blue, and we have addressed each of\nyour comments in detail below. We hope these revisions have adequately\naddressed your concerns.\n1) The authors claim that an ambiguous density appearing above ECL2 and in\nthe groove formed by the intracellular sides of TM3, TM4, and TM5\ncorresponds to LTE . However, the same density is also present in the map of\n4\noxoglutarate-bound GPR99 (see Fig. 1 of reviewing), raising doubts about\nwhether this is truly LTE or simply an endogenous lipid density. In fact, many\nGPR174 (8izb), PTHR (6nbf), and CB2 (6pt0). Therefore, the authors need to\nprovide solid evidence that the observed density is indeed from LTE .\ndensity corresponds to LTE or endogenous lipids. Although X-ray\n4\ncrystallography could clearly identify GPCR ligand bind to the transmembrane\nsurface (see an example of P2Y1 receptor, PDB ID: 4XNV), it is challenging to\naccurately determine the precise identity of a small molecule ligand and\ndistinguish it from other molecules with similar shapes solely based on cryo-\ntechnologies, especially when dealing with flexible ligands binding to\nnoncanonical sites on receptor surface. However, by integrating prior\nknowledge, structural indications, and functional analyses, we have arrived at\na reasonable assignment of the small molecule ligand. Several lines of\nevidence support our assignment of the observed density to LTE :\n4\n1. Structural considerations.\nThe cryo-EM maps of the LTE -saturated GPR99–Gq complex reveal no\n4\nligand density within the canonical orthosteric pocket, suggesting that LTE\n4\nbinds at a noncanonical site. Additionally, the chemical properties of LTE —\n4\nparticularly its molecular size, hydrophobicity, and distinctive branched “Y”\nshape, are inconsistent with binding in the relatively small and hydrophilic\nbinding pocket that accommodates oxoglutarate. Instead, a distinct density is\nobserved in a hydrophobic pocket formed above ICL2 and between the\nintracellular portions of TM3, TM4, and TM5, a location that aligns well with\nLTE ’s hydrophobic character (see figure below). The presence of chemically\n4\nplausible interactions, such as the salt bridge between the carboxyl group of\nLTE and R1484.40, further supports the stable occupancy of LTE in this\n4 4\npocket. No alternative pocket that is more chemically suitable for LTE has\n4\nbeen identified in the resolved structure. It is plausible that, in the absence of\na suitable ligand, a lipid such as cholesterol might occupy the hydrophobic\nnoncanonical pocket on the GPCR surface, primarily serving to stabilize the\npocket and the receptor’s overall structure rather than acting as a\nconventional ligand. Moreover, several studies have implicated similar\npockets in allosteric modulation by lipid-like molecules 1,2.\n4\nbinding pocket of GPR99.\n2. Functional analysis.\nThe interactions between LTE and its surrounding residues in the pocket are\n4\nstrongly supported by our functional data. Extensive mutagenesis on residues\nlining the proposed binding pocket revealed that mutations in this region\nmarkedly diminished LTE -mediated signaling while having minimal effects on\n4\noxoglutarate-mediated response. In contrast, mutations in the orthosteric\npocket primarily disrupted oxoglutarate binding, with only a slight impact on\nthe LTE response. This differential effect supports our assignment of the\n4\nobserved density to LTE and underscores its role in receptor activation via a\n4\nnoncanonical site.\nWe have updated the revised manuscript to include all these additional data.\nMoreover, we have put an emphasis on the LTE binding site which is\n4\nnoncanonical when compared with the oxoglutarate site surrounded by TM\ndomains. Because LTE and oxoglutarate are different ligands sharing the\n4\nsame receptor, we have refrained from using the term “allosteric” which\ncompares ligands of the same class that act at different sites of the receptor.\n2) The density of oxoglutarates is also very poor.\nResponse: We appreciate the valuable comments. In our updated structural\ndata, the cryo-EM densities for both oxoglutarate molecules are clearly\ndefined and reliably modeled as small‑molecule ligands for GPR99 in Coot\n(see figure below). Moreover, ligand–receptor interactions were confirmed by\nfunctional assays, reinforcing our confidence that these densities support a\nrobust conclusion. We hope that the concerns have been addressed through\nthe revision.\nmolecules.\n3) The structure of oxoglutarate-bound GPR99 is almost identical to the LTE -\n4\nbound GPR99 with only 0.259 Å of r.m.s.d., making it suspicious whether this\nis the self-activation conformation of GPR99. An assessment of the\nautonomous activity of GPR99 is needed.\nResponse: We thank the reviewer for the insight comments. We agree that\nthe similarities in structures between oxoglutarate-bound and LTE -bound\nrepresent the self-activation state of GPR99.\nTo address this concern, we have conducted additional experiments to assess\nthe autonomous activity of GPR99. Specifically, we evaluated the constitutive\nactivity of GPR99 using a NanoBiT-based G protein dissociation assay\n(reflecting G protein activation). The receptor’s basal activity was determined\nin the absence of ligands. In this system, a reduction in NanoBiT\nluminescence represents separation of Gα subuit from Gβγ subunits,\n4\nindicating constitutive activity of the receptor without agonist stimulation. No\nconstitutive activity of GPR99 was detected as shown in the figure below. In\ncomparison, KSHV-GPCR, a receptor known to activate autonomously,\ndisplayed a declining curve indicating constitutive activity without agonist\nstimulation. Therefore, the results indicate that GPR99 exhibits little intrinsic\nactivity. We have added this data to Supplemental Figure S9.\nFig. R3 (Fig. S9b). The basal activity of GPR99 was compared with a GPCR\nknown for constitutive activation (KSHV-GPCR) as a function of increasing\nreceptor expression levels (more DNA used in cell transfection). The NanoBiT-\nbased G protein dissociation assay was used and dissociation of the G\nprotein subunits (α from βγ, with reduced values) was an indicator of receptor\nactivation. Data shown were values from five independent experiments, each\nwith triplicates.\nComparing our results with those of other GPCRs activated by different\nligands reveals that, in some cases, high structural similarity can be expected\nfor the same receptor under various activation conditions. For example, the\nsuccinate receptor (SUCNR1), independently reported by three groups,\nconsistently demonstrates this phenomenon with r.m.s.d. values of 0.744 Å\n(Group 1, 8JPN and 8JPP), 0.251Å (Group 2, 8YKW and 8YKX), and 0.276 Å\n(our group, 8WOG and 8WP1). Additionally, GPR88 activated by its allosteric\nmodulator and orthosteric ligand shows an r.m.s.d. value of 0.577 (7EJX and\n7WZ4). Therefore, the r.m.s.d. between structures of GPR99 bound to\ndifferent ligands are reasonable.\n4) No lipids (including cholesterol) were built on clear density.\nResponse: We acknowledge that, due to technical limitation, it is still difficult\nto build lipids with clear density. This is mostly due to the intrinsic flexibility of\nlipids, making it difficult to be identified unambiguously. We also acknowledge\n5\nthe presence of a lipid-like density around the LTE -binding region in the map\n4\nof oxoglutarate-bound GPR99, but it is difficult to reliably model this as\nspecific lipids based on our map. This is true to several other GPCRs with\nsimilar densities appearing in GPR35 (PDB ID: 8H8J), GPR174 (PDB ID:\n8IZB), PTHR (PDB ID: 6NBF), and CB2 (PDB ID: 6PT0). In these cases, the\nlipids were modeled as cholesterol or palmitic acid respectively. Comparison\nwith additional structures has led to the observation that the site of interest is\nnot conserved for specific lipids. However, in regions with clear density for\ncholesterol in other structures, we have incorporated the lipids into our models\naccording our density and the updated the models.