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Summary
Lysophosphatidylserine (LysoPS) is a naturally occurring lipid mediator concerned in varied physiological and pathological processes particularly these associated to the immune system. GPR34, GPR174, and P2Y10 have been recognized because the receptors for LysoPS, and its analogues have been developed as agonists or antagonists for these receptors. Nevertheless, the dearth of structural info hinders the drug growth with novel traits, resembling nonlipid ligands and allosteric modulators. Right here, we decided the buildings of human GPR34 and GPR174 in advanced with LysoPS and G protein by cryo-EM. Mixed with structural evaluation and practical research, we elucidated the lipid-binding modes of those receptors. By structural comparability, we recognized the structural options of GPR34 and GPR174 in energetic state. Taken collectively, our findings present insights into ligand recognition and signaling of LysoPS receptors and can facilitate the event of novel therapeutics for associated inflammatory illnesses and autoimmune illnesses.
Quotation: Liu G, Li X, Wang Y, Zhang X, Gong W (2023) Structural foundation for ligand recognition and signaling of the lysophosphatidylserine receptors GPR34 and GPR174. PLoS Biol 21(12):
e3002387.
https://doi.org/10.1371/journal.pbio.3002387
Educational Editor: Raimund Dutzler, College of Zurich, SWITZERLAND
Acquired: Might 5, 2023; Accepted: October 17, 2023; Printed: December 4, 2023
Copyright: © 2023 Liu et al. That is an open entry article distributed below the phrases of the Artistic Commons Attribution License, which allows unrestricted use, distribution, and replica in any medium, supplied the unique writer and supply are credited.
Information Availability: The atomic coordinates of LysoPS (18:1)-GPR34-Gi and LysoPS (18:1)-GPR174-Gs complexes have been deposited within the Protein Information Financial institution (PDB) below accession codes 8WRB and 8IZB, respectively. The EM maps of LysoPS (18:1)-GPR34-Gi and LysoPS (18:1)-GPR174-Gs complexes have been deposited within the Electron Microscopy Information Financial institution (EMBD) below accession codes EMD-37771 and EMD-35838, respectively.
Funding: W.G. is supported by the grant 2019YFA0904100 from the Ministry of Science and Expertise of China (https://www.most.gov.cn/), and the grant T2221005 from the Pure Science Basis of China (https://www.nsfc.gov.cn/). The funders had no position in research design, knowledge assortment and evaluation, choice to publish, or preparation of the manuscript.
Competing pursuits: The authors have declared that no competing pursuits exist.
Abbreviations:
CCKAR,
cholecystokinin A receptor; CHS,
cholesteryl hemisuccinate; EDG,
endothelial differentiation gene; FSC,
Fourier shell correlation; LMNG,
lauryl maltose neopentyl glycol; LPA,
lysophosphatidic acid; LPC,
lysophosphatidylcholine; LPL,
lysophospholipid; LysoPS,
lysophosphatidylserine; PAFR,
platelet activating issue receptor; S1P,
sphingosine 1-phosphate; TGFα,
remodeling progress factor-α; TMD,
transmembrane area; Treg,
regulatory T cell
Introduction
Lysophospholipids (LPLs) are single-chain lipid mediators generated by phospholipid metabolism and regulate varied physiological processes together with cell survival, proliferation, and migration and play a task in lots of illnesses of vascular, neurological, and metabolic programs [1,2]. Lysophosphatidic acid (LPA), sphingosine 1-phosphate (S1P), lysophosphatidylserine (LysoPS), and lysophosphatidylcholine (LPC) signify essentially the most extensively studied LPLs (S1A Fig). Of those, LysoPS has been proven to be necessary in cell migration [3,4], cell differentiation [5], and oxidative stress [6]. Moreover, LysoPS has been reported to induce mast cell degranulation [7,8] and promote the phagocytosis of apoptotic cells [9]. In human, 3 G protein–coupled receptors have been recognized as LysoPS receptors, specifically, GPR34, GPR174, and P2Y10. The primary LysoPS receptor GPR34 was found by the statement of LysoPS-induced inhibition of cAMP accumulation in GPR34-expressing CHO cells [8]. Subsequently, GPR174 and P2Y10 had been discovered to activate Gα12/13 in response to LysoPS, as demonstrated by the remodeling progress factor-α (TGFα) shedding assay [10].
GPR34 is a Gi-coupled LysoPS receptor that reveals excessive expression in microglia, mast cells, lymphocytes, and macrophages [11] and is related to immune and neurological problems (ref). Along with its involvement in delayed hypersensitivity [12] and dendritic cell apoptosis [13], GPR34 can be implicated in tissue restore, because it senses LysoPS launched by apoptotic neutrophils on sort 3 innate lymphoid cells [14]. Activation of GPR34 has been linked to neuropathic ache [15] and the event of salivary gland MALT lymphoma [16], indicating its potential as a therapeutic goal in these situations. GPR174 is extremely expressed in lymph nodes and thymus and has been related to autoimmune illnesses resembling Graves’ illness [17] and Addison’s illness [18]. GPR174 negatively regulates regulatory T cell (Treg) proliferation and performance by a Gαs-dependent pathway in response to LysoPS and inhibits CD4 T cell proliferation and activation by decreasing IL-2 manufacturing. A latest research additionally gives proof for the position of GPR174 in regulating gene expression and performance of B cells through Gαs [19]. As well as, GPR174 was discovered to induce B cell migration in response to CCL21, which promotes GPR174-Gαi coupling in a sex-dependent method [20]. Nevertheless, the position of Gα12/13 pathway in GPR174 signaling is poorly understood, and the coupling of Gαi to GPR174 is comparatively restricted [21]. The Gα12/13-coupled P2Y10 expressed in immune cells has not been extensively studied. Particularly, P2Y10 has been reported to mediate eosinophils degranulation [22] and facilitate chemokine-induced CD4 T cell migration [23]. Notably, P2Y10 additionally responds to ATP as a purinergic receptor [23] and may reply to S1P [24], which distinguishes it from GRP34 and GPR174.
