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*

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 Phe22 into a hydrophobic pocket on the GLP-1R.

Secretion of insulin is considerably higher in response to oral administration of glucose compared with intravenous delivery (1). This phenomenon, known as the "incretin effect," is primarily mediated by two peptide hormones called incretins: glucagon like peptide 1 (GLP-1) 5 (2) and glucose-dependent insulinotropic polypeptide (3). The incretin effect is often greatly reduced in patients suffering from type 2 diabetes mellitus (4), and a possible therapeutic approach for this condition is incretin supplement therapy. Because type 2 diabetes mellitus sufferers continue to respond to GLP-1, but not to glucose-dependent insulinotropic polypeptide (5), recent therapeutic supplement strategies have focused on GLP-1 and the development of synthetic analogs (6).
GLP-1 exerts its physiological activity in the 0.1-1 nM range (7) by signaling through the glucagon-like peptide-1 receptor (GLP-1R), a secretin/family B of G protein-coupled receptors (GPCR) (8). Primary GLP-1R signaling occurs via binding to G ␣ subunits that activate the intracellular cAMP pathway. Additional signaling also occurs via both ␤-arrestin and mobilization of intracellular calcium. Indeed, the level of variability in response reflects the diverse physiological functions of GLP-1R in different tissues (9). As with other members of GPCR receptor family, the GLP-1R has a large extracellular N-terminal domain (NTD) made up of one ␣-helix and two antiparallel ␤-sheets (10) and is expected to have a seven-transmembrane bundle domain like other class B GPCRs (11). The crystal structure of the extracellular NTD of the GLP-1R in complex with GLP-1 revealed that the ligand binds to the NTD of the receptor through a C-terminal helix, whereas the N-terminal portion of the peptide is unstructured (Fig. 1A) (12). Binding and signaling through the GLP-1R is proposed to occur through a two-do-* This work was supported in part by Australian Research Council (ARC) Linkage Grant LP110200213 and a Queensland Government Department of Science, Information Technology, Innovation, and the Arts Co-investment Fund grant. The authors declare that they have no conflicts of interest with the contents of this article. The atomic coordinates and structure factors ( main model; the helical C terminus of the ligand first binds the NTD of the receptor, dictating binding affinity and specificity (Fig. 1A), before the ligand N terminus interacts with the seventransmembrane bundle domain core of the receptor to affect signaling potency and specificity (13). The mechanism by which the N terminus of GLP-1R interacts with the binding pocket of the seven-transmembrane bundle domain and how this initiates receptor activation is presently unknown. An exogenous GLP-1R agonist with a similar pharmacological profile to GLP-1, named exendin-4 (Ex-4; Fig. 1B), has been isolated from the saliva of the Gila monster (Heloderma suspectum) (14). Ex-4 and GLP-1 share 50% sequence identity, with Ex-4 being a slightly more potent agonist (15). A comparison of the crystal structures of the NTD of the GLP-1R in complex with either GLP-1 or Ex-4 revealed that they bind at the same site of the extracellular domain (10,12), although comprehensive structure and function studies have revealed mechanistic differences in binding and signaling (16). These studies are primarily based upon binding affinity and cAMP signaling and include Ala scans, N-terminal truncations, and chimeras between GLP-1 and Ex-4 (15). The effect of C-terminal truncation of GLP-1 and Ex-4 has been much less extensively investigated. Removing the last two C-terminal residues of GLP-1 resulted in 40% reduction in insulin release from perfused rat pancreas at 100 pM (7) and a 10-fold reduction in binding affinity for the GLP-1R (17). Consistent with a role for the C terminus of GLP-1 in GLP-1R binding and signaling, replacing the C-terminal sequence VKGR of GLP-1 with the corresponding MNT of glucagon caused a 475-fold reduction in affinity (18), and removal of the last 10 C-terminal residues resulted in no binding or cAMP signaling at 10 M (19).
