Structural Determinants of Binding the Seven-transmembrane Domain of the Glucagon-like Peptide-1 Receptor (GLP-1R)

Abstract

The glucagon-like peptide-1 receptor (GLP-1R) belongs to the secretin-like (class B) family of G protein-coupled receptors. Members of the class B family are distinguished by their large extracellular domain, which works cooperatively with the canonical seven-transmembrane (7TM) helical domain to signal in response to binding of various peptide hormones. We have combined structure-based site-specific mutational studies with molecular dynamics simulations of a full-length model of GLP-1R bound to multiple peptide ligand variants. Despite the high sequence similarity between GLP-1R and its closest structural homologue, the glucagon receptor (GCGR), nearly half of the 62 stably expressed mutants affected GLP-1R in a different manner than the corresponding mutants in GCGR. The molecular dynamics simulations of wild-type and mutant GLP-1R·ligand complexes provided molecular insights into GLP-1R-specific recognition mechanisms for the N terminus of GLP-1 by residues in the 7TM pocket and explained how glucagon-mimicking GLP-1 mutants restored binding affinity for (GCGR-mimicking) GLP-1R mutants. Structural analysis of the simulations suggested that peptide ligand binding mode variations in the 7TM binding pocket are facilitated by movement of the extracellular domain relative to the 7TM bundle. These differences in binding modes may account for the pharmacological differences between GLP-1 peptide variants.

Keywords: G protein-coupled receptor (GPCR); glucagon; molecular dynamics; peptide interaction; site-directed mutagenesis.

FIGURE 1.

Differential effects of human GLP-1R and human GCGR point mutations on GLP-1 and glucagon binding. A, comparison of the effects of 66 GLP-1R point mutations on GLP-1 binding and 66 GCGR mutations on glucagon binding, based on IC₅₀ values in ¹²⁵I-GLP-1 (for GLP-1R) and ¹²⁵I-glucagon (for GCGR) displacement assays (including 50 GCGR mutants determined previously (11)). Mutated residues that had <4-fold change (blue), 4–10-fold increase (orange), and >10-fold increase (red) in IC₅₀ values for GLP-1 binding to GLP-1R or glucagon binding to GCGR are shown. Mutant receptors with expression <30% of wild-type are colored gray. For each position, the results of mutations that show the most distinct differences between GLP-1R and GCGR are reported (see Table 1). The effects of previously reported human GLP-1R mutants are presented in supplemental Table S1 (7, 8, 19, 20, 25, 28–30, 32) and rat GLP-1R mutation data in supplemental Table S2 (26, 27). The most conserved residues in TM helices 1–7 of class B GPCRs (19) are boxed. B–D, representative ¹²⁵I-GLP-1/GLP-1R (B and D) and ¹²⁵I-glucagon/GCGR (C) displacement dose-response curves. Data are expressed as a percentage of specific ¹²⁵I-GLP-1 or ¹²⁵I-glucagon binding in the presence of 3.57 pm unlabeled peptide. Each point (±S.E.) represents the mean value of at least three independent experiments done in triplicate (IC₅₀ data presented in Table 1 and supplemental Tables S1 and S3).

FIGURE 2.

GLP-1R structural model explaining GLP-1R/GCGR-specific and ligand-dependent site-directed mutagenesis data. Mutations of homologous residues/positions in GLP-1R and GCGR but with differential effects on GLP-1 binding to GLP-1R versus glucagon binding to GCGR (Fig. 1A and supplemental Table S1) are mapped on a three-dimensional GLP-1R model and color-coded according their relative effects (ΔIC₅₀) on GLP-1R versus GCGR binding as follows: >4-fold relative decrease (cyan), 4–10-fold relative increase (orange), and >10-fold relative increase (red) in IC₅₀ values for GLP-1R mutant compared with the corresponding GCGR mutants. ΔIC₅₀, the effect of a point mutant on binding of GLP-1 to GLP-1R or glucagon to GCGR, is defined as the ratio of IC₅₀ values of wild-type and mutant. The relative effect of a mutation on ligand binding to GLP-1R versus GCGR is defined as the ratio of ΔIC₅₀ (GLP-1R) and ΔIC₅₀ (GLP-1R) values for the same residue position.

FIGURE 3.

