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Structural basis for ligand recognition and signaling of the lysophosphatidylserine receptors GPR34 and GPR174 [1]

['Guibing Liu', 'Division Of Life Sciences', 'Medicine', 'University Of Science', 'Technology Of China', 'Hefei', 'People S Republic Of China', 'Xiu Li', 'Yujing Wang', 'Xuan Zhang']

Date: 2023-12

Notably, the lipid-binding pocket of both GPR34 and GPR174 is covered by a lid formed by ECL2, which is stabilized by the conserved disulfide bond with TM3 ( Fig 1B and 1D ). Additionally, ECL2 also serves as part of the lipid-binding pocket, as seen in other lipid receptors.

( A ) Cryo-EM density map and model of GPR34-Gi complex. Density of LysoPS is shown as mesh in the middle and colored plum. ( B ) The map and model of ECL2 of GPR34-Gi complex (extracellular view). ( C ) Cryo-EM density map and model of GPR174-Gs complex. Density of LysoPS is shown as mesh in the middle and colored salmon. ( D ) The map and model of ECL2 of GPR174-Gs complex (extracellular view). GPR34 is colored marine green. GPR174 is colored blue. ECL2 is colored orange. Gα i , Gα s , Gβ, and Gγ subunits are colored gold, cyan, pink, and light green, respectively. ScFv16 and Nb35 are colored grey.

We coexpressed the GPR34-Gi and GPR174-Gs signaling complexes in Sf9 insect cells and purified them in the presence of LysoPS (18:1), one the most widely used LysoPS in functional studies of LysoPS receptors. ScFv16 and Nb35 were incorporated to stabilize the Gi and Gs complex, respectively. The NanoBiT tethering strategy [ 26 ] was also utilized to facilitate the formation of the complexes and improve the quality of cryo-EM samples. As a result, we successfully acquired high-quality structures of LysoPS-bound GPR34-Gi complex and LysoPS-bound GPR174-Gs complex at resolution of 2.91 Å and 3.06 Å, respectively (Figs 1A, 1C , S2 and S3 and S1 Table ). The well-defined density allowed for precise modeling of GPR34-Gi, GPR174-Gs complexes, and the binding ligands. In both structures, we modeled LysoPS (18:1) into the density and found that it fits well except that the last 6 carbons of the oleic acid have no density.

Binding modes of LysoPS to GPR34 and GPR174

LysoPS binds to GPR34 by a specific interaction network between the ligand and the receptor’s transmembrane domain (TMD) and ECL2 (Fig 2A). The polar head group of LysoPS is accommodated in the central cavity of the TMD, where it forms hydrogen bonds and salt bridges with several key residues. Specifically, the L-serine moiety of LysoPS interacts with TM6 and TM7, with the carboxyl group forming a salt bridge with R2866.55 and hydrogen bonds with Y1353.33 and N3097.35, while the amino group forms a salt bridge with E3107.36. The phosphate group of LysoPS interacts with R208ECL2 via a salt bridge. Additionally, the ester linkage between the fatty acid and the polar head forms a hydrogen bond with N2205.40. The formation of the central pocket also involves hydrophobic interactions with several other residues, including T1323.30, Y2896.58, F205ECL2, and L3137.39. The acyl tail of LysoPS is bound in a hydrophobic subpocket formed by the sidechains of Y1393.37, L1814.52, A1824.53, M1894.60, F2195.39, L2235.43, and M2265.46. Mutagenesis studies using a Gi-dissociation assay in HEK293T cells with both 18:1 and 18:0 LysoPS (Figs 2B and S4) revealed that several residues in contact with the polar head of LysoPS are critical for LysoPS-induced activation of GPR34, including Y1353.33, F205ECL2, R2866.55, Y2896.58, E3097.35, and E3107.36, which severely impaired receptor activation when mutated to alanine (S4C and S4D Fig). L3137.39 mutation moderately affected receptor activity. In contrast, R208ECL2 mutation has little effect on receptor activation, suggesting that this site may not be crucial for LysoPS-induced activation and could potentially be substituted by surrounding positively charged residues such as K1283.26, H199ECL2, H206ECL2, and K210ECL2 in ligand binding. Mutations of most residues in the hydrophobic subpocket have no effect on receptor activation, except for Y1393.37A, A1824.53V, and M1894.60A (S4E Fig). These findings indicate that the recognition of LysoPS by GPR34 is primarily mediated by specific interactions with the polar head group, while the hydrophobic interactions with the acyl tail provide additional stability to the binding.

