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Peptides 41 (2013) 94–100

Contents lists available at SciVerse ScienceDirect

Peptides

j our na l ho me p age : www.elsev ier .com/ locate /pept ides

Anopheles gambiae, Anoga-HrTH hormone, free and bound structure – A nuclearmagnetic resonance experiment

Grace Mugumbatea, Graham E. Jacksona,!, David van der Spoelb, Katalin E. Kövérc, László Szilágyi c

a Department of Chemistry, University of Cape Town, Private Bag, Rondebosch, Cape Town, 7701, South Africab Department of Cell and Molecular Biology, Uppsala University, Box 596, 75124 Uppsala, Swedenc Department of Chemistry, University of Debrecen, H-4032 Debrecen, Egyetem tér 1, Hungary

a r t i c l e i n f o

Article history:Received 5 July 2012Received in revised form 7 January 2013Accepted 8 January 2013Available online 21 February 2013

Keywords:Anopheles gambiaeAnoga-HrTHMolecular dynamicsGROMACSAUTODOCK

a b s t r a c t

The spread of malaria by the female mosquito, Anopheles gambiae, is dependent, amongst other things, onits ability to fly. This in turn, is dependent on the adipokinetic hormone, Anoga-HrTH (pGlu-Leu-Thr-Phe-Thr-Pro-Ala-Trp-NH2). No crystal structure of this important neuropeptide is available and hence NMRrestrained molecular dynamics was used to investigate its conformational space in aqueous solutionand when bound to a membrane surface. The results showed that Anoga-HrTH has an almost cyclicconformation that is stabilized by a hydrogen bond between the C-terminus and Thr3. Upon docking ofthe agonist to its receptor, this H-bond is broken and the molecule adopts a more extended structure.Preliminary AKHR docking calculations give the free energy of binding to be "47.30 kJ/mol. There is aclose correspondence between the structure of the docked ligand and literature structure–activity studies.Information about the 3D structure and binding mode of Anoga-HrTH to its receptor is vital for the designof suitable mimetics which can act as insecticides.

© 2013 Elsevier Inc. All rights reserved.

1. Introduction

The mosquito, Anopheles gambiae, is the World’s most efficientvector for the malaria parasite, Plasmodium falciparum. Malaria isendemic to large parts of Africa and in 2010 was responsible for216 million cases of illness; 81% of these occurring in Sub-SaharanAfrica. An estimated 655 000 people died of malaria in 2010 with86% of the victims being children under the age of 5 years [39].Current methods of mosquito control are dependent on a singleclass of insecticides, the pyrethroids. Resistance to pyrethroids hasbeen noted in 27 countries and is being monitored in 78 countries[27,28,39]. There is, therefore, an urgent need for a new class ofinsecticides.

Crucial to the mosquito’s vector capacity is its ability to fly. Insectflight is under hormonal control via the adipokinetic hormones(AKHs) [10,35]. AKHs are a class of octa-, nona- or decapeptides pro-tected with N-(pyroglutamate) and C-(amidate) termini. Most AKHhormones contain Phe, Pro and Trp at positions 4, 6 and 8 respec-tively. These residues are postulated to be important in determiningthe hormone structure and receptor binding [6]. AKHs con-tribute to hemolymph sugar homeostasis and are involved in the

! Corresponding author. Tel.: +27 21 6502531; fax: +27 21 6505195.E-mail address: Graham.Jackson@uct.ac.za (G.E. Jackson).

mobilization of sugar and lipids from the fat body during energy-demanding activities, such as flight and locomotion [15,19]. TheAKH octapeptide, Anoga-HrTH (pQLTFTPAWa) was identified infreshly hatched larvae, heads and thoraces of pupae and adult A.gambiae [16]. Anoga-HrTH is involved in energy generation duringflight and hence enhances flight activity of the female mosquito[15].

Binding of the AKHs to their G-protein coupled receptor (GPCRs)triggers a number of coordinated signal transduction processes.These ultimately result in the mobilization of carbohydrates andlipid reserves fueling flight activity. Therefore, conformationalstudies of these hormones, that play a pivotal role in processescentral to vector capacity, may contribute to the control of malaria.

