Alpha-helix or beta-turn? An investigation into N-terminally

Subscriber access provided by Kaohsiung Medical University

Article

Alpha-helix or beta-turn? An investigation into N-terminally constrained analogs of glucagon-like peptide 1 (GLP-1) and exendin-4 Alberto Oddo, Sofia Mortensen, Henning Thøgersen, Leonardo De Maria, Stephanie Hennen, James McGuire, Jacob Kofoed, Lars Linderoth, and Steffen Reedtz-Runge Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.8b00105 • Publication Date (Web): 07 Jun 2018 Downloaded from http://pubs.acs.org on June 9, 2018

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 22 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

Alpha-helix or beta-turn? An investigation into Nterminally constrained analogs of glucagon-like peptide 1 (GLP-1) and exendin-4 Alberto Oddo,‡ Sofia Mortensen, ‡ Henning Thøgersen, Leonardo De Maria, Stephanie Hennen, James N. McGuire, Jacob Kofoed, Lars Linderoth and Steffen Reedtz-Runge* Global Research, Novo Nordisk A/S, Novo Nordisk Park, 2760, Måløv, Denmark

KEYWORDS: GLP-1, Diabetes, Exendin, GPCR, Incretin

ABSTRACT Peptide agonists acting on the glucagon-like peptide 1 receptor (GLP-1R) promote glucosedependent insulin release and therefore represent important therapeutic agents for Type 2 diabetes (T2D). Previous data indicated that an N-terminal type II β-turn motif might be an important feature for agonists acting on GLP-1R. In contrast, recent publications reporting the structure of full-length GLP-1R have shown the N-terminus of receptor-bound agonists in an αhelical conformation. In order to reconcile these conflicting results we prepared N-terminally constrained analogs of glucagon-like peptide 1 (GLP-1) and exendin-4 and evaluated their

ACS Paragon Plus Environment

1

Biochemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 2 of 22

receptor affinity and functionality in vitro; we then examined their crystal structures in complex with the ECD of GLP-1R and used molecular modeling and molecular dynamics simulations for further investigations. We report that the peptides’ N-termini in all determined crystal structures adopted a type II β-turn conformation, but in vitro potency varied thousand-folds across the series. Potency correlated better with α-helicity in our computational model, although we have found that the energy barrier between the two mentioned conformations is low in our most potent analogs and flexibility of the N-terminus is highlighted by the dynamic simulations.

Introduction Type 2 diabetes (T2D) is a condition characterized by chronic hyperglycemia due to insufficient insulin release or resistance to insulin action.1 Glucose-sensitive insulin release in the important postprandial state is mediated by peptide hormones belonging to the incretin family: glucagonlike peptide 1 (GLP-1, Fig. 1a) and glucose-dependent insulinotropic polypeptide (GIP).2 As sensitivity to GIP is reduced in T2D patients,3 agonists acting on the GLP-1 receptor (GLP-1R) have traditionally been the preferential target of research efforts by the pharmaceutical industry.4 Besides the endogenous hormone GLP-1, also exendin-4 (EX4, Fig. 1b), a peptide isolated from the saliva of Heloderma suspectum, was found to be a potent activator of GLP-1R and insulin secretagogue.5 The two peptides are thought to derive from a common ancestor and share a 53% sequence homology (Fig. 1).6 Activation of the GLP-1R is generally thought to occur via a two-domain mechanism: the Cterminal portion of the agonist binds with high affinity to the receptor’s extracellular domain (ECD), thus directing the N-terminal portion toward its binding pocket in the receptor’s


2

Page 3 of 22 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

transmembrane domain (TMD).7 This second interaction is thought necessary for triggering the intracellular signaling cascade. Consistently, N-terminally truncated EX4(9-39) is a potent and well characterized competitive GLP-1R antagonist, whereas synthetic C-terminally truncated analogs of GLP-1, although entirely lacking an ECD-binding region, retained potent agonist activity on GLP-1R.8 We have previously shown that the C-terminal portions of EX4 and human GLP-1 adopt an αhelical conformation when bound to the receptor’s ECD.9 However, the inner environment of the TMD is very different from that of the water-soluble ECD. Unfortunately, being deeply embedded in the TMD, studying the receptor-bound conformation of the important pharmacophore region consisting of the six N-terminal residues has proven an arduous task. Studies of solution-phase structures and computational models have produced a considerable amount of evidence suggesting that a distinctive N-terminal type II β-turn motif could be an essential feature in the active conformation of GLP-1R agonists.10 This hypothesis was supported by the turn-like structures already reported for endogenous ligands of other GPCRs (e.g. PACAP and apelin receptors).11 In contrast, the structure recently obtained by cryo-electron microscopy (cryo-EM) shows the N-terminus of receptor-bound GLP-1 in an α-helical conformation.12 The possibility of N-terminal helix capping has been previously hypothesized and is further supported by the recently published structure of receptor-bound salmon calcitonin and of GLP-1R bound to a short peptide agonist.13 Clarifying the activation process of GLP-1R and identifying novel structural determinants for modulating their activity would facilitate the design of better medicines. Here, we attempted to clarify whether an N-terminal α-helix or a β-turn correlates best with potency at the GLP-1R. To


3

Biochemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 4 of 22

this end we prepared a set of N-terminally constrained analogs of both human GLP-1 and EX4, and investigated their receptor potency and binding affinity in vitro. Furthermore, we examined their crystal structures in complex with the ECD of GLP-1R. We also used the published cryoEM structure as a basis for molecular modeling and performed molecular dynamics simulations of selected analogues in complex with the ECD. We report that the N-termini in all determined crystal structures were found in a type II β-turn conformation, but in vitro potency varied thousand-folds across the series. Figure 1. (a) Sequences of human GLP-1(7-37) (1) and (b) EX4(1-39) (7) with respective amino acid numbering; conserved positions are shown in blue. (c) General sequence of the analogs presented in this study; the grey chain represents either human GLP-1(12-37) (2-6) or EX4(6-39) (8-12).

