Molecular features of the ligand-free GLP-1R, GCGR and GIPR in complex with Gs proteins

Abstract

Class B1 G protein-coupled receptors (GPCRs) are important regulators of many physiological functions such as glucose homeostasis, which is mainly mediated by three peptide hormones, i.e., glucagon-like peptide-1 (GLP-1), glucagon (GCG), and glucose-dependent insulinotropic polypeptide (GIP). They trigger a cascade of signaling events leading to the formation of an active agonist-receptor-G protein complex. However, intracellular signal transducers can also activate the receptor independent of extracellular stimuli, suggesting an intrinsic role of G proteins in this process. Here, we report cryo-electron microscopy structures of the human GLP-1 receptor (GLP-1R), GCG receptor (GCGR), and GIP receptor (GIPR) in complex with G_s proteins without the presence of cognate ligands. These ligand-free complexes share a similar intracellular architecture to those bound by endogenous peptides, in which, the G_s protein alone directly opens the intracellular binding cavity and rewires the extracellular orthosteric pocket to stabilize the receptor in a state unseen before. While the peptide-binding site is partially occupied by the inward folded transmembrane helix 6 (TM6)-extracellular loop 3 (ECL3) juncture of GIPR or a segment of GCGR ECL2, the extracellular portion of GLP-1R adopts a conformation close to the active state. Our findings offer valuable insights into the distinct activation mechanisms of these three important receptors. It is possible that in the absence of a ligand, the intracellular half of transmembrane domain is mobilized with the help of G_s protein, which in turn rearranges the extracellular half to form a transitional conformation, facilitating the entry of the peptide N-terminus.

Fig. 1. Cryo-EM structures of the ligand-free GLP-1R, GCGR, and GIPR in complex with G_s proteins.

a, c, e Cryo-EM density maps of the GLP-1R–G_s (a), GCGR–G_s (c), and GIPR–G_s (e) complexes are shown from two viewpoints. GLP-1R, GCGR, GIPR, Gα_s, Gβ, Gγ, and Nb35 are shown in orange, pale green, hot pink, slate gray, powder blue, khaki and light gray, respectively. The maps reveal several lipid densities around the receptors consistent with the shape of cholesterol (olive), palmitate (brown) or phosphatidylinositol 4,5-bisphosphate (purple). There is a strong unassigned cylindrical density in the shape of helix (green) dropping down along the TM4 and TM5 of GCGR, tentatively named as chain X (c). b, d, f Structural models of the GLP-1R–G_s (b), GCGR–G_s (d) and GIPR–G_s (f) complexes are constructed from the respective cryo-EM maps and shown in ribbon.

Fig. 2. General features of the ligand-free receptors by G_s coupling.

a–c Comparison of ligand-free and peptide-bound GLP-1R (a), GCGR (b) and GIPR (c) with inactive receptor structures reveals a similar G_s coupling interface in the absence or presence of an agonist. Black arrows show the movements of TM6 and H8 by indicating the distances of Cα atoms of T^6.42b, R^6.37b, and R/H^8.60b residues. d–f Structural comparison of the TMD bundles of GLP-1R (d), GCGR (e) and GIPR (f) indicates the requirement of G_s protein for receptor activation. Conformational changes are shown for the conserved HETY inactive motif (H^2.50b–E^3.50b–T^6.42b–Y^7.57b) and cytoplasmic polar network (R^2.46b–R^6.37b–N^7.61b–E^8.49b). Black arrows indicate the hallmark conformational changes of TM6. The Gβ and Gγ subunits are omitted for clarity. PDB IDs: 6LN2 (inactive GLP-1R), 5XEZ (inactive GCGR), 6X18 (GLP-1-bound GLP-1R), 6LMK (GCG-bound GCGR), and 7DTY (GIP-bound GIPR). The position of N^7.61b in d, e and f refers to N^8.47b in GPCRdb numbering.

