Peptides 53 (2014) 270–277

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Structural studies of adipokinetic hormones in water and DPC micelle solution using NMR distance restrained molecular dynamics Graham E. Jackson a,∗ , Riedaa Gamieldien a , Grace Mugumbate a , Gerd Gäde b a b

Department of Chemistry, University of Cape Town, Private Bag, Rondebosch, Cape Town 7701, South Africa Biological Sciences, University of Cape Town, Private Bag, Rondebosch, Cape Town 7701, South Africa

a r t i c l e

i n f o

Article history: Received 15 October 2013 Received in revised form 29 December 2013 Accepted 30 December 2013 Available online 18 January 2014 Keywords: Adipokinetic hormones (AKHs) Melme-CC Declu-CC Molecular dynamics simulations and receptor docking

a b s t r a c t Melme-CC (pGlu-Leu-Asn-Tyr-Ser-Pro-Asp-Trp amide) and Declu-CC (pGlu-Leu-Asn-Phe-Ser-Pro-AsnTrp-Gly-Asn amide) are members of the insect adipokinetic hormone family with very different activities in the locust bioassay. The conformations of both peptides were determined in water and in a phospholipid (DPC) micelle solution using nuclear magnetic resonance (NMR) restrained molecular dynamics simulations. In water, Melme-CC has one dominant conformation while in DPC solution it has two preferred conformation. In water, Declu-CC has two conformations but in DPC solution it has one preferred conformation, which is similar to one of the water conformations. All the conformations have type IV ␤-turn between residues 4 and 7. The binding of the two peptides to the DPC micelle is different. Melme-CC does not bind strongly to the surface and is oriented with the ␤-turn facing the surface. Declu-CC interacts more strongly with the ␤-turn facing away from the surface. Both termini having hydrophobic interactions with the surface. In Declu-CC the side chain of Asn7 projects away from the chain while in Melme-CC the Asp7 side chain is folded inside the chain. The different orientation of these side chains may account for the much higher biological activity of Declu-CC in mobilizing lipids in the locust compared to the poor biological effect of Melme-CC in this bioassay. Receptor binding of Declu-CC was tested using a model AKH receptor from Anopheles gambiae. A free energy of binding of −38.5 kJ mol−1 was found. © 2014 Elsevier Inc. All rights reserved.

1. Introduction With more than one million species known, insects account for more than 50% of all existing organisms on earth. They have highly developed and diverse forms and were the first animals during evolution that became truly terrestrial and also developed the ability to fly. The latter development caused the invention of an efficient hormonal system to mobilize substrates from energy stores in the fat body which are subsequently used by oxidation in the exclusively aerobic flight muscles to provide the enormous amount of energy necessary to contract the flight muscles [12,13]. These hormones, involved in substrate mobilization, are peptides and are generically called adipokinetic hormones (AKHs), although they are also involved in the regulation of circulating concentration of carbohydrates and proline [13,19]. AKHs are produced in modified neurons, so-called neurosecretory cells, which are located in the corpora cardiaca of insects [17,19]. At present, approximately 60 members of this family are structurally recognized as being peptides

∗ Corresponding author. Tel.: +27 21 6502531; fax: +27 21 6505195. E-mail address: [email protected] (G.E. Jackson). 0196-9781/$ – see front matter © 2014 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.peptides.2013.12.019

of a chain length of 8–10 amino acids with characteristically posttranslationally modified termini (a pyroglutamate [pGlu] residue at the N-terminus and a carboxyamide at the C-terminus), aromatic amino acids at position 4 (phenylalanine mostly but in some cases tyrosine) and a glycine residue at position 9 in the longer peptides (reviewed in [15,16]). AKHs act via G-protein coupled receptors of which only a few are completely known yet; the second messengers cyclic AMP and Ca2+ are involved for the activation of lipases and the second messengers inositoltrisphosphate (IP3 ), diacylglycerol and Ca2+ play a role in the activation of glycogen phosphorylases (reviewed by [17]). To understand and study the binding of AKH molecules to their receptors and to use that information for the design of peptide mimetics, it is important to have a firm database of the conformational properties of AKH peptides. To date, a few studies have addressed this problem by using nuclear magnetic resonance (NMR) [25,43], computational methods [30,42,44] or circular dichroism spectroscopy [8]. The most comprehensive investigation used a set of AKH peptides that differed in the amino acid at position 4 and 7 as well as in chain length [31,32] (Table 1). In these studies the solution conformation of the AKH peptides were investigated in dimethylsulfoxide (DMSO) solution, applying NMR restrained

