Molecular modelling of the adipokinetic hormone receptor from the stick insect Carausius morosus, and its endogenous agonist

Molecular modelling of the adipokinetic hormone receptor from the stick insect Carausius morosus, and its endogenous agonist | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Molecular modelling of the adipokinetic hormone receptor from the stick insect Carausius morosus, and its endogenous agonist G. E. Jackson, H. G. Marco, M.-A. Sani, F. Separovic, G. Gäde This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9099089/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 10 You are reading this latest preprint version Abstract This study combines NMR spectroscopy, molecular dynamics (MD), and docking simulations to elucidate the structure, dynamics, and receptor interactions of the decapeptide Carmo-HrTH-II, from the stick insect, Carausius morosus . NMR analysis revealed a flexible peptide adopting a β-turn between Pro⁶ and Asn⁷, a feature retained during MD simulations and critical for receptor recognition. Docking Carmo-HrTH-II to the Carmo-AKHR homology model identified a stable binding mode in which Phe⁴ and Trp⁸ lie within the binding pocket, consistent with their known importance for GPCR activation. Alanine-scanning mutagenesis and residue scanning demonstrated strong agreement between calculated binding energies and experimental EC₅₀ values, confirming the essential roles of Phe⁴, Pro⁶, Asn⁷, and Trp⁸ in receptor activation. Comparative studies of Carmo-HrTH-I and other AKH analogues showed that position 10 substitutions have minimal influence on binding, whereas position 3 substitutions significantly alter affinity. In silico screening of insecticide-like compounds identified several potential receptor antagonists that bind to the same site as Carmo-HrTH-II. These ligands interact predominantly through π–π stacking and hydrogen bonding, disrupting the conserved salt bridge essential for receptor activation. Together, these results provide a molecular basis for AKH receptor activation and suggest a new route for the rational design of AKH-targeted insecticides. Biological sciences/Biochemistry Biological sciences/Chemical biology Physical sciences/Chemistry Biological sciences/Computational biology and bioinformatics Biological sciences/Drug discovery Biological sciences/Structural biology Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction In insects, adipokinetic hormone (AKH), corazonin (Crz), and adipokinetic hormone–corazonin-related peptide (ACP), together with their cognate receptors, are structurally related to vertebrate gonadotropin-releasing hormone (GnRH) and collectively form a large superfamily of neuropeptides. In the Indian stick insect Carausius morosus , all three neuropeptides are present, ( 1 ) but to date only the AKH system has been investigated in detail. Two AKH decapeptides have been identified in the corpora cardiaca of C. morosus , designated Carmo-HrTH-I and Carmo-HrTH-II according to their order of elution (hydrophobicity) in reverse-phase high-performance liquid chromatography. ( 2 , 3 ) Both peptides induce hypertrehalosaemia in ligated stick insects. ( 4 ) Sequence elucidation using fast atom bombardment mass spectrometry and nuclear magnetic resonance (NMR) spectroscopy revealed that the two peptides are almost identical. The less hydrophobic Carmo-HrTH-I is distinguished by the presence of an unusual C-bonded mannose moiety attached to the tryptophan residue at position 8, giving the sequence pGlu-Leu-Thr-Phe-Thr-Pro-Asn-Trp(Man)-Gly-Thr-amide. ( 5 ) More recently, the complete functional receptor of C. morosus has been cloned. ( 6 ) In accordance with its membership in this peptide family, it is referred to here as an adipokinetic hormone (AKH) receptor. The receptor is encoded by a 1587-base-pair open reading frame, producing a protein of 528 amino acids. It was characterised as a class A (rhodopsin-like) G protein-coupled receptor (GPCR), comprising seven hydrophobic transmembrane domains, an extracellular N-terminus, and an intracellular C-terminus. ( 6 ) Structure–activity relationship studies have previously been conducted both in vivo , by measuring carbohydrate release into the haemolymph, and in vitro , using heterologous expression of the receptor in vertebrate cells. ( 6 , 7 ) These studies employed strategically modified Carmo-HrTH peptide analogues (alanine replacement series) as well as selected AKH bioanalogues, providing initial insights into the roles of the peptide termini and individual amino-acid side chains in receptor interaction. In the present study, we extend these investigations to further elucidate the molecular interaction between ligand and receptor. First, we obtain detailed secondary structural information on the ligand Carmo-HrTH-II through comprehensive NMR analysis. Second, this structural information is combined with existing knowledge of the receptor primary sequence to construct a molecular model of the C. morosus AKH receptor using molecular dynamics (MD) simulations. Third, we analyse the ligand–receptor complex in detail to identify key interactions governing binding and activation. Comparable studies have been reported for AKH systems in several arthropods, including the mosquitoes Anopheles gambiae and Aedes aegypti (including the ACP system), the water flea Daphnia pulex , the vinegar fly Drosophila melanogaster , and the desert locust Schistocerca gregaria . ( 8 – 11 ) Previous modelling work on the stick insect AKH system has also been published, allowing direct comparison with the results presented here. ( 12 ) Understanding ligand–receptor interactions in the AKH system is of particular interest for the rational design of non-peptidic superagonists or antagonists that could serve as future “green insecticides”. ( 6 , 13 ) Such compounds would ideally be species- or group-specific, biodegradable, and environmentally benign, with minimal effects on non-target organisms. Notably, earlier work on the AKH system of the desert locust identified a non-peptidic compound that not only exhibited favourable binding energies in modelling studies but also acted as a competitive inhibitor of the endogenous AKH peptide in an in vivo bioassay. ( 14 ) Material and Method Solution-state NMR Spectroscopy The synthetic peptide Carmo-HrTH-II (pELTFTPNWGT-amide, Synpeptide Co. Ltd., Shanghai, China) was dissolved in perdeuterated d₃₈-dodecylphosphocholine (DPC) micelles (10 mM phosphate buffer, pH 5.0, containing 0.05 mM sodium trimethylsilylpropanesulfonate (DSS) and 10% v/v D₂O) to a final peptide concentration of 2 mM and a DPC-to-peptide molar ratio of 60:1. NMR spectra were acquired at 298 K on a 700 MHz Bruker Neo equipped with a 5 mm TCI cryoprobe. Two-dimensional 1 H- 1 H TOCSY (τ mix = 80 ms) and NOESY (τ mix = 120 and 200 ms) spectra were recorded with 512 and 1024 complex points in the F₁ dimension, respectively, and 2048 points in F₂. Between 8 and 32 transients were accumulated using a 1.5 s recycle delay. Spectra were apodized with a squared sine-bell window shifted by 90° and a 1 H spectral width of 9090 Hz. 13 C- 1 H HSQC spectra were collected with 256 × 2048 complex points, 256 transients, and a 2 s recycle delay. The 13 C spectral window was set to 29050 Hz. 15 N- 1 H HSQC spectra were recorded with 128 × 2048 points, 256 transients, a 1.5 s recycle delay, and a 15 N spectral width of 5675 Hz. All data were zero-filled to twice the number of acquired points. 