Nuclear magnetic resonance (NMR) spectroscopy derivedinter-proton distance restraints have been successfully appliedduring molecular dynamic conformational searches of peptidesin water [42], dimethyl sulphoxide (DMSO) [24,25], and in mem-brane mimicking environments like dodecylphosphocholine (DPC)micelles [12,31,41]. Information obtained from both one and two-dimensional spectra have been used to determine the peptidesecondary structures and orientations in solution and bound to amembrane.

In this work, NMR was used to study the solution and DPCmicelle bound structure of Anoga-HrTH. Changes in the resultingpreferred conformation, induced by receptor binding, were alsostudied.

0196-9781/$ – see front matter © 2013 Elsevier Inc. All rights reserved.http://dx.doi.org/10.1016/j.peptides.2013.01.008

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G. Mugumbate et al. / Peptides 41 (2013) 94–100 95

2. Experimental

2.1. NMR

2.1.1. Sample preparationAnoga-HrTH was synthesized by GL Biochem (Shanghai) Ltd.,

China (98% pure) and used without further purification. The sol-vents used were either water/D2O (9:1 (v/v%)) or a membranemimicking solution of deuterated dodecylphosphocholine (DPC-d38).

For the aqueous studies, 1.6 mg of Anoga-HrTH was dissolved in0.5 ml of 9:1 (v/v) H2O:D2O solution buffered at pH 4 with 50 mMsodium phosphate buffer solution. In the membrane studies, 1.6 mgof Anoga-HrTH was dissolved in approximately 150 mM DPC-d38micelle solution also buffered at pH4 with 50 mM sodium phos-phate buffer. In both cases trimethylsilylpropionate (TSP) was usedas internal chemical shift reference.

2.1.2. 1H NMR experiments1H NMR experiments were performed on a Bruker Avance

400 MHz (Department of Chemistry, University of Cape Town)and an Avance II 500 MHz (Department of Chemistry, Universityof Debrecen). TOCSY (mixing time, 60 ms) [5], NOESY (mixingtime, 100 ms) [13] and ROESY (mixing time, 150 ms) [1,38] spec-tra of the peptide, in water and DPC, were collected in the phasesensitive mode using the WATERGATE sequence to suppress thewater resonance [30]. Spectral assignments were based on themethod of Wüthrich [38,40]. Inter-proton distances from the 2DNOE cross-peak intensities were estimated by the isolated spin pairapproximation (ISPA) [33]:

rij = rref

✓aref

aij

◆1/6

,

where rij is the inter-proton distances to be estimated and aij is thecorresponding 2D NOE cross-peak intensity.

2.2. Restrained MD simulations of Anoga-HrTH

The starting structure for the conformational search of Anoga-HrTH was built using InsightII 2005 and energy minimized for 3000steps using a steepest descent algorithm. Molecular dynamic sim-ulations in vacuum and water were performed using GROMACSversion 4.0.4 [36]. All simulations were performed with a time stepof 2 fs, using the OPLS-AA/L all – atom [14] force field and con-stant temperature, volume and number of particles (NVT). A 1.0 nmcut-off was used for van der Waals interactions and electrostaticinteractions.

2.2.1. In vacuumAnoga-HrTH was inserted into a simulation box and energy min-

imized for 500 steps using the l-BFGS minimizer. Distance restraintswere applied according to calculated NMR inter-proton distances.The listed distances were used as lower limits and two upper lim-its were obtained by increasing the values by #10% and 20%. Themolecule was subjected to 10 ns of distance restrained MD simula-tion at 600 K, and 100 structures collected. Using a high simulationtemperature allowed the molecule to overcome energy barriers andcover a larger conformational space [12]. Each structure collectedat 600 K was then cooled to 300 K using a simulated annealing pro-tocol. The final structures were energy minimized and clustered,using the g cluster program in GROMACS, with a cut-off distance of<0.2 nm on the backbone atoms. The conformation of lowest energywas identified from the largest cluster.

2.2.2. In waterThe minimum energy conformation, from the simulation in

vacuum, was used as the starting structure of Anoga-HrTH for10 ns MD simulation in water. The water atoms were based onthe SPC water model [2]. To allow the water to soak the peptide,position-restrained MD simulations were performed for 0.1 ns. Allsimulations used periodic boundary conditions [9]. The peptideand solvent were coupled separately using the Berendsen couplingmethod to a temperature of 300 K using a coupling constant, tau(!) t, of 1 $ 10"4 ns.

