Truncated Glucagon-like Peptide-1 and Exendin-4�-Conotoxin pl14a Peptide Chimeras Maintain Potency and�-Helicity and Reveal Interactions Vital for cAMP Signalingin Vitro*

Received for publication, March 4, 2016, and in revised form, April 18, 2016 Published, JBC Papers in Press, May 10, 2016, DOI 10.1074/jbc.M116.724542

Joakim E. Swedberg‡1, Christina I. Schroeder‡2, Justin M. Mitchell‡, David P. Fairlie‡3, David J. Edmonds§,David A. Griffith§, Roger B. Ruggeri§, David R. Derksen¶, Paula M. Loria¶, David A. Price§, Spiros Liras§,and David J. Craik‡4

From the ‡Institute for Molecular Bioscience, The University of Queensland, Brisbane, 4072 Queensland, Australia, §WorldwideMedicinal Chemistry, Cardiovascular and Metabolic Diseases Research Unit, Pfizer Inc., Cambridge, Massachusetts 02139, and¶Pharmacokinetics, Dynamics, and Metabolism, Worldwide Research and Development, Pfizer Inc., Groton, Connecticut 06340

Glucagon-like peptide-1 (GLP-1) signaling through the gluca-gon-like peptide 1 receptor (GLP-1R) is a key regulator of nor-mal glucose metabolism, and exogenous GLP-1R agonist ther-apy is a promising avenue for the treatment of type 2 diabetesmellitus. To date, the development of therapeutic GLP-1R ago-nists has focused on producing drugs with an extended serumhalf-life. This has been achieved by engineering synthetic ana-logs of GLP-1 or the more stable exogenous GLP-1R agonistexendin-4 (Ex-4). These synthetic peptide hormones share theoverall structure of GLP-1 and Ex-4, with a C-terminal helicalsegment and a flexible N-terminal tail. Although numerousstudies have investigated the molecular determinants underpin-ning GLP-1 and Ex-4 binding and signaling through the GLP-1R, these have primarily focused on the length and compositionof the N-terminal tail or on how to modulate the helicity of thefull-length peptides. Here, we investigate the effect of C-termi-nal truncation in GLP-1 and Ex-4 on the cAMP pathway. Toensure helical C-terminal regions in the truncated peptides, weproduced a series of chimeric peptides combining the N-termi-nal portion of GLP-1 or Ex-4 and the C-terminal segment of thehelix-promoting peptide �-conotoxin pl14a. The helicity andstructures of the chimeric peptides were confirmed using circu-lar dichroism and NMR, respectively. We found no direct cor-relation between the fractional helicity and potency in signalingvia the cAMP pathway. Rather, the most important feature forefficient receptor binding and signaling was the C-terminal hel-

ical 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 oraladministration of glucose compared with intravenous delivery(1). This phenomenon, known as the “incretin effect,” is pri-marily mediated by two peptide hormones called incretins: glu-cagon like peptide 1 (GLP-1)5 (2) and glucose-dependent insuli-notropic polypeptide (3). The incretin effect is often greatlyreduced in patients suffering from type 2 diabetes mellitus (4),and a possible therapeutic approach for this condition is incre-tin supplement therapy. Because type 2 diabetes mellitus suf-ferers continue to respond to GLP-1, but not to glucose-depen-dent insulinotropic polypeptide (5), recent therapeuticsupplement strategies have focused on GLP-1 and the develop-ment 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. Addi-tional signaling also occurs via both �-arrestin and mobiliza-tion of intracellular calcium. Indeed, the level of variability inresponse reflects the diverse physiological functions of GLP-1Rin different tissues (9). As with other members of GPCR recep-tor family, the GLP-1R has a large extracellular N-terminaldomain (NTD) made up of one �-helix and two antiparallel�-sheets (10) and is expected to have a seven-transmembranebundle domain like other class B GPCRs (11). The crystal struc-ture of the extracellular NTD of the GLP-1R in complex withGLP-1 revealed that the ligand binds to the NTD of the receptorthrough a C-terminal helix, whereas the N-terminal portion ofthe peptide is unstructured (Fig. 1A) (12). Binding and signalingthrough the GLP-1R is proposed to occur through a two-do-

* This work was supported in part by Australian Research Council (ARC) Link-age Grant LP110200213 and a Queensland Government Department ofScience, Information Technology, Innovation, and the Arts Co-investmentFund grant. The authors declare that they have no conflicts of interest withthe contents of this article.

The atomic coordinates and structure factors (codes 2NAV and 2NAW) have beendeposited in the Protein Data Bank (http://wwpdb.org/).

1 A National Health and Medical Research Council ECR (Early Career Fellow-ships) Fellow (APP1069819).

2 An Institute for Molecular Bioscience Industry Fellow.3 A National Health and Medical Research Council Senior Principal Research

Fellow (APP1027369) and funded by the Australian Research Council Cen-ter of Excellence in Advanced Molecular Imaging (CE140100011).