1 H chemical shifts were referenced to DSS (0 ppm), and 13 C and 15 N shifts were indirectly referenced to the 1 H frequency. Data were processed in TopSpin 4.0 (Bruker) and analyzed using CCPNmr Analysis ( 11 ). Backbone and side-chain assignments were completed using the combined datasets. Structure Calculations NOESY cross-peaks (τ mix = 120 ms) were used to derive interproton distance restraints, supplemented with dihedral angle restraints predicted by TALOS-N ( 15 ) from H α , N H, C α , C β and H N chemical shifts. Structure calculations were performed using Xplor-NIH with standard protocols (16, 17) accessed via the CCPNmr interface. The averaged structure of the ten lowest-energy conformers was selected for molecular dynamics (MD) simulations and validated with MolProbity ( 18 ). Computational modelling Ligand preparation and micelle simulation Carmo-HrTH-II was built and optimized using the Builder module in Maestro ( 19 ), exported as a PDB file, and imported into GROMACS (17). The peptide was placed in a simulation box containing a pre-equilibrated DPC micelle ( 18 ). The micelle was positioned at the bottom of the box, with Carmo-HrTH-II centered so that, under periodic boundary conditions, the micelle spanned both top and bottom boundaries. The micelle was taken from a previous study on adipokinetic hormones in water and DPC micelle solution ( 20 ) Simulations were performed using the OPLS-AA force field ( 19 , 20 ). Following energy minimization, a 500 ns NVT simulation was conducted, saving trajectory frames every 100 ps. Cluster analysis was performed using the g_cluster algorithm in GROMACS with a 0.2 nm cutoff. Carmo-HrTH-I, the second native AKH peptide of Carausius morosus , was generated computationally by adding a C-linked mannose to the Trp residue of the Carmo-HrTH-II model. Receptor homology modelling and docking The primary sequence of the C. morosus adipokinetic hormone receptor (Carmo-AKHR) ( 3 ) was used for homology modelling ( 21 ). The β₂-adrenergic receptor (PDB ID: 5D5A) ( 22 ) was chosen as a template based on resolution (2.48 Å), sequence identity (21.6%), coverage (0.52), and GMQE score (0.27) ( 23 ). The resulting model was refined in Maestro ( 16 ) using the Protein Preparation Wizard at pH 7.0. The receptor was inserted into a 1-palmitoyl-2-eleoyl-sn-glycero-3-phosphocholine (POPC) membrane using CHARMM-GUI (24), and a 50 ns MD simulation performed using GROMACS with CHARMM parameters ( 25 ). The final frame from the simulation was used for docking. A conformational search in Maestro ( 26 ) generated 100 Carmo-HrTH-II conformers, which were docked into the receptor using Glide ( 27 ) in SP-Peptide mode (scaling factor 0.8, partial charge cutoff 0.15 nm). The top-scoring poses were subjected to MD simulation in a POPC membrane using Desmond ( 28 ). Systems were solvated with SPC water, neutralized, and adjusted to 0.15 M NaCl. Binding free energies were calculated using MM/GBSA ( 29 ), and ligand–receptor interactions were calculated by Maestro . In silico Insecticide Screening An insecticide-like compound library comprising 295,011 structures was obtained from Otava Chemicals Ltd. A total of 415,677 tautomers and ionization states were generated using LigPrep (Schrödinger Release 2021-1). Compounds were filtered using QikProp ( 21 ) for pharmacokinetic and physicochemical properties, yielding 320,689 candidates. High-throughput virtual screening (HTVS) was performed against the Carmo-AKHR model. From 24,941 initial poses, 1,970 were refined by extra-precision docking, yielding 197 final ligand–receptor complexes. Free energies of binding were computed via MM/GBSA, and the highest-scoring candidates were embedded into POPC membranes for 50 ns MD simulations as described above. Results and Discussions NMR and structural data of the ligand Carmo-HrTH-II The 1 H, 15 N and 13 C chemical shifts and assignments in DPC micelle solution of Carmo-HrTH-II are summarized in Table 1. Structure-induced chemical shift deviations were calculated by subtracting the random coil values from the observed chemical shifts (Fig. 1a,b). Positive deviations in C α chemical shifts are characteristic of β-strand formation, while α-helices typically produce negative deviations (22). Figure 1a demonstrates that Carmo-HrTH-II adopts a coiled conformation in DPC micelle solution. Although proton chemical shifts are less sensitive to structural changes than 13 C chemical shifts, all 1 H deviations were below 0.3 ppm (26), consistent with a turn structure following the first two residues. Together, the 13 C and 1 H chemical shift data indicate that Carmo-HrTH-II adopts a turn conformation in DPC micelle solution. This observation aligns with previous findings that adipokinetic hormones (AKHs) typically form β-turn structures in micellar environments. Indeed, Tyndal et al. (23) reported that over 100 mammalian GPCR ligands exhibit β-turns, and Cusinato et al. (24) showed by CD spectroscopy that AKH/RPCH peptides form β-turns in 0.6% SDS solution. Table 1 NMR assignments of Carmo-HrTH-II in DPC micelle solution. Residue δ(N)/ppm δ(HN)/ppm δ(Hα)/ppm δ(Hβ)/ppm δ(Hγ)/ppm δ(Cα)/ppm δ(Cβ)/ppm δ(Cγ)/ppm δ(Other)/ppm 1-pGlu 125.0 7.99 4.38 2.50, 1.98 2.35* 58.9 27.5 31.4 2-Leu 121.6 8.45 4.39 1.61, 1.49 1.61 54.5 41.8 26.7 H δ1 0.90, H δ2 0.87, C δ1 24.7, C δ2 23.5 3-Thr 114.4 8.10 4.30 4.15 1.10 61.0 69.3 20.9 4-Phe 121.2 8.09 4.67 3.04, 3.01 - 56.6 39.6 - H δ 7.16, H ε 7.21, H ζ 7.16, C δ 131.5, C ε 130.4, C ζ 128.8 5-Thr 116.9 8.00 4.47 4.04 1.12 58.7 69.1 20.9 6-Pro 121.1 - 4.21 1.87, 1.41 1.76, 1.69 62.4 31.0 26.7 H δ 3.51, C δ 50.0 7-Asn 118.4 8.41 4.64 2.78, 2.69 - 52.4 38.0 - N δ2 112.5, H δ1 7.61, H δ2 6.85 8-Trp 120.9 7.98 4.64 3.32, 3.21 - 56.6 29.1 - N ε 130.3, H δ1 7.26, H ε1 10.51, H ε3 7.56, H ζ2 7.43, H ζ3 7.02, H η2 7.07, C δ1 126.6, C ε3 120.3, C ζ2 113.8, C ζ3 120.8, C η2 123.3 9-Gly 108.7 8.41 3.95, 3.90 - - - - - 10-Thr 112.6 7.96 4.26 4.26 1.18 61.1 69.1 21.2 N η2 109.1, H N1 7.67, H N2 7.16 Chemical shift in ppm, referenced from DSS, experiments run at 298K in DPC micelles - Not observed Munte et al.