G.E. Jackson et al. / Peptides 53 (2014) 270–277

molecular mechanic simulations. The major results were that all peptides had an “extended” structure for the first 4 residues and a ␤-turn between residues 4 and 8. However, whereas the Asn residue at position 7 in the peptides Tenmo-HrTH and Declu-CC project outwards from the turn, the side chain of this residue folds inside the turn in the Melme-Asn7 -analogue peptide; Melme-CC does not have an Asn7 . It was also calculated that the ␤-turn was tighter in Tenmo-HrTH and Declu-CC than in the two Melme peptides. Thus, it was concluded that Phe4 and Asn7 are the preferred amino acids to result in a conformation able to bind to the locust AKH receptor [31,32]. Since experiments conducted in DMSO may be somewhat artificial, the present study takes the investigations a step further and analyzes the secondary structures in water and in a membrane mimetic, dodecylphosphocholine (DPC). The peptides studied were restricted to Melme-CC and Declu-CC, which exhibit vastly different biological activities in mobilizing lipids in the migratory locust [11,14]. Since the peptides are poorly soluble in water their conformation in this medium was investigated using computational methods (Table 1). In DPC micelle solution both NMR and computational methods were used.

2. Materials and methods 2.1. Sample preparation The peptide hormones, Melme-CC and Declu-CC, were previously synthesized by Dr R. Kellner of Merck KGaA, Darmstadt, Germany. The synthesis involved the standard protocol for solidphase peptide synthesis using Fmoc-amino acid chemistry. The peptides were taken up in an acetonitrile/water mixture, purified by high performance liquid chromatography in the presence of 0.1% trifluoroacetic acid and then freeze dried. 1–3 mM solutions of Melme-CC and Declu-CC were prepared in 600 ␮L of 50 mM sodium phosphate buffer (90:10, v/v, H2 O:D2 O, Cambridge Isotopes) and 150 mM DPC-d38 (Cambridge Isotopes, 98.6% d). The pH of the solutions was between 5.2 and 5.8 without correction for the deuterium isotope effect. Trimethylsilylpropionate (TSP) was added to all solutions as a chemical shift reference. Possible peptide–peptide interactions were minimized by maintaining a peptide to micelle ratio of 1:3 assuming 50 molecules of DPC per micelle.

2.2. NMR spectroscopy NMR experiments were performed on a Varian Inova (MelmeCC) or Bruker AMX (Declu-CC) spectrometer operating at 14.1 T. Both were fitted with a 5 mm indirect detection pulse field gradient probe. TOCSY [5] experiments included 1024 F2 and 640 F1 points with a mixing time of 80 ms. Phase sensitive TOCSY, NOESY [2] and ROESY [26] spectra were recorded between 298 and 318 K using the WATERGATE solvent suppression pulse sequence for the H2 O signal. The spectral widths were 7200–7800 Hz in both dimensions, with 2048 and 640 data points in F2 and F1 respectively. Data processing was performed using NMRPipe [9], while spectral assignments and NOE crosspeak integrations were made using SPARKY [21]. NMR assignments were performed using the classical Wüthrich approach [41]. NOESY cross-peaks were integrated using the box integration method and then converted into distances using the ISPA approximation [34,35]. A reference distance of 1.78 A was used for the geminal proton–proton distance of the ␤-methylene groups of the proline residue [25,27]. Adding 20% or subtracting 10% from the originally determined distance approximated the upper and lower bound distances.

271

Table 1 Primary sequences of adipokinetic hormones studied by NMR [31,32]. Neurohormone code name

Primary sequence

Biological Activity as ED50 in pmol

Reference

Melme-CC Melme-CC (N7-analogue) Tenmo-HrTH Declu-CC

pELNYSPDW - - amide pELNYSPNW - - amide

*

203

10 10

pELNFSPNW - - amide pELNFSPNWGN amide

6.8 6.5

10 13

*

Atypical dose–response curve, no ED50 calculable.