4 An Australian Research Council Australian Laureate Fellow (FL150100146).To whom correspondence should be addressed: Institute for MolecularBioscience, Bldg. 80, Services Rd., The University of Queensland, St. LuciaQLD 4072, Australia. Tel.: 61-7-3346-2019; E-mail: d.craik@imb.uq.edu.au.

5 The abbreviations used are: GLP-1, glucagon-like peptide-1; GLP-1R, GLP-1receptor; GPCR, G protein-coupled receptor; Ex-4, exendin-4; NTD, N-ter-minal domain; TOCSY, total correlation spectroscopy; NOESY, nuclearOverhauser effect (NOE) spectroscopy; ECOSY, exclusive correlation spec-troscopy; HSQC, heteronuclear single quantum correlation; r.m.s.d., rootmean square deviation.

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© 2016 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A.

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main model; the helical C terminus of the ligand first binds theNTD of the receptor, dictating binding affinity and specificity(Fig. 1A), before the ligand N terminus interacts with the seven-transmembrane bundle domain core of the receptor to affectsignaling potency and specificity (13). The mechanism bywhich the N terminus of GLP-1R interacts with the bindingpocket of the seven-transmembrane bundle domain and howthis initiates receptor activation is presently unknown.

An exogenous GLP-1R agonist with a similar pharmacolog-ical profile to GLP-1, named exendin-4 (Ex-4; Fig. 1B), has beenisolated from the saliva of the Gila monster (Heloderma suspec-tum) (14). Ex-4 and GLP-1 share 50% sequence identity, withEx-4 being a slightly more potent agonist (15). A comparison ofthe crystal structures of the NTD of the GLP-1R in complexwith either GLP-1 or Ex-4 revealed that they bind at the samesite of the extracellular domain (10, 12), although comprehen-sive structure and function studies have revealed mechanisticdifferences in binding and signaling (16). These studies are pri-marily based upon binding affinity and cAMP signaling andinclude Ala scans, N-terminal truncations, and chimeras

between GLP-1 and Ex-4 (15). The effect of C-terminal trunca-tion of GLP-1 and Ex-4 has been much less extensively investi-gated. Removing the last two C-terminal residues of GLP-1resulted in 40% reduction in insulin release from perfused ratpancreas at 100 pM (7) and a 10-fold reduction in binding affin-ity for the GLP-1R (17). Consistent with a role for the C termi-nus of GLP-1 in GLP-1R binding and signaling, replacing theC-terminal sequence VKGR of GLP-1 with the correspondingMNT of glucagon caused a 475-fold reduction in affinity (18),and removal of the last 10 C-terminal residues resulted in nobinding or cAMP signaling at 10 �M (19).

It has been suggested that the helical structures of class BGPCR ligands, including GLP-1 and Ex-4, are important forreceptor binding and signaling (20), and it may be that C-ter-minal truncations or mutations result in reduced helicity andthereby signaling efficacy. There are a number of chemicalmethods available for the stabilization of truncated helices,including lactam or disulfide bridges, hydrocarbon staples, andhydrogen bond surrogate approaches (21, 22). Lactam bridgeshave been used extensively to test the extent to which helicity in

FIGURE 1. Structures, models, sequences, and strategies for GLP-1/Ex-4 and �-conotoxin pl14a chimeric peptides. A, GLP-1 bound to the NTD of theGLP-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 cagethat 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, havinga 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 peptideconstruct 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 romannumerals. Disulfide connectivity is shown with black lines.

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GLP-1 affects receptor binding and signaling (23–26); however,to date there has been no investigation into the effect of C-ter-minal truncation of GLP-1 or Ex-4 on peptide helicity nor hasthere been research into the effect of inducing helicity in trun-cated analogs on receptor binding and signaling.

Another approach to stabilizing helices is grafting of thesesecondary structural motifs into highly constrained helical pep-tide scaffolds (27). One such class of scaffolds is the disulfide-rich conotoxins isolated from marine cone snail venoms thatnaturally target nicotinic acetylcholine receptors. We recentlyused an �-conotoxin (pc16a) to engineer a potent GLP-1R ago-nist by combining the conotoxin disulfide bond connectivitywith features of a previously published 11-amino acid peptido-mimetic 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 ofpeptides have a flexible N termini followed by an �-helix andboth target membrane integrated receptors. However, in con-trast to the incretins, the �-helix in pl14a is highly constrainedvia two disulfide bonds (29), making the secondary structureless dependent on the amino acid sequence. These propertiesmake pl14a an ideal scaffold for grafting peptides that haveC-terminal helices and flexible N termini.