(25) reported that Carmo-HrTH-II in D₂O does not adopt a random coil structure but rather exists as an ensemble of conformations containing transient ordered species. In our study, binding to the DPC micelle appears to stabilize these conformers. Munte et al., (25) also measured random coil chemical shifts for the tryptophan C-mannosylated analogue, Carmo-HrTH-I, finding them nearly identical to those of Carmo-HrTH-II, indicating that the sugar moiety does not substantially alter peptide conformation. A comparison of random coil shift differences between Carmo-HrTH-I and -II is shown in Figure S1 Both 1 H and 13 C shift trends were similar, although Carmo-HrTH-II in DPC micelles showed larger C α deviations than Carmo-HrTH-I in water, consistent with greater conformational stabilization in the micellar environment. Using all chemical shift data, the model free order parameter, S 2 (26), was calculated to assess peptide flexibility (Fig. 1c). An S ² value of 1 indicates complete rigidity, whereas 0 represents full flexibility. Carmo-HrTH-II exhibited S ² values of 0.5–0.6, indicating a structured but flexible conformation. The peptide is more rigid than some other members of this superfamily such as Aedae-ACP ( S ² = 0–0.36) (27), Aedae-AKH ( S ² = 0.1–0.3) (27), Schgr-AKH-II ( S ² = 0.1–0.4) (9), and Dappu-RPCH ( S ² = 0.1–0.25) (10), but less ordered than Melme-CC ( S ² = 0.85) (20), Declu-CC ( S ² = 0.7–0.9) (20), Locmi-AKH-I ( S ² = 0.9) (9), and Anoga-HrTH ( S ² = 0.7–0.8) (8). The NMR-derived RMSD values (Fig. 1d) similarly indicate moderate flexibility, with deviations ranging from 1.5 Å in the central region to 2.5 Å at the termini. By contrast, Aedae-ACP and Dappu-RPCH display RMSDs of 3–5 Å centrally and up to 8–9 Å at the termini, whereas the more rigid Melme-CC, Declu-CC, and Anoga-HrTH have RMSDs between 0.3 Å and 1.7 Å. The biological significance of these differing flexibilities remains unclear. The 15 N HSQC spectrum of Carmo-HrTH-II in DPC micelle solution (Figure S2A) displayed all 12 15 N resonances. Distance restraints derived from NOESY spectra (mixing time = 120 ms), combined with dihedral restraints from TALOS-N analysis of 15 N and 13 C shifts (Figure S2B), were used to calculate the peptide structure using the Xplor-NIH simulated annealing protocol. Due to force-field limitations, pyroglutamate and C-amidated residues were modeled as glutamic acid and a free C-terminus, respectively. A total of 244 restraints were applied. The 10 lowest-energy structures refined in a high-dielectric environment exhibited a backbone RMSD of 0.54 Å, with only four violations exceeding 0.4 Å. The averaged structure revealed two turns at Thr3 and Asn7, and a central proline-induced kink (Figure S2C), in agreement with the S ² and chemical shift data (Fig. 1). Comparison with Locmi-AKH-I, which differs from Carmo-HrTH-II only at position 3 (Asn → Thr), shows a strong correlation between their C α and H α random coil shift deviations (9). Both peptides share similar S ² profiles, though Locmi-AKH-I is slightly more rigid (S² = 0.8–0.9). Molecular dynamics simulation of Carmo-HrTH-II The solution-state NMR structure served as an initial configuration for molecular dynamics simulation, with pyroglutamate (pGlu) and C-amidated Thr10 residues implemented. The simulation was started with the ligand emersed in water, above the surfaces of a DPC micelle. During the 100 ns simulation the ligand rapidly diffused and made contact with the micelle surface. This is shown by the contact area between Carmo-HrTH-II and the DPC micelle (Figure S3). Initial contact with the DPC micelle is made by the sidechains of Leu2 and Phe4 (inset snapshot 1). Thereafter, the rest of Carmo-Hr-TH-II makes contact with the DPC micelle (inset snapshot 2). The contact between Carmo-HrTH-II and the DPC surface fluctuated between 10 and 14 Å 2 , which is more contact than was seen for Declu-CC, Melme-CC, Dappu-RPCH (20), Aedae-AKH-I,(27) and Schgr-AKH-II. (9) However, the contact is very similar to that of Locmi-AKH-I (9) and Phote-HrTH. (11) Both Carmo-HrTH-II and Locmi-AKH-I (9) lie in a cleft on the surface of the micelle. Contact between the ligand and the cell surface helps the ligand find the receptor.(28) Cluster analysis of the simulation of Carmo-HrTH-II in DPC micelle solution gave several clusters (1–3 members) but only one main cluster of 49 members (Figure S4b). This cluster has a β-turn at proline, very similar to Locmi-AKH-I. The U-shaped structure is stabilised by intra- and inter-residue H-bonds. Comparison of the two conformations of Carmo-HrTH-II from NMR and MD simulation (Figures S4a and b) shows that they are very similar; both having a turn structure commonly found in G-protein coupled receptor ligands and both have the sidechain of Trp8 projecting inside the turn. The two conformers do differ in the orientation of the C-terminus, but this is expected as structure S4a has a free acid, while structure S4b is amidated. Another difference is that S4a has the Phe4 sidechain outside the turn, while S4b has the Phe4 sidechain inside the turn. Again there is a great similarity between the structure of Carmo-HrTH-II and Locmi-AKH-I (Figure S5). (9) The predominant conformations of both peptides show bends at position 3, Asn7, and Trp8, and a central proline kink. The principal difference lies in the tightness of the bend at residue 3: in Carmo-HrTH-II, the bend is sharper and extends to Phe4. Additionally, Locmi-AKH-I exhibits a subtle backbone twist, altering Trp8 orientation relative to Carmo-HrTH-II. Nevertheless, in both peptides, the aromatic rings of Trp8 and Phe4 are positioned to enable π-stacking interactions. Modelling of the Carausius AKH receptor The primary sequence of Carmo-AKHR, provided by Paluzzi (6) has 528 amino acid residues. During homology modelling the 189 residues of the N- and C-termini were automatically excluded in the generation of the trans-membrane helix structures as they are unstructured and not resolved in the crystal structure. However, after generating the model, the N- and C-termini were added. The two models of the receptor, with and without the termini are shown in Fig. 2. A Ramachandran plot of the results had no torsion angles in a disallowed region, which means that the model is computationally acceptable. The homology model of Carmo-AKHR has long N- and C-termini, seven transmembrane helices, with the third helix tilted relative to the others, and an eighth helix, running parallel to the inner membrane surface, all of which are typical of GPCRs (Fig. 2). The constructed model has all the conserved residues, typical of the rhodopsin superfamily of GPCRs, namely (using the Ballesteros and Weinstein numbering system (29)): N 1.50 ; D 2.50 , P 2.59 ; C 3.25 , the DR 3.50 Y motif, W 4.50 ; P 5.50 ; F 6.44 , the CWxPY motif (CWTP 6.50 Y), the NPxxY (NP 7.50 IVY) motif, and a disulfide bridge between Cys262 3.25 on helix 3 and Cys338 on ECL2. These conserved residues are essential for maintaining the structure of the receptors and for their activation. When viewed from the extracellular side, the seven transmembrane helices have an anticlockwise arrangement, typical of class A GPCRs.