\nFig. R4. Lipid density assignment in different receptors. a. Lipid models at\nthe pocket above ICL2 surrounded by TM3, TM4 and TM5, where LTE binds\n4\nto GPR99. b. Lipid models at the pocket above ICL1, surrounded by TM1,\nTM2 and TM3 which seems to be conserved for cholesterol binding.\n5) Poor refinement (MP>2, clash>20).\nResponse: We have carefully reviewed the refinement process and further\noptimized the model. The updated refinement statistics show a significant\nreduction in both MP and clash scores, thereby enhancing the overall model\nvalidation metrics. For the structure of LTE -bound GPR99, the MP was\n4\nupgraded to 1.60 and the clash was minimized to 3.70. For the oxoglutarate-\nGPR99-Gq, the MP was upgraded to 1.69 and the clash was minimized to\n6.85. The revised statistics have been incorporated into the revised\nmanuscript.\n6) No expression level of mutants.\nResponse: We have incorporated experimental data assessing the expression\nlevels of all site-directed mutants into the revised manuscript (Fig. S9). All\nthese mutants were expressed well, demonstrating that the observed\nfunctional changes were not due to altered receptor expression levels.\nwere transfected with the plasmids for WT or mutant GPR99s carrying an N-\nterminal FLAG tag. Flow cytometry analysis were performed using an anti-\nFLAG M2 antibody. Fluorescence intensities were calculated, and shown\nas % of the WT GPR99. Data in triplicates were obtained from three\nindependent experiments and plotted.\n7) A functional assay, such as a BRET assay, that directly links ligand binding\nto G-protein coupling is needed to assess the dose-response relationship\nbetween the ligand and G-protein activation.\nResponse: We appreciate the constructive comments. Accordingly, we have\nconducted NanoBiT-based assays to evaluate G protein coupling and\nreceptor-mediated activation. The results of these assays have been\nincorporated into the revised manuscript. The first assay measured which G\nprotein (alpha subunit) preferentially couples to GPR99 when stimulated with\noxoglutarate or LTE . In this dissociation assay, GPR99-SmBiT and Gi/Gq-\nLTE at various concentrations. The results (Response Fig. 3) demonstrate a\n4\npreference of GPR99 for Gq coupling with either oxoglutarate or LTE\n4\nstimulation. The second assay generated dose-response curves for\noxoglutarate- and LTE -induced Gq protein dissociation (dissociation between\nGPR99. A NanoBiT-based GPR99-G protein coupling assay was used to\nexamine the dose-response relationships between ligand binding to GPR99\nand G protein activation. Vectors encoding GPR99-SmBiT and Gi/Gq-\nLgBiT are respectively transfected to HEK293T cells. Upon oxoglutarate and\nLTE treatment, NanoBiT signals are reduced, implying the dissociation of G\n4\nprotein from the receptor. Data are shown as ratios of negative control (cells\ntreated without ligands) and obtained from three independent experiments,\neach with duplicates.\nFig. R7 (Fig. 4c,d). Dose-response relationship of G protein dissociation\ninduced by GPR99 activation. A NanoBiT-based Gq dissociation assay is\nintroduced to examine the dose-response relationships of GPR99-induced G\nprotein activation. Vectors encoding Gq-LgBiT and G-SmBiT are\ntransfected to HEK293T cells, and cells are co-transfected with GPR99-\nencoding plasmids and vehicles, respectively. Upon oxoglutarate and LTE\n4\ntreatment, NanoBiT signals are reduced, implying the dissociation of Gq and\nG. Data are shown as ratios of negative control (cells treated without\nligands) and obtained from three independent experiments, each with\nduplicates.\nThe study investigates the molecular mechanisms of GPR99 by examining\nhigh-resolution structures of the receptor bound to its endogenous ligands,\noxaloacetate and LTE . This analysis uncovers the basis for ligand\n4\nrecognition and the activation of specific signaling pathways, offering valuable\nstructural and functional insights into GPR99 that will inform subsequent\nresearch. To further enhance the manuscript's quality, the following\nrecommendations are proposed:\nResponse: We thank the reviewer for the thoughtful assessment of our study\nand for the supportive and constructive comments. We greatly appreciate the\ninsightful suggestions. In response, we have conducted additional\nexperiments to further strengthen the work. Below, we address each of the\ncomments and recommendations in detail.\n1. It is recommended to include the structure of GPR99 coupled with Gαi\nprotein to elucidate the molecular mechanisms underlying the G protein\nselectivity preferences of the two ligands, oxaloacetate and LTE . Additionally,\n4\ndiscussing whether these preferences are influenced by varying physiological\nenvironments, such as tissue and cell specificity, and their implications for\nphysiological significance and drug development, would be beneficial. An in-\ndepth discussion is advised.\nResponse: We appreciate the comments and conducted an additional\nexperiment to determine the preference of GPR99 for the two G proteins (see\nfigure below).\nFig. R8 (Fig. 4a,b). Dose-response relationship of G protein coupling to\nGPR99. A NanoBiT-based GPR99-G protein coupling assay was used to\nexamine the dose-response relationships between ligand binding to GPR99\nand G protein activation. Vectors encoding GPR99-SmBiT and Gi/Gq-\nLgBiT are respectively transfected to HEK293T cells. Upon oxoglutarate and\n4\nprotein from the receptor. Data are shown as ratios of negative control (cells\ntreated without ligands) and obtained from three independent experiments,\neach with duplicates.\nAs shown in Fig. R8 above, GPR99 preferentially couples to the Gq protein\nover Gi. The low coupling to Gi has made it difficult to obtain high quality\nreceptor-Gi complex for cryo-EM analysis. Considering that the current work\nfaces a challenge in deciphering the structural basis for GPR99 to interact with\ntwo different ligands, we choose to stay with Gq which is the primary G protein\nfor GPR99 signaling. It is however interest to explore whether coupling to Gi\ncan lead to different functions. Moreover, the G protein selectivity may be\naffected by cellular and tissue environment3-8. These points have now been\ndiscussed in the revised manuscript. In future studies, the Gi-coupled GPR99\nmay be studied in more detail including structural determination.\n2. The manuscript currently lacks data on other potential downstream signals,\nsuch as β-arrestin signaling or receptor internalization, which may limit a\ncomprehensive understanding of the receptor's functionality. It is suggested to\ninclude comparative analyses of the effects of both ligands on these\nalternative signaling pathways.\nResponse: We appreciate the constructive comments and have now included\na comparative analysis of the effects of both ligands on β-arrestin recruitment.\nInterestingly, while oxoglutarate and LTE induced both β-arrestin1 and β-\n4\narrestin2 recruitment to GPR99, oxoglutarate exhibited a higher E for the\nmax\nrecruitment of β-arrestin2, while LTE stimulation recruited more β-arrestin1.\nβ-arrestin recruitment by liganded GPR99 was in a NanoBiT-based β-arrestin\nrecruitment assay. Vectors encoding GPR99-SmBiT and β-arrestin1/β-\narrestin2-LgBiT were transfected to HEK293T cells. Upon oxoglutarate and\nLTE stimulation, NanoBiT signals were increased, implying association of β-\n4\narrestins to GPR99. Data are shown as fold increase over basal (cells treated\n10\nwithout ligands), based on three independent experiments, each with\nduplicates.\n3. While the manuscript mentions allosteric sites as potential drug targets,\nit does not provide a detailed discussion on how structural information can be\nutilized to design effective small molecules or antibody-based therapeutics. A\ncomparative analysis with other GPCRs possessing similar allosteric binding\nsites and mechanisms, such as S1PR3 and GPR88, would enrich the\ndiscussion.\nResponse: We have followed this suggestion by including a more detailed\nanalysis in the revised manuscript, highlighting how our findings on the\nnoncanonical site of GPR99 could inform drug design strategies9-12. We believe\nthat these additions strengthen the manuscript and provide a more\ncomprehensive perspective on the therapeutic potential of targeting GPR99.