In this study we produced a series of C-terminally truncatedGLP-1 and Ex-4 peptides both in their linear form and with thesequences grafted into the N-terminal flexible and central hel-ical portions of the �-conotoxin pl14a. These peptides werestructurally 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 induceincreased helicity in GLP-1 and Ex-4 sequences, signalingpotency strongly correlated with the peptide length of thegrafted peptide rather than overall helicity. Furthermore, thestrongest determinant of GLP-1R agonist signaling throughthe cAMP pathway was found to be the presence or absence ofPhe22. These findings provide new insights into the mechanismof GLP-1R activation and may guide future development ofminimized and disulfide-constrained GLP-1 or Ex-4 analogsfor the treatment of type 2 diabetes mellitus.

Experimental Procedures

Peptide Synthesis, Purification, and Mass Determination—Peptides were assembled on either rink-amide (0.59 mmol/g;Chem-Impex) or 2-chlorotrityl resins (0.80 mmol/g; Chem-Im-pex) at a 0.25-mmol scale using Fmoc (N-(9-fluorenyl)me-thoxycarbonyl) solid-phase peptide synthesis on a SymphonyMultiplex Synthesizer. Reagents and methods of peptide syn-thesis, purification, and mass spectroscopy were as previouslydescribed (30).

Circular Dichroism—Peptides were solubilized in eitherwater or 10 –20% acetonitrile (pH 3– 4) at concentrationsbetween 50 and 100 �M. Far ultraviolet spectra were recorded atroom temperature using a Jasco J-810 spectropolarimeter and a1-mm path length. Spectral data were collected from 3 scansfrom 260 to 190 nm with a scan speed of 100 nm min�1 and0.5-nm wavelength steps. Solvent signal was subtracted beforesmoothing of the data (31) using the JASCO Spectra Managersoftware. Millidegree values were converted to mean residue

ellipticity with units of degree�cm2�dmol�1. Fractional helicitywas calculated from the mean molar ellipticity at 220 nM, aspreviously described (32), assuming that the ellipticity of acompletely helical peptide of infinite length is �37,000degrees�cm2�dmol�1.

CHO cAMP Accumulation Assay—CHO cells stably trans-fected with hGLP-1R were grown at 37 °C, 95% O2, 5% CO2 in75-cm flasks containing DMEM/F-12 (1:1) medium with added1% GlutaMAX™ (Gibco�), 1% PenStrep, and 1% Geneticin�(Gibco�) and grown until 90% confluent. Cells were thenwashed with PBS, lifted (cell dissociation solution; Sigma),counted, and used for cAMP accumulation assays and/or pas-saged (1:10). Following the manufacturer’s instructions for theLANCE� Ultra cAMP assay (PerkinElmer Life Sciences), cellstransfected with hGLP-1R were centrifuged (1500 rpm, 5 min),resuspended in cAMP assay buffer (Hanks’ balanced salt solu-tion, 5.56 mM glucose, 0.1% BSA, 0.5 mM isobutylmethylxan-thine, 5 mM HEPES), and seeded at 1000 cells per well in aProxiPlate-384 Plus plate (PerkinElmer Life Sciences). Cellswere treated with compounds diluted in assay buffer over arange of concentrations (10 �M to 100 fM) and incubated for 30min. Cell lysis buffers (Tracer (1:50) and Ulight (1:150)) wereadded to each well and incubated at room temperature for 2 hbefore reading the plates on a PHERAstar FS (BMG Labtech).Raw signals from three technical replicates were normalizedas a percentage of GLP-1 maximum before determining EC50values using GraphPad Prism 6 from three independentexperiments.

NMR Spectroscopy—NMR experiments were carried out asdescribed by Conibear et al. (33). Briefly, Ex-4[1–16]/pl14a andEx-4[1–27]/pl14a were dissolved in 90% H2O/10% D2O or99.96% D2O (Cambridge Isotope Laboratories) at a concentra-tion of 0.2 mM and pH �3.6 (uncorrected for isotope effects).Spectra were recorded on a Bruker Avance-600 equipped with acryoprobe at 280 K (Ex-4[1–16]/pl14a) and 298 K (Ex-4[1–27]/pl14a). NMR experiments included TOCSY using a MLEV-17spin lock sequence with a 80-ms mixing time, NOESY, ECOSY,13C,1H HSQC, and 1H,15N HSQC. Spectra were recorded with4096 data points in the F2 dimension and 512 increments in theF1 dimension. The spectra were referenced to water at 4.97 ppmat 280 K and 4.76 ppm at 298 K. No 3JNH-H� coupling constantscould be measured from the one-dimensional spectra due tooverlap of the peaks. 3JH�-H� coupling constants for Ex-4[1–16]/pl14a were measured from the ECOSY spectrum, andtogether with intensities of intra-residual NOEs, these wereused for stereo-specific assignments. Temperature coefficientswere derived from a series of TOCSY experiments run at 280 –310 K for Ex-4[1–16]/pl14a.

