Structure and folding of glucagon‐like peptide‐1‐(7–36)‐amide in aqueous trifluoroethanol studied by NMR spectroscopy

Abstract

The conformational changes of free, monomeric glucagon-like peptide-1-(7-36)-amide (GLP-1) in aqueous solution with increasing concentrations of 2,2,2-trifluoroethanol (TFE) were monitored by NMR spectroscopy. It was found that GLP-1 gradually assumes a stable, single-stranded helical structure in water solution when the TFE concentration is increased from 0 to 35% (v/v). No further structural changes were observed at higher TFE concentrations. The structure of GLP-1 in 35% TFE was determined from 292 distance restraints and 44 angle restraints by distance geometry, simulating annealing and restrained energy minimization. The helical structure extends from T7 to K28, with a less well-defined region around G16 and a disordered six-residue N-terminal domain. The folding process of GLP-1 from random coil (in water) to helix (in 35% TFE) is initiated by the formation of the C-terminal segment of the helix that is extended gradually towards the N-terminus of the peptide with increasing concentration of TFE. The exchange rates of the slow exchanging amide protons indicate that the C-terminal part of the helix is more stable than the N-terminal part.

The Isolated N-terminal Domain of the Glucagon-like Peptide-1 (GLP-1) Receptor Binds Exendin Peptides with Much Higher Affinity than GLP-1

“…Hence, the fact that exendin-4 binding does not depend greatly on the extracellular loops does not necessitate that it requires an activation pocket different from that of GLP-1. The increased affinity of exendin-4 and the GLP-1R N terminus may be provided by the C-terminal Trp-cage motif (20) or perhaps by the increased helicity of the middle region of this peptide compared with GLP-1 (36,37). Increasing peptide helicity has been proposed to improve binding to the N-terminal domain of the calcitonin receptor (38).…”

Three Distinct Epitopes on the Extracellular Face of the Glucagon Receptor Determine Specificity for the Glucagon Amino Terminus

“…ChTM2, ChECL2, and ChECL3 did not compromise glucagon binding or potency, which confirmed the structural integrity of the chimeric receptors. Two nonexclusive explanations may account for these results: 1) the GLP-1R segments of ChTM2, ChECL2, and ChECL3 interact directly with Glu 3 , Ser 12 , and Ala 2 , respectively, and/or 2) the GLP-1R segments of ChTM2, ChECL2, and ChECL3 are required to maintain a local binding site conformation that accommodates the interaction with Glu 3 , Ser 12 , and Ala 2 , respectively. Nevertheless, it is difficult to explain the results without considering the proximity of the GLP-1R segments in ChTM2, ChECL2, and ChECL3 with Glu 3 , Ser 12 , and Ala 2 of the glucagon analogs, respectively.…”

“…ECL2 of the secretin and VPAC 2 receptors has been implicated in ligand binding, and specifically four residues in the N-terminal half of ECL2 contribute significantly to the secretin/VIP selectivity profile of the secretin receptor (17,20). On the basis of the results presented here, we will attempt to identity the specific residue(s) in ECL2, TM4, and/or TM5 of GluR that determines specificity for Lys 12 of glucagon.…”

“…Furthermore, His 7 , Gly 10 , Phe 12 , Thr 13 , and Asp 15 of the GLP-1 N terminus are important for optimal binding and activation of GLP-1R, and they are all conserved in exendin-4 (11). NMR structures of GLP-1, glucagon, and PACAP-(1-38) in lipophilic solvents or dodecylphosphocholine micelles agree that the central and C-terminal parts are ␣-helical, often with a central distortion of the helix geometry, whereas the N terminus is a flexible random coil (12)(13)(14). Interestingly, the N terminus of receptor-bound PACAP-(1-21) forms a specific ␤-coil structure, and the PACAP N terminus is important for receptor activation (8,14).…”

“…Furthermore, the second extracellular loop and/or proximal segments of TM4 and/or TM5 are close to and determine specificity for Lys 12 of glucagon.…”