Materials & Methods Peptide synthesis and purification. Peptides were prepared by microwave-assisted solid-phase synthesis using the Fmoc/tBu strategy. All syntheses were carried out on a Liberty Blue instrument (CEM). After release from the solid support in their reduced state, peptides were purified by preparative HPLC. Selected fractions were pooled and diluted, then the oxidizing


4

Page 5 of 22 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

agent Aldrithiol-4 was added in moderate excess to form the desired intramolecular disulfide bond. When LC-MS analysis showed reaction completion, the mixture was loaded once again onto a preparative HPLC to isolate the disulfide analogs in ≥95% purity (exceptions: 4, 7, 8 and 12 were >94% pure). Purity and identity were verified by analytical UPLC and LC-MS.

Isolation and crystallization of complexes of ECD with analogs. The ECD of human GLP-1R (hGLP-1R) was expressed and purified as previously described.14 Aliquots of 1000 nmol of freeze-dried peptide analogs were dissolved in 1 mL of 50 mM Tris buffer (pH 8.0). The ECD in 10 mM Tris (pH 7.5), 100 mM Na2SO4, 2% v/v glycerol was mixed with the peptide solutionat molar ratio ECD:peptide=1:3. The mixtures were incubated at 4ºC for 30 min and then loaded on a Superdex75 16/60 column equilibrated with 10 mM Tris (pH 7.5). Fractions containing the ECD-peptide complex were pooled and used for crystallization experiments using the vapour diffusion technique in sitting drops. The best diffracting crystals were obtained at 20℃ using the Morpheus® crystallization screen from Molecular Dimensions (for detailed description of crystallization conditions see the Supporting Information file). Crystals were harvested and flashcooled in liquid nitrogen.

Structure determinations and model building. Diffraction data from crystals containing 2, 3, 4, 6, 11 were collected in house using a RIGAKU FR-X X-ray source equipped with a DECTRIS PILATUS3 R 1M detector. The data from crystals containing 5 were collected at the X06DA beamline at Swiss Light Source, Villigen, Switzerland. The data were processed with XDS.15 S Phase information was obtained using molecular replacement with Phaser.16 The models were


5

Biochemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 6 of 22

built by iterative cycles of manual building in Coot17 and refining in Phenix.refine.18 Data collection, processing and model refinement statistics are available in the Supporting Information. Figures were prepared with PyMol (www.pymol.org). Coordinates and structure factors have been deposited at the RCSB protein data bank www.rcsb.org with accession codes: 5OTX (2), 5OTV (3), 5OTW(4), 5OTU(5), 5OTT(11).

In vitro receptor binding assays. Experiments were carried out as previously described.19 Briefly, the ability of tested analogs to displace radiolabeled [125I]GLP-1 was measured in a scintillation proximity assay (SPA, Perkin Elmer). Membranes isolated from whole BHK cells overexpressing the hGLP-1R were suspended in a buffer (pH = 7.4) consisting of 50 mM HEPES, 5 mM EGTA, 5 mM MgCl2, 0.005% Tween 20 to a final protein concentration of 0.2 mg/ml. The membranes were added to 96-well microtiter plates followed by SPA beads (0.5 mg/well) and the peptide dilution series. The GLP-1 tracer (0.06 nM [126I]GLP-1) was added and the plates were incubated for 2 hours at 30 ºC. After incubation, SPA beads were pelleted by centrifugation and scintillation intensities were measured in a TopCount reader (Perkin Elmer). Reported values are the average of three or more independent experiments.

In vitro assay for cAMP accumulation. Experiments were carried out using the CRE-luc approach as previously described.19 Briefly, intracellular cAMP accumulation was measured in BHK cells stably co-expressing hGLP-1R and a cAMP reporter element (CRE) coupled to firefly luciferase gene (luc). Peptide solutions pre-arranged in a dilution series in a 96-well plate were transferred to the cell suspension and incubated for 3 hours at 37 ºC (5% CO2). Plates were


6

Page 7 of 22 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

allowed to cool down for 15 min, then the Steadylight Plus reagent (Perkin Elmer) was added and the plates were shaken gently for 30 min in the dark. Luminescence readings were carried out in a Synergy Multimode reader (BioTek). Data from the plate reader were transferred to GraphPad Prism 6 (GraphPad Software, San Diego) to perform non-linear regression analyses (three-parameter fit). Reported values are the average of three or more independent experiments performed in duplicates.