A 200-ms NOESY spectrum run at 280 K and 298 K forEx-4[1–16]/pl14a and Ex-4[1–27]/pl14a, respectively, was usedfor assignment, integration, and measurement of H� chemicalshifts. A complete list of interproton distances was generatedfrom chemical shifts and NOE intensities using the AUTOfunction in CYANA 3.0 (34). Several rounds of AUTO were runto ensure correct assignments of the peaks for both peptides.When �90% of all the picked peaks (463/483 for Ex-4[1–16]/pl14a and 651/731 for Ex-4[1–27]/pl14a) were appropriatelyassigned, the chemical shifts and distance restraints list gener-

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ated by the AUTO function in CYANA was used for furtherstructure calculations. Constraints for the � and the � back-bone dihedral angles were generated using TALOS-N (35) fromH�, C�, C�, HN, and N chemical shifts derived from 13C HSQCand 15N HSQC. In total, 11 �, 13 � backbone dihedral angles,and 5 �1 side-chain dihedral angles for Ex-4[1–16]/pl14a, and19 � and 19 � backbone dihedral angles were included in thestructure calculation for Ex-4[1–27]/pl14a. Disulfide connec-tivity of CysI-CysIII and CysII-CysIV (Cys7-Cys22 and Cys11-Cys24 for Ex-4[1–16]/pl14a Cys21-Cys36 and Cys25-Cys38 forEx-4[1–27]/pl14a) previously reported for pl14A (29) wereincluded as restraints. For Ex-4[1–16]/pl14a, amide protons forresidues 12–17 and 25 shifted ��4.6 ppb/K, were consideredto be shielded, and were included as hydrogen bond restraints.

Due to extensive flexibility observed in the N terminus ofEx-4[1–27]/pl14a, only the structure of Ex-4[1–16]/pl14a wasrefined using protocols from the RECOORD database (36) tocalculate an ensemble of 50 structures within the CNS software(37) using the force-field distributed with Haddock 2.0 (38); the50 structures generated were then further refined in a watershell, as previously described (33). A set of 20 structures withthe lowest energy and no NOE violations �0.2 Å and few dihe-dral violations �3° was selected for MolProbity analysis (39)Table 1.

Molecular Modeling—The lowest energy NMR structures ofEx-4[1–27]/pl14a and Ex-4[1–16]/pl14a were aligned withEx-4 bound to the NTD of the GLP-1R structure (PDB 3C5T).For Ex-4[1–27]/pl14a, the coordinates of residues 21–39 werebased on the NMR constraints, although residues 9 –20 weretaken from the crystal structure. For Ex-4[1–27]/pl14a andEx-4[1–30], the last eight N-terminal residues originated froman Ex-4 solution structure (PDB 1JRJ). Systems were solvatedwith TIP3P water and neutralized by Na�/Cl� counterionsusing VMD1.9.1. This generated systems of 22,000 –26,000atoms, including 19,000 –23,000 water molecules. Each proteincomplex was equilibrated using a stepwise relaxation proce-dure over 2.5 ns before production runs of 5 ns were carried outfor each system using NAMD 2.9 CUDA and CHARMM27force-field parameters, as previously described (30). Coordi-nates were saved every 500 simulation steps, producing the5000 frames per simulation trajectories used for analysis inVMD. Interaction energies were calculated using the NAMDEnergy plugin in VMD.

Results

Design of Truncated GLP-1 and Ex-4 Conotoxin Chimeras—Although previous work suggested that the C-terminal seg-ment of GLP-1 is important for efficient cAMP signaling via theGLP-1R (7, 17–19), the underlying mechanism(s) of action ispoorly understood and may be a result of loss of either C-ter-minal helical structure, binding interactions, or both. To inves-tigate the role of the C-terminal helical segment in GLP-1Rsignaling, a series of C-terminally truncated GLP-1 and Ex-4peptides was produced. These were either linear or grafted intothe bicyclic, helical �-conotoxin pl14a to stabilize the C-termi-nal helix (Fig. 1, B–E). Binding of the C-terminal helix of bothGLP-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 wereselected for synthesis (Fig. 1B).

GLP-1 and Ex-4 Conotoxin pl14a Chimeras Maintain cAMPActivity—Grafting full-length GLP-1 into the conotoxin pl14ascaffold as GLP-1[7–36]/pl14a v1 and GLP-1[7–36]/pl14a v2resulted in 6-fold or 15-fold reductions in cAMP signaling,respectively, compared with wild-type GLP-1 (GLP-1[7–36];EC50 � 0.040 � 0.004 nM) (Table 2). The three-residue C-ter-minally truncated GLP-1 analogs showed 5.8-fold and 10-foldreductions in cAMP signaling for the linear (GLP-1[7–33]) andgrafted forms (GLP-1[7–33]/pl14a), respectively. Removing sixresidues from the C terminus of GLP-1 resulted in 23,000-foldand 75,000-fold reductions in cAMP signaling for the linear(GLP-1[7–30]) and grafted (GLP-1[7–30]/pl14a) forms,respectively. Interestingly, the removal of additional C-termi-nal residues did not further reduce cAMP signaling, andGLP-1[7–29]/pl14a and GLP-1[7–26]/pl14a recovered somepotency, with an approximate 5000-fold reductions in EC50compared with the wild-type GLP-1. Grafting more severelyC-terminally truncated GLP-1 fragments into pl14a resulted ina dramatic loss of potency (�30,000-fold) with the exception ofGLP-1[7–22]/pl14a, which showed similar cAMP signaling tothat of GLP-1[7–29]/pl14a and GLP-1[7–26]/pl14a.