\n4. Some functional experiments lack clear information on the number of\nreplicates and statistical significance, which may affect the credibility of the\nresults. Additionally, the impact of site-directed mutations on receptor\nexpression levels has not been addressed, and corresponding data are\nabsent. Providing this information would strengthen the manuscript's validity.\nResponse: In response to these comments, we have carefully examined the\nfigures and added necessary information on the number of replicates.\nStatistical analysis for all functional experiments is now updated with specific\np-values and statistical methods in the revised manuscript.\nAdditionally, we appreciate the comments on the expression levels of the site-\ndirected mutants. We have performed flow cytometry analysis to determine\nthe expression levels of these mutants, and included the results as Fig. S9a\n(in Supplementary Figures). The data indicate that expression of these\nmutants are about equal, and the observed differences are not due to poor\nreceptor expression.\nReferences:\ndynamics for receptor activation, signalling bias and allosteric modulation. Br\nJ Pharmacol (2024). https://doi.org/10.1111/bph.16495\ndynamics data uncovers allosteric sites and lateral gateways. Nat Commun\n16, 2020 (2025). https://doi.org/10.1038/s41467-025-57034-y\n11\nreceptors in immune cell-related functions. Adv Immunol 142, 65-84 (2019).\nhttps://doi.org/10.1016/bs.ai.2019.04.002\ninflammatory diseases. Int Immunol 31, 607-615 (2019).\nhttps://doi.org/10.1093/intimm/dxz044\nInflammation. Annu Rev Pathol 20, 115-141 (2025).\nhttps://doi.org/10.1146/annurev-pathmechdis-111523-023509\nhyperresponsiveness to LTE4, as compared with normal airways, but not to\nLTC4, LTD4, methacholine, and histamine. Am Rev Respir Dis 142, 1112-\n1118 (1990). https://doi.org/10.1164/ajrccm/142.5.1112\nan intrarenal paracrine mechanism. J Clin Invest 123, 3166-3171 (2013).\nhttps://doi.org/10.1172/JCI67562\nReceptor Expression, Tubular Angiotensin II Formation, and Na(+)\nReabsorption During High Glucose Conditions. Front Cardiovasc Med 8,\n644797 (2021). https://doi.org/10.3389/fcvm.2021.644797\ndeterminants of G protein-subtype bias. Sci Adv 7 (2021).\nhttps://doi.org/10.1126/sciadv.abf5325\nvia automated path searching. Proc Natl Acad Sci U S A 121, e2317893121\n(2024). https://doi.org/10.1073/pnas.2317893121\nand ligand selectivity of S1PR3-Gi signaling complexes. Cell Res 32, 218-221\n(2022). https://doi.org/10.1038/s41422-021-00567-w\nsignaling complex. Nat Commun 13, 2375 (2022).\nhttps://doi.org/10.1038/s41467-022-30081-5\n12"}, {"round": 2, "reviewer_comments": {"reviewer_1": "The authors did not address my concerns. The key question of this study is whether the density appeared above ICL2 is the\ndensity of LTE, as it also appeared in the map of oxoglutarate-bound GPR99, where LTE was not added. In the revision,\nthere are still similar density appear in both maps (see review_fig 1), the author did not provide any solid evidence to prove\nthe density belongs to LTE, those arguments of technical issues make no sense.\nAlso the ligand densities of oxoglutarates are still very poor (review_fig2), it is very hard to distinguish it from background\nnoise.\nBased on above observations, I cannot recommend publication of this work.\nSee attachment.", "reviewer_2": "The authors have adequately addressed all my comments and suggestions.\nHowever, a few minor issues require further attention:\n1. The number of decimal places for EC₅₀ and Eₘₐₓ values should be standardized throughout the manuscript.\n2. Figure 3C indicates that Eₘₐₓ for oxoglutarate is also impaired in the C141A and C149A mutants. However, these\nresidues reside outside the oxoglutarate binding pocket, and the mutation did not significantly alter surface expression\nlevels. Please clarify the mechanistic basis for these observed effects.", "reviewer_3": "The paper by Aijun Liu, Yezhou Liu et al. features structures of the oxoglutarate receptor (OXGR1), also known as cysteinyl\nleukotriene receptor 3 (CysLT3R), in the active state, solved under two different experimental conditions. These are\nsupported by functional data, including extensive mutagenesis. Publishing structures of this receptor represents a valuable\nadvance in understanding the function of class A GPCRs. The paper is clearly written, and the data are well presented,\nparticularly following revisions based on reviewer comments.\nIt is intriguing that the proposed pose of the LTE4 molecule is located on the outer surface of the receptor, while oxoglutarate\nis suggested to bind in the orthosteric binding pocket, which is known to bind membrane lipids.\nPrevious studies on cysteinyl leukotriene receptors (e.g., Luginina et al., 2019) have shown that the shape and volume of the\nligand-binding pocket can change depending on the bound ligand. Therefore, it is plausible that the pocket could expand to\naccommodate a larger molecule such as LTE4.\nIf a mass spectrometry experiment detecting LTE4 in the OXGR1-LTE4 complex could be conducted, it would provide strong\nevidence for the presence of LTE4 in the complex.\nAdditionally, sequence comparison of key orthosteric ligand-binding residues across all three CysLTRs shows that many\nare conserved (e.g., K1.31, N5.39 in both CysLT2R and OXGR1; Y2.64 in all three), raising the question of whether LTE4\ncould still bind to OXGR1 in the orthosteric pocket under different experimental conditions.\nThe paper provides convincing data that mutations in the proposed allosteric LTE4 binding site do not affect oxoglutarate-\ninduced signalling. However, there are no data showing how mutations in the orthosteric binding pocket impact -induced\nsignalling (K1.31A, Y2.64A, etc.). Such data would help assess whether LTE4 binding in the orthosteric pocket is possible in\nOXGR1.\nI appreciate that the paper was written and submitted before the active CysLT2R structure with leukotrienes was published\n(https://doi.org/10.1073/pnas.2417148122), but it would be valuable to include at least a paragraph at the end comparing this\nstructure with the active OXGR1 structures.\nMinor comments:\nLines 82-83: all the listed drugs are known as selective CysLT1R antagonists and are not known to effectively block\nCysLT2R (e.g. https://doi.org/10.1074/jbc.M003490200). The statement should be supported with references to say\notherwise.\nTo sum up, I recommend the paper for publishing after all the concerns about LTE4 binding in the orthosteric pocket are\naddressed and the corresponding sections of the paper corrected."}, "author_response": "NCOMMS-24-76881 R2\nAuthor responses\nGeneral response to all review comments:\nWe appreciate the insightful comments from all reviewers. We are encouraged by the positive\ncomments from 2 of the reviewers who recommended the revised manuscript. We are fully aware of\nthe concerns raised by Reviewer #1 on the poor density at the alternative site for LTE binding. After\n4\ncareful consideration, we agree with the reviewers that our results did not convincingly locate the\nLTE binding site outside the orthosteric site, the same binding pocket for oxoglutarate. Besides, we\n4\nnow named the receptor as GPR99/OXGR1 according to IUPHAR nomenclature.\nIn this revised manuscript, we provide experimental data that demonstrate LTE does not fit into the\n4\northosteric site for oxoglutarate. With an EM map showing the EM density of oxoglutarate\nmolecules at a contour level of 0.3 Å, we have shown tandem binding of two oxoglutarate molecules\nin the orthosteric site, using an arginine network similar to that of the succinate receptor 1 (PDB ID:\n8WOG). The orthosteric site, however, is too small (and insufficiently expandable) to accommodate\nthe branched LTE molecule. A recent PNAS paper (PMID: 40193607), published after our manuscript\n4\nwas submitted, allows for direct comparison of the LTD -bound CysLT R structure with our\n4 2\nGPR99/OXGR1 structure (Fig. R1 below).