Cryo-EM structure of activated bile acids receptor TGR5 in complex with stimulatory G protein

Dear Editor, Takeda G protein-coupled receptor 5 (TGR5), also known as G protein-coupled bile acids (BAs) receptor 1 (GPBAR1), belongs to the class A GPCR subfamily. The major TGR5-dependent actions of BAs include maintaining energy homeostasis, regulating glucose/ lipids metabolism, as well as immunosuppressive properties. TGR5 is identified as a potential therapeutic target for protecting hepatocytes from bile acid overload, preventing atherosclerosis, and inhibiting macrophage inflammation due to its critical role in bile acid sensitization. Thus, elucidation of structural characteristics of TGR5 and its activation mechanism would benefit the discovery of therapeutic drugs for these metabolic disorders. TGR5 activity is governed by endogenous unconjugated or glycine-/taurine-conjugated primary and secondary BAs, semisynthetic derivatives, and some synthetic nonsteroid molecules (Fig. 1a, left panel). Here we report the near-atomic resolution cryo-EM structure of activated TGR5 in complex with the synthetic nonsteroid agonist 23H and Gs protein (Fig. 1b, Supplementary Fig. 1a). For cryo-EM structure determination, we engineered human TGR5 protein (Supplementary Fig. 1b, c). The modified TGR5 retains comparable nanomolar efficacy to several agonists as the wild-type receptor (Fig. 1a, right panel). Vitrified complexes were imaged and processed to yield the map of TGR5-Gs complex at an overall resolution of 3.9 Å (Fig. 1b, Supplementary Figs. 2–3, and Table 1). Backbones of transmembrane helices (TMs) are resolved as well as residues with bulky side-chains. The TGR5 interfaces with Gαs, including α5-helix of Gαs, were also well defined (Supplementary Fig. 4). The density representing 23H was observed adjacent to the extracellular base of TM3, TM5, and TM6 (Fig. 1b and Supplementary Fig. 5a). Due to the limited quality of density map, 23H cannot be precisely modeled in the structure. A sketchy docking was applied to confirm that, the omitted density in the putative TGR5 orthosteric site can accommodate the entire 23H (Supplementary Fig. 5b). By structural analysis combining with intracellular cAMP measurement studies, we extensively screened and identified clusters of residues in the orthosteric site that are critical for 23H induced TGR5 activation (Fig. 1c, d, Supplementary Fig. 5, and Table 2). Within the orthosteric site, TGR5 established interactions with 23H through residues on TM2, TM3, TM5, and TM6. L71W decreased the potency of 23H by two orders of magnitude, indicating the possible stereo clash between the bulky side-chain and 23H. P69/72A double mutation also reduced the potency of 23H by two orders of magnitude, suggesting that this unique PXXP kink located on the cytosolic half of TM2 may stabilize 23H bound conformation of the orthosteric site. N93Q decreased the potency of 23H by two orders of magnitude, indicating possible hydrogen bond formation between N93 and 23H. F96A caused reduced agonist potency with 23H by two orders of magnitude, which might be partly contributed by reducing the hydrophobic interaction with 23H. Bulky side-chain residues substitution of L97 to Trp and Phe reduced agonist potency by two orders and one order of magnitude, respectively, raising the possibility that bulky sidechains may have the stereo clash with 23H indicative of hydrophobic interaction with 23H. L166W and E169W caused reduced cAMP response, indicating that bulky side-chains may clash with 23H. Y240 to Ala but not Phe reduced agonist potency by two orders of magnitude, indicating hydrophobic interaction between Y240 and 23H. Other residues, which reduced the potency of 23H by one order of magnitude, are described in Supplementary Text. 23H has divergent chemical structure comparing to bile acids yet initiate convergent Gs coupling and signal transduction through TGR5. To unveil the molecular mechanism of convergence, we examined the potency of agonist LCA to TGR5 mutants in cAMP assays (Supplementary Fig. 7, and Table 2). Consistently, L71W, L74W, L166W, E169W, and Y240A compromised the potency of LCA. Y89A, which have little effect on the potency of 23H, also decrease the potency of LCA by one order of magnitude. W75, as a “lid”, made the orthosteric binding site occluded. However, W75A did not affect potencies of 23H and LCA. Notably, F96A compromised the potency of 23H but not of LCA. These data suggested that 23H and LCA to a great extent shared the same binding site but had slight differences in recognition details. TGR5 possesses the same fold of class A GPCRs. Since TGR5 and β2AR share an overall 22% sequence identity (Supplementary Fig. 8), structural alignments of active TGR5 with that of inactive (PDB code: 2RH1) and active (PDB code: 3SN6) β2AR 4 were performed, respectively (Fig. 1e and Supplementary Fig. 9). In the superposition of