GPR174 primarily {couples} to the stimulatory G protein Gs, whereas GPR34 solely {couples} to the inhibitory G protein Gi/o [21,25], each of which exert their results by regulation of cAMP degree in cells. Nevertheless, the idea underlying G protein selectivity is just not understood. Moreover, GPR174 shares a excessive homology (as much as 48.2%) with the putative P2Y receptor P2Y10, however a comparatively low homology (28.5%) with GPR34, which was initially categorised as a P2Y-like orphan receptor. It’s unclear whether or not GPR34 and GPR174 share the identical recognition mechanism for LysoPS, which hinders rational drug design for LysoPS receptors. Subsequently, we used cryo-EM to find out the buildings of GPR34 and GPR174 in advanced with LysoPS and their appropriated G proteins. Together with mutagenesis and practical assays, these buildings reveal the idea for ligand recognition and G protein coupling of LysoPS receptors. Our research gives invaluable insights for structure-based drug design concentrating on LysoPS receptors.
Outcomes
Buildings of LysoPS-signaling complexes
We coexpressed the GPR34-Gi and GPR174-Gs signaling complexes in Sf9 insect cells and purified them within the presence of LysoPS (18:1), one essentially the most extensively used LysoPS in practical research of LysoPS receptors. ScFv16 and Nb35 had been integrated to stabilize the Gi and Gs advanced, respectively. The NanoBiT tethering technique [26] was additionally utilized to facilitate the formation of the complexes and enhance the standard of cryo-EM samples. Consequently, we efficiently acquired high-quality buildings of LysoPS-bound GPR34-Gi advanced and LysoPS-bound GPR174-Gs advanced at decision of two.91 Å and three.06 Å, respectively (Figs 1A, 1C, S2 and S3 and S1 Desk). The well-defined density allowed for exact modeling of GPR34-Gi, GPR174-Gs complexes, and the binding ligands. In each buildings, we modeled LysoPS (18:1) into the density and located that it matches properly besides that the final 6 carbons of the oleic acid haven’t any density.
Fig 1. Total buildings of GPR34-Gi advanced and GPR174-Gs advanced.
(A) Cryo-EM density map and mannequin of GPR34-Gi advanced. Density of LysoPS is proven as mesh within the center and coloured plum. (B) The map and mannequin of ECL2 of GPR34-Gi advanced (extracellular view). (C) Cryo-EM density map and mannequin of GPR174-Gs advanced. Density of LysoPS is proven as mesh within the center and coloured salmon. (D) The map and mannequin of ECL2 of GPR174-Gs advanced (extracellular view). GPR34 is coloured marine inexperienced. GPR174 is coloured blue. ECL2 is coloured orange. Gαi, Gαs, Gβ, and Gγ subunits are coloured gold, cyan, pink, and light-weight inexperienced, respectively. ScFv16 and Nb35 are coloured gray.
Notably, the lipid-binding pocket of each GPR34 and GPR174 is roofed by a lid fashioned by ECL2, which is stabilized by the conserved disulfide bond with TM3 (Fig 1B and 1D). Moreover, ECL2 additionally serves as a part of the lipid-binding pocket, as seen in different lipid receptors.
Binding modes of LysoPS to GPR34 and GPR174
LysoPS binds to GPR34 by a particular interplay community between the ligand and the receptor’s transmembrane area (TMD) and ECL2 (Fig 2A). The polar head group of LysoPS is accommodated within the central cavity of the TMD, the place it types hydrogen bonds and salt bridges with a number of key residues. Particularly, 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, whereas the amino group types a salt bridge with E3107.36. The phosphate group of LysoPS interacts with R208ECL2 through a salt bridge. Moreover, the ester linkage between the fatty acid and the polar head types a hydrogen bond with N2205.40. The formation of the central pocket additionally entails hydrophobic interactions with a number of different residues, together with T1323.30, Y2896.58, F205ECL2, and L3137.39. The acyl tail of LysoPS is certain in a hydrophobic subpocket fashioned by the sidechains of Y1393.37, L1814.52, A1824.53, M1894.60, F2195.39, L2235.43, and M2265.46. Mutagenesis research utilizing a Gi-dissociation assay in HEK293T cells with each 18:1 and 18:0 LysoPS (Figs 2B and S4) revealed that a number of residues in touch with the polar head of LysoPS are essential for LysoPS-induced activation of GPR34, together with 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 reasonably affected receptor exercise. In distinction, R208ECL2 mutation has little impact on receptor activation, suggesting that this website will not be essential for LysoPS-induced activation and will doubtlessly be substituted by surrounding positively charged residues resembling K1283.26, H199ECL2, H206ECL2, and K210ECL2 in ligand binding. Mutations of most residues within the hydrophobic subpocket haven’t any impact on receptor activation, aside from Y1393.37A, A1824.53V, and M1894.60A (S4E Fig). These findings point out that the popularity of LysoPS by GPR34 is primarily mediated by particular interactions with the polar head group, whereas the hydrophobic interactions with the acyl tail present extra stability to the binding.
Fig 2. Recognition of LysoPS by GPR34 and GPR174.
