Truncated Glucagon-like Peptide-1 and Exendin-4 α-Conotoxin pl14a Peptide Chimeras Maintain Potency and α-Helicity and Reveal Interactions Vital for cAMP Signaling in Vitro

Abstract

Glucagon-like peptide-1 (GLP-1) signaling through the glucagon-like peptide 1 receptor (GLP-1R) is a key regulator of normal glucose metabolism, and exogenous GLP-1R agonist therapy is a promising avenue for the treatment of type 2 diabetes mellitus. To date, the development of therapeutic GLP-1R agonists has focused on producing drugs with an extended serum half-life. This has been achieved by engineering synthetic analogs of GLP-1 or the more stable exogenous GLP-1R agonist exendin-4 (Ex-4). These synthetic peptide hormones share the overall structure of GLP-1 and Ex-4, with a C-terminal helical segment and a flexible N-terminal tail. Although numerous studies have investigated the molecular determinants underpinning GLP-1 and Ex-4 binding and signaling through the GLP-1R, these have primarily focused on the length and composition of the N-terminal tail or on how to modulate the helicity of the full-length peptides. Here, we investigate the effect of C-terminal truncation in GLP-1 and Ex-4 on the cAMP pathway. To ensure helical C-terminal regions in the truncated peptides, we produced a series of chimeric peptides combining the N-terminal portion of GLP-1 or Ex-4 and the C-terminal segment of the helix-promoting peptide α-conotoxin pl14a. The helicity and structures of the chimeric peptides were confirmed using circular dichroism and NMR, respectively. We found no direct correlation between the fractional helicity and potency in signaling via the cAMP pathway. Rather, the most important feature for efficient receptor binding and signaling was the C-terminal helical segment (residues 22-27) directing the binding of Phe(22) into a hydrophobic pocket on the GLP-1R.

Keywords: G protein-coupled receptor (GPCR); conotoxin pl14a; cyclic AMP (cAMP); cyclic peptide; diabetes; exendin-4; glucagon-like peptide-1; peptide hormone.

FIGURE 1.

Structures, models, sequences, and strategies for GLP-1/Ex-4 and α-conotoxin pl14a chimeric peptides. A, GLP-1 bound to the NTD of the GLP-1R (PDB 1IOL) showing residues 10–35 of GLP-1 as a ribbon plot (green) on the electrostatic surface of the NTD of the GLP-1R (blue, positive; red, negative: white, neutral). B, the NMR structure of Ex-4. The flexible nature of the peptide N terminus, the highly structured central helix, and C-terminal tryptophan cage that folds back over the peptide to stabilize the helix are all clearly defined in this structure. C, the α-conotoxin pl14a shares structural features with Ex-4, having a flexible N terminus, a central helix, and a C-terminal segment that folds back over the peptide to stabilize the helix. D, a model of the chimera peptide construct between the first 30 N-terminal residues of Ex-4 and the C terminus of conotoxin pl14a. E, sequences of linear and chimera peptides between GLP-1 (blue), Ex-4 (green), and conotoxin pl14a (black). Mutated residues are shown in red. Cys residues are highlighted with yellow and are numbered with roman numerals. Disulfide connectivity is shown with black lines.

FIGURE 2.

Representative circular dichroism spectrums for GLP-1 and Ex-4 variants. Circular dichroism spectrums for grafted (A) and linear (B) GLP-1 variants as well as for grafted (C), linear (D), and residue 22 Ala mutant (E) Ex-4 variants. F, plot showing the relationship between fraction helicity and cAMP EC₅₀ values.

FIGURE 3.

Secondary Hα chemical shifts and solution structures of selected Ex-4/pl14a chimeras. A, Ex-4[1–16]/pl14a (PDB 2NAV) secondary Hα chemical shifts (left) and ribbon plots of solution structures (right) with α-helices shown in blue, and disulfide bonds are in yellow. B, Ex-4[1–27]/pl14a (PDB 2NAW) secondary Hα chemical shifts (left) and solution structures (right). Residues with secondary Hα chemical shifts indicating helical structure are indicated with gray helices above the residue numbering.

FIGURE 4.

Molecular modeling of the GLP-1R NTD and Ex-4/Ex-4 pl14a chimeras. A, overlay of the average molecular dynamics simulation secondary structures of Ex-4[1–30] (light blue), Ex-4[1–30] F22A (cyan) and a previous crystal structure (PDB 3C5T; dark blue). Phe²² is labeled and shown in stick model. B, overlay of the average molecular dynamics simulation secondary structures of Ex-4[1–27]/pl14a (light blue) and PDB 3C5T (dark blue). The area delineated by the gray dotted rectangle is shown in greater detail for the overlay of Ex-4[1–27]/pl14a and PDB 3C5T (C) as well as for Ex-4[1–27]/pl14a (D) and PDB 3C5T (E) alone, where the ligand's secondary structure (green) and stick models (green, carbon: blue, nitrogen; red, oxygen; yellow, sulfur) are shown bound to the electrostatic surface of the GLP-1R NTD (blue, positive; red, negative: white, neutral).

FIGURE 5.

Molecular determinants of cAMP signaling. A, correlation between the number of residues of GLP-1 or Ex-4 grafted into conotoxin pl14a and the resulting cAMP signaling Log EC₅₀ values. B, alignment of various Ex-4 (blue) and conotoxin pl14a (black) chimeras and their EC₅₀ values with residue 22 are bound by a black box. Mutated and Cys residues are highlighted in red and yellow, respectively. C, ribbon plot of a crystal structure (PDB 3C5T) of Ex-4 (green) bound to the NTD of the GLP-1R (blue, showing residues interacting in the hydrophobic patch as stick models.