Removal of the nine C-terminal residues in Ex-4 (Ex-4[1–30]) had little effect on cAMP signaling compared with the

TABLE 1Structural statistics for the family of 20 lowest-energy Ex-4[1–16]/pl14a structuresData are based on structures with highest overall MolProbity score (39).

Energies (kcal/mol)Overall �894.3 � 55.4Bonds 10.6 � 1.3Angles 30.0 � 5.2Improper 14.7 � 2.7van der Waals �82.0 � 5.6NOE 0.1 � 0.03cDih 0.4 � 0.3Dihedral 121.7 � 1.9Electrostatic �989.9 � 55.2

MolProbity statisticsClashes (�0.4 Å/1000 atoms) 8.25 � 2.2Poor rotamers 0.05 � 0.2Ramachandran outliers (%) 0.6 � 1.4Ramachandran favored (%) 85.0 � 5.6MolProbity score 2.1 � 0.2MolProbity score percentile 70 � 9.5

Atomic r.m.s.d. (Å)Mean global backbone (9–16) 0.25 � 0.1Mean global heavy (9–16) 1.30 � 0.3Mean global backbone (9–28) 1.79 � 0.2Mean global heavy (9–28) 2.09 � 0.5

Distance restraintsIntraresidue (i-j � 0) 116Sequential (/i-j/ � 1) 134Medium range (/i-j/ � 5) 72Long range (/i-j/ � 5) 11Hydrogen bonds 14Total 347

Dihedral angle restraints� 11 13�1 5Total 29

Violations from experimental restraintsNOE violations exceeding 0.3 Å 0Dihedral violations exceeding 3.0° 0

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wild-type Ex-4[1–39], with both peptides having EC50 poten-cies �20 pM, which is in agreement with previous studies (15).Grafting Ex-4[1–30] into pl14a (Ex-4[1–30]/pl14a) resulted in a4-fold reduction in cAMP signaling, which is significantly lessthan that of the GLP-1 equivalent, GLP-1[7–36]/pl14a v2. Con-sistent with this, the removal of three additional C-terminalresidues from Ex-4 resulted in no reduction in cAMP EC50 forthe grafted (Ex-4[1–27]/pl14a) form, whereas the linear coun-terpart (Ex-4[1–27]) was 12-fold less potent. After the removalof another three C-terminal residues from Ex-4, a similarreduction in cAMP signaling was observed for the linear (Ex-4[1–24]) and conotoxin grafted (Ex-4[1–24]/pl14a/Ex-4[1–23]/pl14a) variants. Further C-terminal truncations of Ex-4resulted in dramatic losses of cAMP potency, with EC50 valuesclearly in the micromolar range, with the exception of Ex-4[1–16] (cAMP EC50 � 100 � 10 nM).

Phe22 in Ex-4 Plays a Major Role in cAMP Signaling—Com-paring the overall EC50 values of grafted and truncated analogsof both GLP-1 and Ex4 revealed that removal of residues 27–30(numbering from the N terminus) had little effect on cAMPsignaling, whereas removal of residues 24 –30 resulted in dra-matic potency losses. Further truncations had lesser impacts oncAMP signaling, suggesting that the segment-spanning resi-dues 24 –27 plays a particularly important role. Furthermore, itwas noticeable that peptides including the native Phe22 weremore potent in general. This phenomenon was particularlyapparent for Ex-4 variants. To verify the importance of residue22, we produced a series of linear and chimeric residue 22 Alamutants, all of which exhibited greatly reduced cAMP potencycompared with their parent molecules. For Ex-4[1–30] F22A

there was a �500-fold reduction in cAMP potency comparedwith Ex-4[1–30], whereas for the linear Ex-4[1–27] F22A,Ex-4[1–24] F22A, and chimera Ex-4[1–27]/pl14a F22A, thereductions were 1700-, 45-, and 190-fold, respectively. Interest-ingly, Ex-4[1–16]/pl14a, which has a Tyr residue at position 22(from the conotoxin scaffold), showed much more potentcAMP activity than other short chimeras lacking an aromaticresidue at this position (Ex-4[1–19]/pl14a and Ex-4[1–20]/pl14a). Consistent with this, substituting Tyr22 with Ala22 inEx-4[1–16]/pl14a resulted in a peptide (Ex-4[1–16]/pl14aY22A) with a 30-fold lower cAMP activity.