active TGR5 and inactive β2AR, the overall r.m.s.d is 2.9 Å over 145 residues majorly located on the TM region. The Nterminus of TM6 in TGR5 swing outward about 9 Å (the distance between Cα of residue K267 in TGR5 and the corresponding residue R216 in β2AR), resulting in the elevation of intracellular terminal of TM6 for GαsRas interaction. Two helical turns extension of TM5 helix, which contributed to the interaction between TGR5 and GαsRas, was observed (Fig. 1e, upper panel). These structural features are coincident with previous studies in β2AR activation. Viewing towards the membrane plane from the intracellular side, the TMs at cytoplasmic half of activated TGR5 and β2AR assume similar topology (Fig. 1e, lower panel). Thus, both TGR5 and β2AR form a similar cavity recognizing the C-terminal of the α5-helix of GαsRas domain. The structural superposition of TGR5-Gs with β2AR-Gs reveals that the G protein adopts almost identical conformation (Supplementary Fig. 10). The main differences of Gαs between the two complexes are located at β2, β6, α4, and N-terminal of α5 in GαsRas. The main differences of Gβγ are located at some β


Dear Editor,
Takeda G protein-coupled receptor 5 (TGR5), also known as G protein-coupled bile acids (BAs) receptor 1 (GPBAR1), 1 belongs to the class A GPCR subfamily. The major TGR5-dependent actions of BAs include maintaining energy homeostasis, regulating glucose/ lipids metabolism, as well as immunosuppressive properties. 2 TGR5 is identified as a potential therapeutic target for protecting hepatocytes from bile acid overload, preventing atherosclerosis, and inhibiting macrophage inflammation due to its critical role in bile acid sensitization. Thus, elucidation of structural characteristics of TGR5 and its activation mechanism would benefit the discovery of therapeutic drugs for these metabolic disorders.
TGR5 activity is governed by endogenous unconjugated or glycine-/taurine-conjugated primary and secondary BAs, semisynthetic derivatives, and some synthetic nonsteroid molecules (Fig.  1a, left panel). Here we report the near-atomic resolution cryo-EM structure of activated TGR5 in complex with the synthetic nonsteroid agonist 23H 3 and G s protein (Fig. 1b, Supplementary  Fig. 1a). For cryo-EM structure determination, we engineered human TGR5 protein ( Supplementary Fig. 1b, c). The modified TGR5 retains comparable nanomolar efficacy to several agonists as the wild-type receptor (Fig. 1a, right panel). Vitrified complexes were imaged and processed to yield the map of TGR5-G s complex at an overall resolution of 3.9 Å (Fig. 1b, Supplementary Figs. 2-3, and Table 1). Backbones of transmembrane helices (TMs) are resolved as well as residues with bulky side-chains. The TGR5 interfaces with G αs , including α5-helix of G αs , were also well defined ( Supplementary Fig. 4).
The density representing 23H was observed adjacent to the extracellular base of TM3, TM5, and TM6 ( Fig. 1b and Supplementary Fig. 5a). Due to the limited quality of density map, 23H cannot be precisely modeled in the structure. A sketchy docking was applied to confirm that, the omitted density in the putative TGR5 orthosteric site can accommodate the entire 23H ( Supplementary Fig. 5b). By structural analysis combining with intracellular cAMP measurement studies, we extensively screened and identified clusters of residues in the orthosteric site that are critical for 23H induced TGR5 activation (Fig. 1c, d, Supplementary  Fig. 5, and Table 2). Within the orthosteric site, TGR5 established interactions with 23H through residues on TM2, TM3, TM5, and TM6. L71W 2.60 decreased the potency of 23H by two orders of magnitude, indicating the possible stereo clash between the bulky side-chain and 23H. P69 2.58 /72A 2.61 double mutation also reduced the potency of 23H by two orders of magnitude, suggesting that this unique PXXP kink located on the cytosolic half of TM2 may stabilize 23H bound conformation of the orthosteric site. N93Q 3.33 decreased the potency of 23H by two orders of magnitude, indicating possible hydrogen bond formation between N93 3.33 and 23H. F96A 3.36 caused reduced agonist potency with 23H by two orders of magnitude, which might be partly contributed by reducing the hydrophobic interaction with 23H. Bulky side-chain residues substitution of L97 3.37 to Trp and Phe reduced agonist potency by two orders and one order of magnitude, respectively, raising the possibility that bulky sidechains may have the stereo clash with 23H indicative of hydrophobic interaction with 23H. L166W 5.40 and E169W 5.43 caused reduced cAMP response, indicating that bulky side-chains may clash with 23H. Y240 6.51 to Ala but not Phe reduced agonist potency by two orders of magnitude, indicating hydrophobic interaction between Y240 6.51 and 23H. Other residues, which reduced the potency of 23H by one order of magnitude, are described in Supplementary Text.
