Crystal structure of glucagon-like peptide-1 in complex with the extracellular domain of the glucagon-like peptide-1 receptor

Abstract

GLP-1 (glucagon-like peptide-1) is an incretin released from intestinal L-cells in response to food intake. Activation of the GLP-1 receptor potentiates the synthesis and release of insulin from pancreatic beta-cells in a glucose-dependent manner. The GLP-1 receptor belongs to class B of the G-protein-coupled receptors, a subfamily characterized by a large N-terminal extracellular ligand binding domain. Exendin-4 and GLP-1 are 50% identical, and exendin-4 is a full agonist with similar affinity and potency for the GLP-1 receptor. We recently solved the crystal structure of the GLP-1 receptor extracellular domain in complex with the competitive antagonist exendin-4(9-39). Interestingly, the isolated extracellular domain binds exendin-4 with much higher affinity than the endogenous agonist GLP-1. Here, we have solved the crystal structure of the extracellular domain in complex with GLP-1 to 2.1 Aresolution. The structure shows that important hydrophobic ligand-receptor interactions are conserved in agonist- and antagonist-bound forms of the extracellular domain, but certain residues in the ligand-binding site adopt a GLP-1-specific conformation. GLP-1 is a kinked but continuous alpha-helix from Thr(13) to Val(33) when bound to the extracellular domain. We supplemented the crystal structure with site-directed mutagenesis to link the structural information of the isolated extracellular domain with the binding properties of the full-length receptor. The data support the existence of differences in the binding modes of GLP-1 and exendin-4 on the full-length GLP-1 receptor.

FIGURE 1.

Structure of the GLP-1-bound ECD of the GLP-1R. A, stereoview of GLP-1 (blue) bound to the ECD of the GLP-1R (α-helix in black, β-strands in red, and loops in gray). Disulfide bridges are shown as orange sticks. Residues Cys⁶²–Asp⁶⁷ (β₁) and Ala⁷⁰–Gly⁷⁵ (β₂) constitute the first region of antiparallel β-sheets, and the second region is comprised of residues Gly⁷⁸–Ser⁸⁴ (β₃) and His⁹⁹–Thr¹⁰⁵ (β₄), which is shown in red. Our final structure contains GLP-1 residues Gly¹⁰*–Gly³⁵*. The residues that interact with GLP-1R ECD lie within Ala²⁴* and Val³³*, which are shown as sticks. B, sequence alignment of GLP-1, exendin-4, GIP, GLP-2, glucagon, and PACAP(1–27). Fully conserved residues are highlighted in yellow, and partially conserved residues are highlighted in green. The residues of GLP-1 and exendin-4 that interact with GLP-1R ECD are colored blue. The underlined residues symbolize residues of GLP-1 in α-helical conformation when bound to the ECD. Residue number 1 of exendin-4 corresponds to residue number 7 of GLP-1.

FIGURE 2.

Interactions between GLP-1 and GLP-1R ECD. A, ribbon diagram of GLP-1 and its hydrophilic interactions with GLP-1R ECD. GLP-1 is colored in cyan, and residues Gln²³*, Lys²⁶*, Glu²⁷*, Trp³¹*, and Val³³* are illustrated as sticks. Receptor residues Arg¹²¹, Leu¹²³, Glu¹²⁷, and Glu¹²⁸ are shown as sticks. The surface of the hydrophilic binding cavity of ECD is illustrated in gray. B, ribbon diagram of GLP-1 and its hydrophobic interactions with GLP-1R ECD. GLP-1 residues Ala²⁴*, Glu²⁷*, Phe²⁸*, Trp³¹*, and Leu³²* are illustrated as sticks, and so are ECD residues Leu³², Trp³⁹, Asp⁶⁷, and Arg¹²¹. The surface of the hydrophobic binding cavity of ECD is illustrated in gray. C, ribbon diagram illustrating a common motif found in the GLP-1R ECD and in the GIP receptor ECD. The side chain of Arg¹²¹ interacts with the backbone carbonyls of Asp⁶⁷ and Leu³²* through a water molecule. GLP-1 residues Leu³²* and Val³³* are illustrated as sticks, and so are ECD residues Asp⁶⁷ and Arg¹²¹.

FIGURE 3.

Differences between the GLP-1- and exendin-4(9–39)-bound structure of ECD. Ribbon diagrams showing significant differences in side chain conformations between the GLP-1-bound structure and the exendin-4(9–39)-bound structure of GLP-1R ECD. Receptor and ligand residues are highlighted in blue for the GLP-1-bound structure and in orange for the exendin-4(9–39)-bound structure. Water molecules in orange are present only in the exendin-4(9–39)-bound structure. A, one diverging residue, Val³³* of GLP-1 and Lys²⁷** of exendin-4(9–39), causes a shift in the conformations of four residues, namely Glu¹²⁷, Leu¹²³, Arg¹²¹, and Pro¹¹⁹. B, GLP-1-specific conformations affect the conserved core of the ECD by rotating the guanidine group of Arg¹⁰² and by decreasing the distance between Asp⁶⁷ and Arg¹⁰² compared with the exendin-4(9–39)-bound structure without affecting the relative position of Trp⁷² and Trp¹¹⁰.

FIGURE 4.

Functional and binding properties of the L32A GLP-1R mutant. Upper panel, stimulation of cAMP production by transiently transfected HEK293 cells expressing the L32A mutant by GLP-1 (squares, A) and exendin-4 (circles, B). Dashed dose-response curves represent cAMP production by GLP-1 and exendin-4 at the wild-type GLP-1R, respectively. Lower panel, competition binding assay on plasma membranes from transiently transfected HEK293 cells expressing the L32A mutant. GLP-1 binding curves are presented with squares (C) and exendin-4 curves with circles (D). Dashed binding curves represent ¹²⁵I-GLP-1 displacement by GLP-1 and exendin-4 at the wild-type GLP-1R. Data are normalized according to ¹²⁵I-GLP-1 binding and correspond to three independent experiments performed in duplicate.

FIGURE 5.

Crystal packing. A, superposition of ECD-bound GLP-1 (blue) and exendin-4(9–39) (cyan). GLP-1 residue Gly²²* denotes a kink in the α-helix, which is situated in close proximity to Leu³² of the ECD. B, crystal packing involving symmetry-related complex molecules. C, ribbon diagram of GLP-1 (blue) and its interactions with the ligand of a symmetry-related molecule. Residues Tyr¹⁹*, Gln²³*, and Glu²⁷* are shown as sticks, and the surface of the GLP-1R ECD is shown in gray. The packing of complex molecules allows Tyr¹⁹* to interact with Gln²³* (3 Å) and Glu²⁷* (2.5 Å) in a symmetry-related ligand molecule. D, interactions between GLP-1 and residues of symmetry-related ECD molecules. GLP-1 residues Thr¹¹*_, Thr¹³*, Ser¹⁴*, and Glu²¹* are shown as sticks. The backbone carbonyl of Thr¹¹* could form a weak hydrogen bond (3.2 Å) to the backbone amide of Gln¹¹², the backbone amide of Thr¹³* could form a hydrogen bond (2.9 Å) to the backbone carbonyl of Gln¹¹², and the backbone amide of Ser¹⁴* may form a hydrogen bond (3 Å) to the side chain of Asp¹¹⁴. The side chain of Glu²¹* forms a hydrogen bond to the backbone amide of Phe⁸⁰ (2.6 Å).