Molecular recognition of an acyl-peptide hormone and activation of ghrelin receptor

Ghrelin, also called “the hunger hormone”, is a gastric peptide hormone that regulates food intake, body weight, as well as taste sensation, reward, cognition, learning and memory. One unique feature of ghrelin is its acylation, primarily with an octanoic acid, which is essential for its binding and activation of the ghrelin receptor, a G protein-coupled receptor. The multifaceted roles of ghrelin make ghrelin receptor a highly attractive drug target for growth retardation, obesity, and metabolic disorders. Here we present two cryo-electron microscopy structures of G q -coupled ghrelin receptor bound to ghrelin and a synthetic agonist, GHRP-6. Analysis of these two structures reveals a unique binding pocket for the octanoyl group, which guides the correct positioning of the peptide to initiate the receptor activation. Together with mutational and functional data, our structures define the rules for recognition of the acylated peptide hormone and activation of ghrelin receptor, and provide structural templates to facilitate drug design targeting ghrelin receptor.

Structural determination We fused thermostabilized BRIL at the N-terminus of the ghrelin receptor and applied the NanoBiT tethering strategy to improve complex stability and homogeneity (Supplementary Fig. 1a, b ) 25 . These modifications have little effect on the pharmacological properties of the ghrelin receptor (Supplementary Fig. 2 ). 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 (Supplementary Fig. 1c ), and such an analogous approach had been used to obtain structures of the G q -bound 5-HT 2A receptor 26 and G 11 -bound M1 receptor 27 . 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 28 , 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 (Supplementary Fig. 3 ). 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 (Fig. 1a–d , Supplementary Fig. 3 , Supplementary Table 1 ). 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 (Supplementary Fig. 4 ). Fig. 1 Cryo-EM structures of the G q -coupled ghrelin receptor bound to ghrelin and GHRP-6. 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.

