Food intake is one of the most fundamental processes required for sustaining human life. It is primarily regulated by two endogenous hormones with opposite physiological functions: leptin, the energy surfeit hormone, and ghrelin, the hunger hormone, both of which are involved in controlling energy balance and obesity. Ghrelin is an orexigenic peptide hormone secreted from stomach in response to fasting situations and stimulates the ghrelin receptor in the brain to initiate appetite. One unique feature of ghrelin is the fatty acid modification, with its third amino acid Ser being modified with an octanoyl group, catalyzed by ghrelin O-acyltransferase (GOAT). Although less than 10% of ghrelin is acylated in the blood, this acyl-modification is essential for its activity. Both ghrelin and synthesized growth hormone secretagogues show potent growth hormone-releasing activity and serve as potential candidates for the treatment of growth hormone deficiency (GHD). The growth hormone-releasing activity also makes these hormones attractive performance-enhancing substances, whose usages are banned by the World Anti-Doping Agency in competitive sports.
The pleiotropic functions of ghrelin are mediated through the ghrelin receptor, also known as the growth hormone secretagogue receptor, which was first identified in the pituitary gland and the hypothalamus. As a G protein-coupled receptor (GPCR), the ghrelin receptor couples to G q protein and modulates diverse physiological processes upon binding to ghrelin and other synthetic agonists. LEAP2, an intestinally derived hormone, is identified as an endogenous antagonist of the ghrelin receptor, which fine-tunes ghrelin action via an endogenous counter-regulatory mechanism. Ghrelin receptor is characterized by its high basal activity, with approximately 50% of its maximal capacity in the absence of a ligand. This high level of basal activity may serve as a “signaling set point” to counterbalance the inhibitory input from leptin and insulin in appetite regulation. Several naturally occurring mutations of ghrelin receptor, such as A204E and F279L, decrease the basal activity of the receptor and have been found to associate with obesity, diabetes, and short stature, which led to the idea of ghrelin receptor being an attractive therapeutic target for these diseases. However, there are only two orally active synthetic agonists, pralmorelin and macimorelin, been approved as diagnostic agents for GHD to date.
Extensive efforts have been devoted to examining the structural basis for the potential binding sites of ghrelin and synthetic agonists and the basal activity of ghrelin receptor. Nevertheless, compared to leptin and its receptor, which structures are known, much less are known about the structures of ghrelin and ghrelin-bound receptor. In this study, we reported two cryo-electron microscopy (cryo-EM) structures of the active ghrelin receptor–G q complexes bound to ghrelin and GHRP-6, respectively.
We fused thermostabilized BRIL at the N-terminus of the ghrelin receptor and applied the NanoBiT tethering strategy to improve complex stability and homogeneity. These modifications have little effect on the pharmacological properties of the ghrelin receptor. An engineered Gα q was designed based on the mini-Gα s scaffold with its N-terminus replaced by corresponding sequences of Gα i1 to facilitate the binding of scFv16, and such an analogous approach had been used to obtain structures of the G q-bound 5-HT 2A receptor and G 11-bound M1 receptor. Unless otherwise specified, G q refers to the engineered G q, which is used for further structure study. The ghrelin receptor was co-expressed with Gα q and Gβγ, and incubated with ghrelin in the presence of Nb35 to stabilize the receptor-G protein complex, allowing the efficient assembly of the ghrelin receptor–G q complex. The scFv16 was additionally added to assemble the GHRP-6–ghrelin receptor–G q–scFv16 complex.
The complex structures of the G q-coupled ghrelin receptor bound to ghrelin and GHRP-6 were determined by cryo-EM to the resolutions of 2.9 Å and 3.2 Å, respectively. For both ghrelin receptor–G q complexes, the majority of the amino acid side chains of receptor and G q protein were well-resolved in the final models, which are refined against the EM density map with excellent geometry. Both peptides, ghrelin (Gly 1P-Arg 15P) with octanoylated modification and GHRP-6, were clearly identified, thus providing reliable models for the mechanistic explanation of peptide recognition and activation of ghrelin receptor.
a, b Orthogonal views of the density map (a) and model (b) for the ghrelin–ghrelin receptor–G q–Nb35 complex. The density map is shown at 0.104 threshold. c, d Orthogonal views of the density map (c) and model (d) for the GHRP-6–ghrelin receptor–G q–Nb35–scFv16 complex. The density map is shown at 0.1 threshold. e Structural superposition of ghrelin-bound and GHRP-6-bound ghrelin receptors. f Binding poses of ghrelin and GHRP-6. Two peptides occupy a similar orthosteric binding pocket with opposite orientation. g Binding pocket of ghrelin receptor is bifurcated into two cavities by a salt bridge between E124 3.33 and R283 6.55. Salmon oval, cavity I; yellow oval, cavity II. Ghrelin is shown in magenta, ghrelin-bound ghrelin receptor in slate blue. GHRP-6 is displayed in green, and GHRP-6 bound ghrelin receptor in salmon. Compound 21-bound ghrelin receptor (PDB: 6KO5) is colored gray. The G q heterotrimer is colored by subunits. Gα q: peru, Gβ: light sky blue, Gγ: sea green, Nb35: orchid, scFv16: light pink.
Both ghrelin–ghrelin receptor–G q and GHRP-6–ghrelin receptor–G q complexes present canonical folds of seven transmembrane segments with the TMD of the receptors surrounded by an annular detergent micelle mimicking the natural phospholipid bilayer. Within the micelle, two cholesterols are clearly visible and hydrophobically bind around the helix bundles of both ghrelin receptor complexes. Both complexes display highly identical overall conformations with the root mean square deviation (RMSD) of 0.8 Å for all atoms.