It has been suggested that the helical structures of class B GPCR ligands, including GLP-1 and Ex-4, are important for receptor binding and signaling (20), and it may be that C-terminal truncations or mutations result in reduced helicity and thereby signaling efficacy. There are a number of chemical methods available for the stabilization of truncated helices, including lactam or disulfide bridges, hydrocarbon staples, and hydrogen bond surrogate approaches (21,22). Lactam bridges have been used extensively to test the extent to which helicity in  ). 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 ␣-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.
GLP-1 affects receptor binding and signaling (23)(24)(25)(26); however, to date there has been no investigation into the effect of C-terminal truncation of GLP-1 or Ex-4 on peptide helicity nor has there been research into the effect of inducing helicity in truncated analogs on receptor binding and signaling.
Another approach to stabilizing helices is grafting of these secondary structural motifs into highly constrained helical peptide scaffolds (27). One such class of scaffolds is the disulfiderich conotoxins isolated from marine cone snail venoms that naturally target nicotinic acetylcholine receptors. We recently used an ␣-conotoxin (pc16a) to engineer a potent GLP-1R agonist by combining the conotoxin disulfide bond connectivity with features of a previously published 11-amino acid peptidomimetic GLP-1R agonist (28). Another ␣-conotoxin pl14a (Fig.  1C), first isolated from the cone snail Conus planorbis (29), shares several properties with GLP-1 and Ex-4 as both classes of peptides have a flexible N termini followed by an ␣-helix and both target membrane integrated receptors. However, in contrast to the incretins, the ␣-helix in pl14a is highly constrained via two disulfide bonds (29), making the secondary structure less dependent on the amino acid sequence. These properties make pl14a an ideal scaffold for grafting peptides that have C-terminal helices and flexible N termini.
In this study we produced a series of C-terminally truncated GLP-1 and Ex-4 peptides both in their linear form and with the sequences grafted into the N-terminal flexible and central helical portions of the ␣-conotoxin pl14a. These peptides were structurally characterized using circular dichroism and NMR, and their ability to elicit cAMP signaling in vitro was evaluated. We found that although the pl14a scaffold could induce increased helicity in GLP-1 and Ex-4 sequences, signaling potency strongly correlated with the peptide length of the grafted peptide rather than overall helicity. Furthermore, the strongest determinant of GLP-1R agonist signaling through the cAMP pathway was found to be the presence or absence of Phe 22 . These findings provide new insights into the mechanism of GLP-1R activation and may guide future development of minimized and disulfide-constrained GLP-1 or Ex-4 analogs for the treatment of type 2 diabetes mellitus.
Circular Dichroism-Peptides were solubilized in either water or 10 -20% acetonitrile (pH 3-4) at concentrations between 50 and 100 M. Far ultraviolet spectra were recorded at room temperature using a Jasco J-810 spectropolarimeter and a 1-mm path length. Spectral data were collected from 3 scans from 260 to 190 nm with a scan speed of 100 nm min Ϫ1 and 0.5-nm wavelength steps. Solvent signal was subtracted before smoothing of the data (31) using the JASCO Spectra Manager software. Millidegree values were converted to mean residue ellipticity with units of degree⅐cm 2 ⅐dmol Ϫ1 . Fractional helicity was calculated from the mean molar ellipticity at 220 nM, as previously described (32), assuming that the ellipticity of a completely helical peptide of infinite length is Ϫ37,000 degrees⅐cm 2 ⅐dmol Ϫ1 .