(30) Carmo-AKH-II docked to Carmo-AKHR. Attempts to dock Carmo-HrTH-II to the Carmo-AKHR homology model using Glide were initially unsuccessful, showing only surface binding or limited entry into the binding site. Examination of the putative pocket indicated that the decapeptide ligand could not fit within the binding site. Consequently, molecular dynamics (MD) simulations of the receptor alone were performed in a POPC membrane for 500 ns. During the simulation, the extracellular helices moved apart, producing a more open structure (Figure S6a). Using this ‘open’ model for Glide docking yielded several successful poses with scores ranging from − 10.3 to − 6.4. Only peptide conformers containing a β-turn docked successfully, consistent with the hypothesis that a β-turn is essential for GPCR binding (23). The highest-scoring pose was subjected to a further 50 ns of MD simulation in a POPC membrane, giving a binding free energy of − 132 (± 13) kJ mol⁻¹. In the resulting complex (Fig. 3), the ligand spans the entrance of the binding pocket, with p Glu located deep within the pocket and the N-terminus oriented toward the receptor surface. This orientation contrasts with that reported by Iyison et al. (12) for Carmo-HrTH-I, where only the N-terminus entered the binding pocket. Because their model was based on the active-state β₂-adrenergic receptor structure, the pocket was likely too small to accommodate the full peptide. Similar behaviour was observed when docking to the closed conformation of our receptor model. The bound peptide structure (Figure S4c) closely resembles its predominant conformation in DPC micelles, differing mainly in a slightly more extended N-terminus. The free and bound conformations of Carmo-HrTH-II both exhibit a β-turn between Pro6 and Asn7, with Phe4 and Trp8 positioned within the turn, similar to the conformation reported for Locmi-AKH-I bound to its receptor.(9) During MD simulation of the docked complex, the receptor transitioned from an inactive to an active state. In the inactive ‘open’ form (Fig. 4b), ECL2 lies to the side of the helical bundle, permitting ligand entry, while H6 is displaced outward and ICL3 occludes the G-protein binding site Fig. 4d. Activation involves inward movement of H6 (extracellularly) and outward displacement (intracellularly) in a scissors-like motion, accompanied by ECL2 folding over the ligand and ICL3 shifting to expose the intracellular binding surface (Figs. 4a and c). This conformational rearrangement is consistent with activation mechanisms described for Aedae-ACPR and Aedes-AKH-I. (27) RMSD analysis (Figure S7a) shows that the protein shifted by ~ 3 Å over the simulation, mainly due to ECL2 closure, while the ligand reoriented without major conformational change. Ligand RMSF data (Figure S7b) indicate flexibility at the N-terminus and in the sidechains of Leu2, Phe4, and Asn7. Protein RMSF plots (Figs. 5a) reveal high mobility in loop regions and rigidity in transmembrane helices. ECL2 undergoes substantial movement, whereas ECL3, which interacts directly with the ligand, remains comparatively stable. The interacting residues, identified by green vertical lines, (Figs. 5a) are consistent with previous AKH/ACP receptor studies (7). Key receptor–ligand interactions (Figs. 5b) include persistent hydrogen bonds (> 75% of the simulation) with Glu246²· 61 , Asn353⁵·³⁹, Asn356⁵·⁴², Ser436 (ECL3), and Phe341 (ECL2). Bridging water is important in stabilizing the ligand in the binding site (31) and we see bridging waters with many of the protein residues but particularly with Val339 of ECL2. Hydrophobic contacts involve Trp249 and Phe341, and a conserved salt bridge links Glu246²·⁶¹, Arg269³·³², and Lys446⁷·³⁶, consistent with the model of Iyison et al. . Additional interactions among Asn⁵·⁴², Arg³·³², Tyr⁶·⁵², and Phe⁷·³⁹ correspond to those proposed for β₂-adrenergic receptor binding (32). The Arg³·³²–Trp⁸ interaction observed here also parallels that seen in the Aedae-ACPR/Aedae-ACP complex (27). The interaction diagram (Figs. 3b) summarizes ligand residues maintaining receptor contact for > 30% of the simulation. Trp8 is involved in a stable hydrogen bond (84%) with Glu246²·⁶¹ and a π–π interactions with Trp249²·⁶⁴. Experimental data confirm that Trp8 is essential for hypertrehalosaemic activity (6, 7). There is a hydrogen bond (75%) between Phe341²·⁵⁶ and the amide CO of Asn7 and a water bridge a water bridge with Val339 for 59% of the simulation, although in vitro results suggest this residue is not critical for activation. Leu2 shows no significant receptor contact, consistent with our in vitro results (6) and the in vivo findings of Katali et al. (7). This is reminiscent of the situation in B. discoidalis where Ala2 substitution for Val2 is well tolerated (33). Substitution of Leu2 with its β-branched isomer, isoleucine, was not tested here but was tested using the natural peptide of the cockroach Polyphaga aegyptiaca , Polae-HrTH (see later). Carmo-HrTH-II forms several internal hydrogen bonds that stabilize its β-turn conformation, though fewer than observed in Aedae-ACP,(27) likely because the Pro6 residue intrinsically induces a turn without requiring additional hydrogen bonding. Comparison of Carmo-HrTH-I and II Since Iyison et al (12) investigated the docking of Carmo-HrTH-I to their model of Carmo-AKHR, it was of interest to compare the binding of Carmo-HrTH-I to our inactive receptor model. Munte et al. (25) reported that addition of a mannose moiety to Trp8 does not significantly alter peptide conformation. Accordingly, the NMR-derived structure of Carmo-HrTH-II was used as a template, and a C-linked mannose group was added to Trp8 to generate the Carmo-HrTH-I model. It should be noted that the mannose used by Iyison et al. (12) lacked the CH₂OH substituent at C5 and was therefore not a true sugar. Docking and MD simulation of Carmo-HrTH-I bound to Carmo-AKHR in a POPC membrane yielded a binding free energy (ΔG binding ) of − 146 ± 12 kJ mol⁻¹, compared with − 159.7 kJ mol⁻¹ reported by Iyison et al. (12) An overlay of the Carmo-HrTH-I and Carmo-HrTH-II complexes revealed that both peptides adopt essentially the same conformation within the binding site. Minor adjustments in receptor helices were observed to accommodate the bulkier Carmo-HrTH-I ligand, but both peptides occupy the same binding pocket (Figures S8). The ligand interaction diagram (Figures S9) shows that the mannose moiety resides in a hydrophilic pocket and forms several interactions mediated by water molecules. Alanine-Scanning Mutagenesis and Computational Correlation In an in vivo study, Katali et al. (7) used a calcium bioluminescence reporter assay to measure the activity of several alanine-substituted analogues of Carmo-HrTH-II. To interpret these