Distance restrained MD simulations, based on the NMR resultswere performed for 10 ns, and 100 structures collected for analysis.Cluster analysis of the structures was performed based on a cut-offvalue of <0.2 nm for superimposing backbone atoms. The minimumenergy conformation from the largest cluster gave the structure ofAnoga-HrTH.

2.3. Docking calculations

Docking calculations of Anoga-HrTH were used to estimate thefree energy of binding and the conformation of the ligand boundto an adipokinetic hormone receptor (AKHR) model. The AKHRmodel was built by homology modeling the helices, which werethen joined to separately built loops, as described by Mugumbateet al. [23]. The Lamarckian genetic algorithm (LGA), incorporated inAutodock4 was used in 30 docking trials with 24 free torsion anglesto generate bound conformations of the ligand within the bind-ing pocket [21,22]. In each trial, a total of 5 $ 108 generations andenergy evaluations were applied for a population of 300. Clusteranalysis was executed at a cut-off of 0.2 nm, and the conformationof lowest estimated free energy of binding was identified from thehighest ranked cluster.

3. Results and discussion

3.1. NMR

3.1.1. Spectral assignment and inter-proton distancesThe 1H and 13C chemical shift assignments for Anoga-HrTH in

water and DPC are given in Tables 1 and 2. These assignments weredone by referring to chemical shift tables, the primary sequenceof Anoga-HrTH, and by identifying the individual amino acid spinsystems and their sequential order based on the combined use ofthe TOCSY and NOESY/ROESY spectra [38,40]. Line broadening inDPC solution, indicates that the ligand readily binds to the micelle.There was a close relationship between the chemical shifts in waterand DPC micelle solution, indicating that the molecule has a similarstructure in the two different media. This is unusual, as previousstudies on insect peptides, have indicated that they are unstruc-tured in water but structured in micelle solution [8].

To investigate this further, random coil deviation of the chem-ical shifts were plotted (Fig. 1). With the exception of Ileu2 andAla7, all the amide (HN) protons are shifted downfield 0.2–0.6 ppmrelative to their random coil values. Similarly the H! protons areshifted downfield. Studies have shown that, with respect to theirrandom coil values, both H! and amide (HN) 1H chemical shifts areshifted upfield by "0.30 ppm, on average, in helices and downfieldby ca. 0.6 ppm in "-sheets [32] Thus the experimental chemicalshifts indicate that the dominant conformation of the peptide hasa "-strand or "-turn.

The chemical shifts were also used to determine the flexibilityand order parameter, S2, of the peptide (Fig. 1) [3]. The N-terminalend from pGlu1 to Phe4 has higher backbone deviations and fluc-tuations (MD RMSDs ranging from 1.4 to 1.7) than the C-terminalend (Thr5-Trp8) which has much lower MD RMSDs of 1.1–1.2

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Table 11H and 13C chemical shift (ppm) assignments for Anoga-HrTH, pGlu-LTFTPAW-NH2 peptide in H2O–D2O 9:1 pH ca. 4 (phosphate buffer), T = 290 K.

Residue NH H! H" H"% H# H#% H$ H$% Other

pGlu1 7.752 4.183 2.228 1.868 2.355Leu2 8.275 4.236 1.445 1.355 0.750 0.702Thr3 7.946 4.233 3.949 0.958Phe4 8.154 4.494 2.853 Aromatics 7.08–7.16Thr5 7.961 4.314 3.797 0.968Pro6 – 4.0100 1.219 1.856 1.656 3.407Ala7 8.200 4.004 1.145Trp8–NH2 7.592 4.457 3.121 Aromatics; 7.031(2%), 7.508(4%),

6.97–7.03(5%6%); 7.337(7%);CONH2; 7.183, 6.925

Residue C! C" C# C$ Other

pGlu1 56.6 25.1 29.1Leu2 52.4 39.5 24.1 20.6; 22.0Thr3 58.6 67.1 18.5Phe4 54.6 37.1 127.0; 128.5; 129.0Thr5 56.4 67.1 18.5Pro6 60.0 29.0 24.3 48.1Ala7 49.8 16.1Trp8-NH2 53.7 26.6 111.8; 118.1; 119.2; 121.8; 124.4

(Fig. 1c). The molecule is ordered with order parameters rangingfrom #0.72 for the N-terminus to #0.80 for the C-terminus (Fig. 1d).As expected, the highly flexible N-terminus is less ordered than theC-terminus.