The N-terminal amino acids from Gly 1P to Pro 7P of ghrelin occupied nearly the entire receptor TMD binding pocket (Fig. 2a ). The peptide fragment from Glu 8P to Arg 15P is enriched with polar and charged amino acids and adopts an α-helical conformation, which sits above the orthosteric pocket and interacts with the solvent (Fig. 2a ). Fig. 2 The ghrelin-binding pocket of ghrelin receptor. a Cross-section of the ghrelin-binding pocket in ghrelin receptor. The cryo-EM density of ghrelin is highlighted. Ghrelin is shown in cartoon presentation. Side chains of the residues are displayed as sticks. b The binding pocket for octanoyl group. The cryo-EM density of the octanoyl group is shown. The hydrogen bonds are depicted as yellow dashed lines. c Effects of mutations of residues in the octanoyl group-binding pocket on calcium response. ΔpEC50 represents the difference between pEC 50 values of the mutant ghrelin receptor and the wild-type (WT) receptor. Data are presented as mean ± S.E.M. of three independent experiments performed in technical triplicate. *** P < 0.001, **** P < 0.0001 vs. WT receptor, NS, no significant difference. All data were analyzed by two-side, one-way ANOVA with Tukey’s test ( P < 0.0001, P = 0.0002, P < 0.0001, P = 0.9871, P < 0.0001, P = 0.9888, P < 0.0001, P = 0.0028, P = 0.0006, P = 0.0001, P < 0.0001, and P < 0.0001 from top to bottom). d , e Detailed interactions of ghrelin (Gly 1P -Pro 7P ) with residues in ghrelin-binding pocket. Ghrelin is shown in magenta, and ghrelin receptor in slate blue. The clearly visible map allows us to locate Ser 3P and its octanoyl group accurately. A density connects the side-chain of Ser 3P and stretches horizontally toward the gap between TM4 and TM5, occupying cavity II of the binding pocket. Five carbons of the eight-carbon fatty acid modification can be placed in the density (Fig. 2b ). F286 6.58 covers the side-chain of Ser 3P , while the fatty acid chain of ghrelin forms hydrophobic contacts with I178 4.60 and L181 4.63 (Figs. 2 b, 3e ). Most substitutions of I178 4.60 , L181 4.63 , and F286 6.58 with alanine or polar and charged amino acids (Ser, Asp, or Lys) display dramatically diminished receptor activation compared with wild-type (WT) ghrelin receptor (Fig. 2c , Supplementary Table 2 ). It is worth noting that F286 6.58 makes a greater contribution to ghrelin’s activity. Mutating F286 6.58 to Asp produces an over 250-fold decreased activity of ghrelin (Fig. 2c ), while mutating L178 4.60 or I181 4.63 to Asp diminishes the activities by only ~5-fold and 20-fold, respectively (Fig. 2c , Supplementary Table 2 ). These findings highlight the critical role of the potent receptor hydrophobic environment for binding of the octanoyl group. These results are consistent with the previous report that replacement of Ser 3P by a charged amino acid (Lys) or small hydrophobic residues (Val, Leu, or Ile) significantly decreased ghrelin’s activity 29 . Conversely, replacing Ser 3P with aromatic amino acids, such as Trp or β-Nal (2-naphtylalanine), preserved ghrelin’s activity 29 . Fig. 3 The GHRP-6-binding pocket of ghrelin receptor. a Cross-section of the GHRP-6-binding pocket in ghrelin receptor. The cryo-EM density of GHRP-6 is highlighted. GHRP-6 is shown in a cartoon presentation. Side chains of the residues are displayed as sticks. b , c Detailed interactions of GHRP-6 with residues in GHRP-6 binding pocket. The binding site of D-Trp 2P , Trp 4P , Lys 6P ( b ) and D-Phe 5P ( c ) are shown. The hydrogen bond is depicted as a yellow dashed line. GHRP-6 is shown in green, and ghrelin receptor in salmon. d Effects of mutations of residues in D-Phe 5P binding pocket on calcium response. ΔpEC50 represents the difference between pEC 50 values of the mutant ghrelin receptor and the wild-type (WT) receptor. Data are presented as mean ± S.E.M. of three independent experiments performed in technical triplicate. *** P < 0.001, **** P < 0.0001 vs. WT receptor, NS, no significant difference. All data were analyzed by two-side, one-way ANOVA with Tukey’s test ( P < 0.0001, P < 0.0001, P < 0.0001, P < 0.0001, P < 0.0001, P = 0.0009, P < 0.0001, P < 0.0001, P = 0.6335, P < 0.0001, P = 0.8310, and P < 0.0001 from top to bottom). e Interaction residues in the ligand-binding pocket of the ghrelin–ghrelin receptor–G q and the GHRP-6–ghrelin receptor–G q complexes. Compared with WT receptor, the alanine replacement of residues showed comparable impacts on peptide’s activity are indicated by white circles. The alanine mutation of residues showed 2-10-fold, 10-100-fold, 100-1000-fold, and over 1000-fold decreased peptide’s activity are indicated by yellow, green, pink, and salmon circles, respectively. Besides these hydrophobic residues surrounding the octanoyl group, other pocket residues also make substantial contributions to ghrelin binding and receptor activation. R283 6.55 forms a stabilizing salt bridge with E