Increased Helicity Does Not Correlate with Higher Potency—To evaluate the effect of grafting the truncated GLP-1 and Ex-4peptides into the helical conotoxin pl14a on overall helicity,circular dichroism was measured, and helicity was estimatedfor all peptides (Table 2; Fig. 2, A–E). The fraction helicity ofGLP-1 and Ex-4 corresponded well with those determined pre-viously in aqueous buffer at pH 3.5 (40). Overall, incorporationof the truncated GLP-1 and Ex-4 into the helical pl14a resultedin increased helicity. A comparison of the potency of cAMPsignaling for the various GLP-1 and Ex-4 variants did not showany correlation with fraction helicity (Table 2; Fig. 2F), which isin agreement with previous reports for lactam-bridged GLP-1analogs (23, 24).

NMR Spectroscopy—Comparison of the C-terminal sectionof Ex-4[1–16]/pl14a (PDB 2NAV) and Ex-4[1–27]/pl14a (PDB2NAW) with the native pl14a showed an r.m.s.d. of 0.32 Å and0.40 Å across the heavy backbone atoms of the �-helix (Ex-4[1–16]/pl14a residues 9 –16, Ex-4[1–27]/pl14a residues 20 –27,and pl14a residues 6 –13), suggesting that conotoxin pl14a can

TABLE 2Helicity and cAMP signaling for truncated and chimera GLP-1 and Ex-4 variants

Peptide Calculated mass Determined mass Helicity cAMP EC50 � S.E. n EC50a

% nM -Fold changeGLP-1[7–36] 3353.66 3353.67 22.3 0.040 � 0.004 19GLP-1[7–36]/pl14a v1 4653.21 4653.80 54.5 0.24 � 0.06 3 6GLP-1[7–36]/pl14a v2 4724.25 4724.80 60.7 0.61 � 0.10 3 15GLP-1[7–33]/pl14a 4239.97 4239.95 38.5 0.40 � 0.03 3 10GLP-1[7–33] 2955.42 2955.43 26.7 0.23 � 0.03 3 5.8GLP-1[7–30]/pl14a 3938.86 3938.74 22.8 3000 � 200 3 75,000GLP-1[7–30] 2557.19 2557.20 16.1 930 � 170 3 23,000GLP-1[7–29]/pl14a 3828.73 3828.71 26.7 180 � 20 3 4,500GLP-1[7–26]/pl14a 3512.51 3512.49 41.8 220 � 30 3 5,500GLP-1[7–25]/pl14a 3277.42 3277.40 26.8 1200 � 300 3 30,000GLP-1[7–22]/pl14a 3086.25 3086.23 63.8 300 � 70 3 7,500GLP-1[7–21]/pl14a 2963.30 2963.27 42.9 � 10000 3GLP-1[7–19]/pl14a 2811.18 2811.16 16.0 1300 � 400 3 33,000Ex-4[1–39] 4184.02 4184.03 70.4 0.019 � 0.009 3Ex-4[1–30]/pl14a 4764.23 4764.20 74.6 0.084 � 0.006 3 4.4Ex-4[1–30] 3407.64 3407.65 57.9 0.017 � 0.005 3 0.89Ex-4[1–30] F22A 3331.60 3331.61 63.8 8.6 � 1.2 3 450Ex-4[1–27]/pl14a 4480.06 4480.04 77.9 0.019 � 0.004 3 1Ex-4[1–27]/pl14a F22A 4404.03 4404.01 51.3 3.6 � 0.15 3 190Ex-4[1–27] 3179.55 3179.56 40.3 0.23 � 0.02 3 12Ex-4[1–27] F22A 3103.52 3103.53 36.8 390 � 40 3 21,000Ex-4[1–24]/pl14a 4133.86 4133.84 73.6 46 � 4 3 2,400Ex-4[1–24] 2752.30 2752.30 23.2 67 � 6 3 3,500Ex-4[1–24] F22A 2676.26 2676.27 36.8 3000 � 1400 3 160,000Ex-4[1–23]/pl14a 3980.80 3980.78 25.1 33 � 17 3 1,700Ex-4[1–20]/pl14a 3647.61 3647.59 25.2 6600 � 300 3 350,000Ex-4[1–19]/pl14a 3436.49 3436.47 60.2 3100 � 300 3 160,000Ex-4[1–16]/pl14a 3150.32 3150.30 50.3 100 � 10 3 5,300Ex-4[1–16]/pl14a Y22A 3058.29 3058.27 50.8 3000 � 100 3 160,000Ex-4[1–15]/pl14a 3022.31 3022.29 59.1 1900 � 400 3 100,000Ex-4[1–13]/pl14a 2817.24 2817.21 45.1 2300 � 700 3 120,000Wild-type pl14a 2909.44 2909.60 61.7

a -Fold change refers to the parent molecule GLP-1[7–36] or EX-4[1–39].