3.1.2. Temperature coefficients and hydrogen bond formationThe temperature coefficients of the amide proton chemical

shifts were determined from a plot of the chemical shifts againsttemperature (Fig. 2). The temperature coefficient is used as anindicator of amide proton hydrogen bond formation [7]. Thetemperature coefficients, 1H–1H coupling constants (Hz) and thecorresponding calculated phi (") angles are given in Table 3. Theobserved temperature coefficients indicate that the NH of mostresidues participates in the formation of intra-molecular, hydro-gen bonds. The temperature coefficient of Ala7 was the lowest,"1.6 ppb/K, which indicates that this NH is involved in strong intra-molecular H-bonding or is protected from the solvent. From themolecular dynamics results (see latter) we find that there are no

intra-molecular hydrogen bonds to Ala7:NH but that the motion ofthe Trp8 aromatic side-chain prevents water access.

3.1.3. Inter-proton distancesInter-proton distances from the NOE cross-peaks were cal-

culated by the Isolated Spin Pair Approximation (ISPA) method[33], with pseudoatom corrections according to Wüthrich, 1986[40]. ISPA is a suitable approximation since short mixing times of100 ms and 150 ms were used in the NOESY and ROESY experi-ments respectively, and so effects of spin diffusion were small [26].Since NOE measurements are biased toward short inter-nucleardistances, the first and second distance upper limits were obtainedby adding #10% and 20% to each of the measured distances [12].The Pro6 H"2–H"3 inter-nuclear distance of 0.18 nm was used as areference. 17 inter-nuclear distances ranging from #0.26–0.43 nmwere obtained in water and 32 inter-nuclear distances ranging from#0.27 (Leu2 NH – pGlu1 NH)–0.74 (Leu2 Hd2 – Phe4 H2,6) nm inDPC solution. The slow molecular tumbling of the peptide, bound

Table 21H and 13C chemical shift (ppm) assignments for Anoga-HrTH, pGlu-LTFTPAW-NH2 peptide H2O–D2O 9:1 150 mM DPC micelle solution, pH ca. 4 (phosphate buffer), T = 290 K.

Residue NH H! H" H"% H# H#% H$ H$% Other

pGlu1 7.914 4.266 1.880 2.245 2.395Leu2 8.408 4.300 1.391 1.513 0.754 0.777Thr3 8.092 4.226 4.052 0.995Phe4 8.013 4.564 2.933 Aromatics 7.11(2%6%); 7.04 (3%4%5%)Thr5 7.933 4.338 3.914 0.984Pro6 – 4.118 1.294 1.793 1.637 1.562 3.391Ala7 8.166 4.152 1.198Trp8-NH2 7.687 4.490 3.10 3.16 Aromatics 7.154(2%); 7.452(4%); 6.962(5%);

9.886(6%); 7.333(7%); CONH2 7.32, 7.15

Residue C! C" C# C$ Other

pGlu1 56.6 25.3 29.2Leu2 52.2 39.6 24.4 21.2; 22.4Thr3 58.7 67.0 18.6Phe4 54.4 37.3 126.6; 128.2; 129.2Thr5 56.7 66.8 18.6Pro6 60.1 28.8 24.5 47.8Ala7 49.6 16.4Trp8-NH2 53.6 27.0 111.7

118.1118.6121.0124.3

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Fig. 1. (a) and (b) Random coil chemical shift deviations [32] of Anoga-HrTH in water and DPC micelle solution respectively. (c) Molecular dynamics backbone root meansquare deviation and (d) order parameter, S2, of Anoga-HrTH calculated using RCI server [3].

to the large DPC micelle, increases the rate of cross relaxation andhence the larger number of NOE’s detected. Where correspondingdistances were found in both media, they had similar values indi-cating again, that the conformation of the molecule was similar in

Fig. 2. (a) NMR spectra of Anoga-HrTH in water at varying temperatures. (b) Vari-ations of HN chemical shifts in water as a function of temperature.

the two solutions. These inter-nuclear distances were applied indistance restrained MD simulation.

