Molecular mechanism of agonism and inverse agonism in ghrelin receptor

Abstract

Much effort has been invested in the investigation of the structural basis of G protein-coupled receptors (GPCRs) activation. Inverse agonists, which can inhibit GPCRs with constitutive activity, are considered useful therapeutic agents, but the molecular mechanism of such ligands remains insufficiently understood. Here, we report a crystal structure of the ghrelin receptor bound to the inverse agonist PF-05190457 and a cryo-electron microscopy structure of the active ghrelin receptor-Go complex bound to the endogenous agonist ghrelin. Our structures reveal a distinct binding mode of the inverse agonist PF-05190457 in the ghrelin receptor, different from the binding mode of agonists and neutral antagonists. Combining the structural comparisons and cellular function assays, we find that a polar network and a notable hydrophobic cluster are required for receptor activation and constitutive activity. Together, our study provides insights into the detailed mechanism of ghrelin receptor binding to agonists and inverse agonists, and paves the way to design specific ligands targeting ghrelin receptors.

Fig. 1. Overall structures of ghrelin receptors bound to inverse agonist and agonist.

a Dose-dependent responses of endogenous agonist ghrelin, inverse agonist PF-05190457 and antagonist compound 21 containing 10 nM ghrelin peptide at wild-type ghrelin receptor measured by cellular IP1 accumulation assays. The ghrelin peptide represents high activation potency as a full agonist, and PF-05190457 functions as an inverse agonist to significantly reduce basal activity (dotted line). Data represent the mean ± SEM from n = 3 biologically independent experiments performed in triplicate. b View from within the plane of the membrane. The inverse agonist-bound ghrelin receptor is represented as a wheat cartoon, and PF-05190457 is shown as a marine blue sphere. Cryo-EM structure of the agonist ghrelin-bound ghrelin receptor-Gαo1-scFv16 complex. c Ghrelin peptides and receptors are shown as deep salmon spheres and aquamarine cartoons, respectively. The Go heterotrimer and scFv16 are shown in the gray cartoon. d View from the extracellular side of the membrane. The structural comparison of inverse agonist-bound (wheat) with agonist-bound structures (aquamarine) reveals that notable outward movements occur in the extracellular ends of TM domains.

Fig. 2. The binding mode of PF-05190457 with the ghrelin receptor.

a The detailed binding mode of PF-05190457 in the orthosteric pocket of the ghrelin receptor. The |Fo| − |Fc| omit map (contoured at 3.0 σ) for PF-05190457 (shown as marine blue sticks). In particular, the herein described cavities of the ghrelin receptor named cavity III and cavity IV. b The structure of the neutral antagonist (gray stick)-bound ghrelin receptor (forest cartoon) (PDB: 6KO5) reveals a bifurcated ligand-binding pocket, which is referred to as cavities I and II. c Two-dimensional structures of the inverse agonist PF-05190457 and the groups of ligands are termed the diazaspiro core, arm-1 and arm-2. The diazaspiro core is shown in pink, arm-1 is sky blue, and arm-2 is light green. The red solid lines indicate hydrogen bonds involved in interactions with the side chains of key residues. d–f Key residues of the ghrelin receptor (wheat sticks) involved in ligand binding in cavity III (d), cavity I (e), and cavity IV (f). The binding pocket is highly hydrophobic, and the particular polar network is shown as black dotted lines. g IP1 accumulation induced by the inverse agonist PF-05190457 and antagonist compound 21. Bars represent the difference in the calculated potency (pIC₅₀) of the inverse agonist for mutations relative to the WT ghrelin receptor. Data are colored according to extent of effect. nd not detected, **P < 0.01, ***P < 0.001 (one-way analysis of variance (ANOVA) followed by Dunnett’s test, compared with the response of WT, P < 0.001, P < 0.001, P < 0.001, P > 0.999, nd, nd, P = 0.05, nd, P < 0.001, P < 0.001, nd, nd, nd, nd, nd, nd, P < 0.01, P = 0.264, P < 0.001, nd, P < 0.001, nd from left to right). Data represent the mean ± SEM from n = 3 biologically independent experiments performed in triplicate. See also Supplementary Figs. 6 and 7. h Structural superimposition of inverse agonist-bound with neutral antagonist-bound structures shows significant displacements of the critical residues in the orthosteric pocket. The salt bridges between E124^3.33 and R283^6.55 as well as between R102^2.63 and Q120^3.29, are broken upon PF-05190457 binding.

