Differential Structural Properties of GLP-1 and Exendin-4 Determine Their Relative Affinity for the GLP-1 Receptor N-Terminal Extracellular Domain

Abstract

Glucagon-like peptide-1 (GLP-1) and exendin-4 (Ex4) are homologous peptides with established potential for treatment of type 2 diabetes. They bind and activate the pancreatic GLP-1 receptor (GLP-1R) with similar affinity and potency and thereby promote insulin secretion in a glucose-dependent manner. GLP-1R belongs to family B of the seven transmembrane G-protein coupled receptors. The N-terminal extracellular domain (nGLP-1R) is a ligand binding domain with differential affinity for Ex4 and GLP-1: low affinity for GLP-1 and high affinity for exendin-4. The superior affinity of nGLP-1R for Ex4 was previously explained by an additional interaction between nGLP-1R and the C-terminal Trp-cage of Ex4. In this study we have combined biophysical and pharmacological approaches thus relating structural properties of the ligands in solution to their relative binding affinity for nGLP-1R. We used both a tracer competition assay and ligand-induced thermal stabilization of nGLP-1R to measure the relative affinity of full length, truncated, and chimeric ligands for soluble refolded nGLP-1R. The ligands in solution and the conformational consequences of ligand binding to nGLP-1R were characterized by circular dichroism and fluorescence spectroscopy. We found a correlation between the helical content of the free ligands and their relative binding affinity for nGLP-1R, supporting the hypothesis that the ligands are helical at least in the segment that binds to nGLP-1R. The Trp-cage of Ex4 was not necessary to maintain a superior helicity of Ex4 compared to GLP-1. The results suggest that the differential affinity of nGLP-1R is explained almost entirely by divergent residues in the central part of the ligands: Leu10-Gly30 of Ex4 and Val16-Arg36 of GLP-1. In view of our results it appears that the Trp-cage plays only a minor role for the interaction between Ex4 and nGLP-1R and for the differential affinity of nGLP-1R for GLP-1 and Ex4.

Crystal Structure of the Ligand-bound Glucagon-like Peptide-1 Receptor Extracellular Domain

“…Crystallization and Structure Determination-The nGLP-1R was expressed in E. coli inclusion bodies, refolded, and purified as described previously (24). We synthesized a selenium derivative of the ligand, where Met 14 and Leu 21 were substituted by SeMet: [SeMet 14,21 ]Ex4(9 -39).…”

“…Protein and Peptide Preparation-The nGLP-1R was prepared as earlier described (24). Briefly, N-terminal His 6 -tagged nGLP-1R was expressed in Escherichia coli inclusion bodies, isolated as inclusion body protein, solubilized in guanidine HCl and dithiothreitol, dialyzed against guanidine HCl to remove the dithiothreitol, and refolded using L-Arg and a 1:5 molar ratio of reduced and oxidized glutathione.…”

“…The Trp cage was suggested to interact with nGLP-1R, thereby explaining the superior affinity of Ex4 compared with GLP-1 (23). However, recent results suggest that the Trp cage plays only a minor role in receptor binding (24). Instead, the superior affinity of Ex4 was explained concomitantly by its superior ␣-helical propensity in solution and by superior interactions, due to specific divergent residues in the C-terminal part of GLP-1 and Ex4 (24).…”

“…However, recent results suggest that the Trp cage plays only a minor role in receptor binding (24). Instead, the superior affinity of Ex4 was explained concomitantly by its superior ␣-helical propensity in solution and by superior interactions, due to specific divergent residues in the C-terminal part of GLP-1 and Ex4 (24). Therefore, the role of the Trp cage in receptor binding is not crystal clear.…”

Isolation of Positive Modulator of Glucagon-like Peptide-1 Signaling from Trigonella foenum-graecum (Fenugreek) Seed

“…7, A and D). GLP-1 and exendin 4 share 50% sequence identity but have distinct secondary structure, order, and stability characteristics (30,35). Second, N55, but not SEA (19), binds GLP-1 in a dosedependent manner (Fig.…”