\nFig. R1. Comparison of the structures of GPR99/OXGR1 and CysLT R. The LTD -CysLT R (PDB ID:\n2 4 2\n9IXX) in active conformation (gold) is used as a reference model. a, overall superimposed structures\nof GPR99/OXGR1 (cyan, this paper) and CysLT R, both in active state. For display clarity, the ligands\n2\nare removed from the orthosteric pocket. b, the transmembrane (TM) binding pocket of CysLT R\n2\nwith bound LTD (yellow) and the superimposed structure of GPR99/OXGR1. Notably, CysLT R\n4 2\nresidues important for interaction with LTD are either not present in GPR99/OXGR1 or changed to\n4\namino acids of different properties. For example, L188, E189 and L190 in the ECL2 of CysLT R, that\n2\ninteract with LTD , are not found in the equivalent position in GPR99/OXGR1. Critical amino acids of\n2 4\nposition (K1.31, Y2.64, H7.32) or substituted with different amino acids (R2.60 to L, Y3.33 to H, /Q5.35 to I,\nN5.38 to Y, H6.58 to R, and L7.39 to R) in GPR99/OXGR1.\nWe further compared the sequences of GPR99/OXGR1, CysLT receptors and P2Y receptors with\nphylogenetic analysis. As shown in Fig. R2, GPR99/OXGR1 is in the subfamily of the P2Y nucleotide\nreceptors, while CysLT1R and CysLT2R are members of a different receptor subfamily.\nFig. R2. Sequence analysis of GPR99, CysLTs and P2Y1. a, phylogenetic tree of amino acid sequences\nof GPR99, CysLT receptors and P2Y receptors. GPR99, CysLT2R and P2RY1 are boxed. b, sequence\nalignment of GPR99, CysLT2R, and P2RY1.\nThe differences are reflected in their ligand binding pockets, with a much larger orthosteric pocket in\nsolved structures of CysLT1R 1 and CysLT2R 2 when compared with our structure of GPR99/OXGR1. In\nCysLT1R and CysLT2R, a hydrophobic orifice between the TM4 and TM5 allows lateral insertion of\nthe hydrophobic tail of CysLTs; the polar network region at the top of the receptor TM pocket favors\nthe binding of carboxylic head of CysLTs. However, GPR99/OXGR1 lacks such a hydrophobic opening\nbetween TM4 and TM5.\nWe conducted additional mutagenesis experiments and found little effect of alanine substitutions of\nthe oxoglutarate interaction site on LTE -induced GPR99/OXGR1 signaling (new Fig. 5). Analysis of\n4\nthe geometry of the orthosteric site also excludes its accommodation of LTE , that would be visible\n4\ninside the solved structure if LTE binding is allowed through expansion of the receptor’s binding\n4\npocket.\nWe have substantially revised the manuscript, to emphasize our major findings: 1) a solved cryo-EM\nstructure of GPR99/OXGR1 with the simultaneous binding of two oxoglutarate molecules; 2) an\northosteric binding pocket which is too small to accommodate LTE , suggesting the presence of a\n4\ndistinct binding site; 3) functional assays confirming response of GPR99/OXGR1 to both oxoglutarate\nand LTE stimulation; 4) a projected alternative site for LTE above intracellular loop 2 of\n4 4\nGPR99/OXGR1; 5) mutagenesis data showing that substitutions of key residues for oxoglutarate\nbinding do not significantly alter LTE -induced response, and vice versa.\naccomplished in oxoglutarate binding to GPR99/OXGR1, and what still need to be investigated in\nLTE interaction at the distinct site of GPR99/OXGR1. We also hope that the information presented is\n4\nvaluable to the research community interested in oxoglutarate and LTE signaling.\nREVIEWER COMMENTS\nReviewer #1 (Remarks to the Author):\nThe authors did not address my concerns. The key question of this study is whether the density\nappeared above ICL2 is the density of LTE, as it also appeared in the map of oxoglutarate-bound\nGPR99, where LTE was not added. In the revision, there are still similar density appear in both maps\n(see review_fig 1), the author did not provide any solid evidence to prove the density belongs to LTE,\nthose arguments of technical issues make no sense.\nAlso the ligand densities of oxoglutarates are still very poor (review_fig2), it is very hard to\ndistinguish it from background noise.\nBased on above observations, I cannot recommend publication of this work.\nResponse:\nWe thank the reviewer for the critical feedback. Regarding the oxoglutarate ligand densities,\nwe realized that the small size of the dicarboxylate molecule makes it difficult to identify against\nbackground. We have re-examined the EM map and found a clean fit for both oxoglutarate\nmolecules at the contour level of 0.3 Å (Fig. R3 below). The placement of both oxoglutarate\nmolecules is consistent with our functional data. The figures are now included in the revised\nmanuscript as Supplemental Fig. S4.\nFig. R3. EM map density of oxoglutarate molecules at a contour level of 0.3 Å. Left, the EM map\ndensity of the two oxoglutarate molecules. Right, the EM map density of the oxoglutarate molecules\n(colored in magenta) and the GPR99 TM binding pocket (colored in light cyan) in a different angle.\nRegarding LTE density at the alternative site, a direct comparison of both maps (with LTE or with\n4 4\noxoglutarate as ligand) at the same 0.5 Å contour level reveals a distinct difference at this proposed\nsite above ICL2. While the oxoglutarate-bound map displays an amorphous density likely\nattributable to a co-purified detergent or lipid molecule, the map resolved with LTE shows a sharper\n4\nand more defined density (Fig. R4 below). This feature is consistent with the specific size and shape\nof the LTE molecule at this site.\n4\n0.5 Å. Left, the EM map density at the alternative binding site in LTE -GPR99/OXGR1-Gq complex.\nIn our previous revision, we showed that mutations of the projected LTE binding site had little effect\n4\non oxoglutarate signaling (previous main Fig. 3b). In this revision, we conducted alanine substitution\nof key residues in the oxoglutarate binding pocket (the TM binding pocket) of GPR99, and\ndemonstrated that these mutations produced minimal effect on LTE activation of the receptor (see\nthe notion that LTE and oxoglutarate occupy different binding pockets.\nfunctional assays. Transfected cells (HeLa) expressing either WT or mutant receptors were\nstimulated with oxoglutarate or LTE , the induced responses were measured to reflect G protein\n4\nactivation (cAMP reduction for Gαi, IP1 accumulation for Gαq). a, 6 mutants with alanine\nsubstitutions of key residues for oxoglutarate binding were tested against WT in IP1 accumulation.\nFour of the 6 mutants abrogated GPR99/OXGR1 signaling for Gαq activation (IP1 accumulation), and\nthe other 2 mutants reduced the response. b, the same mutants had no or little effect on LTE -\n4\ninduced IP1 accumulation. c and d, 3 alanine-substituted mutants of residues suspected to interact\nwith LTE at the projected site were tested against WT in IP1 accumulation assay. All 3 mutants\n4\nabrogated the LTE -induced IP1 accumulation (c) while having little or much smaller effect on\n4\noxoglutarate-induced response (d). In e, LTE , but not oxoglutarate, stimulated cells expressing the\nbacterial toxin that blocks Gαi activation, abrogated LTE -induced cAMP reduction, supporting\n4\ncoupling of GPR99/OXGR1 with Gi proteins in addition to Gq. In g, PTX had no effect on LTE -induced\nas mean ± SEM from three independent experiments.\nWhile the mutational studies support the use of separate binding sites by the two ligands,\ncomputational approaches were taken to further test whether LTE binding uses the alternative site.\n4\n(Table in Fig. R6a). In addition, 3 independent 1-µs MD simulations were carried out and a stable\nbinding pose at this location was captured (Fig. R6b-c below).\nFig. R6 (updated Supplementary Fig. S11). Molecular docking and MD simulations of LTE binding\n4\nto GPR99. a, molecular docking-computed binding energies of LTE docked to the orthosteric binding\n4\nsite and the alternative binding site above ICL2 of GPR99/OXGR1, respectively. Three independent\nbinding poses were generated for binding energy computation. b, stable poses of LTE in the\n4\nalternative binding site from 3 independent 1-µs MD simulations (colored in magenta, salmon pink\nand yellow), superimposed with LTE (cyan) in the LTE -GPR99-Gq structure. c, root mean square\n4 4\ndistance (RMSD) of LTE from three independent 1-µs MD simulations.