in vitro (6) and in vivo (7) results, each residue of Carmo-HrTH-II was systematically replaced with alanine, and the docking recalculated. The computational results were compared with the in vitro data in Figs. 6a, where the computed change in ligand affinity (relative to the native peptide) is plotted alongside the experimentally determined EC₅₀ values. A positive change in affinity indicates that the mutated ligand binds less strongly than the native AKH. Overall, the correlation between calculated and experimental data is strong, though not perfect. EC₅₀ values for the Phe4 and Trp8, Ala mutants were not determined because both peptides exhibited minimal activity even at the highest concentration tested. Consistently, these two substitutions produced the largest decreases in computed binding affinity, with Trp8 showing a 21.7 kJ mol⁻¹ reduction relative to the native peptide. Mutations at Thr3, Thr5, Pro6, and Asn7 resulted in moderately reduced affinities and correspondingly higher EC₅₀ values. Substitution of Leu2 decreased affinity by 9 kJ mol⁻¹, an unexpected result given that Leu2 forms no direct receptor interactions; this suggests an entropic contribution to the binding free energy. Ligand RMSF data (Figures S7b) show that the Leu2 side chain fluctuates by ~ 2.5 Å within the binding pocket, which may contribute to this effect. Experimentally, the Ala2 analogue showed a moderate decrease in activity.(6) In contrast, substitution of Gly9 by alanine was predicted to slightly increase affinity (by 2.3 kJ mol⁻¹), whereas in vitro results showed that Ala9 was approximately two orders of magnitude less active than the native peptide. (6) Because residue-scanning calculations estimate binding free energy changes without accounting for conformational flexibility, each mutant was subsequently subjected to 50 ns of MD simulation in a POPC membrane. Binding free energies were extracted from the trajectories (Figs. 6b). The resulting trend in ΔG binding differed somewhat from that of ΔAffinity, though both showed good overall agreement with experimental activity. In this case, a higher (less negative) ΔG binding corresponds to a lower EC₅₀ and thus greater activity, meaning that the two plots should be approximately mirror images. This relationship holds for Trp8, which has a low (strong) binding energy and is inactive, and for Thr5, Pro6, and Asn7, which exhibit higher ΔG binding values and moderate activity. However, the Phe4 mutant showed a ΔG binding similar to that of the native peptide despite being inactive.(6, 7) As Phe4 is known to be essential for receptor activation, this discrepancy suggests that binding affinity alone cannot fully account for activity. Similarly, Carmo-HrTH-II itself does not have the highest ΔG binding value, yet it is the most active ligand. These results highlight that receptor activation involves additional steps beyond initial ligand binding, of which only the binding event was modeled here. Gäde et al. (6) and Katali et al. (7) also measured the activity of several endogenous AKH peptides, allowing comparison with our computational results (Table 2). While the agreement between calculated and experimental data is not exact, the overall correlation is strong, supporting the validity of the docking and MD models. Table 2 Calculated DG binding of AKH peptides bound to Carmo-AKHR, and EC 50 values from in vitro bioluminescence assays. Peptide name Native source Peptide sequence a EC 50 value (mol dm − 3 ) b DG binding (kJ/mol) Carmo-HrTH-II Carausius morosus pELTFTPNWGT amide 7.36E-08 -132 (7) Triin-AKH Triatoma infestans pELTFTPNWG - amide 5.48E-07 -101 (9) Peram-CAH-II Periplaneta americana pELTFTPNW– amide 3.88E-07 -118 (9) Phyle-CC Phymateus leprosus pELTFTPNWG S amide 1.80E-08 -136 (8) Locmi-AKH-I Locusta migratoria pEL N FTPNWGT amide 4.14E-06 -112 (14) Phymo-AKH Phymateus morbillosus pEL N FTPNWG S amide 5.05E-06 -114 (14) Polae-HrTH Polyphaga aegyptiaca pE I TFTPNW– amide 8.76E-06 -119 (15) Aedae-AKH Aedes aegypti pELTFTP S W– amide 1.03E-06 -118 (7) Peram-CAH-I Periplaneta americana pE VN F S PNW– amide ND* -94 (9) Panbo-RPCH Pandalus borealis pEL N F S P G W– amide 3.35E-05 -122 (8) Rommi-CC pE VN FTPNWGT amide 1.5E-7 d -107 (14) a Amino acid residues in bold text indicate a substitution relative to Carmo-HrTH-II. b Data from (6) c Standard deviation is given in parentheses. d Estimated from carbohydrate bioassay (7) Phyle-CC differs from Carmo-HrTH-II only by substitution of Thr10 with Ser. As both residues are hydrophilic, this change was expected to have little effect on receptor binding or activity—consistent with our results. Phyle-CC had the same ΔG binding and essentially the same bioactivity as Carmo-HrTH-II. Similarly, replacement of Thr10 with the more hydrophobic Ala10 produced no measurable change in binding or activity, indicating that position 10 is not critical for receptor activation. Comparable biological activity results were reported for L. migratoria (34) and B. discoidalis (33). The importance of residue 3 is illustrated by comparison of Carmo-HrTH-II and Locmi-AKH-I, where Thr3 is replaced by Asn3. This substitution reduces the binding free energy by 20 kJ mol⁻¹ and increases the EC₅₀ from 7.36 × 10⁻⁸ to 4.14 × 10⁻⁶ mol dm⁻³, indicating a substantial loss of potency. Also, the tolerance at position 10 is further demonstrated by comparison of Locmi-AKH-I and Phymo-AKH, which differ only at this position but have nearly identical binding energies and activities. Together, these data indicate that Thr10 does not contribute significantly to receptor activation, whereas position 3 is critical, requiring threonine for Carmo-AKHR, but asparagine for Locmi-AKHR. According to the in vitro activation data (6) position 2 plays a limited role in receptor activation. Consistent with this result, the residue-scanning experiment shows that substituting the bulky Leu2 with alanine had minimal effect on binding as well. However, in the bioassay data set (7) it was measured that replacement of Leu2 by valine in Locmi-AKH-I (yielding Rommi-CC) increased hypertrehalosaemic activity from 19% to 48% relative to Carmo-HrTH-II, suggesting that steric effects at this position may be receptor- or species-specific. Carmo-HrTH-II is a decapeptide; deletion of one residue, as in Triin-AKH, reduced the binding free energy by 31 kJ mol⁻¹ and caused a one-order-of-magnitude loss in activity. Removal of two terminal residues, as in Peram-CAH-II, produced a smaller decrease in ΔG binding (14 kJ mol⁻¹) yet a similar reduction in activity.