(A) Interactions between LysoPS and GPR34. Polar interactions are depicted as black dashed traces. (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 traces. The π–π interplay is depicted as a pink dashed line. (D) Web cAMP accumulation of CHO cells expressing GPR174 mutants in response to 10 μM LysoPS. WT-basal represents web cAMP accumulation of cells expressing wild-type GPR174 within the absence of exogenous LysoPS. Information signify imply ± SEM from 3 unbiased experiments. Mutants with expression lower than half that of the wild-type receptor are labeled with pink stars. The information used to generate graphs in Fig 2B and 2D can be found in S1 Information.
Earlier evolutionary research discovered that GPR34 receptors have existed for greater than 450 million years and recognized residues conserved throughout GPR34 evolution [27,28]. Of the residues concerned in LysoPS recognition, Y1353.33, Y1393.37, R208ECL2, N2205.40, R2866.55, Y2896.58, N3097.35, E3107.36, and L3137.39 of GPR34 are extremely conserved throughout vertebrate evolution. Notably, all 6 residues related to LysoPS by polar interactions are conserved. Most mutations of those residues impaired GPR34 activation besides R208ECL2 and N2205.40 (Fig 2B).
GPR174 binds the polar head of LysoPS in a central pocket by in depth polar interactions (Fig 2C). Particularly, the carboxyl group of the serine moiety types salt bridges with R752.60 and K983.32, and a hydrogen bond with Y993.33, whereas the amino group types a hydrogen bond with Y792.64. Moreover, the phosphate group types 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 take part within the formation of the central pocket by hydrophobic interactions, with F169 additional stabilizing the binding of the polar head by a cation–π interplay with R752.60. The acyl tail of LysoPS binds in a slender cleft fashioned by hydrophobic residues from transmembrane helices 3 to six, together with Y1033.37, F1524.60, M1855.38, and F2506.55, with the double bond of the oleoyl group in touch with Y1033.37 by a π–π interplay. G1895.42 and G1935.46 on TM5 present area for the binding of the acyl group. cAMP accumulation assays had been carried out in CHO cells to evaluate the contributions of the residues concerned in LysoPS binding (Figs 2D and S5). Mutations to alanine on the polar interplay websites considerably impaired and even abolished LysoPS-induced cAMP accumulation, whereas mutations of hydrophobic websites like F152A and Y103A have related results.
We observed the lately reported construction of the LysoPS (18:0)-GPR174-Gs advanced [29] and in contrast it with our LysoPS (18:1)-bound advanced. Curiously, the identical polar head of the two forms of LysoPS binds to the central pocket with some surprising variations (Fig 3A). In our construction, the carboxyl group of the serine moiety doesn’t type a hydrogen bond with the primary chain carbonyl of F169ECL2, and the hydrogen bond between the carbonyl group of the ester linkage and R1564.64 can be absent. Moreover, we noticed a further π–π interplay between the double bond on the acyl group of LysoPS (18:1) and the phenol ring of Y103, which can lead to tighter binding (Fig 3B).
Fig 3. Comparability 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 coloured pink; the corresponding single bond in LysoPS (18:0) is coloured orange. (C) Charged interactions between LysoPS and GPR34 within the positively charged pocket. (D) Charged interactions between LysoPS and GPR174 within the positively charged pocket. (E) Structural superposition of LysoPS certain GPR34 and GPR174 (extracellular view). (F) Comparability between acyl tails of LysoPS binding in GPR34 and GPR174. Polar or charged interactions are depicted as black dashed traces.
The interplay patterns of LysoPS with GPR34 and GPR174 are completely different. In each receptors, the polar head binds in a positively charged central pocket (Fig 3C and 3D), however in contacts with completely different units of residues. In GPR34, the serine carboxyl and phosphate group of LysoPS type ionic interactions with R2866.55 and R208ECL2, respectively (Fig 3C), whereas in GPR174, their orientations are decided by K983.32, R752.60, R1564.64, and K2576.62 (Fig 3D). In GPR34, amino group of the serine in LysoPS types a salt bridge with E3107.38, whereas in GPR174, it types a hydrogen bond with Y792.64. The polar head of LysoPS makes extra charged interactions with GPR174, suggesting a tighter binding than with GPR34. Relating to the hydrophobic tail, its orientation is principally decided by the association of TM4 and TM5 (Fig 3E and 3F), that are differentiated by prolines on them. In GPR34, there are not any prolines on TM4/5, together with the conserved P5.50 amongst class A GPCRs. A curved path extends towards TM3 and TM4, additional restricted by L2235.43. In GPR174, P1534.61 and P1975.50 lead to a definite association of TM4 and TM5, forming a straight path with hydrophobic residues from TM3-6. L5.43 is changed by a glycine at place 5.42 in GPR174.
Total, our findings counsel that whereas GPR34 and GPR174 share related binding modes for LysoPS, they exhibit variations within the particular binding residues and the hydrophobic tail’s orientation.