Structural comparison of two G q -coupled ghrelin receptors with other G q/11 -coupled class A GPCRs reveals similar receptor conformations. TM6 and TM7 of ghrelin receptor adopt nearly identical conformations to G q -coupled 5-HT 2A receptor and G 11 -coupled M1 receptor (Supplementary Fig. 8a, b ). Unlike M1R, the cytoplasmic end of TM5 of the ghrelin receptor does not show an inward movement (Supplementary Fig. 8a ). In addition, the Gα α5 helix displays slightly shifts and the distal end of Gα q α5 helix shows more notable shifts across these receptor complexes (Supplementary Fig. 8c ). Furthermore, structural comparison of two G q -coupled ghrelin receptors with the antagonist-bound ghrelin receptor (PDB: 6KO5) 18 supports the contention that these two complexes are indeed in the active state (Fig. 4a, b ). These two ghrelin receptor complexes display pronounced outward displacements of TM6 cytoplasmic end (~8 Å, measured at Cα of H258 6.30 ), the hallmark of GPCR activation, and ~3 Å inward shift of cytoplasmic end of TM7 (measured at Cα of Y323 7.53 ) (Fig. 4a, b ). Fig. 4 Activation mechanism of ghrelin receptor. a , b Structural superposition of the active and antagonist-bound ghrelin receptor. ( a ) side view; ( b ) cytoplasmic view. c , d Conformational changes of the salt bridge comprising of E124 3.33 and R283 6.55 upon peptide activation. Ghrelin and GHRP-6 push the side-chain of R283 6.55 to swing away from the receptor helical core due to steric clash. The swing orientations of E124 3.33 and R283 6.55 were indicated by red arrows. The clashes were highlighted as red oval dashed lines. e-h Conformational changes of the conserved “micro-switches” upon receptor activation. ( e ) Toggle switch, ( f ) ERY motif, ( g ) PIF motif, ( h ) NPxxY motif. The outward displacement of TM6 of the active receptor is shown as a red arrow ( e ). The conformational changes of residue side chains are shown as red arrows upon receptor activation. i The hydrophobic cluster comprising F279 6.51 , F309 7.39 , F312 7.42 , and Y313 7.43 stabilizes the inter-helical hydrophobic contacts between TM6 and TM7. 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. The compound 21-bound ghrelin receptor (PDB: 6KO5), is colored in gray. Although ghrelin and GHRP-6 differ in their chemical scaffolds, they induce the activation of ghrelin receptor through similar conformational changes in TM6 as well as TM7. Compared to the antagonist-bound receptor, the extracellular end of TM7 of these two active receptors laterally move ~5 Å towards TM6 (measured at Cα of Q302 7.32 ), presenting a unique TM7 conformational change across class A GPCRs solved to date (Fig. 4a , Supplementary Fig. 9 ). The notable movement of the extracellular end of TM7 may translate to the inward shift of its cytoplasmic end. Furthermore, structural comparison of both active and antagonist-bound receptors supports that R283 6.55 is the determinant for peptide-induced ghrelin receptor activation. The salt bridge between R283 6.55 and E124 3.33 exists in both antagonist-bound and active ghrelin receptor structure, however, in contrast to antagonist compound 21, ghrelin or GHRP-6 pushes the side-chain of R283 6.55 to swing away from the receptor helical core, which induces rotation of TM6 that initiates the cascade of conformational changes in receptor activation (Fig. 4c, d ). Concomitantly, the swing of R283 6.55 leads to the rotameric switches of F279 6.51 and W276 6.48 , a residue in conserved “micro-switch”. These conformational changes lead to the swing of F272 6.44 and the pronounced outward displacement of the cytoplasmic end of TM6 (Fig. 4e ). The other conserved residues in “micro-switches” (ERY, PIF, and NPxxY) also undergo active-like conformational changes relative to the antagonist-bound receptor and transmit the peptidic agonism signaling to the cytoplasmic end to facilitate receptor-G protein coupling. (Fig. 4f-h ). Meanwhile, the conformational changes of F279 6.51 and W276 6.48 also cause rotameric switches of F312 7.42 and Y313 7.43 and the repacking of the inter-helical hydrophobic contacts between TM6 and TM7, which leads to the inward shift of the cytoplasmic end of TM7 (Fig. 4i ). Our structures also provide clues for understanding the mechanism of the basal activity of the ghrelin receptor. The hydrophobic residues F279 6.51 , F312 7.42 , and Y313 7.43 are closely packed and form a “hydrophobic lock” to packing TM6 and TM7 (Fig. 4i ). As aforementioned, these hydrophobic residues primarily transmit peptide agonism signaling with limited impact on peptide binding. This finding raises a hypothesis that this hydrophobic lock may be responsible for the basal activity of the receptor. This hypothesis is further supported by our mutagenesis analysis that substituting F279 6.51 and F312 7.42 with alanine dramatically diminishes the basal activity of