(6) Structural comparison revealed that both peptides occupy the same binding pocket as Carmo-HrTH-II but adopt different orientations. In Peram-CAH-II, the N-terminus lies deep within the binding pocket and the C-terminus faces the receptor surface, resulting in minimal interaction between Gly9 and Thr10, consistent with the small effect of their removal. By contrast, Triin-AKH binds in the reverse orientation, with the C-terminus buried in the pocket. This reversal, along with the lower binding energy, may explain its reduced but not abolished activity. The influence of position 2 was examined by comparing Peram-CAH-II and Polae-HrTH, which differ only at this site: Leu2 versus Ile2. Both peptides exhibit similar binding energies and activities. Examination of the receptor surface (Figure S10) shows that Leu2 resides in a hydrophobic pocket capable of accommodating isoleucine, explaining the tolerance at this position. A comparable pattern is observed for position 7. Peram-CAH-II and Aedae-AKH differ only by Asn7 versus Ser7 yet have nearly identical ΔG binding and bioactivity. These data suggest that residue 7 is not critical for receptor activation. Finally, Peram-CAH-I, an octapeptide that differs from Carmo-HrTH-II by three residues and the shorter length, showed no in vitro activity and the lowest ΔG binding of all peptides examined. Conversely, Panbo-RPCH displayed a slightly higher ΔG binding (comparable to Carmo-HrTH-II and Phyle-CC) but lower activity. This underscores that strong binding is necessary but not sufficient for receptor activation. In Peram-CAH-I, Phe4 and Trp8, both essential for activity, show weak interactions with the receptor and move continuously within the binding pocket. By contrast, in Panbo-RPCH, Phe4 and Trp8 align closely with the corresponding residues in Carmo-HrTH-II and interact with the same receptor residues (Figure S11), explaining the residual activity despite the overall sequence divergence. Because all known endogenous AKH peptides retain Phe4 and Trp8, direct experimental testing of these positions was not possible. However, residue-scanning analysis confirmed that both residues are indispensable for receptor activation and reversing the order of these two essential amino acids in a peptide such as Peram-CAH-I (thus pEVN W SPN F amide instead of pEVN F SPN W amide) resulted in no hypertrehalosaemic activity in the American cockroach. (35) Insecticide Screening To identify potential antagonists capable of blocking receptor activation by endogenous ligands, in silico screening was conducted using the Octavia database of commercially available insecticide-like molecules. From an initial library of 295,011 compounds, 415,677 conformers were generated and docked to the Carmo-AKHR model. Of these, 197 met the screening criteria, yielding Glide scores from − 10.7 to − 1.4 and ΔG binding binding energies between − 118 and − 3 kJ mol⁻¹. The top-scoring candidates were subsequently subjected to 50 ns MD simulations in a POPC membrane. Results are summarized in Table 3, which also includes reference data from the study of Iyison (12) for comparison. All but one of the compounds has an amide bond and the best two are carboxylic acids. Hydrophobic π-π stacking is an important contributor to the binding of Carmo-HrTH-II to Carmo-AKHR and all the compounds have multiple aromatic rings, which are able to form π-π interactions. All compounds bound within the Carmo-HrTH-II binding pocket of the receptor. Among these, compound 2429 achieved the highest Glide docking score (–10.75), while compound 9072 exhibited the most favourable free energy of binding (–98 kJ mol⁻¹). These two ligands are discussed in detail below; results for the remaining compounds are provided in the Supplementary Information. Table 3 Best hit compounds found after after in silico screening of Otava database of insecticide-like molecules using our model of Carmo-AKHR. For comparison top hits from Iyison (12) are included. Code Structure Glide score kcal/mol DG binding kJ/mol 2429 -10.75 -91 7831 -9.82 -83 9072 -9.64 -98 7727 -9.56 -73 6026 -8.75 -118 P7715460398 -8.44 -83 P771556530 -7.31 -85 P2187686 -7.77 -75 Compound 2429 The RMSF plot for compound 2429 (Figure S12a) shows that the aromatic portions of the molecule are tightly bound (RMSF ≈ 0.8 Å), whereas the aliphatic butane segment fluctuates by ~ 3 Å, consistent with limited receptor contacts. Throughout the simulation, the ligand forms persistent hydrogen-bond and ionic interactions with Lys446 (Figure S12b). Additional interactions include an H-bond and water bridge with Phe344, and hydrophobic contacts with Phe449, Trp420, Tyr423, and Phe273. The ligand interaction diagram (Figure S13) reveals that the phenyl group of compound 2429 occupies a hydrophobic pocket, engaging in π–π stacking with Trp420, Phe449, and Phe273. Arg346 forms a π–cation interaction with the benzene ring, while the ligand’s carboxylic acid and amide carbonyl groups hydrogen-bond to Lys446, Gln337, and Trp249. Compound 9072 Compound 9072 contains two phenyl rings linked by a thiadiazole and amide bridge, with one hydrophobic (alkyl-substituted) and one hydrophilic (ether-substituted) aromatic ring. The ligand exhibits limited mobility in the binding pocket, with all atoms fluctuating by approximately 1 Å (Figure S14). Its key receptor interactions largely overlap with those of compound 2429, including hydrogen bonding to Lys446 and hydrophobic contacts with Phe273, Trp420, Tyr423, and Phe449. In addition, compound 9072 interacts with Arg269, Arg346, and Tyr424, further stabilizing the complex (Figure S13). Comparison and Functional Implications Although both ligands occupy the same binding pocket as Carmo-HrTH-II, their receptor contacts differ markedly. Apart from Trp249 and Phe341, most peptide-specific interactions are absent. Notably, both compounds disrupt the conserved salt bridge between Glu246²·⁶¹, Arg269³·³², and Lys446⁷·³⁶ by forming persistent interactions with Lys446 and a water-mediated bridge to Arg269. A critical requirement for antagonist design is that a compound binds to the receptor without triggering activation. None of the tested compounds induced the conformational change associated with receptor activation, as indicated by the distance between Arg287 and Leu406. In the active state this distance is ~ 15 Å, whereas in the inactive state it is ~ 7 Å; for all docked insecticides, the measured distance remained between 7.2 and 7.9 Å, consistent with the inactive conformation. All but one of the docked ligands contain an amide bond, and the two highest-affinity compounds are carboxylic acids. Consistent with the dominant role of hydrophobic π–π stacking in the binding of Carmo-HrTH-II to Carmo-AKHR, all hit compounds possess multiple aromatic rings capable of such interactions. Conclusion In this study, the structural, dynamic, and functional properties of Carmo-HrTH-II were characterized using NMR, molecular dynamics, and docking studies, providing a comprehensive view of its interaction with the Carmo-AKH receptor. NMR and MD data indicate that Carmo-HrTH-II adopts a stable β-turn conformation, with flexibility primarily at the termini, consistent with other AKH peptides and suitable for GPCR recognition. Docking and MD simulations reveal that successful receptor binding requires this β-turn, positioning key residues Phe4 and Trp8 within the binding pocket, consistent with their essential roles in receptor activation. Alanine-scanning and computational free energy analyses demonstrate that positions 3 and 8 are critical for activity, whereas positions 2, 7, and 10 are more tolerant to substitutions, highlighting structural determinants of receptor specificity and potency. Comparison with Carmo-HrTH-I shows that glycosylation minimally affects peptide conformation and receptor binding which is consistent with the in vitro activation studies.