Lipid-binding modes of lysophospholipid receptors
The binding modes of a number of different LPLs have been illustrated by earlier structural research, together with LPA receptors [30–32], S1P receptors [33–39], and the LPC receptor GPR119 [40]. S1P receptors (S1P1-5) are referred to as the endothelial differentiation gene (EDG) household. LPA1-3 additionally belong to the EDG household, whereas LPA4-6 belong to the P2Y household (S1B Fig). In the meantime, GPR34 and GPR174 are thought of P2Y-like orphan receptors [1]. Notably, the binding pocket of LysoPS in GPR34 or GPR174 is distinct from that of S1P, LPA, and LPC of their respective receptors. Particularly, within the construction of GPR34 and GPR174, the polar head of LysoPS is enveloped by the 7-TM bundles, whereas the hydrophobic tail extends to the cleft fashioned by TM3-5 (Fig 4A and 4B), thereby making a semi-open pocket for LysoPS. Conversely, within the buildings of endogenous ligand-bound S1P1, LPA1, and GPR119, the ligand-binding pockets are virtually occluded, whereby S1P, LPA, and LPC are surrounded by TM helices (Fig 4C–4E). Curiously, within the crystal construction of LPA6, one of many monoolein molecules, though incomplete, binds in a pocket much like that of LysoPS in GPR34 and GPR174 (Fig 4F). Moreover, the platelet activating issue receptor (PAFR), one other P2Y-like receptor, is extremely related in sequence to GPR34. Docking evaluation revealed that PAFR employs the same lipid-binding mode to that of GPR34 and GPR174 [41]. By evaluating the ligand-binding modes of various LPL receptors, we advise that the semi-open lipid-binding pocket could also be shared by different P2Y-like lipid receptors together with LPA4-6 and PAFR.
Fig 4. Comparability of lysophospholipid-binding modes.
The ligand-binding pockets of GPR34 (A), GPR174 (B), S1P1 (C, PDB: 7td3), LPA1 (D, PDB: 7dt0), GPR119 (E, PDB: 7xz5), and LPA6 (F, PDB: 5xsz). GPR34, GPR174, S1P1, LPA1, GPR119, and LPA6 are coloured marine inexperienced, blue, darkish gold, orange, plum, and light-weight blue, respectively. The ligands are proven as sticks. The blobs signify the floor of the atomic fashions.
G protein coupling
GPR34 and GPR174 modulate cAMP degree in cells through Gi and Gs pathways, respectively. We analyzed the G protein–coupling profiles of those 2 receptors to achieve insights into the idea of their G protein selectivity.
The coupling mode between GPR34 and Gi resembles that of many Gi/o-coupled class A GPCRs resembling rhodopsin and cannabinoid receptor CB2. The construction of GPR34-Gi was in contrast with the buildings of rhodopsin-Gi and CB2-Gi (Fig 5A and 5B). All of them undertake the same conformation on the α5 helix of Gαi. Nevertheless, TM6 of GPR34 reveals a smaller outward displacement on the intracellular finish of TM6, which leads to a extra upright pose of the α5 helix. TM5 is just not concerned within the coupling between GPR34 and Gαi subunit. The C terminus of α5 inserts right into a cavity primarily fashioned by TM3, TM6, and the linkage between TM7 and H8. Particularly, R1523.50, N2676.36, and S3327.58 type hydrogen bonds with important chain atoms of C351, L353, and F354 of Gαi, respectively (Fig 5C). As well as, I892.39, K1553.53, I1563.54, T2646.33, M3307.56, and S3317.57 work together with α5 by hydrophobic interactions. ICL3 and the intracellular finish of TM6 binds to Gαi by extra interactions with α5 and β6. Particularly, N2576.26 and Y2616.30 type hydrogen bonds with K345 and D341, respectively. Hydrophobic interactions between F255 and P256 of ICL3 and I344, D337, Y320, and E318 of Gαi additional stabilize the interface (Fig 5D). Most mutations on the G protein–coupling websites impaired exercise of GPR34 (Figs 5E, S6A and S6B). Particularly, R152A and Y261A dramatically impaired GPR34 activation.
Fig 5. G protein coupling of GPR34 and GPR174.
(A) Structural superposition of GPR34-Gαi and rhodopsin-Gαi (PDB: 6cmo). Rhodopsin is coloured pink, and Gi coupled to it’s coloured cyan. (B) Structural superposition of GPR34-Gαi and CB2-Gαi (PDB: 6pt0). CB2 is coloured brown, and Gαi coupled to it’s coloured cyan. The displacements are indicated by arrows. (C) Interactions between the C terminus of α5 and GPR34. (D) Interactions between α5/β6 of Gαi and ICL3 of GPR34. (E) Relative intrinsic exercise (RAi) plot of GPR34 mutants of the G protein–coupling websites in Gi-dissociation assays. RAi was calculated as [Emax(test)/EC50(test)]/[Emax(WT)/EC50(WT)]. Emax and EC50 are from the averaged concentration-response curves of three unbiased experiments. (F) Superposition of GPR174-Gαs, β2AR-Gαs (PDB: 3sn6), and CCKAR-Gαs (PDB: 7ezk). GPR174, β2AR, and CCRKAR are coloured blue, mild yellow, and magenta, respectively. Gαs subunits in these buildings are coloured mild blue, yellow, and plum, respectively. (G) Interactions between the C terminus of α5 helix and GPR174. (H) Interactions between Gαs and ICL3 of GPR174. (I) Interactions between Gαs and ICL2 of GPR174. Polar interactions are depicted as black dashed traces. (J) Basal exercise and maximal LysoPS-induced exercise of GPR174 mutants in cAMP accumulation assays. Information are from the averaged concentration-response curves of three unbiased experiments. The information used to generate graphs in Fig 5E and 5J can be found in S1 Information.