2.3. Molecular dynamics simulations and experimental conditions The peptides were built using the builder module of the program InsightII [24] and converted to GROMACS [39] coordinate and topology files. GROMACS was used for searching conformational space and molecular dynamics. The dynamic simulations were performed in an SPC water box or a box containing a micelle consisting of 53 DPC molecules. The neuropeptide was placed such that its hydrophobic side chains were oriented toward the DPC micelle. The neuropeptide and DPC micelle were placed into a cubic simulation box, with dimensions of 7 nm and a volume of 343 nm3 , and then solvated with approximately 10,300 water molecules. All simulations were performed with periodic boundary condition, the OPLS-AA force field, commonly used for peptides, and the SPC model for water. A search of conformational space was performed using a simulated annealing protocol involving dynamics at 600 K for 4 ps, cooling to 300 K in 2 ps, followed by an equilibration period of 2 ps at 300 K and finally minimized, using the Steepest Descent Method. This protocol was repeated 100 times. Cluster analysis, based on the backbone conformation was performed and the lowest energy (root) conformer of the most populous family subjected to 20 ns dynamic simulation at 300 K. This is a relatively short simulation time but analysis of the data showed that very little structural change occurred after 10 ns. The dihedral angles of the central residues were used to classify any secondary structural elements [40]. Hydrogen bond strengths were based on the hydrogen bond geometry and classified according to Disiraju [10]. Docking calculations were done using AutoDock4 [28] and the 3D structure of AKHR [29,30]. Docking parameters were set based on those used before in blind docking [30]. A spacing of 0.05 nm was used in AutoGrid with grid points of 64 × 64 × 50 in xyz. The flexible ligand had 30 active torsion angles. For a population size of 150, the maximum number of generations and evaluations was set at 100 × 106 , in addition to 10 quaternion steps, 10 torsion steps, and 0.5 translation steps. All other parameters were set to default AutoDock4 values (Table S4) [23]. The molecule was subjected to 50 trials of blind docking [22] to search for the binding site. Once the binding site was identified, the protein–ligand complex with lowest estimated free energy of binding was then used for further docking calculations. Based on the blind docking results, docking calculations were performed for 30 runs using a rigid receptor molecule and a flexible ligand with 32 released torsion angles. The number of generations and evaluations were 500 × 106 in all trials for a population of 300. 3. Results and discussion 3.1. NMR The 1 H chemical shift assignments for Melme-CC and DecluCC in DPC are given in Tables 2 and 3. These assignments were done by referring to chemical shift tables, the primary sequence

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Table 2 1 H chemical shift assignment for Melme-CC in DPC micelle solution (ppm) relative to TSP. Residue

NH

␣H

␤CH2

Other

pE1 L2 N3 Y4 S5 P6 D7 W8

7.90 8.24 8.44 7.80 7.68 – 8.16 7.43

4.06 4.18 4.56 4.28 4.50 4.18 4.43 4.50

1.89 1.53 2.74, 2.66 2.74 3.55, 3.45 1.89 2.49, 2.35 3.20, 3.10

2.35, 2.19 – ␥ CH2 1.62 – ␤ CH2 , 0.89 – ␦ CH2 6.80-7.50 – NH2 – – 1.45 – ␥ CH2 , 3.32, 3.38–␦ CH2 – 7.06 – 5-membered-ring C H

of the peptides, and by identifying the individual amino acid spin systems and their sequential order based on the combined use of the TOCSY [5] and NOESY [26]/ROESY [2] spectra (supplementary data Figs. S3–S6). The solubility of the peptides in DPC solution and the NMR line broadening indicates that the ligand readily binds to the micelle. The line broadening precluded the measurement of 3 JNH coupling constants. The calculated inter-nuclear distances based on the ISPA are given as supplementary data (Tables S1 and S2). Berjanskii [3] and Tremblay [37] have shown that proton NMR chemical shifts can be used to probe the solution structure and flexibility of peptides. Comparing the measured chemical shifts to literature values, for a random coil structure, does this. For MelmeCC, in DPC micelle solution, the results (Fig. 1B) show that, with the exception of Ile2 and Asp7 , all the amide (HN ) and H␣ protons are shifted down-field 0.2–0.6 ppm relative to their random coil values. Similarly, for Declu-CC (Fig. 1A) most of the protons are shifted down-field relative to their random coil values. For peptides which form helices, H␣ and amide (HN ) are shifted up-field by ca. −0.30 ppm, while for peptides in a ␤-sheet conformation the protons are shifted down-field ca. 0.6 ppm [36]. Thus the experimental chemical shifts indicate that Declu-CC has an extended (random coil) structure at the N-terminus but that the dominant conformation of the rest of the peptide is a ␤-strand or ␤-turn; the indication for Melme-CC is that the whole peptide seems to be structured. Remarkably similar random coil deviations were obtained for Melme-CC and Declu-CC in DMSO [32] (supplementary data Fig. S1) indicating similar structures for the two peptides in the different media. Chemical shifts can also be used to determine the flexibility and order parameter, S2 , of the peptide (Fig. 1D) [3]. An order parameter of 1 means that the structure is completely ordered, while a value of 0 means the structure is completely disordered. For Melme-CC all the residues have an order parameter of ca. 0.85 again indicating that the whole chain length is ordered. For Declu-CC the N-terminus has a lower order but after the 4th residue the peptides is structured. A similar effect is shown by the NMR-RMSD plot (Fig. 1C) which shows the flexibility of the molecules. A low value indicates that the residue is inflexible, while a high value indicates that the