Due to extensive flexibility observed in the N terminus of Ex-4 /pl14a, only the structure of Ex-4[1-16]/pl14a was refined using protocols from the RECOORD database (36) to calculate an ensemble of 50 structures within the CNS software (37) using the force-field distributed with Haddock 2.0 (38); the 50 structures generated were then further refined in a water shell, as previously described (33). A set of 20 structures with the lowest energy and no NOE violations Ͼ0.2 Å and few dihedral violations Ͼ3°was selected for MolProbity analysis (39)   , the last eight N-terminal residues originated from an Ex-4 solution structure (PDB 1JRJ). Systems were solvated with TIP3P water and neutralized by Na ϩ /Cl Ϫ counterions using VMD1.9.1. This generated systems of 22,000 -26,000 atoms, including 19,000 -23,000 water molecules. Each protein complex was equilibrated using a stepwise relaxation procedure over 2.5 ns before production runs of 5 ns were carried out for each system using NAMD 2.9 CUDA and CHARMM27 force-field parameters, as previously described (30). Coordinates were saved every 500 simulation steps, producing the 5000 frames per simulation trajectories used for analysis in VMD. Interaction energies were calculated using the NAMD Energy plugin in VMD.

Design of Truncated GLP-1 and Ex-4 Conotoxin Chimeras-
Although previous work suggested that the C-terminal segment of GLP-1 is important for efficient cAMP signaling via the GLP-1R (7,(17)(18)(19), the underlying mechanism(s) of action is poorly understood and may be a result of loss of either C-terminal helical structure, binding interactions, or both. To investigate the role of the C-terminal helical segment in GLP-1R signaling, a series of C-terminally truncated GLP-1 and Ex-4 peptides was produced. These were either linear or grafted into the bicyclic, helical ␣-conotoxin pl14a to stabilize the C-terminal helix (Fig. 1, B-E). Binding of the C-terminal helix of both GLP-1 and Ex-4 occurs in a groove in the GLP-1R NTD (Fig.  1A). To prevent steric hindrance upon binding, all possible chi-meras for residues 13-30 of GLP-1 and Ex-4 were modeled (data not shown), and variants with minimal steric clashes were selected for synthesis (Fig. 1B).
GLP-1 and Ex-4 Conotoxin pl14a Chimeras Maintain cAMP Activity-Grafting full-length GLP-1 into the conotoxin pl14a scaffold as GLP-1 /pl14a v1 and GLP-1 /pl14a v2 resulted in 6-fold or 15-fold reductions in cAMP signaling, respectively, compared with wild-type GLP-1 (GLP-1 ; EC 50 ϭ 0.040 Ϯ 0.004 nM) ( Table 2). The three-residue C-terminally truncated GLP-1 analogs showed 5.8-fold and 10-fold reductions in cAMP signaling for the linear (GLP-1 ) and grafted forms (GLP-1[7-33]/pl14a), respectively. Removing six residues from the C terminus of GLP-1 resulted in 23,000-fold and 75,000-fold reductions in cAMP signaling for the linear (GLP-1 ) and grafted (GLP-1 /pl14a) forms, respectively. Interestingly, the removal of additional C-terminal residues did not further reduce cAMP signaling, and GLP-1    JULY 22, 2016 • VOLUME 291 • NUMBER 30 wild-type Ex-4 , with both peptides having EC 50 potencies Ͻ20 pM, which is in agreement with previous studies (15). Grafting Ex-4  into pl14a (Ex-4[1-30]/pl14a) resulted in a 4-fold reduction in cAMP signaling, which is significantly less than that of the GLP-1 equivalent, GLP-1[7-36]/pl14a v2. Consistent with this, the removal of three additional C-terminal residues from Ex-4 resulted in no reduction in cAMP EC 50 for the grafted (Ex-4[1-27]/pl14a) form, whereas the linear counterpart (Ex-4 ) was 12-fold less potent. After the removal of another three C-terminal residues from Ex-4, a similar reduction in cAMP signaling was observed for the linear (Ex-4  Increased Helicity Does Not Correlate with Higher Potency-To evaluate the effect of grafting the truncated GLP-1 and Ex-4 peptides into the helical conotoxin pl14a on overall helicity, circular dichroism was measured, and helicity was estimated for all peptides ( Table 2; Fig. 2, A-E). The fraction helicity of GLP-1 and Ex-4 corresponded well with those determined previously in aqueous buffer at pH 3.5 (40). Overall, incorporation of the truncated GLP-1 and Ex-4 into the helical pl14a resulted in increased helicity. A comparison of the potency of cAMP signaling for the various GLP-1 and Ex-4 variants did not show any correlation with fraction helicity (Table 2; Fig. 2F), which is in agreement with previous reports for lactam-bridged GLP-1 analogs (23,24).