Unexpectedly, GPR174 {couples} to Gs in a noncanonical method in contrast with the well-studied Gs-coupled receptor β2AR (Fig 5F). Particularly, the displacement of TM6 in GPR174 is considerably smaller than in β2AR, with a distance of 12.7 Å on the Cα atom of the residue at place 6.29. Moreover, Gαs adopts a definite conformation on the C terminus of α5 helix in contrast with the β2AR-Gs advanced. As an alternative of turning again to work together with TM5 and TM6, the C terminus of α5 helix reaches out like a bended finger to bind right into a deeper cavity fashioned by the TM7-H8 linkage, TM1-3, ICL1, and ICL2. Inside this pocket, F1193.53 of GPR174 prevents the formation of a cation–π interplay between R1163.50 and Y391 of Gαs (S6C and S6D Fig). Y391 rotates to the route of ICL2 and types a hydrogen bond with D128 (Fig 5G). Cholecystokinin A receptor (CCKAR) was additionally reported to undertake a noncanonical Gs-coupling mode. It shares the same extent of TM6 displacement with GPR174 upon Gs binding (Fig 5F). Nevertheless, in CCKAR-Gs advanced, the interplay between R3.50 and Y391 persists because of the absence of a phenylalanine at place 3.53 (S6E Fig), which is exclusive to GPR174 and P2Y10 amongst class A GPCRs. Within the GPR174-Gs advanced, the C terminus of α5 helix binds to the receptor by in depth polar interactions and hydrophobic interactions throughout the pocket (Fig 5G). The ICL3 loop, together with the intracellular finish of TM5 and TM6, types in depth polar interactions with residues at α5 and β6 of Gαs, together with Y358, D381, Q384, and R385 (Fig 5H). Moreover, the ICL2 loop of GPR174 binds to the αN-α5 area of Gαs primarily by hydrophobic interactions, together with the binding of F12434.51 within the hydrophobic pocket fashioned by H41, V217, F376, C379, R380, and I383 (Fig 5I), as seen in β2AR-Gs advanced. For GPR174, some mutations on the G protein–coupling websites additionally impaired receptor activation (Figs 5J, S6F and S6G). Notably, F124ICL2A virtually abolished each spontaneous exercise and LysoPS-induced exercise. R53ICL1A and K225ICL3A additionally dramatically impaired GPR174 activation, suggesting the necessary roles of the intracellular loops of GPR174 in Gs coupling. Total, the GPR174-Gs advanced reveals a novel coupling mode characterised by the dearth of R3.50–Y391 interplay and an uncommon binding pocket for the C terminus of α5 helix, which can have implications for drug discovery efforts concentrating on this receptor.
Dialogue
LysoPS is a lipid mediator implicated in varied pathological situations, together with inflammatory and autoimmune illnesses, which makes LysoPS receptors promising drug targets.
Structural evaluation of GPR34 and GPR174 reveals a novel binding pocket for LysoPS, comprising an interior polar subpocket and a lateral hydrophobic subpocket. Based mostly on structural and practical evaluation, we discover that GPR34 and GPR174 use completely different mixtures of residues to acknowledge the polar head of LysoPS. GPR174 possesses a extra positively charged pocket that accommodates the polar head of LysoPS, which can partially account for its spontaneous exercise [42].
In a preprint paper [43], the authors reported 2 cryo-EM buildings of GPR34-Gi advanced certain with the LysoPS analogue S3E-LysoPS or its derivate M1. S3E-LysoPS is a sn-3 LysoPS containing an ethoxy group on the sn-1 place, whereas what we utilized in our research is sn-1 LysoPS. M1 is a S3E-LysoPS derivate with the oleic acid substituted by an fragrant fatty acid. Equally, in addition they discovered that the ligand-binding pocket of GPR34 is laterally open towards the membrane, and recognition of the serine moiety was realized by the charged cluster. Furthermore, they discovered that the fragrant fatty acid surrogate of M1 types steady hydrophobic interactions with the ligand-binding pocket, which makes M1 a super-agonist. It’s fascinating to see the structural foundation underlying which GPR34 responds to each endogenous LysoPS and LysoPS analogues from our works. In actual fact, S3E-LysoPS and sn-1 LysoPS share the same construction, besides that the sn-1 carbon of S3E-LysoPS lies on the sidechain, shortening the spine of S3E-LysoPS. And the carbonyl of the ester linkage is nearer to R2866.55, with which it types a further hydrogen bond. The authors additionally discovered that LysoPS produced on the outer leaflet of the plasma membrane by PS-PLA1 is related to the cells and enters the binding pocket of GPR34 laterally to activate it. This supplied convincing proof that LysoPS can enter the ligand-binding pocket from the lateral cleft between TM4 and TM5. And we expect this ligand-entry mechanism may be shared by different P2Y-like receptors.
Our buildings additionally present insights into the structure-activity relationships of LysoPS analogs [44,45]. From the construction, the truth that analogs with a methyl group on the β-position of the serine moiety haven’t any exercise on GPR34 could presumably end result from the steric hindrance with Y1353.33. Then again, substitution of the ester linkage with an amide bond confers selectivity for GPR174, which can end result from the completely different conformations of LysoPS molecules within the buildings of GPR34 and GPR174. The ester linkage of LysoPS in GPR174 is nearly an identical to the trans configuration of an amide bond in conformation, whereas this isn’t the case in GPR34 (Fig 2). Lack of flexibility prevents formation of the optimum binding conformation and results in lowered efficiency in the direction of GPR34. Moreover, substitution of the fatty acid with particular 3-(alkoxyphenyl)propionic acid enhances efficiency in the direction of LysoPS receptors presumably by forming hydrophobic and fragrant interactions with particular residues in GPR34 and GPR174. In GPR34, the benzene ring of the alkoxyphenyl group is predicted to type hydrophobic interactions with M189 and F219. In GPR174, the benzene ring may type fragrant interactions with F152 and F250.
Collectively, these outcomes present invaluable insights into the mechanisms of ligand recognition and signaling of LysoPS receptors, in addition to a structural framework for rational drug design concentrating on LysoPS receptors.