\n4 2\nmanuscript2. CysLT R is quite different in primary sequence from GPR99/OXGR1 (CysLT R), with only\n2 3\n36% identical amino acids and representing two different subfamilies of Class A GPCRs (3-5, also the\nsequence alignment in Fig. R2). We compared the solved structure of LTD -bound CysLT R in active\n4 2\nconformation with our structure (Fig. R7, also in the updated Supplementary Fig. S7), paying special\nattention to the ligand binding pockets. LTD binds to the canonical TM pocket of CysLT R, with the\n4 2\npolar tail of LTD inserted into the bottom of the pocket and nonpolar interactions supporting the\n4\nbinding of the bulky head of LTD . Comparison of the sliced receptor TM binding pockets at the same\n4\nplane identified a much smaller binding pocket with different shape in GPR99/OXGR1. Together,\nthese independent lines of evidence suggest that it is unlikely that LTE binds to the same orthosteric\n4\npocket surrounded by the TM domains.\nFig. R7 (updated Supplementary Fig. S7). Comparison of the transmembrane (TM) binding pockets\nbetween GPR99/OXGR1 and CysLT R. The LTD4-CysLT R (PDB ID: 9IXX) is used as a reference model.\n2 2\nPanels a, b, d, e are the receptor structures sliced at the TM binding pocket in the same plane. In\npanels d-e, the LTD molecule in the 9IXX model is retained and shown in the aligned receptor\n4\nstructure of GPR99/OXGR1. Panels a and d show the electrostatic surface of the receptors, and\npanels b and e show the hydrophobicity surface of the receptors. In panels c and f, the volumes of\nTM binding pocket are measured using CASTpFold. The computed binding pockets are shown as\nsurface.\nIn summary, the added experimental data support the notion that LTE does not fit into the\n4\northosteric binding pocket for oxoglutarate. It is highly likely that LTE occupies an alternative site on\n4\naffirmative conclusion for the exact location of LTE binding. We have made marked changes to the\n4\ntext to indicate this limitation while presenting additional evidence for the presence of the\nalternative binding site in GPR99/OXGR1.\nReviewer #2 (Remarks to the Author):\nThe authors have adequately addressed all my comments and suggestions.\nHowever, a few minor issues require further attention:\n1. The number of decimal places for EC₅₀ and Eₘₐₓ values should be standardized throughout the\nmanuscript.\n2. Figure 3C indicates that Eₘₐₓ for oxoglutarate is also impaired in the C141A and C149A mutants.\nHowever, these residues reside outside the oxoglutarate binding pocket, and the mutation did not\nsignificantly alter surface expression levels. Please clarify the mechanistic basis for these observed\neffects.\nResponse:\nWe thank the reviewer for the kind suggestions. We have now updated the manuscript to correct\nthese minor issues.\n1. The decimal places for all values have been standardized throughout the manuscript.\n2. As these two residues are particularly close to the ICL2 region, their substitutions may alter the\noverall stability of ICL2 region where the receptor extensively interacts with G proteins. We have\nadded a short discussion on the decreased Emax for oxoglutarate in the C141A and C149A\nmutants (Lines 282-288, also below).\n… We have also noticed that some mutants (C141ICL2A, C1494.41A) exhibited a reduced E\nmax\nalbeit having comparable cell surface expression with the WT GPR99/OXGR1. As C141ICL2 and\nC1494.41 are proximal to the ICL2 region where the receptor extensively interacts with G\nproteins, we suspect that mutants of these sites may alter the overall stability of ICL2 and\nconsequently the recruitment and activation of G proteins, and affect the efficiency of Gq\nresponse. …\nReviewer #3 (Remarks to the Author):\nThe paper by Aijun Liu, Yezhou Liu et al. features structures of the oxoglutarate receptor (OXGR1),\nalso known as cysteinyl leukotriene receptor 3 (CysLT3R), in the active state, solved under two\ndifferent experimental conditions. These are supported by functional data, including extensive\nmutagenesis. Publishing structures of this receptor represents a valuable advance in understanding\nthe function of class A GPCRs. The paper is clearly written, and the data are well presented,\nparticularly following revisions based on reviewer comments.\nResponse:\nWe thank the reviewer for the encouraging comments.\nIt is intriguing that the proposed pose of the LTE4 molecule is located on the outer surface of the\nreceptor, while oxoglutarate is suggested to bind in the orthosteric binding pocket, which is known\nto bind membrane lipids.\nPrevious studies on cysteinyl leukotriene receptors (e.g., Luginina et al., 2019) have shown that the\nshape and volume of the ligand-binding pocket can change depending on the bound ligand.\nTherefore, it is plausible that the pocket could expand to accommodate a larger molecule such as\nLTE4.\nIf a mass spectrometry experiment detecting LTE4 in the OXGR1-LTE4 complex could be conducted,\nit would provide strong evidence for the presence of LTE4 in the complex.\nAdditionally, sequence comparison of key orthosteric ligand-binding residues across all three\nCysLTRs shows that many are conserved (e.g., K1.31, N5.39 in both CysLT2R and OXGR1; Y2.64 in all\nthree), raising the question of whether LTE4 could still bind to OXGR1 in the orthosteric pocket\nunder different experimental conditions.\nResponse:\nWe thank the reviewer for the constructive comments. Published reports indicate that the binding\npockets in GPCRs are quite limited in space due to the 7-transmembrane structure, thus conferring\nligand specificity. The presence of more than 800 GPCR-encoding genes testifies the high specificity\nof GPCRs. The CysLT receptors, however, may be exceptional in that these receptors (CysLT R and\n2\ncysteinyl leukotrienes. This expandable space, however, is not found in our solved structure of\nGPR99/OXGR1.\nWe compared sequences and structure of recently reported CysLT receptors, as well as the local\nelectrostatic properties and hydrophobicity at the orthosteric binding pocket. While several key\nresidues are conserved, our analysis reveals that the orthosteric pocket of GPR99/OXGR1 is\nmarkedly smaller and more polar than that of CysLT R (PDB ID: 9IXX)2 (Fig. R7 above), creating a\n2\nchemically unfavorable environment for the large, amphipathic CysLT molecules. Moreover, a\nhydrophobic orifice is found between TM4 and TM5 in CysLT R that can accommodate the nonpolar\n2\nhead of LTD . In GPR99/OXGR1, however, the space is occupied by residues with larger side chains\n4\n(Fig. R7, panel h).\nWe acknowledge that mass spectrometry can be useful in confirming the presence of LTE in the\n4\nreceptor complex. The receptor-G protein complex formation, however, is dependent on agonist\nbinding. Among the few ligands we tried, only oxoglutarate and LTE (but not the antagonist\n4\nmontelukast for example) induced formation of the ligand-receptor-Gq complex. We also\ndemonstrated, in the R1 revision, that GPR99/OXGR1 is not a self-activating receptor to complex\nwith Gq in the absence of an agonist. Given the limited time for revision, we chose to focus on\nexperiments that confirm whether LTE utilizes the orthosteric sites rather than experiments\n4\nshowing whether LTE is a part of the receptor complex.\nnot affect oxoglutarate-induced signalling. However, there are no data showing how mutations in\nthe orthosteric binding pocket impact -induced signalling (K1.31A, Y2.64A, etc.). Such data would\nhelp assess whether LTE4 binding in the orthosteric pocket is possible in OXGR1.\nResponse:\nWe thank the reviewer for the valuable suggestion. We have already shown functional data of IP1\naccumulation in cells expressing the alanine substituted mutants based on the projected LTE\n4\nbinding site. We now have added new results showing whether mutants based on oxoglutarate\nbinding site can affect LTE signaling. As shown in Fig. R8a, neither of the 6 mutants had significant\n4\neffect on LTE signaling. Additionally, we introduced new mutations to the TM pocket of\n2\nmutations are readily expressed on the cell surface and signal similarly to the WT receptor when\nstimulated by LTE (Fig. R8b).