GLP-1 and Ex-4 ␣-Conotoxin pl14a Chimeras
be used as a stabilizing scaffold and replace the Trp-cage present in native exendin-4. A comparison of the H␣ chemical shifts to random coil values (41) showed stretches of negative secondary shifts, which in agreement with the literature, indicate an ␣-helix secondary structure from residues 8 -16 in Ex-4[1-16]/ pl14a and 8 -28 in Ex-4[1-27]/pl14a (Fig. 3, A and B; Table 3) (42). This also corresponds well with circular dichroism data acquired for these two peptides, which suggests the presence of a large degree of helicity (Fig. 2). This helicity was observed in the three-dimensional structure of Ex-4[1-16]/pl14a, but not Ex-4[1-27]/pl14a, due to the lack of sufficient NOEs in the region despite the presence of several i-iϩ4 NOEs. Molecular Modeling-Molecular dynamics was used to investigate how truncated Ex-4 variants might interact with the NTD of the receptor. In the case of Ex-4 /pl14a, where incomplete NMR constraints were available, solution and crystallography structures were used to create potential models of the bound peptides (see "Experimental Procedures"). Simulations of Ex-4  and Ex-4  F22A bound to the NTD of GLP-1 resulted in average simulation structures that closely aligned with the crystal structures of Ex-4/GLP-1R with 0.72 and 0.74 Å C␣ r.m.s.d., respectively (Fig. 4A). Despite the lack of structural change between Ex-4  and Ex-4[1-30] F22A, a reduction in the interaction energy between the NTD of GLP-1R was observed for the F22A mutant ( Table 3). The average simulation structure of the chimeric peptide Ex-4[1-27]/ pl14a also closely aligned with the crystal structure (C␣ r.m.s.d. 0.75 Å; Fig. 4, A and B), and the corresponding F22A mutation in Ex-4[1-27]/pl14a F22A was associated with a similar reduction in interaction energy as Ex-4  F22A, although Ex-4[1-27] F22A adopted a slight change in the spatial extension of the N-terminal end of the Ex-4 helix, resulting in a somewhat higher degree of deviation compared with the crystal structure (C␣ r.m.s.d. 0.97 Å). These  JULY 22, 2016 • VOLUME 291 • NUMBER 30 reductions in interaction energies in silico for F22A mutants follow the trend seen in the cAMP assays but were not significant or absolutely proportional to the changes in EC 50 values. Attempts were made to model the binding between the NTD of GLP-1 and Ex-4[1-16]/pl14a, but the overlapping binding interface was determined too small to provide a realistic simulation.

Discussion
In this study we produced and characterized a series of C-terminally truncated GLP-1 and Ex-4 peptides, both in their linear forms and as ␣-conotoxin pl14a chimeric peptides. We found that residues 28 -30 of GLP-1 and residues 28 -39 of Ex-4 were not essential for either peptide helicity or GLP-1R signaling via the cAMP pathway in vitro. Further C-terminal truncations were associated with loss of ␣-helicity and GLP-1R signaling potency for both peptide hormones. Incorporating C-terminally truncated peptides into the ␣-conotoxin scaffold often increased helicity but did not similarly improve signaling potency. The most prominent determinant of GLP-1R signaling in vitro was found to be Phe 22 in either peptide hormone. These findings might help to guide the future design of minimized and/or constrained GLP-1 or Ex-4 peptide analogs suitable for the treatment of type 2 diabetes mellitus.