Supplies and strategies
Constructs
For structural research, human GPR34 and GPR174 had been codon optimized for expression in Sf9 insect cells and cloned right into a modified pFastBac1 vector. On the N terminus, a bovine prolactin sign peptide was added, adopted by a FLAG tag (DYKDDDD), an 8× His tag, an N-terminal fragment of β2 adrenergic receptor (BN, residues from 2 to 30), and a TEV website. Two copies of BN had been used on the similar website for GPR174. On the C terminus, an LgBiT subunit linked by a 17-amino acid linker (HMGSSGGGGSGGGGSSG) was inserted earlier than the cease codon. Residues after Q347 and L308 had been truncated in GPR34 and GPR174, respectively. An engineered Gαi1 protein with 4 mutations (DNGαi) [46] and a DN_miniGαs [47] utilized in earlier research had been additionally cloned into the pFastBac1 vector. Human Gβ1 and Gγ2 had been cloned into the pFastBac-Twin vector with a 6× His tag on the N terminus of Gβ1 and a HiBiT subunit linked by a 15-amino acid linker (GSSGGGGSGGGGSSG) on the C terminus of Gβ1. Nb35 was cloned into the pMESy4 vector, with a pelB sign sequence on the N terminus and a 6× His tag on the C terminus. And scFv16 with an AcNPV gp67 sign peptide on the N terminus and a TEV website adopted by an 8× His tag on the C terminus was cloned into the pFastBac1 vector.
For practical assays, GPR34 or GPR174 of full size was cloned into the pcDNA3.1(+) vector with an HA sign adopted by a FLAG tag on the N terminus.
Protein expression and purification
Nb35 and scFv16 had been ready as beforehand reported [48,49]. Briefly, Nb35 was expressed at BL21 E.coli cells and purified by His tag. scFv16 was expressed at Tni (HiFive) insect cells and purified by His tag earlier than digested with TEV enzyme to take away His tag. Purified Nb35 and scFv16 had been saved at −80°C earlier than use.
GPR34-Gi or GPR174-Gs advanced was coexpressed in Sf9 utilizing the Bac-to-Bac system (Invitrogen). Sf9 cells on the density of two.5 × 106 cells per ml had been contaminated with viruses for receptor, Gα, and Gβγ on the ratio of 1:1:1 and cultured at 27°C for 48 hours. Cells had been collected by centrifugation and resuspended in PBS earlier than frozen in liquid nitrogen. Cells had been saved at −80°C earlier than use.
Cells had been thawed in precold lysis buffer consisting of 20 mM HEPES (pH 7.5), 50 mM NaCl, 10 mM MgCl2, 5 mM CaCl2, 25 mU/ml apyrase, 2.5 μg/ml leupeptin, 150 μg/ml benzamidine, and 100 μM TCEP and incubated at room temperature for two hours. Then, 18:1 LysoPS (Avanti polar lipids) was added to the lysis buffer and all the next step on the focus of 1 μM. The pattern was centrifuged at 30,700 × g for half-hour to gather cell membranes. The membranes had been resuspended with a glass Dounce homogenizer in solubilization buffer consisting of 20 mM HEPES (pH 7.5), 100 mM NaCl, 1% (w/v) lauryl maltose neopentyl glycol (LMNG, Anatrace), 0.2% (w/v) cholesteryl hemisuccinate (CHS, Anatrace), 10% (v/v) glycerol, 10 mM MgCl2, 5 mM CaCl2, 12.5 mU/ml apyrase, 2.5 μg/ml leupeptin, 150 μg/ml benzamidine, and 100 μM TCEP and incubated at 4°C for two hours. The supernatant was remoted by centrifugation at 38,900 × g for 45 min, after which incubated with Ni resin at 4°C for two hours. After binding, the resin was washed with 20 column volumes of buffer A consisting of 20 mM HEPES (pH 7.5), 100 mM NaCl, 0.05% LMNG, 0.01% CHS, 2.5 μg/ml leupeptin, 150 μg/ml benzamidine, and 100 μM TCEP with 20 mM imidazole earlier than certain protein was eluted with 5 column volumes of buffer A containing 400 mM imidazole. The eluate was supplemented with 2 mM CaCl2 and incubated with M1 anti-FLAG resin (Sigma Aldrich) in a single day at 4°C. The resin was washed with 10 column volumes of buffer A supplemented with 2 mM CaCl2, and the protein was eluted with 3.5 column volumes of buffer A containing 5 mM EDTA and 200 μg/ml FLAG peptide.
Nb35 or scFv16 was added at a ratio of 1.3:1 over the advanced for GPR174-Gs or GPR34-Gi, respectively. The advanced was concentrated and loaded onto Superdex 200 10/300 GL column preequilibrated with operating buffer containing 20 mM HEPES (pH 7.5), 100 mM NaCl, 0.00075% (w/v) LMNG, 0.00025% (w/v) glyco-diosgenin (GDN, Anatrace), 0.00015% (w/v) CHS, and 100 μM TCEP. Fractions containing monomeric advanced had been pooled and concentrated for electron microscopy experiments.
Cryo-EM knowledge acquisition
For GPR34-Gi advanced, 3 μl purified protein on the focus of 13 mg/ml was utilized onto a glow-discharged holey carbon grid (Quantifoil, Au200 R1.2/1.3). For GPR174-Gs advanced, 3 μl purified protein on the focus of three mg/ml was utilized onto a glow-discharged holey Ni-Ti alloy grid (ANTcryo, Au300 R1.2/1.3). The grids had been plunge frozen in liquid ethane utilizing Vitrobot Mark IV (Thermo Fisher Scientific). Information assortment was carried out on a Titan Krios electron microscope at 300 kV accelerating voltage utilizing a Gatan K3 Summit direct electron detector with an power filter. Micrographs had been collected utilizing the EPU software program in super-resolution mode with a calibrated pixel dimension of 0.535 Å and a defocus vary of −1.2 to −2.2 μm. Every film was comprised of 32 frames with a complete publicity dose of 55 e−/Å2.