Fig. 3. G_s coupling induced rearrangements in the extracellular portion of TMD.

a Structural superimposition of ligand-free and GLP-1-bound GLP-1R–G_s (PDB: 6X18) complexes from side and top views, as well as inactive GLP-1R (PDB: 6LN2) and peptide 5-bound GLP-1R (PDB: 5NX2) from top view, shows different conformational changes induced by agonist binding with or without G_s protein coupling. Gray arrows indicate the movements of TM1, TM6 and TM7 measured by the Cα atoms of E138^1.33b, V370^6.59b and T378^7.33b, respectively. b Structural superimposition of ligand-free and GCG-bound GCGR–G_s (PDB: 6LMK) complexes from side and top views, as well as inactive GCGR (PDB: 5XEZ) and NNC1702-bound GCGR (PDB: 5YQZ) from top view, shows different conformational changes induced by agonist binding with or without G_s protein coupling. Gray arrows indicate the movements of TM1, TM6, and TM7 measured by the Cα atoms of K136^1.34b, V368^6.59b, and T376^7.33b, respectively. c Structural superimposition of ligand-free and GIP-bound GIPR–G_s (PDB: 7DTY) complexes from side and top views shows a unique conformational change in the extracellular region induced by GIP binding. Gray arrows indicate the movements of TM1, TM6 and TM7 measured by the Cα atoms of L128^1.30b, V356^6.55b, and A368^7.33b, respectively. d Superimposition of ligand-free GLP-1R, GCGR and GIPR structures reveals dynamics of the ECLs. The conformational differences are indicated by two-way arrows. e Structures of ligand-free and endogenous ligand-bound receptors are superimposed onto the inactive GLP-1R or GCGR structure, showing different TM6 kink conformations. The kink angle is shown among the Cα atoms of V^6.59b–P^6.47b–D^6.33b in TM6. G_s proteins are omitted for clarity.

Fig. 4. ECL2 of GCGR and ECL3 of GIPR occupy the classic orthosteric binding site for peptide.

a Superimposition of ligand-free and GCG-bound GCGR structures reveals that the GCG-binding pocket is partially occupied by inward moved ECL2 upon G protein coupling. GCG-bound GCGR (PDB: 6LMK) is shown in surface representation. ECL2 residues of ligand-free GCGR are shown in sphere. b Superimposition of ligand-free and GIP-bound GIPR structures reveals that the GIP-binding pocket is partially occupied by inward moved TM6–ECL3 juncture upon G protein coupling. GIP-bound GIPR (PDB: 7DTY) is shown in surface representation. Residues in the TM6–ECL3 juncture of ligand-free GIPR are shown in sphere. c Sequence alignment of ECL2 and ECL3 of GCGR, GLP-1R and GIPR. d Magnified view of the ECL2 within the orthosteric binding pocket (left panel) and its interaction with GCG (right panel). e Magnified view of the TM6–ECL3 juncture in the orthosteric binding pocket (left panel) and its interaction with GIP (right panel). Key interacting residues and their contacts are shown as sticks and dashed lines, respectively.

Fig. 5. Mechanistic implication of ligand-free and G_s-coupled GLP-1R, GCGR and GIPR complex structures.

a Superimposition of inactive GLP-1R and GCGR structures shows an extracellular shutting conformation (yellow shadow) and intracellular closed conformation (gray shadow), thus intercepting peptide binding and G_s coupling. b Superimposition of ligand-free GLP-1R, GCGR and GIPR structures shows that G_s coupling directly causes the formation of an intracellular cavity and stabilizes the intracellular half of TMD, whereas the extracellular portion of the receptor is stabilized in the central and outer layers. c, d Magnified views of different contacting modes in the central (c) and outer (d) layers of GLP-1R, GCGR and GIPR. Key interacting residues are shown as ball-and-sticks, and the interactions are shown as dashed lines. e Superimposition of endogenous ligand-bound GLP-1R, GCGR and GIPR structures shows that peptide binding stabilizes the TM6–ECL3–TM7 conformation and rewires G_s protein. The right panel shows magnified views of the M/K^5.33b–R^5.40b–T/D/E^ECL3 and L^6.48b–Y^7.57b–N^7.61b–E^8.49b–E392^GαH5 interfaces. Key interacting residues are shown as ball-and-sticks. PDB IDs: 6LN2 (inactive GLP-1R), 5XEZ (inactive GCGR), 6X18 (GLP-1-bound GLP-1R), 6LMK (GCG-bound GCGR) and 7DTY (GIP-bound GIPR). The position of N^7.61b in e refers to N^8.47b in GPCRdb numbering.