The results presented in this study have clarified the relative contribution of the C-terminal helical segments of GLP-1 and Ex-4 to GLP-1R binding and signaling. Although there are a few previous studies involving C-terminal truncations of GLP-1 using different methods (7,17,19), ours is the first to systematically explore the effects of C-terminal truncation of GLP-1 and Ex-4. We found that C-terminal removal of three residues from GLP-1 and 12 residues from Ex-4 had only small effects upon cAMP signaling for both linear and chimeric peptides (Fig. 5A). Conversely, removal of three more residues (residues 25-27) from the C terminus of either peptide resulted in a potency loss equal to at least three orders of magnitude. A similar dominating effect was seen for Phe 22 in both GLP-1 and Ex-4, where peptides that included Phe 22 generally showed more potent cAMP signaling. The positive effect of Phe 22 was particularly strong for Ex-4, and a series of F22A mutant peptides confirmed the importance of Phe 22 for cAMP signaling (Fig. 5B). These findings are in line with previous Ala scans of GLP-1, which also found that the F22A substitution caused the largest loss in binding affinity (1300-fold) and cAMP signaling (1000-fold) out of all Ala mutants (43). Combined, these findings suggest that the central portion of both GLP-1 and EX-4 acts as a spacer to position a C-terminal helical segment (residues [22][23][24][25][26][27] to correctly align Phe 22 with a vital binding pocket on the receptor Fig. 5C). However, future studies need to explore to what extent these structural determinants modulating cAMP signaling translates to insulin secretion.     A recent study involving GLP-1R mutants concluded that a complementary "hydrophobic patch" on GLP-1/Ex-4 and the receptor is needed for correct positioning of the N terminus for GLP-1R activation, a feature that may extend to all class B GPCRs (44). This conclusion was based on GLP-1R mutagenesis of L32A (44), on studies showing the importance of Phe 22 / Ile 23 in GLP-1 for receptor activation (43,45) and on the presence of a similar hydrophobic patch on glucagon (needed for efficient binding to the glucagon extracellular domain) (46). Although Phe 22 only forms part of this hydrophobic patch, we have shown that mutating Phe 22 has an impact that is orders of magnitude greater than other hydrophobic residues in this patch.
These findings in conjunction with previous studies provide novel insight into the mechanistic foundations for GLP-1R receptor activation. Considering the size and nature of the binding interface between GLP-1/Ex-4 and the GLP-1R, the mutation of a single residue alone is unlikely to explain such a large change in signaling efficacy; rather, it suggests a mechanistic cause. It may well be that the correct spatial orientation of Phe 22 to the pocket in the receptor triggers conformational change in the receptor that affects the relative orientation of the N-terminal and transmembrane domains. This may also explain why much smaller 11-residue peptides, which share the N-terminal portion of GLP-1/Ex-4 but have various biphenyl and/or phenylalanine derivatives at the C-terminal end, achieve similar activation profiles to that of GLP-1 and Ex-4 (28,47,48). It is possible that, for these compounds, the biphenyl derivatives bind to the same pocket as Phe 22 in GLP-1/Ex-4 and trigger a conformational change in the receptor that allows the N terminus of these shorter peptides to interact with the core of the receptor. If so, one may envision future GLP-1R ligands that consist of a C-terminal hydrophobic anchor segment, a central spacer segment of correct length, and an N-terminal activation segment that binds to the core of the receptor.
Further studies need to be undertaken to fully clarify the role of the hydrophobic patch in receptor activation and signaling. For example, further GLP-1R mutagenesis studies of all residues involved in this hydrophobic interaction may shed further light on the extent of the patch and the relative contribution of various receptor residues. In particular, comparing the effect of GLP-1R mutation on binding affinity and signaling using both full-length peptide agonists and the 11-residue peptides should give a good indication of whether they capitalize on the same interactions. Cross-linking experiments between the 11-residue peptides and the GLP-1R may also be used to further pinpoint the binding area of the biphenyl derivatives.