Molecular modelling. The β-sheet and α-helix conformational preferences for the N-terminal part of 6 and 12 were estimated from the potential energies of NAc-[D-hCys8,hCys11]-GLP-1[811]-NHCH3 having these conformations. These energies were determined by energy minimization of NAc-[D-hCys8,hCys11]-GLP-1[8-11]-NH2 having either the corresponding βsheet conformation of the 11-mer determined by NMR(ref 17, pdb:2n0i) or the α-helix conformation of GLP-1 bound to GLP-1R (ref 21, pdb:5vai). The energy minimizations were done using Macromodel, the OPLS3 force field and a continuum model for water. Macromodel and the OPLS3 force field are available from Schrödinger, LLC, New York, NY, US. Molecular dynamics simulations. Two 25 ns simulations were performed for each of the analogues 1, 2, 5 and 6 in complex with the GLP-1R ECD. In the first one the analogue started from the α-helical configuration of GLP-1 bound to GLP-1R (ref 21, pdb:5vai) with the appropriate substitutions at positions 8 and 11 introduced with Maestro from Schrödinger, LLC, New York, NY, US. The side-chain modifications where minimized while keeping the backbone fixed. The second simulation was performed with the GLP-1 analogues starting from a β-turn configuration. For analogues 2 and 5 the starting structures were taken from those solved in this


7

Biochemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 8 of 22

work; analogue 1 complex was modelled from 5 by back-mutations to the wild-type Ala-8 and Thr-11. For analogue 6 the coordinates from complex 5 of this work were used using those for residues 7-14 from 2N0I (first model). The GLP-1/GLP-1R ECD complexes where placed at the center of a cubic box of side 12.5 nm and optimally oriented using its moments of inertia. The system was solvated with explicit (TIP4P) water molecules and charge neutralized; additional NaCl was added to150 mM.. The solvent (water+ions) was equlibrated for 250 ps while constraining all the protein back-bone atoms to their initial position. The 25 ns production run sampled the isothermal–isobaric ensemble (NPT) with a modified Berendsen thermostat20 for temperature control (300K) and the Berendsen barostat21 for pressure control (1 atm). A configuration was saved every 10 fs. Particle-Mesh-Ewald (PME) was used to treat long-range electrostatics. Simulations were performed with the OPLS force-field22 and prepared and carried on with GROMACS23. The secondary-structure assignments for each of the saved configuration along the trajectories were done with DSSP24.

Results and Discussion Ten N-terminally constrained analogs were synthesized based on both human GLP-1(7-37) (26) and EX4 (8-12) (Fig. 1c). The analogs bear either a cysteine (Cys) or D/L-homocysteine (hCys) engaged in a i,i+3 disulfide bond. All analogs are listed in Table 1 along with their in vitro potency and binding affinity. Table 1. In vitro potency (cAMP accumulation assessed by cAMP-dependent reporter gene assay, EC50) and binding affinity (IC50) of 1-12 on human GLP-1R. Listed values are the average


8

Page 9 of 22 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

of three or more independent experiments performed in duplicates. Standard deviations are reported within brackets where relevant.

No.

Description

hGLP-1R EC50, pM (SEM)

hGLP-1R IC50, nM (SEM)

8.3 (2.2)

0.4 (0.1)

GLP-1 and analogs 1

GLP-1

2

Cys8, Cys11

>104

187 (73)

3

Cys8, hCys11

>104

13 (4.3)

4

hCys8, Cys11

>105

249 (43)

5

hCys8, hCys11

1471 (252)

22 (2.5)

270 (99)

7.8 (1.8)

4.1 (2.1)

0.6 (0.2)

6

D-hCys

8

, hCys11

Exendin-4 and analogs 7

EX4

8

Cys2, Cys5

>104

14 (5.7)

9

Cys2, hCys5

434 (56)

6.5 (3.6)

10

hCys2, Cys5

1020 (176)

11 (3.5)

11

hCys2, hCys5

10 (4.0)

3.0 (1.0)

12

D-hCys

2.9 (1.2)

1.5 (3.4)

2

, hCys5

Consistently with previous findings,10b the most potent disulfide analogs were those featuring a bridge between D-hCys in i and L-hCys in i+3 (6 and 12). However, constrained GLP-1 analogs


9

Biochemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 10 of 22

resulted at least 32-fold less potent than the parent peptide, whereas 12 was found to be equipotent to EX4. This difference is very apparent when examining the full concentrationresponse curves (Fig. 2). The relative potency ranking among the analogs was generally maintained across both series, but with the following exception: the least potent analog in the GLP-1 series was 4 (featuring a hCys-Cys bridge), whereas for the EX4 series it was 8 (featuring a Cys-Cys bridge). Both in the GLP-1 and EX4 scaffolds, in analogs featuring heteromeric bridges between a Cys and a hCys (3-4 and 9-10), positioning the latter in the i+3 position led to more potent agonists. This effect was more pronounced for GLP-1 than for EX4, and for the former we also observed an overall better correlation between binding affinity and potency. Specifically, in the case of EX4 the least potent analog (8) showed only a 10-fold loss in binding affinity compared to the most potent (12), versus a >104-fold loss in potency. This observation correlates well with the fact that the N-terminally truncated EX4(9-39) binds the receptor with affinity similar to that of the full-length peptide, whereas similarly truncated GLP-1 analogs bind with approx. 100-fold lower affinity.14, 25 Our results indicate that the EX4 scaffold is overall more tolerant of N-terminal constraints, as 12 resulted equipotent to EX4 while no constrained GLP-1 analog matched the activity of the parent peptide. These incongruences are difficult to rationalize within the frame of a single receptor activation model, but instead support the postulate that N-terminal interactions play a more crucial role in the binding of GLP-1 than of EX4.26 Nonetheless, subtle N-terminal interactions have proven essential for the intrinsic activity of EX4 analogs as well.