( 6 ) In silico screening of insecticide-like compounds identified several molecules that occupy the same binding pocket as Carmo-HrTH-II, with stable interactions primarily mediated by hydrophobic π–π stacking and hydrogen bonding, without inducing receptor activation. These results validate the binding pocket as a target for antagonist design and provide lead candidates for the development of novel insecticides capable of modulating AKH receptor activity. Overall, this integrative approach links peptide structure, receptor dynamics, and ligand-receptor interactions, offering insights into AKH receptor activation and a framework for rational design of species-specific insecticides. Declarations Informed Consent Statement : Not applicable. Conflicts of Interest: The authors declare no conflict of interest. Funding: This research was funded by the National Research Foundation of South Africa (grant Nos 93450 and 85466 to Graham E. Jackson; grant numbers 85768 [IFR13020116790] to GG and 109204 [IFR170221223270] to HGM, and the University of Cape Town Research Committee. Author Contribution Conceptualization, GG and GEJ; methodology, GEJ, MAS and FS; formal analysis, GEJ, GG, HGM and MAS; investigation, GEJ and MAS; resources, GEJ, GG, HGM, MAS and FS; data curation, GEJ; writing—original draft preparation, GEJ and MAS.; writing—review and editing, GEJ, GG, HGM, MAS and FS; funding acquisition, GEJ, GG, HGM, MAS and FS. All authors have read and agreed to the published version of the manuscript. Acknowledgement MAS and FS acknowledge access to the Bio21 NMR Facility, University of Melbourne. The Centre for High-Performance Computing (CHPC), South Africa, provided computational resources for this research project. Data Availability All data supporting the findings of this study are available within the paper and its Supplementary Information. Raw data is available from the corresponding author upon request. References Liessem, S., Ragionieri, L., Neupert, S., Büschges, A. & Predel, R. Transcriptomic and Neuropeptidomic Analysis of the Stick Insect, Carausius morosus. J. Proteome Res. 17 (6), 2192–2204 (2018). Gäde, G. Adipokinetic and hyperglycaemic factors of different insect species: Separation with high performance liquid chromatography. J. Insect Physiol. 30 , 729–736 (1984). Gäde, G. 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C-mannosylation in the hypertrehalosaemic hormone from the stick insect Carausius morosus. FEBS J. 275 (6), 1163–1173 (2008). Tremblay, M-L., Banks, A. W. & Rainey, J. K. The predictive accuracy of secondary chemical shifts is more affected by protein secondary structure than solvent environment. J. Biomol. NMR. 46 , 257–270 (2010). Jackson, G. E., Sani, M-A., Marco, H. G., Separovic, F. & Gäde, G. The Adipokinetic Hormone (AKH) and the Adipokinetic Hormone/Corazonin-Related Peptide (ACP) Signalling Systems of the Yellow Fever Mosquito Aedes aegypti: Chemical Models of Binding. Biomolecules [Internet] ; 14 (3). (2024). Stone, S. R., Giragossian, C., Mierke, D. F. & Jackson, G. E. Further evidence for a C-terminal structural motif in CCK2 receptor active peptide hormones. Peptides 28 (11), 2211–2222 (2007). Ballesteros, J. A. & Weinstein, H. 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Synthesis and biological activity of locust AKH-I and its analogues with modifications at the threonine residues. 1994(0367–8377 (Print)). Marco, H. G., König, S. & Gäde, G. Predicted novel hypertrehalosaemic peptides of cockroaches are verified by mass spectrometry. Amino Acids . 55 , 1641–1654 (2023). Additional Declarations No competing interests reported. Supplementary Files supfigures.pdf Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Revision requested 09 Apr, 2026 Reviews received at journal 09 Apr, 2026 Reviewers agreed at journal 24 Mar, 2026 Reviews received at journal 24 Mar, 2026 Reviewers agreed at journal 22 Mar, 2026 Reviewers invited by journal 20 Mar, 2026 Editor invited by journal 20 Mar, 2026 Editor assigned by journal 12 Mar, 2026 Submission checks completed at journal 12 Mar, 2026 First submitted to journal 11 Mar, 2026 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Marco","email":"","orcid":"","institution":"University of Cape Town","correspondingAuthor":false,"prefix":"","firstName":"H.","middleName":"G.","lastName":"Marco","suffix":""},{"id":611355774,"identity":"de3c64ea-9d67-47a9-a08a-19fa267034f6","order_by":2,"name":"M.-A. Sani","email":"","orcid":"","institution":"University of Melbourne","correspondingAuthor":false,"prefix":"","firstName":"M.-A.","middleName":"","lastName":"Sani","suffix":""},{"id":611355775,"identity":"ded4552c-fc7a-48c8-ae77-881c37532f7e","order_by":3,"name":"F. Separovic","email":"","orcid":"","institution":"University of Melbourne","correspondingAuthor":false,"prefix":"","firstName":"F.","middleName":"","lastName":"Separovic","suffix":""},{"id":611355776,"identity":"153b272f-d6b6-4bca-8b48-366c2cc80a75","order_by":4,"name":"G. Gäde","email":"","orcid":"","institution":"University of Cape Town","correspondingAuthor":false,"prefix":"","firstName":"G.","middleName":"","lastName":"Gäde","suffix":""}],"badges":[],"createdAt":"2026-03-12 02:09:17","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9099089/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9099089/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":105418596,"identity":"88a18234-6b0c-456e-85e2-f0ee1520c981","added_by":"auto","created_at":"2026-03-25 19:54:43","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":85039,"visible":true,"origin":"","legend":"\u003cp\u003eSee image above for figure legend.\u003c/p\u003e","description":"","filename":"Picture1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9099089/v1/46c7fd200a998a6f2f6bd9a6.jpg"},{"id":105418595,"identity":"e002409b-bf0a-419e-8f98-1cfd354eaff3","added_by":"auto","created_at":"2026-03-25 19:54:42","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":122925,"visible":true,"origin":"","legend":"\u003cp\u003eCarmo-AKHR model (a) showing conserved residues and (b) N- and C-termini.