Cryo-EM knowledge processing
For GPR34-Gi advanced, a complete of 4,381 film stacks had been subjected to Patch movement correction and Patch CTF estimation in CryoSPARC [50]. A complete of 8,530,353 particles had been picked by Template picker after which subjected to 2D classification to discard poorly outlined particles. After 3 rounds of 3D classification with Ab-initio reconstruction and Heterogeneous refinement, a subset with 304,493 particles had been chosen for Non-Uniform refinement [51]. The particles had been imported into Relion [52] and categorised by 3D classification with out alignment. The classification was centered on the receptor by a masks. The perfect class was chosen for Non-Uniform refinement, which generated a map with an indicated world decision of two.91 Å at a Fourier shell correlation (FSC) of 0.143 (S2 Fig).
For GPR174-Gs advanced, 2,043 motion pictures had been imported into CryoSPARC [50] and preprocessed by Patch movement correction and Patch CTF correction. Roughly 1,486 micrographs with cheap whole movement distance and CTF decision had been chosen for additional processing. 2D templates for particle choosing had been created by Blob picker and 2D classification utilizing 100 micrographs. A complete of two,561,330 particles had been picked by Template piker from all micrographs. The particles had been subjected to 2D classification, and 533,772 particles had been chosen for 3D reconstruction. Particles had been categorised by Heterogeneous Refinement utilizing templates generated by Ab-initio Reconstruction. Then, 329,915 particles from the most effective 2 lessons had been chosen for one more spherical of 3D classification. Lastly, 132,808 particles had been chosen and processed by Non-uniform Refinement [51]. The map was additional improved by Native Refinement with a world masks, leading to a map at decision of three.06 Å estimated by gold-standard FSC on the threshold of 0.143 (S3 Fig). Native decision was estimated in CryoSPARC. And the ultimate map was domestically filtered based mostly on native decision for mannequin constructing and visualization.
Mannequin constructing and refinement
Buildings of GPR34 and GPR174 predicted by AlphaFold [53,54] had been used as preliminary fashions for the receptors. The coordinates of DNGi and scFv16 had been derived from the construction of D2R-Gi-scFv16 advanced (PDB: 7JVR) [47], and the coordinates of miniGs and Nb35 had been derived from the construction of D1R-Gs-Nb35 advanced (PDB: 7JVQ) [47]. Completely different subunits had been docked into the density maps in UCSF Chimera. Then, the preliminary fashions had been manually adjusted in Coot [55] and autorefined by Phenix [56]. Ligands had been generated by the eLBOW program in Phenix. The mannequin statistics had been calculated by MolProbity [57] (S1 Desk).
G protein-dissociation assay
The operate of GPR34 and its mutants was analyzed by a G protein-dissociation assay based mostly on the TRUPATH platform [58]. HEK293T cells had been transfected with wild-type or mutant GPR34, Gαi1-Rluc8, Gβ3, Gγ9-GFP2 plasmid on the ratio of 1:1:1:1 in 6-cm dishes utilizing lipofectamine 3000 (Thermo Fisher Scientific). About 40 hours after transfection, cells had been collected by trypsin-EDTA remedy and resuspended in BRET buffer (1× HBSS, 25 mM HEPES (pH 7.4), 0.1% BSA). Cells had been plated in 96-well white wall, white-bottom plates on the density of 100,000 cells in 30 μl per properly. Then, 30 μl substrate answer with 15 μM coelenterazine 400a (Nanolight Expertise) in BRET buffer was added to every properly, and the plate was incubated at room temperature for five minutes. Lastly, 30 μl 3× LysoPS (18:1 or 18:0) diluted in BRET buffer at completely different concentrations was added to every properly. The plate was incubated at room temperature for five minutes earlier than examined on a SpectraMax iD5 (Molecular Gadgets) microplate reader. BRET sign was measured utilizing 515 nm and 410 nm emission filters. BRET ratio (515 nm/410 nm) was normalized to the ligand-free management earlier than additional evaluation. Outcomes of three unbiased assays (every in triplicate) had been used to calculate a concentration-response curve with a 3-parameter logistic operate. All curves had been additional aligned by the “Backside” parameter.
cAMP accumulation assay
The operate of GPR174 and its mutants was analyzed by cAMP accumulation assay with a cAMP HTRF equipment (Cisbio). CHO-K1 cells had been transfected with wild-type or mutant GPR174 plasmids in 12-well plates utilizing lipofectamine 3000 (Thermo Fisher Scientific). About 44 hours after transfection, cells had been collected by trypsin-EDTA remedy and resuspended in Stimulation buffer supplemented with 0.5 mM IBMX. Cells had been plated in 384-well plates on the density of two,500 cells in 5 μl per properly. And 5 μl 2× LysoPS (18:1) diluted in Stimulation buffer with 0.5 mM IBMX at completely different concentrations was added to every properly. The plate was incubated at 37°C for half-hour. Then, 10 μl detection answer with Eu-cAMP and d2-antibody was added to every properly. Then, the plate was incubated at room temperature for 1 hour earlier than examined on a SpectraMax iD5 (Molecular Gadgets) microplate reader. FRET sign was measured on the excitation wavelength of 340 nm and the emission wavelength of 665 nm and 616 nm utilizing filters. Final result of three unbiased assays (every in duplicate) was used for evaluation. The HTRF ratio (665 nm/616 nm) was reworked into the focus of cAMP by a normal curve. The concentration-response curve was calculated with a 3-parameter logistic operate.