10

Page 11 of 22

Figure 2. Concentration-dependent increase of intracellular cAMP accumulation triggered by GLP-1 and analogs (1-6, circles), and EX4 and analogs (7-12, squares), assessed by a whole-cell cAMP response element reporter gene assay (CRE-luc). Reference compounds are displayed in black; pairs of analogs sharing the same disulfide bridge are displayed in the same color for ease of comparison. Depicted values represent mean ± SEM of at least three independent experiments and are normalized to maximum response of GLP-1 (1). Curves have been fitted using GraphPad Prism 6 (GraphPad Software, San Diego).

CREluc response (% of GLP-1)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

In the attempt to identify the structural determinants accountable for the observed differences in in vitro activity, we obtained crystals of the presented analogs in complex with the ECD of GLP-1R. It was possible to acquire diffraction data for 2-5 of excellent quality with respect to the electron density map around the N-termini. This allowed us to build unambiguous models of their turn structures (Fig. 3a). However, as expected, the N-termini are not in contact with the ECD.


11

Biochemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 12 of 22

Figure 3. (a) From left to right, detailed view of the N-terminal structures of 2 to 5. The models of the ring structures are placed in polder maps obtained omitting residues 7-11; the maps are countered at 3 σ. (b) Superposition of the structures of 2-5 in complex with the ECD of hGLP-1R (grey body), with detailed view from orthogonal perspectives. His7 and the side chain of Glu9 have been removed for clarity. Models are aligned on the backbone atoms of residues 8-11. Larger versions of these figures are available in the Supporting Information file.


12

Page 13 of 22 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

The N-terminal region of all these analogs was observed in a type II β-turn structure where i is position 8 and i+3 is position 11 (Table 2).10d,

27

We did not observe an extensive contact

network within the crystal that could enforce this behavior, suggesting instead that the residues had a spontaneous tendency to arrange themselves in this fashion. In all structures we found that the amide proton of residue i and the carbonyl oxygen of residue i+3 were positioned in a way that suggests their engagement in a hydrogen bond. In all cases, we observed only one interaction between atoms in the turn structure and their surroundings; namely, the amide proton of Gly10 is involved in a hydrogen bond with the side chain of Asp15 of the same molecule. The structures of 2-5 are largely superimposable when the backbone atoms of residues 8-11 are aligned, revealing that they all form surprisingly similar βturns (Fig. 3b). The average observed root-mean-square deviation (rmsd) for such alignments was 0.18 ± 0.14 Å, with the largest discrepancy measured between 2 and 3, and the lowest between 3 and 5. The most noticeable differences were found in the orientation of the side chains of Cys/hCys involved in the disulfide bridges (Fig. 3b). In addition, we managed to obtain the structure of the EX4 analog 11. Like GLP-1 analogs 2-5, the backbone conformation observed in 11 is also classifiable as a type II β-turn (Table 2) and is largely superimposable with the structure of 5 (see Supporting Information). Like the turns in 2-5, the turn in 11 was found to form a single contact with surrounding atoms, which was a hydrogen bond between the amide proton of Gly4 and the side chain of Glu17 of a symmetry-related ligand molecule. A structural comparison between the structure of 5 and (PDB codes) 2N0I,10b 5NX213c and 5VAI12 is shown in supplementary figures S4-S6. Interestingly, the N-terminal conformation in 2N0I appears similar to that of 5 presented here. The overall fold adopted by the N-terminus of


13

Biochemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 14 of 22

the 11-mer in 5NX2 displays some similarity to that of 5, however with significant differences of backbone geometry of the distal N-terminal part. We were not able to identify any correlation between propensity to form β-turns and in vitro potency. For example, the measured torsion angles for compound 2, the least active of the series, are remarkably similar to those of 11, the most potent (Table 2).

Table 2. Geometrical characteristics of the N-terminal β-turn structures of 2-5 and 11. The torsion angles are measured for the models' chains which present fully built turn structures. Torsion angles (º) No.

Hydrogen bond lengtha (Å)

Description ϕ i+1

ψ i+1

ϕ i+2

ψ i+2

Ideal type II β-turnb

-60

120

80

0

-c

Average type II β -turnb

-60

131

84

1

-c

-82.2

135.9

78.1

6.4

3.6

3

Cys8-Cys11 hCys8-Cys11

-66.0

124.2

96.6

2.1

3.2

4

Cys8-hCys11

-73.9

139.9

99.0

-3.8

3.7

5

hCys8-hCys11

-55.2

130.1

84.7

4.4

3.0

11

hCys2-hCys5

-78.6

126.7

84.2

20.6

3.1

2

a

Expressed as distance between the carbonyl oxygen in i and the amide nitrogen in i+3. From reference.27c c Hydrogen bonds may or may not be present. b