Construct cloning The full-length human ghrelin receptor (residues 1–366) was sub-cloned into pFastBac1 vector with an N-terminal haemagglutinin signal peptide (HA) and His×10 tag followed by BRIL epitope, as well as LgBiT at the C-terminus to facilitate the protein expression and stability. The ghrelin receptor sequence had no additional mutations or loop deletions. The Gα q was designed into a multifunctional chimera based on mini-Gα s skeleton with G i1 N-terminus for the binding of Nb35 and scFv16, respectively. Gα q , rat Gβ1 with C-terminal HiBiT connected with a 15 residues linker, bovine Gγ2 and Ric8A were cloned into pFastBac1 vector, respectively. All constructs were prepared using homologous recombination (CloneExpress One Step Cloning Kit, Vazyme).

Cell pellets were thawed at R.T. and resuspended in 20 mM HEPES pH 7.5, 100 mM NaCl, 10 mM MgCl 2 , 5 mM CaCl 2 , 2 mM KCl, 0.1 mM TCEP, supplemented with Protease Inhibitor Cocktail (TargetMol, 1 mL/100 mL suspension). ScFv16 was applied to stabilize GHRP-6–ghrelin receptor–G q complex, and Nb35 was used to improve the stability of both ghrelin receptor–G q complexes. The monomeric scFv16 and Nb35 were prepared as previously reported 28 , 31 . Both ghrelin receptor–G q complexes were formed on the membrane in the presence of 10 μM ligands (ghrelin or GHRP-6, synthesized by GenScript) and treated with apyrase (25 mU/ml, NEB), Nb35-His (15 μg/ml), or scFv16 followed by incubation for 1 h at R.T. The suspension was then solubilized by 0.5% (w/v) lauryl maltose neopentyl glycol (LMNG, Anatrace) with 0.1% (w/v) cholesteryl hemisuccinate TRIS salt (CHS, Anatrace) for 3 h. Insoluble material was removed by centrifugation at 65,000 g for 40 min and the solubilized complex was incubated overnight at 4 °C with pre-equilibrated Nickel resin (Ni Smart Beads 6FF, SMART Lifesciences) containing 10 mM imidazole. The resin was washed with 15 column volumes of Wash Buffer 1 containing 20 mM HEPES pH 7.5, 100 mM NaCl, 2 mM MgCl 2 , 15 mM imidazole, 0.1% LMNG, 0.02% CHS, 10 μM ligands (ghrelin or GHRP-6) and 15 column volumes of Wash Buffer 2 containing 20 mM HEPES, pH 7.5, 100 mM NaCl, 2 mM MgCl 2 , 25 mM imidazole, 0.01% LMNG, 0.005% GDN (Anatrace), 0.003% CHS, 10 μM ligands (ghrelin or GHRP-6). The complex was then eluted with 5 column volumes of Elution Buffer containing 20 mM HEPES, pH 7.5, 100 mM NaCl, 250 mM imidazole, 2 mM MgCl 2 , 0.01% LMNG, 0.005% GDN, 0.003% CHS and 10 μM ligands (ghrelin or GHRP-6). The complex was concentrated to 0.5 ml using Ultra Centrifugal Filter (ThermoFisher MWCO, 100 kDa) and subjected to size-exclusion chromatography on a Superdex 200 Increase 10/300 column (GE Healthcare) that was pre-equilibrated with 20 mM HEPES pH 7.5, 100 mM NaCl, 2 mM MgCl 2 , 5 μM ligands, 0.00075%(w/v) LMNG, 000025% glyco-diosgenin (GDN, Anatrace) and 0.00015% (w/v) CHS to separate complex from contaminants. Eluted fractions that consisted of receptor and G protein complex were pooled and concentrated to approximately 10 mg/ml for electron microscopy experiments.

