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. 2022 Dec 21;8(1):135.
doi: 10.1038/s41421-022-00499-8.

Insights into divalent cation regulation and G13-coupling of orphan receptor GPR35

Jia Duan #  1   2 , Qiufeng Liu #  1 , Qingning Yuan #  1 , Yujie Ji  1   2 , Shengnan Zhu  3 , Yangxia Tan  1 , Xinheng He  1   2 , Youwei Xu  1 , Jingjing Shi  1 , Xi Cheng  1   2   4 , Hualiang Jiang  1   2   4   5   6 , H Eric Xu  7   8   9   10 , Yi Jiang  11   12
Affiliations

Insights into divalent cation regulation and G13-coupling of orphan receptor GPR35

Jia Duan et al. Cell Discov. .

Abstract

Endogenous ions play important roles in the function and pharmacology of G protein-coupled receptors (GPCRs) with limited atomic evidence. In addition, compared with G protein subtypes Gs, Gi/o, and Gq/11, insufficient structural evidence is accessible to understand the coupling mechanism of G12/13 protein by GPCRs. Orphan receptor GPR35, which is predominantly expressed in the gastrointestinal tract and is closely related to inflammatory bowel diseases (IBDs), stands out as a prototypical receptor for investigating ionic modulation and G13 coupling. Here we report a cryo-electron microscopy structure of G13-coupled GPR35 bound to an anti-allergic drug, lodoxamide. This structure reveals a novel divalent cation coordination site and a unique ionic regulatory mode of GPR35 and also presents a highly positively charged binding pocket and the complementary electrostatic ligand recognition mode, which explain the promiscuity of acidic ligand binding by GPR35. Structural comparison of the GPR35-G13 complex with other G protein subtypes-coupled GPCRs reveals a notable movement of the C-terminus of α5 helix of the Gα13 subunit towards the receptor core and the least outward displacement of the cytoplasmic end of GPR35 TM6. A featured 'methionine pocket' contributes to the G13 coupling by GPR35. Together, our findings provide a structural basis for divalent cation modulation, ligand recognition, and subsequent G13 protein coupling of GPR35 and offer a new opportunity for designing GPR35-targeted drugs for the treatment of IBDs.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1
Fig. 1. Overall structure of the lodoxamide–GPR35–G13–scFv16 complex.
a Schematic diagram of lodoxamide-mediated activation and G protein coupling of GPR35. b Orthogonal views of the density map for the lodoxamide–GPR35–G13–scFv16 complex. GPR35 is shown in orange, Gα13 in slate blue, Gβ in salmon, Gγ in lime, scFv16 in grey, and lodoxamide in magenta. c Orthogonal views of the model of the lodoxamide–GPR35–G13–scFv16 complex.
Fig. 2
Fig. 2. Allosteric agonism of GPR35 by cations.
a Architecture of the cation coordination site. The cation is coordinated by the backbone oxygen of G9NT and S11NT, the side chain of R164ECL2, and a carbonyl oxygen atom of lodoxamide. Cation in GPR35 is displayed as a green sphere, while its EM density is colored in grey. Interactions between the cation and surrounding residue coordinates are indicated as blue dashed lines. b–h Allosteric regulatory effects of different cations on lodoxamide-induced G13 recruitment by GPR35, including Mg2+ (b), Ca2+ (c), Mn2+ (d), Co2+, Zn2+, Cu2+ (e), K+ (f), Li+ (g), and Fe3+ (h). The concentrations of different cations are indicated. The physiological concentrations of Co2+, Zn2+, Cu2+, and Fe3+ are much lower than those of Mg2+ and Ca2+ (WHO Vitamin and Mineral Nutrition Information System, VMNIS). The maximum cell safety concentrations of these cations under our experimental conditions were used.
Fig. 3
Fig. 3. Lodoxamide recognition by GPR35.
a Cross-section of the lodoxamide-binding pocket in GPR35. The pocket is colored by electrostatic surface potential, with the positive potential colored in blue. b, c The extracellular view of GPR35. The N-terminus (NT), all ECLs (ECL1, ECL2, and ECL3), which cover the ligand-binding pocket, are shown in a surface presentation (b). The polar interactions between these extracellular receptor components are indicated by blue dashed lines. ECL2 stretches into the ligand-binding pocket and stuffs the space embraced by lodoxamide and the extracellular portion of TM3, TM4, and TM5 (c). d Detailed interactions that contribute to lodoxamide binding in GPR35. The polar interactions are depicted by blue dashed lines. e 2D presentation of the interactions between lodoxamide and receptor. f Effects of pocket residue mutations on lodoxamide-induced G13 recruitment by GPR35.
Fig. 4
Fig. 4. Activation mechanism of GPR35 by lodoxamide.
a Structural superposition of GPR35 with the inactive β2AR. The movement directions of TM6 and TM7 of GPR35 (orange) relative to the inactive β2AR (grey, PDB: 2RH1) are indicated by black arrows. b The potential steric hindrance, caused by R1003.36 and S2657.42, pushes F2306.48 moving downward. The potential steric hindrance is highlighted in a red dashed circle. c The polar interaction network at the bottom of the ligand-binding pocket of GPR35 and the gain-of-inter-helical hydrophobic contacts between F2306.48 and two conserved PIF residues, I1043.40 and F2266.44. d, e Effects of mutation of residues in the polar interaction network on lodoxamide-induced G13 recruitment by GPR35.
Fig. 5
Fig. 5. Mechanism of GPR35–G13 coupling.
a Structural comparison of the GPR35–G13 complex with representative class A GPCRs coupled to Gs, Gi/o, and Gq/11 proteins, including the β2AR–Gs (PDB: 3SN6), MC1R–Gs (PDB: 7F4F), 5-HT1A–Gi (PDB: 7E2Y), 5-HT1B–Go (PDB: 6G79), H1R–Gq (PDB: 7DFL), and M1R–G11 complexes (PDB: 6OIJ). b Structural comparison of the GPR35–G13 complex with reported G13-coupled GPCRs, including class A GPCR S1PR2 (PDB: 7T6B) and two adhesion (class B2) GPCRs, GPR56 (PDB: 7SF8) and latrophilin 3 (LPHN3, PDB: 7SF7). The movement orientations of the cytoplasmic end of TM6 and the extreme C-terminal end of α5 helix of the Gα subunit in the GPR35–G13 complex compared with those of other listed GPCR–G protein complexes are indicated by black arrows. The black dashed line indicates the sharp kink in GPR56 and LPHN3. The hinge residue G6.50 was labeled. The GPCR–G protein complexes are colored as indicated. c The rotation of the α5 helix of the GPR35–G13 complex relative to that of the S1PR2–G13 complex. The rotation angle of 18° is indicated by a black arrow. The rotation probably arises from the extensive contacts between S1PR2 ICL2 and the α5 helix, which induces the noncanonical loop-like ICL2 to face inward towards the α5 and its resulting rotation. d Sequence alignment of C-terminal sequences of α5 helix of different G protein subtypes. The residues at the –3 position are labeled by a black triangle. e The methionine pocket in GPR35. M375 (–3) of G13 α5 helix forms hydrophobic interactions with residues F451.57, M49ICL1, and M592.43, which constitute a featured methionine pocket of GPR35. f Effects of mutations of residues in the methionine pocket on G13 recruitment by GPR35.
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