Fig. 3. Recognition of ghrelin by ghrelin receptor.

a The cutaway surface of the ghrelin receptor bound to the ghrelin structure reveals that ghrelin occupies the bifurcated ligand-binding pocket with the residues Gly⁺¹ and Ser⁺² filling up cavity I and its octanoyl group of Ser⁺³ inserting into cavity II. b Detailed polar interaction of both residues Gly⁺¹ and Ser⁺² (aquamarine sticks) with the key residues from cavity I. c Ghrelin-induced Gαo1-Gγ dissociation assay. Bars represent differences in calculated potency (pEC₅₀) of ghrelin for mutations relative to the WT ghrelin receptor. Data are colored according to the extent of the effect. *P < 0.1, **P < 0.01 and ***P < 0.001. ns no significant difference, nd not detected. All data were analyzed by one-way analysis of variance (ANOVA) followed by Dunnett’s multiple comparison test, compared with the response of WT (P = 0.002, P = 0.002, P = 0.015, P < 0.001, P < 0.001, P = 0.732, P < 0.001, P = 0.843, P = 0.012, P < 0.001, P = 0.003, P < 0.001, P < 0.001 and P < 0.001 from left to right). Data represent the mean ± SEM from n = 3 biologically independent experiments performed in triplicate. See also Supplementary Figs. 6 and 7. d Detailed interaction of the octanoyl group of Ser⁺³ with the key residues from cavity II and salt bridges between E124^3.33 and R283^6.55 are clearly observed in the ghrelin-bound structure.

Fig. 4. Mechanism of ghrelin action on ghrelin receptor.

a, b Structural comparison of agonist-bound with neutral antagonist-bound structures (PDB: 6KO5) reveals remarkable displacements in the extracellular side (a) and intracellular side (b), resulting in contracting of the orthosteric binding pocket in the extracellular side as well as making a cavity to couple G protein in the intracellular side. c Superposition of ghrelin-bound (aquamarine cartoon) and neutral antagonist-bound (forest cartoon) structures shows that the WFF cluster exhibits obvious conformational displacements upon ghrelin binding. d The rearrangement of local residues in the backbone of TM6 between ghrelin-bound (aquamarine cartoon) and neutral antagonist-bound (forest cartoon) structures (PDB: 6KO5) shows the cascade changes of residues R283^6.55, F272^6.44, and the WFF cluster. e Mutations of W276^6.48, F279^6.51, and F312^7.42 in the ghrelin receptor decreased the activation potency induced by ghrelin. Data represent the mean ± SEM from n = 3 biologically independent experiments performed in triplicate. See also Supplementary Figs. 6 and 7.

Fig. 5. Detailed mechanism of inverse agonism action on the ghrelin receptor.

a The arm-2 group of PF-05190457 was observed to insert into the core of the receptor and was sandwiched by side chains from the hydrophobic cluster W276^6.48-F279^6.51-F312^7.42 (WFF cluster). b Superposition of PF-05190457-bound (wheat cartoon) and ghrelin-bound (aquamarine cartoon) structures shows that the WFF cluster exhibits obvious conformational displacements upon inverse agonist binding. c, d The inverse agonist PF-05190457 pushes outward movements of the extracellular end of TM6 (c), destroying the key polar interactions involved in activation (d). The polar networks are shown as black dotted lines (ghrelin-bound) and orange dotted lines (PF-05190457-bound). e Mutations of W276^6.48, F279^6.51, and F312^7.42 in the ghrelin receptor significantly reduced the potency of inverse agonism. Data represent the mean ± SEM from n = 3 biologically independent experiments performed in triplicate. See also Supplementary Fig. 6.

Fig. 6. Summary model of ghrelin receptor bound with inverse agonist, agonist, and antagonist.

Simple model for PF-05190457 (a), ghrelin (b), and compound 21 (c) bound to the ghrelin receptor. Some of the essential residues and modules are marked with different colors and patterns. The cascade motifs referred to the combination of the P–I-F motif, E-R-Y motif, and NPxxY motif.