Molecular modeling was performed on the basis of the published cryo-EM structure of receptor-bound GLP-1.12 Specifically, we wished to evaluate the compatibility of the presented

α-helical structure with the N-terminal constraints investigated here. It is known from previous


14

Page 15 of 22 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

literature that in a disulfide bond the ideal S-S distance is approx. 2.0 Å and the preferred S-S torsion angle is approx. ±90º.28 We proceeded as follows: first, the side-chains of Ala8 and Thr11 in the published GLP-1 structure were replaced with those of Cys, hCys or D-hCys as described in Table 1. Then, the torsion angles of the side-chains of these residues (but not the helical backbone) were manually adjusted to achieve an S-S distance as close as possible to 2.0 Å. However, it was possible to place the two sulfur atoms within the desired distance only for 6, featuring a D-hCys/L-hCys disulfide bridge. For this peptide it was additionally possible to finetune the S-S torsion angle. Results are reported in Table 3.

Table 3. Calculated geometrical characteristics of the disulfide bonds in 2-6 in the assumption of a rigid α-helical backbone based on the structure reported in ref. 22. No.

a

Description

S-S distance (Å)

S-S torsion angle (º)

EC50 increase (folds)a

2

Cys8, Cys11

5.6

93

103

3

Cys8, hCys11

4.9

-28

103

4

hCys8, Cys11

4.7

-162

104

5

hCys8, hCys11

3.5

-111

102

6

D-hCys

2.0

-92

10

8

, hCys11

Expressed as order of magnitude of the EC50 ratio between the entry and native GLP-1 (1).


15

Biochemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 16 of 22

A better correlation with potency emerged here. Only 6 was able to accommodate the αhelical structure of the peptide backbone while also satisfying the geometrical requirements of the S-S bond, thus resulting the most potent GLP-1 analog in the series. Hence, peptides with different constraints are expected to destabilize the α-helix and indeed showed reduced potency. We estimated the potential energy levels of the α-helix and the β-turn conformations based on the truncated N-terminus of 6 and 12. The calculated potential energies in a simulated aqueous environment showed that the type II β-turn is favored over the α-helical conformation by approx. 10 kJ/mol. This preference for the β-turn in aqueous solution is in line with previous observations10. Since this result has been obtained from examining a truncated N-terminus, the helix-stabilizing effect of H-bonding between adjacent turns was not accounted for. Hence, the real energetic difference is likely to be lower. In order to investigate the N-terminal conformation further we performed molecular dynamics simulations of analogues 1, 2, 5 and 6 in complex with the GLP-1R ECD starting both from an α-helical and a β-turn configuration. The α-helical configuration of GLP-1 was modelled on the full length cryo-EM structure (pdb 5VAI, chain P); for the β-turn configuration we used the crystal structures reported in this work. The results of the simulations highlight the flexibility of the N-terminal region and show no preference for an α-helical conformation but some tendency to form a β-turn type structure as observed in our X-ray crystal structures (see Supplementary Material for details).


16

Page 17 of 22 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

Molecular dynamic simulations demonstrated that the N-terminal portions of all peptides were very flexible, and showed some preference for a β-turn conformation as observed in our Xray crystal structures, but essentially no tendency to form a helical conformation. The initial αhelical configuration of the N-terminus was rapidly lost in favor of coil, bend or turn configurations as from the DSSP classification. In the simulations that started from the type II βturn, the N-terminus did not display any α-helical character in the simulated timeframe. From a pragmatic perspective, a deeper understanding of the receptor’s activation process would facilitate the design of agonists with improved pharmacodynamic profiles. This has proven challenging because the N-terminal region of GLP-1 and related hormones is typically very sensitive to modifications. The identification of analogs (i.e. 12) capable of retaining full potency despite major chemical modifications at the N-terminus is therefore remarkable and opens up to new strategies in medicinal chemistry. On the other hand, it should also be considered that constraining the N-terminus of GLP-1R agonists did not lead to any potency benefits, either in this study or in previous literature,10a,

10b

even when the geometrical

characteristics of the constraint (e.g. in 6 and 12) were virtually ideal. It is possible that the expected entropic advantages are nullified by concomitant steric hindrance issues, and/or that a higher structural flexibility is required at a previous stage of the interaction. In conclusion, this study provides a basis to reconcile currently conflicting literature around the N-terminal structure of GLP-1R agonists. Specifically, we have shown that α-helicity is the N-terminal secondary structure that correlates best with agonist activity on the GLP-1R. The Nterminus of GLP-1 is highly flexible in solution, however, and the ECD structures, modelling and the molecular dynamic simulations support a preference by the free N-terminus for a β-turn


17

Biochemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 18 of 22

conformation. We hypothesize that the agonist changes conformational preference during the transition from solvated to receptor-bound state, as result of the different interaction network and chemical environment. In addition, we have presented the first instance of an N-terminally constrained GLP-1R agonist retaining potency on par with the native peptide.

ASSOCIATED CONTENT Supporting Information. Detailed descriptions of experimental conditions and crystallographic parameters, analytical chromatograms, mass spectra, enlarged figures, DSSP secondary-structure assignments from the molecular dynamics simulations. AUTHOR INFORMATION Corresponding Author *[email protected] Author Contributions A.O. synthetized and purified the peptides, and analyzed the data. S.M. performed ECD complexes purification, crystallization, and determined the structures. S.H. and J.N.M. conducted the in vitro experiments. H.T.T. has carried out the modeling work. L.D.M. performed and analyzed the molecular dynamics simulations. J.K., L.L., and S.R.R. outlined the research methods and objectives, secured internal funding and provided scientific advice. The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. ‡These authors contributed equally.


