Exendin-4 and Glucagon-like-peptide-1:  NMR Structural Comparisons in the Solution and Micelle-Associated States

Abstract

Exendin-4, a 39 amino acid peptide originally isolated from the oral secretions of the lizard Heloderma suspectum, has been shown to share certain activities with glucagon-like-peptide-1 (GLP-1), a 30 amino acid peptide. We have determined the structuring preferences of exendin-4 and GLP-1 by NMR in both the solution and dodecylphosphocholine (DPC) micelle-associated states. Based on both chemical shift deviations and the pattern of intermediate range NOEs, both peptides display significant helicity from residue 7 to residue 28 with greater fraying at the N-terminus. Thornton and Gorenstein [(1994) Biochemistry 33, 3532-3539] reported that the presence of a flexible, helix-destabilizing, glycine at residue 16 in GLP-1 was an important feature for membrane and receptor binding. Exendin-4 has a helix-favoring glutamate as residue 16. In the micelle-associated state, NMR data indicate that GLP-1 is less helical than exendin-4 due to the presence of Gly16; chemical shift deviations along the peptide sequence suggest that Gly16 serves as an N-cap for a second, more persistent, helix. In 30 vol-% trifluoroethanol (TFE), a single continuous helix is evident in a significant fraction of the GLP-1 conformers present. Exendin-4 has a more regular and less fluxional helix in both media and displays stable tertiary structure in the solution state. In the micelle-bound state of exendin-4, a single helix (residues 11-27) is observed with residues 31-39 completely disordered and undergoing rapid segmental motion. In aqueous fluoroalcohol or aqueous glycol, the Leu21-Pro38 span of exendin-4 forms a compact tertiary fold (the Trp-cage) which shields the side chain of Trp25 from solvent exposure and produces ring current shifts as large as 3 ppm. This tertiary structure is partially populated in water and fully populated in aqueous TFE. The Leu21-Pro38 segment of exendin-4 may be the smallest protein-like folding unit observed to date. When the Trp-cage forms, fraying of the exendin-4 helix occurs exclusively from the N-terminus; backbone NHs for the C-terminal residues of the helix display H/D exchange protection factors as large as 10(5) at 9 degrees C. In contrast, no tertiary structure is evident when exendin-4 binds to DPC micelles. An energetically favorable insertion of the tryptophan ring into the DPC micelle is suggested as the basis for this change. With the exception of exendin-4 in media containing fluoro alcohol cosolvents, NMR structure ensembles generated from the NOE data do not fully reflect the conformational averaging present in these systems. Secondary structure definition from chemical shift deviations may be the most appropriate treatment for peptides that lack tertiary structure.

Exendin-4, but not glucagon-like peptide-1, is cleared exclusively by glomerular filtration in anaesthetised pigs

“…This susceptibility of GLP-1 towards degradation in vivo, compared to peptides that, judged from their sequence and in vitro kinetic data, ought to be cleaved at a similar rate, indicates that, in addition to differences in primary structure, other features such as differences in secondary structure may render GLP-1 particularly prone to cleavage. With regard to exendin-4, it could be speculated that the tertiary structure, formed by the C-terminal extension [25], may help protect it from degradation not only because of the increased size of the peptide (i.e. NEP has a preference for smaller substrates [26]), but also by simply hindering the access of substrate to enzymes.…”

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

“…Although the N termini of GLP-1 and exendin-4 are almost identical, exendin-4 contains 9 extra amino acids at the C terminus, which have been shown by NMR analysis to form a compact folding unit termed a "Trp-cage" (20). The NMR analysis also reveals that the central region of exendin-4 is largely helical, whereas the N terminus is rather more disordered (20).…”

“…The NMR analysis also reveals that the central region of exendin-4 is largely helical, whereas the N terminus is rather more disordered (20). Although the central region of exendin-4 (comprising residues 10 -30) only shares 8 identical residues with GLP-1, they all lie on the same face of an ideal ␣-helix, suggesting that it is this face of the helix which makes the critical contact with the receptor (21).…”

An investigation of the evolution of the anguimorph lizard venom system

“…NMR-study revealed a well-converged helix over residues 18-27 in both GLP-1 and exendin-4 in the conditions mimicking that just prior to binding to the receptor. Exendin-4 was shown to display a greater intrinsic propensity toward the formation of a monomeric helix than GLP-1, which can be attributed to the absence in exendin-4 of the helix-interrupting glycine residue at position 16 of GLP-1 (Neidigh et al, 2001). This allows for the helix to go much further toward the N-terminus in exendin-4 that, along with other structural differences between GLP-1 and exendin-4, may reduce the entropic cost of binding and be responsible for the higher potency of exendin-4 in some organisms.…”

“…Exendin-4 was shown to stimulate secretion of insulin in the presence of elevated blood glucose concentrations, but not during hypoglycemic periods, which led to its pharmacological use in the treatment of type 2 diabetes (Neidigh et al, 2001). Treatment with exendin-4 increases cell proliferation and neuroblast differentiation in the dentate gyrus, potentially through promoting structural plasticity (Li et al, 2010).…”