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TIFF original image Download: Fig 2. Recognition of LysoPS by GPR34 and GPR174. (A) Interactions between LysoPS and GPR34. Polar interactions are depicted as black dashed lines. (B) Normalized ΔBRET of GPR34 mutants in response to 10 μM LysoPS relative to wild-type GPR34 in Gi-dissociation assays. (C) Interactions between LysoPS and GPR174. Polar interactions are depicted as black dashed lines. The π–π interaction is depicted as a red dashed line. (D) Net cAMP accumulation of CHO cells expressing GPR174 mutants in response to 10 μM LysoPS. WT-basal represents net cAMP accumulation of cells expressing wild-type GPR174 in the absence of exogenous LysoPS. Data represent mean ± SEM from 3 independent experiments. Mutants with expression less than half that of the wild-type receptor are labeled with red stars. The data used to generate graphs in Fig 2B and 2D are available in S1 Data. https://doi.org/10.1371/journal.pbio.3002387.g002

Previous evolutionary studies found that GPR34 receptors have existed for more than 450 million years and identified residues conserved during GPR34 evolution [27,28]. Of the residues involved in LysoPS recognition, Y1353.33, Y1393.37, R208ECL2, N2205.40, R2866.55, Y2896.58, N3097.35, E3107.36, and L3137.39 of GPR34 are highly conserved during vertebrate evolution. Notably, all 6 residues associated with LysoPS by polar interactions are conserved. Most mutations of these residues impaired GPR34 activation except R208ECL2 and N2205.40 (Fig 2B).

GPR174 binds the polar head of LysoPS in a central pocket through extensive polar interactions (Fig 2C). Specifically, the carboxyl group of the serine moiety forms salt bridges with R752.60 and K983.32, and a hydrogen bond with Y993.33, while the amino group forms a hydrogen bond with Y792.64. Additionally, the phosphate group forms ionic interactions with R1564.64 and K2576.62, and the sn-2 hydroxyl group contacts Y2466.51 by a hydrogen bond. F169 and V170 of ECL2 participate in the formation of the central pocket by hydrophobic interactions, with F169 further stabilizing the binding of the polar head through a cation–π interaction with R752.60. The acyl tail of LysoPS binds in a narrow cleft formed by hydrophobic residues from transmembrane helices 3 to 6, including Y1033.37, F1524.60, M1855.38, and F2506.55, with the double bond of the oleoyl group in contact with Y1033.37 through a π–π interaction. G1895.42 and G1935.46 on TM5 provide space for the binding of the acyl group. cAMP accumulation assays were performed in CHO cells to assess the contributions of the residues involved in LysoPS binding (Figs 2D and S5). Mutations to alanine at the polar interaction sites substantially impaired or even abolished LysoPS-induced cAMP accumulation, while mutations of hydrophobic sites like F152A and Y103A have similar effects.

We noticed the recently reported structure of the LysoPS (18:0)-GPR174-Gs complex [29] and compared it with our LysoPS (18:1)-bound complex. Interestingly, the same polar head of the 2 types of LysoPS binds to the central pocket with some unexpected differences (Fig 3A). In our structure, the carboxyl group of the serine moiety does not form a hydrogen bond with the main chain carbonyl of F169ECL2, and the hydrogen bond between the carbonyl group of the ester linkage and R1564.64 is also absent. Furthermore, we observed an additional π–π interaction between the double bond on the acyl group of LysoPS (18:1) and the phenol ring of Y103, which may result in tighter binding (Fig 3B).

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TIFF original image Download: Fig 3. Comparison of LysoPS recognition by GPR34 and GPR174. (A) Interactions between GPR174 and the polar head of LysoPS (18:0) or LysoPS (18:1). (B) Interactions between GPR174 and the acyl tail of LysoPS (18:0) or LysoPS (18:1). The double bond of LysoPS (18:1) is colored red; the corresponding single bond in LysoPS (18:0) is colored orange. (C) Charged interactions between LysoPS and GPR34 in the positively charged pocket. (D) Charged interactions between LysoPS and GPR174 in the positively charged pocket. (E) Structural superposition of LysoPS bound GPR34 and GPR174 (extracellular view). (F) Comparison between acyl tails of LysoPS binding in GPR34 and GPR174. Polar or charged interactions are depicted as black dashed lines. https://doi.org/10.1371/journal.pbio.3002387.g003

The interaction patterns of LysoPS with GPR34 and GPR174 are different. In both receptors, the polar head binds in a positively charged central pocket (Fig 3C and 3D), but in contacts with different sets of residues. In GPR34, the serine carboxyl and phosphate group of LysoPS form ionic interactions with R2866.55 and R208ECL2, respectively (Fig 3C), while in GPR174, their orientations are determined by K983.32, R752.60, R1564.64, and K2576.62 (Fig 3D). In GPR34, amino group of the serine in LysoPS forms a salt bridge with E3107.38, whereas in GPR174, it forms a hydrogen bond with Y792.64. The polar head of LysoPS makes more charged interactions with GPR174, suggesting a tighter binding than with GPR34. Regarding the hydrophobic tail, its orientation is mainly determined by the arrangement of TM4 and TM5 (Fig 3E and 3F), which are differentiated by prolines on them. In GPR34, there are no prolines on TM4/5, including the conserved P5.50 among class A GPCRs. A curved path extends against TM3 and TM4, further restricted by L2235.43. In GPR174, P1534.61 and P1975.50 result in a distinct arrangement of TM4 and TM5, forming a straight path with hydrophobic residues from TM3-6. L5.43 is replaced by a glycine at position 5.42 in GPR174.

Overall, our findings suggest that while GPR34 and GPR174 share similar binding modes for LysoPS, they exhibit differences in the specific binding residues and the hydrophobic tail’s orientation.

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[1] Url: https://journals.plos.org/plosbiology/article?id=10.1371/journal.pbio.3002387

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