Probing the 7TM-binding site of GLP-1R with truncated peptide ligands. A, top view of effects of GLP-1R mutations on ¹²⁵I-exendin-(9–39) displacement by GLP-1 (dark green), truncated GLP-1-(15–36), and exendin-4 (light green) (see Table 2) mapped on the three-dimensional GLP-1R model, color-coded according their effects (ΔIC₅₀) on GLP-1R binding as follows: >4-fold decrease (cyan); 4-fold decrease to 4-fold increase (blue); 4–10-fold increase (orange); and >10-fold increase (red) in IC₅₀ values for GLP-1R mutants. ΔIC₅₀, the effect of a point mutant on binding of a specific peptide ligand to GLP-1, is defined as the ratio of IC₅₀ values of wild-type and mutant. B, representative ¹²⁵I-exendin-(9–39) displacement dose-response curves of GLP-1, exendin-4, and GLP-1-(15–36) at wild-type and mutant GLP-1R mutants used to derive IC₅₀ data presented in Table 2. Data are expressed as a percentage of specific binding in the presence of 3.57 pm unlabeled peptide. Each point (±S.E.) represents the mean value of at least three independent experiments done in triplicate (IC₅₀ data presented in Table 2 and supplemental Table S4).

FIGURE 4.

Analysis of receptor-ligand interactions in GLP-1-bound GLP-1R and glucagon-bound GCGR. Comparison of 1000-ns MD simulations of GLP-1 (green) in GLP-1R (A–C) and glucagon (light green) in GCGR (D–F). Based on the analysis of receptor-ligand interaction patterns in GLP-1R (C) and GCGR (F), the structures of two representative snapshots at similar time frames (MD snapshots 1 and 2, indicated by green arrows and lines) of GLP-1R (A and B) and GCGR (D and E) MD simulations are shown. The analyses of H-bond interactions between positively and negatively charged groups (red), other H-bond interactions in which backbone groups (black) or only side-chain groups (gray) are involved, and apolar contacts (yellow) of specific residues in GLP-1/GLP-1R (C) and between specific residues in glucagon/GCGR (F) are shown.

FIGURE 5.

MD simulations explain combined GLP-1R and GLP-1 mutation studies. GLP-1 binding to GLP-1R mutants R190^2.60bK (A), E387^7.42bD (D), and Q234^3.37bE (G) is restored by combination with glucagon-mimicking GLP-1 mutants Ala⁸Ser (B) and Glu⁹Gln (E and H), is determined by ¹²⁵I-exendin-(9–39) displacement studies (C, F, and I), and is consistent with representative snapshots (A, B, D, E, G, and H) and interaction analyses (C, F, and I) of MD simulations studies (supplemental Fig. S3). Data in ¹²⁵I-exendin-(9–39) displacement curves (C, F, and I) are expressed as a percentage of specific ¹²⁵I-exendin-(9–39) binding in the presence of 3.57 pm unlabeled peptide. Each point (±S.E.) represents the mean value of at least three independent experiments done in triplicate (IC₅₀ data presented in Table 3 and supplemental Table S5).

FIGURE 6.

Alternative orientations of the ECD with respect to the 7TM domain accommodate different binding modes of glucagon-mimicking GLP-1 mutants in GCGR-mimicking GLP-1R mutants. Overlays of representative MD simulation snapshots of Ala⁸Ser GLP-1 mutant-bound E387^7.42bD GLP-1R mutant (A), Glu⁹Gln GLP-1 mutant-bound R190^7.42bK GLP-1R mutant (B), and Glu⁹Gln GLP-1 mutant-bound Q234^3.37bE GLP-1R mutant with wild-type (WT) GLP-1-bound GLP-1R (C) are shown. The GLP-1 mutant (green) and ECD (magenta) and 7TM domain (light yellow) of the GLP-1R mutants are colored to allow comparison with the WT GLP-1 and GLP-1R (dark gray). The structures are superimposed onto the original model of the GLP-1R·GLP-1 complex using the main-chain atoms of residues I146^1.41b–I161^1.56b (TM1), F184^2.53b–F195^2.65b (TM2), L231^3.34b–L245^3.48b (TM3), S271^4.47b–P283^4.59b (TM4), L311^5.41b–R326^5.56b (TM5), K351^6.40b–G361^6.50b (TM6), and F^7.48b–I400^7.55b (TM7). The side views of the receptor·ligand complexes at the top of each panel are similar to the view presented in Figs. 2 and 3. The extracellular views of the receptor·ligand complexes presented in the middle of each panel are rotated vertically 180° clockwise and horizontally 90° counter-clockwise, compared with the top view, and are compatible with the XY plane shown at the bottom of each panel, which shows the average positions (and S.D. error bars) of the COM projections of the extracellular end of transmembrane helices (open squares), the ECD (R²⁴–K¹³⁰) (filled circles), and the helical part of the peptide ligands (S¹⁴–V³³) (filled squares) on the XY plane in the last 500-ns simulations of different receptor-ligand systems.