Table 3 1 H assignments for Declu-CC in DPC micelle solution (ppm) relative to TSP. Residue

NH

␣H

␤CH2

Other

pE1 L2 N3 F4 S5 P6 N7 W8 G9 N10

8.01 8.44 8.48 7.82 8.03 – 8.34 7.54 8.22 7.82

4.29 4.31 4.66 4.60 4.65 4.34 4.24 4.59 3.79 4.51

1.97 1.48 2.76, 2.64 3.28, 3.16 3.67, 3.60 2.50, 2.36 2.77, 2.62 3.18, 3.30 – 2.77, 2.64

2.34 – ␥ CH2 1.61 – ␤ CH2 , ␦.89–␦ CH2 6.80–7.50 – NH2 7.24 – Ar-H – 1.97 – ␥ CH2 , 3.60–␦ CH2 6.80–7.50 – NH2 7.189 – 5-membered-ring C-H – 6.80–7.50 – NH2

residue is flexible. The N-terminus of Declu-CC is flexible but the flexibility decreases towards the C-terminal end. While the chemical shift deviations from random coil values have been interpreted as being indicative of secondary structure, it must be pointed out that this interpretation is not unambiguous. Tremblay et al. [37] have concluded that chemical shifts are more affected by protein secondary structure than solvent environment and indeed similar shifts were found in DMSO and DPC solution, peak shifts could, at least in part, be due to solvent effects. 3.2. Molecular dynamics 3.2.1. Water Cluster analysis of Melme-CC reveals that one dominant family (Fig. 2A) of conformations exists with an average energy of −71.64 kJ mol−1 and a backbone RMSD of 1.79. With the exception of pGlu, all the dihedral angles of the amino acids are in the allowed region of dihedral space. A type IV ␤-turn exists between residues 4 and 7 and an “extended” structure for the first 3 residues. The ␤-turn is due to the Pro6 residue and is stabilized by a moderately strong, transient H-bond between Trp8 (NH) and Tyr4 (CO) as well as numerous H-bonds between the side chains and backbone. There is a significant amount of hydrogen bonding between the peptide and the surrounding water molecules, which indicates structuring of water around the hormone. Cluster analysis of the molecular dynamics trajectory of DecluCC in water gives two, equally populated, families of conformations (Fig. 3A). The first family has an average energy of −132.50 kJ mol−1 and an RMSD of 1.18 and the second family has an average energy of −151.53 kJ mol−1 and an RMSD of 0.94. With the exception of pGlu and Gly, the dihedral angles of all the amino acids fall in the Ramachandran allowed region. Both families have two, type IV ␤-turns between residues 4–7 and 7–10. The difference between the two families is that, in family 1, the turn between residues 7–10 projects above the plane while in family 2 the turn projects below the plane (Fig. 3A). H-bonding between Asn7 (NH) and Asn10 (CO) stabilizes the turn between residues 7 and 10 while an H-bond between Phe4 (NH) and Trp8 (NH) stabilizes the turn between residues 4 and 7. Family 2 is also stabilized by a hydrophobic interaction between Phe4 and Trp8 both of which project into the turn. The distance from Ser5 to Trp8 gives an indication of the tightness of the 4-7 turn, where, for 98% of the simulation, the distance is