Expression evaluation of mutants
The expression of GPR34 or GPR174 and their mutants was analyzed by movement cytometry. In consistence with the practical assays, HEK293T and CHO-K1 cells had been used for GPR34 and GPR174, respectively. Cells had been transfected with wild-type or mutant receptor plasmid in 24-well plates utilizing lipofectamine 3000 (Thermo Fisher Scientific). After 2 days, cells had been collected by trypsin-EDTA remedy and resuspended in HBSS. Cells at a density of 40,000 to 60,000 cells in 20 μl was blended with 20 μl anti-FLAG M2-FITC antibody (Sigma Aldrich, for GPR34) or Alexa Fluor 647 anti-FLAG antibody (Abcam, for GPR174) diluted in TBS buffer with 4% BSA. The pattern was incubated at 4°C for 20 minutes earlier than 160 μl HBSS with 5 mM HEPES (pH 7.4) was added. Fluorescence sign was measured on CytoFLEX (Beckman), and 10,000 cells had been recorded for every pattern. Single cells had been outlined by setting FSC/SSC thresholds, and the median fluorescence depth was used to guage receptor expression. Information had been normalized to the expression degree of wild-type receptor (100%) and mock-transfected cells (0%). Outcomes of three unbiased assays (every in duplicate) had been used for evaluation.
Supporting info
S1 Fig. Lysophospholipids and lysophospholipid receptors.
(A) Lysophospholipids. (B) Phylogenetic tree of lysophospholipid receptors. Sequence similarity evaluation of EDG household receptors, P2Y household receptors, and different lysophospholipid receptors. A number of sequence alignment was achieved with MUSCLE. Phylogenetic tree was calculated by neighbor-joining technique and displayed by iTOL. EDG household and P2Y household are coloured pink and inexperienced, respectively.
https://doi.org/10.1371/journal.pbio.3002387.s001
(TIF)
S2 Fig. GPR34-Gi advanced preparation and cryo-EM knowledge processing.
(A) Cryo-EM picture processing workflow for GPR34-Gi advanced. (B) SDS-PAGE profile of GPR34-Gi-scFv16 advanced. Uncropped gel for S2B is supplied in S1 Uncooked Photographs. (C) Consultant cryo-EM picture (scale bar: 50 nm). (D) Consultant 2D class averages (scale bar: 5 nm). (E) Angular distribution plot of ultimate particles. (F) The “gold-standard” FSC curves of the GPR34-Gi-scFv16 advanced. (G) Cryo-EM density maps and fashions of the 7 transmembrane helices (TM1-7), Helix 8 (H8), α5 helix of Gαi, and the ligand of LysoPS 18:1 certain GPR34-Gi advanced are proven. The EM density is proven on the threshold of 0.3.
https://doi.org/10.1371/journal.pbio.3002387.s002
(TIF)
S3 Fig. GPR174-Gs advanced preparation and cryo-EM knowledge processing.
(A and B) Dimension-exclusion chromatography and SDS-PAGE profiles of GPR174-Gs-Nb35 advanced. Uncropped gel for S3B is supplied in S1 Uncooked Photographs. (C) Consultant cryo-EM picture (scale bar: 50 nm). (D) Movement chart of cryo-EM knowledge processing and native decision of the ultimate map. Density of the ligand within the ultimate map is indicated by a black dashed ellipse. (E) Consultant 2D class averages (scale bar: 5 nm). (F) Angular distribution plot of ultimate particles. (G) The “gold-standard” FSC curves of the GPR174-Gs-Nb35 advanced. (H) Density maps of the 7 transmembrane helices (TM1-7), Helix 8 (H8), α5 helix of Gαs and LysoPS certain in GPR174-Gs advanced are proven. The EM density is proven on the threshold of 0.276.
https://doi.org/10.1371/journal.pbio.3002387.s003
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S4 Fig. Purposeful knowledge of GPR34.
(A) Exercise of GPR34 induced by 18:1 or 18:0 LysoPS within the Gi-dissociation assay. (B) Expression of GPR34 mutants in HEK293T cells. (C–E) Focus-response curves of GPR34 mutants within the Gi-dissociation assay. Information signify imply ± SEM from a minimum of 3 unbiased experiments. The information used to generate graphs in S4A-S4E can be found in S1 Information.
https://doi.org/10.1371/journal.pbio.3002387.s004
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S5 Fig. Purposeful knowledge of GPR174.
(A) Expression of GPR174 mutants in CHO cells. (B–D) Focus-response curves of GPR174 mutants within the cAMP accumulation assay. Information signify imply ± SEM from 3 unbiased experiments. The information used to generate graphs in S5A-S5D can be found in S1 Information.
https://doi.org/10.1371/journal.pbio.3002387.s005
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S6 Fig. Purposeful knowledge of G protein coupling.
(A) Focus-response curves of GPR34 mutants within the Gi-dissociation assay. (B) Expression of GPR34 mutants in HEK293T cells. (C–E) Particulars of Gαs binding by GPR174 (C), β2AR (D, PDB: 3sn6), and CCKAR (E, PDB: 7ezk). (F) Focus-response curves of GPR174 mutants within the cAMP accumulation assay. (G) Expression of GPR174 mutants in CHO cells. All knowledge signify imply ± SEM from a minimum of 3 unbiased experiments. The information used to generate graphs in S6A, S6B, S6F, and S6G can be found in S1 Information.
https://doi.org/10.1371/journal.pbio.3002387.s006
(TIF)
Acknowledgments
We thank the Cryo-EM Middle at College of Science and Expertise of China for the assist of cryo-EM knowledge assortment. We thank Dr Yongxiang Gao for help with cryo-EM knowledge screening and assortment.
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