\n4 4\naccumulation in cells expressing WT and mutant GPR99/OXGR1. The alanine-substituted mutants\nwere based on oxoglutarate binding sites. b, LTE -induced IP1 accumulation in cells expressing WT\n4\nand mutant GPR99/OXGR1, where the mutants were residues in the TM binding pocket conserved\nbetween GPR99/OXGR1 and CysLT R. IP1 levels are calculated as percentage of response of the\n2\ncontrol (without ligand stimulation). Data are shown as mean ± SEM from three independent\nexperiments.\nI appreciate that the paper was written and submitted before the active CysLT2R structure with\nleukotrienes was published (https://doi.org/10.1073/pnas.2417148122), but it would be valuable to\ninclude at least a paragraph at the end comparing this structure with the active OXGR1 structures.\nResponse:\nWe thank the reviewer for the suggestion. We have now included a discussion comparing the\nrecently published CysLT R structure with the active GPR99/OXGR1 structures reported in this work\n2\n(updated Supplementary Fig. S7, Lines 434-456, also below).\n… A recent work of the LTD -bound CysLT R structure, published while this study was under\n4 2\nrevision, provides a solid proof of the binding mode of LTD to its cognate CysLT R receptor2.\n4 2\nLTD binds within the CysLT R transmembrane pocket, with its polar head reaching to the\n4 2\npolar top of the TM pocket and nonpolar interactions spanning TM helices TM4 and TM5\nstabilizing its bulky tail (Supplementary Fig. S7a-c). The long bulky tail of LTD can therefore\n4\nextend through an orifice opening formed between TM4 and TM5 of CysLT R, similar to the\n2\npreviously proposed binding mode for CysLT R and CysLT R 6. In contrast, the GPR99/OXGR1\n1 2\nTM pocket is much smaller, more polar and more hydrophilic, and has no cleft space\nbetween TM4 and TM5 for the accommodation of the long bulky tail of LTE , making it\n4\npoorly suited for LTE binding (Supplementary Fig. S7d-f). While the superimposed active-\n4\nstate structures of GPR99/OXGR1 and CysLT R are generally similar, the TM3 of CysLT R\n2 2\nslightly shifts outwards (Supplementary Fig. S7g), and their binding pockets are very\ndifferent. By examining the geometry of residues surrounding the TM binding pocket of\nCysLT R, an opening is observed around the hydrophobic tail of LTD . For the superimposed\n2 4\nGPR99/OXGR1 structure at this site, larger amino acid side chains are found to block such an\nopening (Supplementary Fig. S7h). Despite the ability of being activated by LTE , the\n4\nsequence of GPR99/OXGR1 is quite different from CysLT R and CysLT R receptors, with less\n1 2\nthan 30% sequence identity (Supplementary Fig. S7i)3-5. Besides, as CysLT R and CysLT R can\n1 2\nboth selectively recognize LTC and LTD with high affinity (but only low affinity for LTE ), it is\n4 4 4\nplausible that the binding mode of LTE is distinct from that of LTC and LTD to the\n4 4 4\nrespective cysteinyl leukotriene receptors 7,8. …\nMinor comments:\nLines 82-83: all the listed drugs are known as selective CysLT1R antagonists and are not known to\neffectively block CysLT2R (e.g. https://doi.org/10.1074/jbc.M003490200). The statement should be\nsupported with references to say otherwise.\nResponse:\nWe thank the reviewer for pointing this out. We have removed this description in the updated\nmanuscript.\nTo sum up, I recommend the paper for publishing after all the concerns about LTE4 binding in the\northosteric pocket are addressed and the corresponding sections of the paper corrected.\nResponse:\nWe thank the reviewer for the encouraging comments.\nLiterature cited:\ninhibition by antiasthmatic drugs. Sci Adv 5, eaax2518 (2019).\nhttps://doi.org/10.1126/sciadv.aax2518\nby LTD4. Proc Natl Acad Sci U S A 122, e2417148122 (2025).\nhttps://doi.org/10.1073/pnas.2417148122\nreceptors. Nature 429, 188-193 (2004). https://doi.org/10.1038/nature02488\nLXXXVIII. G protein-coupled receptor list: recommendations for new pairings with\ncognate ligands. Pharmacol Rev 65, 967-986 (2013).\nhttps://doi.org/10.1124/pr.112.007179\npotential third cysteinyl leukotriene receptor with a preference for leukotriene E4\nligand. J Biol Chem 288, 10967-10972 (2013).\nhttps://doi.org/10.1074/jbc.C113.453704\nBiol Chem 299, 105247 (2023). https://doi.org/10.1016/j.jbc.2023.105247\nreceptor. Nature 399, 789-793 (1999). https://doi.org/10.1038/21658\nBiol Chem 275, 30531-30536 (2000). https://doi.org/10.1074/jbc.M003490200"}, {"round": 3, "reviewer_comments": {"reviewer_2": "The authors have addressed all my comments and suggestions.", "reviewer_3": "The authors have addressed most of my concerns and added the corresponding sections to the manuscript. The expanded\nfunctional data supports the binding position of the ligands suggested by the authors. However, given that the structures\nleave some room for interpretation, I advise caution when interpreting the nature of the densities. Otherwise, I recommend\nthe paper for publication.\nOne minor suggestion would be to remove the side chains from Figure S7-h and use different colours to differentiate\nconserved and non-conserved amino acids.\nThank you!", "reviewer_4": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature\nCommunications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career\nResearchers who co-review manuscripts.", "reviewer_5": "The article “Molecular insights into ligand recognition and signaling of GPR99/OXGR1” by Liu et al. presents novel cryoEM\nstructures of GPR99/OXGR1 in complex with native ligands, oxoglutarate and LTE4 and trimeric Gq protein. Due to poor EM\ndensity of the LTE4 binding region, the precise location of this ligand was not possible. However, it was suggested that the\nligand binds to the alternative binding site, outside of the receptor, in the membrane above ICL2. The experimental\nmutational analysis and the computational studies were added to confirm the hypothesis. The in silico part covered\nmolecular docking of LTE4 to GPR99/OXGR1 receptor and lengthy molecular dynamics (MD) simulations of the obtained\ncomplex. The MD simulations demonstrated that the binding of the LTE4 ligand to its putative binding site is stable across 3\nreplicates of 1 us each, supporting the presented hypothesis. However, the methodology and results regarding the in silico\npart are insufficiently described in the manuscript and require improvements. In general, the presented work is novel and\nvaluable, but the following comments should be addressed before accepting the manuscript for publication.\nIn the molecular docking & MD simulations part, please address the following questions and suggestions:\nMolecular docking:\n1. How was the binding site defined in the docking procedure in case of TM site and the alternative binding site?\n2. Since LTE4 in an endogenous ligand, the alternative binding site should be named the secondary orthosteric binding site.\nIs it possible that both orthosteric ligands bind at the same time to the primary and secondary binding site?\n3. In the docking procedure, how many poses were generated? This is important to ensure robust sampling. Also, in the line\n749-750: “the top-ranked poses were selected for binding free energy calculations” - how many of them? Did the authors\napply any clustering in the selection of the best poses?\nMD simulations:\n4. Did the system include only the receptor and ligand or also G protein? This should be explicitly written.\n5. More information on the MD simulation setup should be provided, such as: integration algorithms, time step, pressure and\ntemperature control, methods for calculation of nonbonded interactions.\n6. Fig. S11b. The structures of LTE4 from 3 replicates. Are these structures from some selected frames of the simulations?\nHow were they selected? Please specify in the figure description.\n7. Fig. S11c. RMSD of LTE4 from three independent 1-μs MD simulations. The chart shows just one data series, is it the\nmean RMSD from the 3 replicates? It would be better to show explicitly 3 data series, for each replicate separately in one\nchart using different colors to see if RMSD values are consistent across the replicates.