Since it is generally accepted, that short peptides are unstruc-tured in aqueous solution and structured in micelle medium, theseNMR results require further comment. Zubrzycki and Gäde [41],in their study of Emp-AKH found just this, as did Cusinato et al.[8] when looking at a series of adipokinetic peptides using circu-lar dichroism. However, Langelaan et al. [18], in their review ofthe biophysical characterization of G-protein coupled receptor lig-ands list several examples where stabilized structures were foundin aqueous solution. Similarly, over 100 GPCR ligands have beenreported to have a turn structure, albeit, these were all mammalianpeptides [29,34]. More interesting, a recent CD study of pE-apelin-13 suggested a random coil structure but in an exhaustive NMRinvestigation a turn structure was found in aqueous solution [17].In micelle solution the peptide became more structured. Notwith-standing these divergent observations, our NMR results, in terms ofchemical shift, the temperature dependence of the amide chemicalshifts and the measured NOE’s, point unequivocally to the peptidehaving some structure in aqueous solution.

3.2. MD simulations of Anoga-HrTH

3.2.1. Conformational searchInter-proton distances and restraints obtained from NMR exper-

iments in water were used during 10 ns MD simulations at 600 Kin vacuum. This high temperature was used to allow the moleculeto explore high-energy conformers [12,20,31]. 100 structures were

Table 3HN temperature coefficients ("ppb/K), 1H–1H couplings (Hz) and the calculated "angles.

Residue HN temperaturecoefficient("ppb/K)

3JHNH! couplingconstant (Hz)

Calculated " angles(&)

pGlu1 – – –Leu2 3.4 6.8 "68Thr3 4.2 8.0 "90Phe4 2.8 7.5 "86Thr5 3.6 8.0 "90Pro6 – – –Ala7 1.6 5.5 "70Trp8 – – –

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Fig. 3. Conformation of Anoga-HrTH. (a) An overlay of 100 structures from MD simulations in water at 300 K. (b) Overlay of the minimum energy conformation in water andDPC micelle solution. The red dotted lines indicate H-bonding between Trp8 and Thr3. (c) Comparison of phi torsion angles calculated from NMR chemical shifts by PREDITOR[4], estimated from MD simulations in water and from calculations using 3JHNH! coupling constants.

collected, subjected to 0.1 ns simulated annealing and then energyminimized. Cluster analysis of the final 100 structures gave only onesignificant cluster. The conformation of lowest energy was furthersubjected to 10 ns MD in water at 300 K. Another set of 100 struc-tures was collected and each structure again energy minimized.

As a check on the molecular dynamic simulations, the pro-gram PREDITOR [4] was used to compare, calculated phi anglesto those determined experimentally from NMR coupling constantsand chemical shifts. Fig. 3c shows a comparison of these phi anglesaveraged over a 10 ns trajectory in water. The standard deviation

Fig. 4. (a) Anoga-HrTH, inside the binding pocket of its receptor, AKHR. (b) Expansion of the binding pocket showing the interaction between the ligand and receptor. H-bondsare indicated by green dotted lines. For clarity, residues Ile106, Thr129, Leu311, Gly308, Phe312, Leu286, Arg512 and Ile207 are omitted. (c) Overlay of bound (gray) and free(pink) conformations of Anoga-HrTH. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)

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G. Mugumbate et al. / Peptides 41 (2013) 94–100 99

was estimated to be ±15& for each residue. With the exceptionof Thr5, there was reasonable agreement between the calculatedand observed values. This result lends confidence to the moleculardynamics protocol used.

Cluster analysis of the water trajectory, using a cut-off of 0.2 nmfor backbone atoms, gave only one cluster indicating that the con-formation of the peptide was conserved (Fig. 3a). The identifiedminimum energy structure has the peptide in a cyclic conforma-tion, H-bonding between Thr3 and Trp8:CONH2 completing thering (Fig. 3b). This conformation is similar to the lowest energyconformation of Gonadotropin releasing hormone (GnRH) [20] andthe Crustacean cardioactive peptide (CCAP) [12] where a H-bondand a disulphide bridge respectively maintained the cyclic struc-ture. Fig. 3b shows an overlay of the lowest energy structure ofAnoga-HrTH found in water and DPC micelle solution. The overlaysupports the NMR results, which indicated that the peptide had asimilar structure in the two media.