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be used as a stabilizing scaffold and replace the Trp-cage pres-ent in native exendin-4. A comparison of the H� chemical shiftsto random coil values (41) showed stretches of negative second-ary 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 dataacquired for these two peptides, which suggests the presence ofa large degree of helicity (Fig. 2). This helicity was observed inthe three-dimensional structure of Ex-4[1–16]/pl14a, but notEx-4[1–27]/pl14a, due to the lack of sufficient NOEs in theregion despite the presence of several i-i�4 NOEs.

Molecular Modeling—Molecular dynamics was used toinvestigate how truncated Ex-4 variants might interact with theNTD of the receptor. In the case of Ex-4[1–27]/pl14a, whereincomplete NMR constraints were available, solution and crys-tallography structures were used to create potential models of

the bound peptides (see “Experimental Procedures”). Simula-tions of Ex-4[1–27] and Ex-4[1–27] F22A bound to the NTD ofGLP-1 resulted in average simulation structures that closelyaligned with the crystal structures of Ex-4/GLP-1R with 0.72and 0.74 Å C� r.m.s.d., respectively (Fig. 4A). Despite the lack ofstructural change between Ex-4[1–30] and Ex-4[1–30] F22A, areduction in the interaction energy between the NTD ofGLP-1R was observed for the F22A mutant (Table 3). The aver-age 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 F22Amutation in Ex-4[1–27]/pl14a F22A was associated with asimilar reduction in interaction energy as Ex-4[1–30] F22A,although Ex-4[1–27] F22A adopted a slight change in thespatial extension of the N-terminal end of the Ex-4 helix,resulting in a somewhat higher degree of deviation com-pared with the crystal structure (C� r.m.s.d. 0.97 Å). These

FIGURE 2. Representative circular dichroism spectrums for GLP-1 and Ex-4 variants. Circular dichroism spectrums for grafted (A) and linear (B) GLP-1variants 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 EC50values.

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reductions in interaction energies in silico for F22A mutantsfollow the trend seen in the cAMP assays but were not sig-nificant or absolutely proportional to the changes in EC50values. Attempts were made to model the binding betweenthe NTD of GLP-1 and Ex-4[1–16]/pl14a, but the overlap-ping binding interface was determined too small to provide arealistic simulation.

Discussion

In this study we produced and characterized a series of C-ter-minally truncated GLP-1 and Ex-4 peptides, both in their linearforms and as �-conotoxin pl14a chimeric peptides. We foundthat residues 28 –30 of GLP-1 and residues 28 –39 of Ex-4were not essential for either peptide helicity or GLP-1R sig-naling via the cAMP pathway in vitro. Further C-terminaltruncations were associated with loss of �-helicity andGLP-1R signaling potency for both peptide hormones.Incorporating C-terminally truncated peptides into the�-conotoxin scaffold often increased helicity but did notsimilarly improve signaling potency. The most prominentdeterminant of GLP-1R signaling in vitro was found to bePhe22 in either peptide hormone. These findings might help

to guide the future design of minimized and/or constrainedGLP-1 or Ex-4 peptide analogs suitable for the treatment oftype 2 diabetes mellitus.

The results presented in this study have clarified the relativecontribution of the C-terminal helical segments of GLP-1 andEx-4 to GLP-1R binding and signaling. Although there are a fewprevious studies involving C-terminal truncations of GLP-1using different methods (7, 17, 19), ours is the first to system-atically explore the effects of C-terminal truncation of GLP-1and Ex-4. We found that C-terminal removal of three residuesfrom GLP-1 and 12 residues from Ex-4 had only small effectsupon cAMP signaling for both linear and chimeric peptides(Fig. 5A). Conversely, removal of three more residues (resi-dues 25–27) from the C terminus of either peptide resulted in apotency loss equal to at least three orders of magnitude. A sim-ilar dominating effect was seen for Phe22 in both GLP-1 andEx-4, where peptides that included Phe22 generally showedmore potent cAMP signaling. The positive effect of Phe22 wasparticularly strong for Ex-4, and a series of F22A mutant pep-tides confirmed the importance of Phe22 for cAMP signaling(Fig. 5B). These findings are in line with previous Ala scans ofGLP-1, which also found that the F22A substitution caused thelargest loss in binding affinity (1300-fold) and cAMP signaling(1000-fold) out of all Ala mutants (43). Combined, these find-ings suggest that the central portion of both GLP-1 and EX-4acts as a spacer to position a C-terminal helical segment (resi-dues 22–27) to correctly align Phe22 with a vital binding pocketon the receptor Fig. 5C). However, future studies need toexplore to what extent these structural determinants modulat-ing cAMP signaling translates to insulin secretion.

FIGURE 3. Secondary H� chemical shifts and solution structures of selected Ex-4/pl14a chimeras. A, Ex-4[1–16]/pl14a (PDB 2NAV) secondary H� chemicalshifts (left) and ribbon plots of solution structures (right) with �-helices shown in blue, and disulfide bonds are in yellow. B, Ex-4[1–27]/pl14a (PDB 2NAW)secondary H� chemical shifts (left) and solution structures (right). Residues with secondary H� chemical shifts indicating helical structure are indicated withgray helices above the residue numbering.