18

Page 19 of 22 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

ORCID Alberto Oddo: 0000-0002-5759-9988 Sofia Mortensen: 0000-0003-1631-6651 Leonardo De Maria: 0000-0002-8061-4242 Funding Sources A.O. and S.M. are postdoctoral researchers funded by Novo Nordisk via the STAR program. All other authors are – or have been – full-time employees and minor shareholders of Novo Nordisk. ACKNOWLEDGMENT The authors wish to thank Louise Kjerulf Christensen, Katja Chandelle Pedersen and Mette Hersom Bien for excellent technical assistance.

ABBREVIATIONS BHK, baby hamster kidney; cAMP, 3’,5’-cyclic adenosine monophosphate; ECD, extracellular domain; EX4, exendin-4; GLP-1, glucagon-like peptide 1; GLP-1R, glucagon-like peptide 1 receptor; GPCR, G-protein coupled receptor; rmsd, root-mean-square deviation (of atom positions); ps, picoseconds; ns, nanoseconds; nm: nanometers. REFERENCES 1. Groop, L., Pathogenesis of type 2 diabetes: the relative contribution of insulin resistance and impaired insulin secretion. Int. J. Clin. Pract. Suppl. 2000, (113), 3-13. 2. Baggio, L. L.; Drucker, D. J., Biology of incretins: GLP-1 and GIP. Gastroenterology 2007, 132 (6), 2131-57. 3. Vilsbøll, T.; Holst, J. J., Incretins, insulin secretion and Type 2 diabetes mellitus. Diabetologia 2004, 47 (3), 357-366. 4. (a) Tomlinson, B.; Hu, M.; Zhang, Y.; Chan, P.; Liu, Z. M., An overview of new GLP-1 receptor agonists for type 2 diabetes. Expert Opin. Investig. Drugs 2016, 25 (2), 145-58; (b) Knudsen, L. B., Glucagon-like peptide-1: the basis of a new class of treatment for type 2


19

Biochemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 20 of 22

diabetes. J. Med. Chem. 2004, 47 (17), 4128-34; (c) Lau, J.; Bloch, P.; Schaffer, L.; Pettersson, I.; Spetzler, J.; Kofoed, J.; Madsen, K.; Knudsen, L. B.; McGuire, J.; Steensgaard, D. B.; Strauss, H. M.; Gram, D. X.; Knudsen, S. M.; Nielsen, F. S.; Thygesen, P.; Reedtz-Runge, S.; Kruse, T., Discovery of the Once-Weekly Glucagon-Like Peptide-1 (GLP-1) Analogue Semaglutide. J. Med. Chem. 2015, 58 (18), 7370-80. 5. Eng, J.; Kleinman, W. A.; Singh, L.; Singh, G.; Raufman, J. P., Isolation and characterization of exendin-4, an exendin-3 analogue, from Heloderma suspectum venom. Further evidence for an exendin receptor on dispersed acini from guinea pig pancreas. J. Biol. Chem. 1992, 267 (11), 7402-5. 6. Fry, B. G.; Roelants, K.; Winter, K.; Hodgson, W. C.; Griesman, L.; Kwok, H. F.; Scanlon, D.; Karas, J.; Shaw, C.; Wong, L.; Norman, J. A., Novel venom proteins produced by differential domain-expression strategies in beaded lizards and gila monsters (genus Heloderma). Mol. Biol. Evol. 2010, 27 (2), 395-407. 7. (a) Donnelly, D., The structure and function of the glucagon-like peptide-1 receptor and its ligands. Br. J. Pharmacol. 2012, 166 (1), 27-41; (b) Hoare, S. R., Mechanisms of peptide and nonpeptide ligand binding to Class B G-protein-coupled receptors. Drug Discov. Today 2005, 10 (6), 417-27. 8. (a) Haque, T. S.; Martinez, R. L.; Lee, V. G.; Riexinger, D. G.; Lei, M.; Feng, M.; Koplowitz, B.; Mapelli, C.; Cooper, C. B.; Zhang, G.; Huang, C.; Ewing, W. R.; Krupinski, J., Exploration of structure-activity relationships at the two C-terminal residues of potent 11mer Glucagon-Like Peptide-1 receptor agonist peptides via parallel synthesis. Peptides 2010, 31 (7), 1353-60; (b) Mapelli, C.; Natarajan, S. I.; Meyer, J. P.; Bastos, M. M.; Bernatowicz, M. S.; Lee, V. G.; Pluscec, J.; Riexinger, D. J.; Sieber-McMaster, E. S.; Constantine, K. L.; Smith-Monroy, C. A.; Golla, R.; Ma, Z.; Longhi, D. A.; Shi, D.; Xin, L.; Taylor, J. R.; Koplowitz, B.; Chi, C. L.; Khanna, A.; Robinson, G. W.; Seethala, R.; Antal-Zimanyi, I. A.; Stoffel, R. H.; Han, S.; Whaley, J. M.; Huang, C. S.; Krupinski, J.; Ewing, W. R., Eleven amino acid glucagon-like peptide-1 receptor agonists with antidiabetic activity. J. Med. Chem. 2009, 52 (23), 7788-99. 9. (a) Underwood, C. R.; Garibay, P.; Knudsen, L. B.; Hastrup, S.; Peters, G. H.; Rudolph, R.; Reedtz-Runge, S., Crystal structure of glucagon-like peptide-1 in complex with the extracellular domain of the glucagon-like peptide-1 receptor. J. Biol. Chem. 2010, 285 (1), 72330; (b) Runge, S.; Thogersen, H.; Madsen, K.; Lau, J.; Rudolph, R., Crystal structure of the ligand-bound glucagon-like peptide-1 receptor extracellular domain. J. Biol. Chem. 2008, 283 (17), 11340-7. 10. (a) Plisson, F.; Hill, T. A.; Mitchell, J. M.; Hoang, H. N.; de Araujo, A. D.; Xu, W.; Cotterell, A.; Edmonds, D. J.; Stanton, R. V.; Derksen, D. R.; Loria, P. M.; Griffith, D. A.; Price, D. A.; Liras, S.; Fairlie, D. P., Helixconstraints and amino acid substitution in GLP-1 increase cAMP and insulin secretion but not beta-arrestin 2 signaling. European journal of medicinal chemistry 2017, 127, 703-714; (b) Hoang, H. N.; Song, K.; Hill, T. A.; Derksen, D. R.; Edmonds, D. J.; Kok, W. M.; Limberakis, C.; Liras, S.; Loria, P. M.; Mascitti, V.; Mathiowetz, A. M.; Mitchell, J. M.; Piotrowski, D. W.; Price, D. A.; Stanton, R. V.; Suen, J. Y.; Withka, J. M.; Griffith, D. A.; Fairlie, D. P., Short Hydrophobic Peptides with Cyclic Constraints Are Potent Glucagon-like Peptide-1 Receptor (GLP-1R) Agonists. J. Med. Chem. 2015, 58 (9), 40805; (c) Miller, L. J.; Chen, Q.; Lam, P. C.; Pinon, D. I.; Sexton, P. M.; Abagyan, R.; Dong, M., Refinement of glucagon-like peptide 1 docking to its intact receptor using mid-region photolabile probes and molecular modeling. J. Biol. Chem. 2011, 286 (18), 15895-907; (d) Petersen, B.; Lundegaard, C.; Petersen, T. N., NetTurnP--neural network prediction of beta-turns by use of