\n8. How did the authors calculate the RMSD? It should be preformed by aligning by the receptor structure first, and then\ncalculate the RMSD for the ligand. Please clarify the procedure in the Methods section.\n9. Did the crucial interactions (C141, R148, C149) remain stable throughout the simulations? Some additional chart showing\nthe stability of these crucial interactions could be provided.\nGeneral comments about the manuscript:\n10. In the abstract, line 46: “LTE4 also induced inhibition of cAMP accumulation that was sensitive to pertussis toxin”. It\nshould be explicitly said that this result indicates signaling through Gi.\n11. Line 81-85: “An early study on LTE4-induced bronchoconstriction suggested that the high stability of LTE4, compared to\nLTC4 and LTD4 that are cysteinyl leukotrienes binding to different receptors, contributes to asthma development due to the\nchronic effect of receptor/ligand-related hyperresponsiveness”. The sentence is very complex; it should be divided into 2\nsentences for clarity.\n12. Fig. 2. The structural alignment is not well visible. I suggest to show the receptor residues in thinner sticks than the\nligands, for clarity.\n13. Lines 215-217 - “Other notable features include the absence of positively charged arginine network, which is important\nfor binding of the bicarboxylic ligands such as oxoglutarate and succinate 3,24,27, from the CysLT receptors“. This sentence\nis not clear. Was it supposed to be “in the CysLT receptors”? Meaning that CysLT receptors lack the arginine network? The\nsame in line 219: “missing from” - missing in\n14. Line 317 - “These results indicate that GPR99/OXGR1 is not an auto-activating GPCR that can complex with G proteins\nwithout agonist binding.“. Such basal activity, which does not require agonist binding, is commonly referred to in the\nliterature as “constitutive activity”. I suggest using this term.\n15. Line 657: CHARMm, correct the typo to CHARMM"}, "author_response": "NCOMMS-24-76881B\nAUTHOR RESPONSE TO REVIEWER COMMENTS\nReviewer #2 (Remarks to the Author):\nThe authors have addressed all my comments and suggestions.\nResponse: We thank the reviewer for the positive comment.\nReviewer #3 (Remarks to the Author):\nThe authors have addressed most of my concerns and added the corresponding sections to\nthe manuscript. The expanded functional data supports the binding position of the ligands\nsuggested by the authors. However, given that the structures leave some room for\ninterpretation, I advise caution when interpreting the nature of the densities. Otherwise, I\nrecommend the paper for publication.\nOne minor suggestion would be to remove the side chains from Figure S7-h and use\ndifferent colours to differentiate conserved and non-conserved amino acids.\nThank you!\nResponse: We thank the reviewer for the suggestion. We have revised the Fig. S7h as\nsuggested (see revised manuscript; also shown below as Fig. R1).\nFig. R1. (Fig. S7h). The transmembrane (TM) binding pocket of CysLT R (orange) with\n2\nbound LTD (yellow) and the superimposed structure of GPR99/OXGR1 (cyan). The\n4\nbinding pocket residues are shown as thin sticks. Non-conserved amino acids are\nhighlighted in light red.\nReviewer #4 (Remarks to the Author):\nI co-reviewed this manuscript with one of the reviewers who provided the listed\nreports. This is part of the Nature Communications initiative to facilitate training in\npeer review and to provide appropriate recognition for Early Career Researchers\nwho co-review manuscripts.\nResponse: We thank the reviewer for the comments and suggestions. Our replies are\nshown below in Response to Reviewer #5.\nReviewer #5 (Remarks to the Author):\nThe article “Molecular insights into ligand recognition and signaling of GPR99/OXGR1” by\nLiu et al. presents novel cryoEM structures of GPR99/OXGR1 in complex with native\nligands, oxoglutarate and LTE4 and trimeric Gq protein. Due to poor EM density of the LTE4\nbinding region, the precise location of this ligand was not possible. However, it was\nsuggested that the ligand binds to the alternative binding site, outside of the receptor, in the\nmembrane above ICL2. The experimental mutational analysis and the computational studies\nwere added to confirm the hypothesis. The in silico part covered molecular docking of LTE4\nto GPR99/OXGR1 receptor and lengthy molecular dynamics (MD) simulations of the\nobtained complex. The MD simulations demonstrated that the binding of the LTE4 ligand to\nits putative binding site is stable across 3 replicates of 1 us each, supporting the presented\nhypothesis. However, the methodology and results regarding the in silico part are\ninsufficiently described in the manuscript and require improvements. In general, the\npresented work is novel and valuable, but the following comments should be addressed\nbefore accepting the manuscript for publication.\nResponse: We thank the reviewer for the comments and suggestions. We now have\nupdated the manuscript in response to these valuable comments.\nIn the molecular docking & MD simulations part, please address the following questions and\nsuggestions:\nMolecular docking:\n1. How was the binding site defined in the docking procedure in case of TM site and the\nalternative binding site?\nResponse: A docking region was defined using a spherical selection with a radius of 20 Å\ncentered on the geometric center of the bound ligand in the cryo-EM structure, LTE for the\n4\ntransmembrane (TM) allosteric site and oxoglutarate for the orthosteric site, respectively.\nThe relatively large radius was intentionally chosen due to the flexibility and length of LTE\n4\nand to allow unbiased sampling of the surrounding space, helping to validate Cryo-EM\nobservations. For the allosteric site, the information of the sphere is: 8.848, -10.3647,\n10.9218, 20; for the orthosteric site, the sphere information is: 130.428, 127.303, 174.853,\n20. Additionally, residues within 5 Å of the bound ligand in the cryo-EM structure were\nselected as flexible for ChiFlex/ChiRotor processing.\n2. Since LTE4 in an endogenous ligand, the alternative binding site should be named the\nsecondary orthosteric binding site. Is it possible that both orthosteric ligands bind at the\nsame time to the primary and secondary binding site?\nResponse: We thank the reviewer for the suggestion and have updated the naming\naccordingly. In terms of whether the two ligands can bind at the same time, this is an\nimportant question but requires substantial amount of work for a full answer. Our\nmutagenesis experiments indicate that alanine substitutions of key residues in one ligand\nbinding pocket did not affect binding of the other ligand. Now we have incorporated the\nNanoBiT Gq protein dissociation assay to determine the effect of sequential addition of the\ntwo ligands. We found cumulative (additive) effect, whether LTE4 is added before or after\noxoglutarate (Fig. R2). This result supports simultaneous occupancy of the two agonist\nbinding sites. In future studies, we plan to further explore simultaneous occupancy of the two\nagonists in a physiologically relevant setting.\nFig. R2. Sequential addition of LTE and oxoglutarate enhances GPR99/OXGR1\n4\nactivation. LTE and oxoglutarate (100 μM) are added sequentially to activate\n5-min intervals after ligand additions. Data shown are means ± SEM from 3 experiments.\n3. In the docking procedure, how many poses were generated? This is important to ensure\nrobust sampling. Also, in the line 749-750: “the top-ranked poses were selected for binding\nfree energy calculations” - how many of them? Did the authors apply any clustering in the\nselection of the best poses?\nResponse: For the orthosteric site case, 37 poses were generated, and all were subjected\nto the binding free energy calculations. In contrast, significantly more poses were generated\nfor the allosteric site—1,018 in total. From these, 217 poses with a -CDOCKER_ENERGY\ngreater than 30 (a commonly used threshold in drug screening pipelines) were selected for\nfurther analysis. Regarding the selection of best poses, no additional clustering was\nperformed after scoring. However, clustering was already incorporated into the docking\nalgorithm: in stage (ii), after ligand conformation generation and rigid placement into each\nprotein structure via LibDock. The poses were energy-minimized and clustered using a\nradius of 0.5 Å as the cut-off value.