3.2.2. Docking calculations3.2.2.1. Binding mode of Anoga-HrTH. During docking calculationsof Anoga-HrTH to its receptor, the hormone successfully occupied abinding pocket formed by the helices and the extracellular domains(Fig. 4a). The ligand residues were in contact with residues in helices2, 3 and 5–7, the second extracellular loop and the N-terminus of theGPCR (Fig. 4b). High binding affinity of Anoga-HrTH was indicatedby a low estimated free energy of binding, #Gb, ("47.3 kJ/mol) andinhibition constant, Ki, (5.2 nM).

3.2.2.2. Conformational changes in Anoga-HrTH. Binding Anoga-HrTH to its receptor, AKHR, induced significant conformationalchanges in the ligand (Fig. 4c). An overlay of the ligand beforeand after docking gave an rmsd of #0.28 nm. Upon binding, themolecule opened and the C-terminal end stretched out giving it anL-shape.

While there are no crystal structures of insect hormonesbound to their GPCR, there is much indirect evidence throughstructure–activity relationships. In an elegant study, Caers et al.used a bioluminescence cellular assay of two expressed AKH recep-tors from Drosophila melanogaster and A. gambiae to study theactivity of a series of single amino acid replacement analogs ofDrome-AKH [6]. Included in this series was Anoga-HrTH. Caers et al.found that the N and C termini were not important for activity andconcluded that they did not interact with the receptor. Residues 2–5were crucial for activity and they postulated that a "-strand wasformed followed by a "-turn at residues 5–8. In this turn residues6 and 8 were essential. Replacement of residue 7 had no effect onactivity and they conclude that this implied it was folded inside thechain and did not interact with the receptor.

Fig. 4 shows Anoga-HrTH bound to its receptor model. Thisbound conformation is stabilized by a network of H-bonds, whichclosely follows the results of Caers et al. Thus, neither the C- nor N-terminal interacts with the receptor; the pyroglutamic acid pointsaway from the receptor and there are no H-bonds to the terminalamide. There is a "-strand between residues 2 and 5 followed by a"-turn at Pro6. There are hydrogen bonds between, Trp8:H%1 andPhe126:O; Thr3:H#1 and Tyr110:OH&; Thr5:H#1 and Tyr285:OH&;Thr3:O#1 and His16:H%2; Trp8:O and Tyr221:H&; Ala7:O andTyr285:H&. All of these H-bonds, with the exception of Ala7, corre-spond to essential residues for activity [6]. In the case of Ala7, theH-bond involves the amide carbonyl oxygen so any small aminoacid would suffice. The absence of any interaction with the sidechain of residue 7 is in line with previous studies where it wasfound that substitution by a smaller residue, Gly, lead to a 46%increase in receptor activation [6]. It has been postulated that aro-matic residues at position 4 and 8 are essential [6,11,37]. In Fig. 4it is clear that the side chain of Trp8 sits in a hydrophobic pocket,

and hydrogen bonds to Phe126:O. Obviously there are no H-bondsto Phe4 but there are hydrophobic interactions with hydrophobicresidues of the receptor N-terminus. Finally, Leu2 is postulated tobe essential for activity [6] and we find it fits into a hydrophobicpocket created by Tyr110 and Ile106.

4. Conclusion

The most stable conformation of the adipokinetic hormone,Anoga-HrTH, in water and DPC micelle solution was determinedusing NMR data and distance restrained molecular dynamics simu-lations. In water, the molecule preferred a cyclic conformation witha H-bond completing the circle. A similar structure was found inDPC micelle solution. AUTODOCK calculations with a model recep-tor showed that Anoga-HrTH had a high binding affinity. Uponreceptor binding the ligand adopted a more extended structure.The C-terminus of the ligand was buried between the helices ofthe receptor, whilst the N-terminus extended toward the extracel-lular domains. The Anoga-HrTH/AKHR complex was stabilized byboth H-bonding and hydrophobic interactions involving receptorresidues in the helices and extracellular loops. There was a highdegree of correspondence between our modeling results and lit-erature structure–activity studies. These results should pave theway for the design of non-peptide mimetics that can be used asalternative insecticides.

Acknowledgements

This work was supported by the National Research Foundationof South Africa, and the Equity Development program (UCT). Finan-cial support from the Hungarian Scientific Research Fund (OTKANK-68578 and K-105459) and the South African-Hungarian (TéTZA-20/2008) scientific exchange program are gratefully acknowl-edged. The support from the Swedish Research Council is greatlyacknowledged. Support for the NMR instrument time from the East-NMR Project No. 228461 is also acknowledged.

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