20

Page 21 of 22 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

evolutionary information and predicted protein sequence features. PLoS One 2010, 5 (11), e15079. 11. (a) Macaluso, N. J.; Glen, R. C., Exploring the 'RPRL' motif of apelin-13 through molecular simulation and biological evaluation of cyclic peptide analogues. ChemMedChem 2010, 5 (8), 1247-53; (b) Inooka, H.; Ohtaki, T.; Kitahara, O.; Ikegami, T.; Endo, S.; Kitada, C.; Ogi, K.; Onda, H.; Fujino, M.; Shirakawa, M., Conformation of a peptide ligand bound to its Gprotein coupled receptor. Nat. Struct. Biol. 2001, 8 (2), 161-5. 12. Zhang, Y.; Sun, B.; Feng, D.; Hu, H.; Chu, M.; Qu, Q.; Tarrasch, J. T.; Li, S.; Sun Kobilka, T.; Kobilka, B. K.; Skiniotis, G., Cryo-EM structure of the activated GLP-1 receptor in complex with a G protein. Nature 2017, 546 (7657), 248-253. 13. (a) Neumann, J. M.; Couvineau, A.; Murail, S.; Lacapere, J. J.; Jamin, N.; Laburthe, M., Class-B GPCR activation: is ligand helix-capping the key? Trends Biochem. Sci. 2008, 33 (7), 314-9; (b) Liang, Y. L.; Khoshouei, M.; Radjainia, M.; Zhang, Y.; Glukhova, A.; Tarrasch, J.; Thal, D. M.; Furness, S. G. B.; Christopoulos, G.; Coudrat, T.; Danev, R.; Baumeister, W.; Miller, L. J.; Christopoulos, A.; Kobilka, B. K.; Wootten, D.; Skiniotis, G.; Sexton, P. M., Phaseplate cryo-EM structure of a class B GPCR-G-protein complex. Nature 2017, 546 (7656), 118123; (c) Jazayeri, A.; Rappas, M.; Brown, A. J. H.; Kean, J.; Errey, J. C.; Robertson, N. J.; FiezVandal, C.; Andrews, S. P.; Congreve, M.; Bortolato, A.; Mason, J. S.; Baig, A. H.; Teobald, I.; Dore, A. S.; Weir, M.; Cooke, R. M.; Marshall, F. H., Crystal structure of the GLP-1 receptor bound to a peptide agonist. Nature 2017, 546 (7657), 254-258. 14. Runge, S.; Schimmer, S.; Oschmann, J.; Schiodt, C. B.; Knudsen, S. M.; Jeppesen, C. B.; Madsen, K.; Lau, J.; Thogersen, H.; Rudolph, R., Differential structural properties of GLP-1 and exendin-4 determine their relative affinity for the GLP-1 receptor N-terminal extracellular domain. Biochemistry 2007, 46 (19), 5830-40. 15. Kabsch, W., Integration, scaling, space-group assignment and post-refinement. Acta Crystallogr., Sect. D: Biol. Crystallogr. 2010, 66 (Pt 2), 133-44. 16. McCoy, A. J.; Grosse-Kunstleve, R. W.; Adams, P. D.; Winn, M. D.; Storoni, L. C.; Read, R. J., Phaser crystallographic software. J. Appl. Crystallogr. 2007, 40 (Pt 4), 658-674. 17. Emsley, P.; Lohkamp, B.; Scott, W. G.; Cowtan, K., Features and development of Coot. Acta Crystallogr., Sect. D: Biol. Crystallogr. 2010, 66 (Pt 4), 486-501. 18. Afonine, P. V.; Grosse-Kunstleve, R. W.; Echols, N.; Headd, J. J.; Moriarty, N. W.; Mustyakimov, M.; Terwilliger, T. C.; Urzhumtsev, A.; Zwart, P. H.; Adams, P. D., Towards automated crystallographic structure refinement with phenix.refine. Acta Crystallogr., Sect. D: Biol. Crystallogr. 2012, 68 (Pt 4), 352-67. 19. Hennen, S.; Kodra, J. T.; Soroka, V.; Krogh, B. O.; Wu, X.; Kaastrup, P.; Orskov, C.; Ronn, S. G.; Schluckebier, G.; Barbateskovic, S.; Gandhi, P. S.; Reedtz-Runge, S., Structural insight into antibody-mediated antagonism of the Glucagon-like peptide-1 Receptor. Sci. Rep. 2016, 6, 26236. 20. Bussi, G.; Donadio, D.; Parrinello, M., Canonical sampling through velocity rescaling. The Journal of chemical physics 2007, 126 (1), 014101. 21. Berendsen, H. J. C.; Postma, J. P. M.; van Gunsteren, W. F.; DiNola, A.; Haak, J. R., Molecular dynamics with coupling to an external bath. The Journal of chemical physics 1984, 81 (8), 3684-3690. 22. Jorgensen, W. L.; Maxwell, D. S.; Tirado-Rives, J., Development and Testing of the OPLS All-Atom Force Field on Conformational Energetics and Properties of Organic Liquids. Journal of the American Chemical Society 1996, 118 (45), 11225-11236.