\nMD simulations:\n4. Did the system include only the receptor and ligand or also G protein? This should be\nexplicitly written.\nResponse: We thank the reviewer for the important comment. The system for MD\nsimulation includes only the receptor and ligand, and without the G protein. This has been\nclarified in the revised Methods section (Lines 695-698).\n5. More information on the MD simulation setup should be provided, such as: integration\nalgorithms, time step, pressure and temperature control, methods for calculation of\nnonbonded interactions.\nResponse: We thank the reviewer for the comment. The detailed information on the MD\nsimulation setup is now included in the Methods section (Lines 703-710).\n6. Fig. S11b. The structures of LTE4 from 3 replicates. Are these structures from some\nselected frames of the simulations? How were they selected? Please specify in the figure\ndescription.\nResponse: The structures shown in Fig. S11b are the final snapshots from each of the three\n1-µs simulations of the receptor-ligand complex, illustrating the stable binding pose of LTE\n4\nto GPR99/OXGR1. The figure legend has been revised accordingly for clarity.\n7. Fig. S11c. RMSD of LTE4 from three independent 1-μs MD simulations. The chart shows\njust one data series, is it the mean RMSD from the 3 replicates? It would be better to show\nexplicitly 3 data series, for each replicate separately in one chart using different colors to see\nif RMSD values are consistent across the replicates.\nResponse: We thank the reviewer for the comment. The original Fig. S11c presents the\nmean  SD RMSD from the 3 replicates. The figure is now updated with 3 explicit data series\nfor clarity (also Fig. R3 below).\nFig. R3 (Fig. S11c). Root mean square distance (RMSD) of LTE from three\n4\nindependent 1-µs MD simulations.\n8. How did the authors calculate the RMSD? It should be preformed by aligning by the\nreceptor structure first, and then calculate the RMSD for the ligand. Please clarify the\nprocedure in the Methods section.\nResponse: Trajectory frames were first aligned to the protein backbone C atoms of the\ninitial structure, and the ligand’s RMSD was then computed relative to its starting pose. The\nprocedure is clarified in the updated Methods section (Lines 711-713).\n9. Did the crucial interactions (C141, R148, C149) remain stable throughout the simulations?\nSome additional chart showing the stability of these crucial interactions could be provided.\nResponse: The pairwise distances of crucial interactions between LTE and GPR99/OXGR1\n4\n(C141, R148 and C149) remain generally consistent throughout the simulation. Pairwise\ndistances of these interactions are included in the updated Fig. S11d (Fig. R4).\nFig. R4 (Fig. S11d). Pairwise distances between LTE and the crucial residues of\nresidues (C141, R148 and C149) of GPR99/OXGR1 were calculated and plotted from the\nMD simulation. Data are shown as mean  SD from 3 replicates.\nGeneral comments about the manuscript:\n10. In the abstract, line 46: “LTE4 also induced inhibition of cAMP accumulation that was\nsensitive to pertussis toxin”. It should be explicitly said that this result indicates signaling\nthrough Gi.\nResponse: We thank the reviewer for the suggestion, and have now revised the text of the\nAbstract section (Line 46).\n11. Line 81-85: “An early study on LTE4-induced bronchoconstriction suggested that the high\nstability of LTE4, compared to LTC4 and LTD4 that are cysteinyl leukotrienes binding to\ndifferent receptors, contributes to asthma development due to the chronic effect of\nreceptor/ligand-related hyperresponsiveness”. The sentence is very complex; it should be\ndivided into 2 sentences for clarity.\nResponse: We thank the reviewer for the suggestion, and have divided the sentence into\ntwo: “Compared to LTC and LTD that are cysteinyl leukotrienes binding to different G\n4 4\nprotein-coupled receptors, LTE is more stable 12. This feature of LTE contributes to asthma\n4 4\ndevelopment due to the chronic effect of receptor/ligand-related hyperresponsiveness 16.”\n(Lines 80-84).\n12. Fig. 2. The structural alignment is not well visible. I suggest to show the receptor\nresidues in thinner sticks than the ligands, for clarity.\nResponse: We have now updated the Fig. 2 accordingly for clarity (below Fig. R5).\nFig. R5 (Fig. 2). Comparison of the dicarboxylate ligand binding pockets of\nGPR99/OXGR1-oxoglutarate and GPR91/SUCNR1-succinate. Side view (a) and top view\n(b) of the transmembrane binding pockets of GPR99/OXGR1 (cyan) bound with oxoglutarate\n(yellow) and GPR91/SUCNR1 (pink) bound with succinate (green). Ligands and key\nresidues for ligand binding are shown as sticks.\n13. Lines 215-217 - “Other notable features include the absence of positively charged\narginine network, which is important for binding of the bicarboxylic ligands such as\noxoglutarate and succinate 3,24,27, from the CysLT receptors“. This sentence is not clear.\nWas it supposed to be “in the CysLT receptors”? Meaning that CysLT receptors lack the\narginine network? The same in line 219: “missing from” - missing in\nResponse: Thanks for pointing out – these should be “missing in”. This is now updated in\nrevised manuscript text (Lines 214, 217).\n14. Line 317 - “These results indicate that GPR99/OXGR1 is not an auto-activating GPCR\nthat can complex with G proteins without agonist binding.“. Such basal activity, which does\nnot require agonist binding, is commonly referred to in the literature as “constitutive activity”.\nI suggest using this term.\nResponse: We now revised the text and use “constitutive activity” in the revised manuscript\n(Line 314).\n15. Line 657: CHARMm, correct the typo to CHARMM\nResponse: We thank the reviewer for pointing out this typo."}, {"round": 4, "reviewer_comments": {"reviewer_4": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature\nCommunications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career\nResearchers who co-review manuscripts.", "reviewer_5": "Fig. S11d requires improvement. The distance between the crucial residues and center-of-mass of the LTE4 ligand is\ninsufficient to reliably evaluate the stability of the interactions. Please prepare a chart with distances between atoms\npotentially involved in the interactions instead of center-of-mass.\nIn lines 671 and 686 there is a typo: CHARMm, it should be CHARMM.\nOnce the comments above are addressed, the article may be accepted for publication."}, "author_response": "NCOMMS-24-76881C\nREVIEWERS' COMMENTS\nReviewer #4 (Remarks to the Author):\nI co-reviewed this manuscript with one of the reviewers who provided the listed\nreports. This is part of the Nature Communications initiative to facilitate training in\npeer review and to provide appropriate recognition for Early Career Researchers\nwho co-review manuscripts.\nReviewer #5 (Remarks to the Author):\nFig. S11d requires improvement. The distance between the crucial residues and\ncenter-of-mass of the LTE4 ligand is insufficient to reliably evaluate the stability of\nthe interactions. Please prepare a chart with distances between atoms\npotentially involved in the interactions instead of center-of-mass.\nAuthor Response: We thank the reviewer for this important comment. We have now\nupdated Supplemental Fig 11d accordingly. The distances between these atoms\ninvolved in hydrogen bond interaction between the receptor and LTE4 is stably close\nthroughout the three independent 1-s MD simulations.\nFig R1. Pairwise distances between hydrogen bond-engaging atoms in LTE and the crucial\n4\nresidues (C141, R148 and C149) of GPR99/OXGR1, namely C141-HG1~LTE -O5, R148-\n4 4\nof 1-s MD simulation runs.\nIn lines 671 and 686 there is a typo: CHARMm, it should be CHARMM.\nAuthor Response: Thanks! We have now corrected the typo.\nOnce the comments above are addressed, the article may be accepted for\npublication.\nAuthor Response: We thank the reviewer for the comments."}]

Decision letter