23H has divergent chemical structure comparing to bile acids yet initiate convergent G s coupling and signal transduction through TGR5. To unveil the molecular mechanism of convergence, we examined the potency of agonist LCA to TGR5 mutants in cAMP assays ( Supplementary Fig. 7, and Table 2). Consistently, L71W 2.60 , L74W 2.63 , L166W 5.40 , E169W 5.43 , and Y240A 6.51 compromised the potency of LCA. Y89A 3.29 , which have little effect on the potency of 23H, also decrease the potency of LCA by one order of magnitude. W75 2.64 , as a "lid", made the orthosteric binding site occluded. However, W75A 2.64 did not affect potencies of 23H and LCA. Notably, F96A 3.36 compromised the potency of 23H but not of LCA. These data suggested that 23H and LCA to a great extent shared the same binding site but had slight differences in recognition details.
TGR5 possesses the same fold of class A GPCRs. Since TGR5 and β 2 AR share an overall 22% sequence identity ( Supplementary Fig.  8), structural alignments of active TGR5 with that of inactive (PDB code: 2RH1) and active (PDB code: 3SN6) β 2 AR 4 were performed, respectively ( Fig. 1e and Supplementary Fig. 9). In the superposition of active TGR5 and inactive β 2 AR, the overall r.m.s.d is 2.9 Å over 145 residues majorly located on the TM region. The Nterminus of TM6 in TGR5 swing outward about 9 Å (the distance between C α of residue K267 in TGR5 and the corresponding residue R216 in β 2 AR), resulting in the elevation of intracellular terminal of TM6 for G αs Ras interaction. Two helical turns extension of TM5 helix, which contributed to the interaction between TGR5 and G αs Ras, was observed (Fig. 1e, upper panel). These structural features are coincident with previous studies in β 2 AR activation. Viewing towards the membrane plane from the intracellular side, the TMs at cytoplasmic half of activated TGR5 and β 2 AR assume similar topology (Fig. 1e, lower panel). Thus, both TGR5 and β 2 AR form a similar cavity recognizing the C-terminal of the α5-helix of G αs Ras domain.
The structural superposition of TGR5-G s with β 2 AR-G s reveals that the G protein adopts almost identical conformation ( Supplementary Fig. 10). The main differences of G αs between the two complexes are located at β2, β6, α4, and N-terminal of α5 in G αs Ras. The main differences of G βγ are located at some β sheets in G β . The total buried interface of the TGR5-G α Ras, which is mediated by extensive hydrogen bonds and hydrophobic interactions, is about 841 Å 2 . This interface is majorly composed by TM3/5/6, ICL1/3 of the TGR5, and α4/5 helices, β6 strand of G α Ras domain. Most of the residues involved in TGR5 interaction are in the carboxyl-terminus of α5-helix of G α Ras, such as Q384, H387, Y391, L393, and F394. It is consistent with the observation in β 2 AR-Gs interaction (Fig. 1f), suggesting the conserved G s binding and activation mechanism.
Sequence analysis revealed that several TGR5 residues involved in the interaction were identical to that in β 2 AR, including E109 3.49 (the most highly conserved amino acids E/DRY, which are located Letter at the cytoplasmic ends of TM3), A113 3.53 , V114 3.54 , V188 5.62 , A192 5.66 , and Q195 5.69 (Fig. 1f and Supplementary Fig. 7). It is worth mentioning that D312 in G β forms hydrogen bonds with R44 ICL1 of TGR5 (Fig. 1f), which was coincident with G s -coupled peptide activated class B GLP-1 receptor 5 but not in β 2 AR. This suggested that other than stabilizing the N-terminal α helix of G αs , G β might also involve in receptor binding. Besides, Nb35 binds to the interface between G β and G αs Ras to stabilize the complex for structure determination (Fig. 1a).
In summary, our studies on TGR5-G s complex structure and mutagenesis analysis revealed the agonist binding mode of TGR5 indicating the convergent activation mechanism, in which the orthosteric binding site could recognize distinct ligands and accommodate the receptor activation. The slight differences in detailed recognition of 23H and LCA will also shed light on the development of therapeutics with improved efficacy and specificity. We firmly believed that TGR5 is a proper prototype on the mechanistic understanding of other GPCRs sensing steroids.

DATA AVAILABILITY
All relevant data are available from the authors and/or included in the manuscript. Atomic coordinates and EM density maps of the human TGR5 have been deposited in the Protein Data Bank (PDB code: 7BW0) and the Electron Microscopy Data Bank (EMDB code: EMD-30221), respectively.
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