Dose-fractionated image stacks for the ghrelin-bound ghrelin receptor–Gα q complex were subjected to beam-induced motion correction using Motion-Cor2.1 32 . Contrast transfer function (CTF) parameters for each micrograph were determined by Ctffind4 33 . Particle selection, 2D and 3D classifications of the ghrelin-bound ghrelin receptor–G q complex were performed on a binned data set with a pixel size of 2.09 Å using RELION-3.0-beta2 34 . For the ghrelin-bound ghrelin receptor–Gα q complex, semi-automated particle selection yielded 4,598,528 particle projections. The projections were subjected to 2D classification to discard particles in poorly defined classes, producing 1,842,606 particle projections for further processing. The map of the D1R–G s complex low-pass filtered to 40 Å was used as a reference model for four rounds of maximum-likelihood-based 3D classifications, resulting in one well-defined subset with 912,636 projections. A map generated by 3D refinement was subsequently post-processed in DeepEMhancer 35 . The final refinement generated a map with an indicated global resolution of 2.9 Å at a Fourier shell correlation of 0.143. For the GHRP-6-bound ghrelin receptor–Gα q complex, semi-automated particle selection yielded 2,728,266 particle projections. The projections were subjected to 2D classification, producing 1,523,752 particle projections for further processing. The map of the D1R–G s complex low-pass filtered to 40 Å was used as a reference model for three rounds of maximum-likelihood-based 3D classifications, resulting in two well-defined subsets with 262,892 projections. A map generated by 3D refinement was subsequently post-processed in DeepEMhancer 35 . The final refinement generated a map with an indicated global resolution of 3.2 Å at a Fourier shell correlation of 0.143. Local resolution for both density maps was determined using the Bsoft package with half maps as input maps 36 .

Ligand binding was performed with a homogeneous time-resolved fluorescence-based assay. N-terminal-SNAP-tagged ghrelin receptor (WT or with various mutations) and full-length ghrelin labeled with the dye A2 on an additional cysteine at the C-terminal end of the peptide (ghrelin-A2, synthesized by Vazyme, China) were used as previously described 41 . HEK293 cells transfected with SNAP-ghrelin receptor (WT or mutants) were seeded at a density of 1 × 10 6 cells into 3 cm dish and incubated for 24 h at 37 °C in 5% CO 2 . Cell culture medium was removed and Tag-lite labeling medium with 100 nM of SNAP-Lumi4-Tb (Cisbio, SSNPTBC) was added, and the cells were further incubated for 1 h at 37 °C in 5% CO 2 . The excess of SNAP-Lumi4-Tb was then removed by washing 4 times with 1 ml of Tag-lite labeling medium. For saturation binding experiments, we incubated cells with increasing concentrations of ghrelin-A2 in the presence or absence of 10 μM unlabeled ghrelin for 1 h at R.T. Signal was detected using the Multimode Plate Reader (PerkinElmer EnVision) equipped with an HTRF optic module allowing a donor excitation at 340 nm and a signal collection both at 665 nm and at 620 nm. HTRF ratios were obtained by dividing the acceptor signal (665 nm) by the donor signal (620 nm). Kd values were obtained from binding curves using Prism 8.0 software (GraphPad Software).

Cell-surface expression for each mutant was monitored by a fluorescence-activated cell sorting (FACS) assay. In brief, the expressed cells were incubated with mouse anti-HA-FITC antibody (Sigma) at a dilution of 1:200 for 20 min at 4 °C, and then a 9-fold excess of PBS was added to cells. Finally, the surface expression of the ghrelin receptor was monitored by detecting the fluorescent intensity of FITC using a BD ACCURI C6. The FACS data were analyzed by BD Accuri C6 software 1.0.264.21. The gating strategy and calculation method of expression are shown in Supplementary Fig. 11 .

All functional study data were analyzed using Prism 8 (GraphPad) and presented as means ± S.E.M. from at least three independent experiments. Concentration-response curves were evaluated with a three-parameter logistic equation. EC 50 is calculated with the Sigmoid three-parameter equation. The significance was determined with two-side, one-way ANOVA with Tukey’s test, and P < 0.05 vs. wild type (WT) was considered statistically significant.

The online version contains supplementary material available at 10.1038/s41467-021-25364-2.