TABLE 3Calculated interaction energies for the GLP-1R NTD and Ex-4 variants

Peptide Interaction energy

kcal/mol � S.D.Ex-1[1–30] �258.0 � 38.4Ex-1[1–30] F22A �215.7 � 30.6Ex-4[1–27]/pI14a �194.0 � 32.8Ex-4[1–27]/pI14a F22A �158.3 � 19.2

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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 theresulting cAMP signaling Log EC50 values. B, alignment of various Ex-4 (blue) and conotoxin pl14a (black) chimeras and their EC50 values with residue 22 arebound 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.

FIGURE 4. Molecular modeling of the GLP-1R NTD and Ex-4/Ex-4 pl14a chimeras. A, overlay of the average molecular dynamics simulation secondarystructures 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 bythe 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 theelectrostatic surface of the GLP-1R NTD (blue, positive; red, negative: white, neutral).

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A recent study involving GLP-1R mutants concluded that acomplementary “hydrophobic patch” on GLP-1/Ex-4 and thereceptor is needed for correct positioning of the N terminus forGLP-1R activation, a feature that may extend to all class BGPCRs (44). This conclusion was based on GLP-1R mutagene-sis of L32A (44), on studies showing the importance of Phe22/Ile23 in GLP-1 for receptor activation (43, 45) and on the pres-ence of a similar hydrophobic patch on glucagon (needed forefficient binding to the glucagon extracellular domain) (46).Although Phe22 only forms part of this hydrophobic patch, wehave shown that mutating Phe22 has an impact that is orders ofmagnitude greater than other hydrophobic residues in thispatch.

These findings in conjunction with previous studies providenovel insight into the mechanistic foundations for GLP-1Rreceptor activation. Considering the size and nature of thebinding interface between GLP-1/Ex-4 and the GLP-1R, themutation of a single residue alone is unlikely to explain such alarge change in signaling efficacy; rather, it suggests a mecha-nistic cause. It may well be that the correct spatial orientation ofPhe22 to the pocket in the receptor triggers conformationalchange in the receptor that affects the relative orientation ofthe N-terminal and transmembrane domains. This may alsoexplain why much smaller 11-residue peptides, which sharethe N-terminal portion of GLP-1/Ex-4 but have variousbiphenyl and/or phenylalanine derivatives at the C-terminalend, achieve similar activation profiles to that of GLP-1 andEx-4 (28, 47, 48). It is possible that, for these compounds, thebiphenyl derivatives bind to the same pocket as Phe22 inGLP-1/Ex-4 and trigger a conformational change in thereceptor that allows the N terminus of these shorter peptidesto interact with the core of the receptor. If so, one may envi-sion future GLP-1R ligands that consist of a C-terminalhydrophobic anchor segment, a central spacer segment ofcorrect length, and an N-terminal activation segment thatbinds to the core of the receptor.

Further studies need to be undertaken to fully clarify the roleof the hydrophobic patch in receptor activation and signaling.For example, further GLP-1R mutagenesis studies of all resi-dues involved in this hydrophobic interaction may shed furtherlight on the extent of the patch and the relative contribution ofvarious receptor residues. In particular, comparing the effect ofGLP-1R mutation on binding affinity and signaling using bothfull-length peptide agonists and the 11-residue peptides shouldgive a good indication of whether they capitalize on the sameinteractions. Cross-linking experiments between the 11-resi-due peptides and the GLP-1R may also be used to further pin-point the binding area of the biphenyl derivatives.

Author Contributions—J. E. S. designed the peptides, planned theexperiments, performed circular dichroism spectroscopy and insilico calculations, and drafted the manuscript. J. M. and D. R. D.performed the cAMP assays. C. I. S. performed the NMR experi-ments and analysis. D. P. F., D. J. E, D. A. G., R. B. R., P. M. L.,D. A. P, S. L., and D. J. C. planned the experiments, analyzed thedata, and drafted the manuscript. All authors approved the finalarticle.

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GLP-1 and Ex-4 �-Conotoxin pl14a Chimeras

JULY 22, 2016 • VOLUME 291 • NUMBER 30 JOURNAL OF BIOLOGICAL CHEMISTRY 15787

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David A. Price, Spiros Liras and David J. CraikJ. Edmonds, David A. Griffith, Roger B. Ruggeri, David R. Derksen, Paula M. Loria,

Joakim E. Swedberg, Christina I. Schroeder, Justin M. Mitchell, David P. Fairlie, Davidin VitrocAMP Signaling

-Helicity and Reveal Interactions Vital forαChimeras Maintain Potency and -Conotoxin pl14a PeptideαTruncated Glucagon-like Peptide-1 and Exendin-4

doi: 10.1074/jbc.M116.724542 originally published online May 10, 20162016, 291:15778-15787.J. Biol. Chem. 

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