21

Biochemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 22 of 22

23. Abraham, M. J.; Murtola, T.; Schulz, R.; Páall, S.; Smith, J. C.; Hess, B.; Lindah, E., Gromacs: High performance molecular simulations through multi-level parallelism from laptops to supercomputers. SoftwareX 2015, 1-2, 19-25. 24. Kabsch, W.; Sander, C., Dictionary of protein secondary structure: Pattern recognition of hydrogen‐bonded and geometrical features. Biopolymers 1983, 22 (12), 2577-2637. 25. (a) Thorens, B.; Porret, A.; Buhler, L.; Deng, S. P.; Morel, P.; Widmann, C., Cloning and functional expression of the human islet GLP-1 receptor. Demonstration that exendin-4 is an agonist and exendin-(9-39) an antagonist of the receptor. Diabetes 1993, 42 (11), 1678-82; (b) Montrose-Rafizadeh, C.; Yang, H.; Rodgers, B. D.; Beday, A.; Pritchette, L. A.; Eng, J., High potency antagonists of the pancreatic glucagon-like peptide-1 receptor. J. Biol. Chem. 1997, 272 (34), 21201-6. 26. (a) Al-Sabah, S.; Donnelly, D., A model for receptor-peptide binding at the glucagon-like peptide-1 (GLP-1) receptor through the analysis of truncated ligands and receptors. Br. J. Pharmacol. 2003, 140 (2), 339-46; (b) Mann, R. J.; Nasr, N. E.; Sinfield, J. K.; Paci, E.; Donnelly, D., The major determinant of exendin-4/glucagon-like peptide 1 differential affinity at the rat glucagon-like peptide 1 receptor N-terminal domain is a hydrogen bond from SER-32 of exendin-4. Br. J. Pharmacol. 2010, 160 (8), 1973-84; (c) Doyle, M. E.; Theodorakis, M. J.; Holloway, H. W.; Bernier, M.; Greig, N. H.; Egan, J. M., The importance of the nine-amino acid C-terminal sequence of exendin-4 for binding to the GLP-1 receptor and for biological activity. Regul. Pept. 2003, 114 (2-3), 153-8. 27. (a) Venkatachalam, C. M., Stereochemical criteria for polypeptides and proteins. V. Conformation of a system of three linked peptide units. Biopolymers 1968, 6 (10), 1425-36; (b) Richardson, J. S., The anatomy and taxonomy of protein structure. Adv Protein Chem 1981, 34, 167-339; (c) Hutchinson, E. G.; Thornton, J. M., A revised set of potentials for beta-turn formation in proteins. Protein Sci. 1994, 3 (12), 2207-16. 28. Thornton, J. M., Disulphide bridges in globular proteins. J. Mol. Biol. 1981, 151 (2), 26187.

GRAPHICS FOR THE TABLE OF CONTENTS


22

Alpha-helix or beta-turn? An investigation into N-terminally

Recommend Documents