\u003c/p\u003e","description":"","filename":"Picture2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9099089/v1/61ebdf8fe0852224eb3d2492.jpg"},{"id":105418583,"identity":"547a74b2-5bf7-4b68-aff9-dc77f79881df","added_by":"auto","created_at":"2026-03-25 19:54:34","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":195732,"visible":true,"origin":"","legend":"\u003cp\u003e(a) Carmo-HrTH-II docked to Carmo-AKHR.( b) Ligand interaction diagram of Carmo-HrTH-II docked to Carmo-AKHR. The interactions are colour coded such that H-bonding is depicted in purple, while p-cation interactions are shown in red. For the duration of the simulation, contacts between the ligand and receptor are formed and broken. This is recorded as a % of the simulation time. Only interactions that persist for more than 30% of the simulation are shown. The surface of the receptor is shown as a solid line, again colour coded, polar, hydrophobic, etc., according to the nature of the receptor residues.\u003c/p\u003e","description":"","filename":"Picture3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9099089/v1/65f5716edc49a3d1bd61a554.jpg"},{"id":105418567,"identity":"5867012a-4b48-4c28-a508-102ddc164177","added_by":"auto","created_at":"2026-03-25 19:54:25","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":86649,"visible":true,"origin":"","legend":"\u003cp\u003ea) Extracellular domain of Carmo-AKH-II showing movement of loops and helices upon ligand binding. Note how H6 has moved away from the helical bundle giving a more ‘open’ structure’ while ECL2 allows ligands access to the binding site in the ‘inactive’ model . In the active conformer (b) ECL2 has moved above the binding site and H6 has closed in trapping the ligand in the binding site. In (c) the intracellular domain is ‘open’, while in (d) ICL3 closes the binding site.\u003c/p\u003e","description":"","filename":"Picture4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9099089/v1/05d21b0c47196cb636422e9d.jpg"},{"id":105418578,"identity":"1a032fad-658f-4c71-8f42-6d8b170e43c1","added_by":"auto","created_at":"2026-03-25 19:54:32","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":85159,"visible":true,"origin":"","legend":"\u003cp\u003e(a) Protein RMSF and (b) Camro_AKHR/CamHrTH-II contact during MD simulation in POPC membrane. Green = H-bonds; Grey = Hydrophobic; Red = Ionic and Blue = Water bridge.\u003c/p\u003e","description":"","filename":"Picture5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9099089/v1/a7d2e7c1c3b16610326f395c.jpg"},{"id":105418591,"identity":"0f6d9ce8-7fe5-4896-8da0-6de72d1a65b8","added_by":"auto","created_at":"2026-03-25 19:54:35","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":79400,"visible":true,"origin":"","legend":"\u003cp\u003e(a) Graph of change in ligand affinity and EC\u003csub\u003e50\u003c/sub\u003e values of Carmo-HrTH-II upon sequential mutation of residues to Ala. The first point is for the unmutated peptide and (b) Free energy of binding as function of alanine mutation.\u003c/p\u003e","description":"","filename":"Picture6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9099089/v1/74ee0d6d58726d8b80eca1aa.jpg"},{"id":105418600,"identity":"89da9f77-448d-4a0b-abc0-cbe9aa498de9","added_by":"auto","created_at":"2026-03-25 19:54:49","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1883348,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9099089/v1/736cd32f-0fbe-4636-b55d-55b07bcbcd4f.pdf"},{"id":105418584,"identity":"4bd91bdd-08a3-433c-a3e4-78127d045810","added_by":"auto","created_at":"2026-03-25 19:54:35","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":3425664,"visible":true,"origin":"","legend":"","description":"","filename":"supfigures.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9099089/v1/4d6d4806a34c57573fe0649f.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Molecular modelling of the adipokinetic hormone receptor from the stick insect Carausius morosus, and its endogenous agonist","fulltext":[{"header":"Introduction","content":"\u003cp\u003eIn insects, adipokinetic hormone (AKH), corazonin (Crz), and adipokinetic hormone\u0026ndash;corazonin-related peptide (ACP), together with their cognate receptors, are structurally related to vertebrate gonadotropin-releasing hormone (GnRH) and collectively form a large superfamily of neuropeptides. In the Indian stick insect \u003cem\u003eCarausius morosus\u003c/em\u003e, all three neuropeptides are present, (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e) but to date only the AKH system has been investigated in detail.\u003c/p\u003e \u003cp\u003eTwo AKH decapeptides have been identified in the corpora cardiaca of \u003cem\u003eC. morosus\u003c/em\u003e, designated Carmo-HrTH-I and Carmo-HrTH-II according to their order of elution (hydrophobicity) in reverse-phase high-performance liquid chromatography. (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e) Both peptides induce hypertrehalosaemia in ligated stick insects. (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e) Sequence elucidation using fast atom bombardment mass spectrometry and nuclear magnetic resonance (NMR) spectroscopy revealed that the two peptides are almost identical. The less hydrophobic Carmo-HrTH-I is distinguished by the presence of an unusual C-bonded mannose moiety attached to the tryptophan residue at position 8, giving the sequence pGlu-Leu-Thr-Phe-Thr-Pro-Asn-Trp(Man)-Gly-Thr-amide. (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e)\u003c/p\u003e \u003cp\u003eMore recently, the complete functional receptor of \u003cem\u003eC. morosus\u003c/em\u003e has been cloned. (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e) In accordance with its membership in this peptide family, it is referred to here as an adipokinetic hormone (AKH) receptor. The receptor is encoded by a 1587-base-pair open reading frame, producing a protein of 528 amino acids. It was characterised as a class A (rhodopsin-like) G protein-coupled receptor (GPCR), comprising seven hydrophobic transmembrane domains, an extracellular N-terminus, and an intracellular C-terminus. (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e) Structure\u0026ndash;activity relationship studies have previously been conducted both \u003cem\u003ein vivo\u003c/em\u003e, by measuring carbohydrate release into the haemolymph, and \u003cem\u003ein vitro\u003c/em\u003e, using heterologous expression of the receptor in vertebrate cells. (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e) These studies employed strategically modified Carmo-HrTH peptide analogues (alanine replacement series) as well as selected AKH bioanalogues, providing initial insights into the roles of the peptide termini and individual amino-acid side chains in receptor interaction.\u003c/p\u003e \u003cp\u003eIn the present study, we extend these investigations to further elucidate the molecular interaction between ligand and receptor. First, we obtain detailed secondary structural information on the ligand Carmo-HrTH-II through comprehensive NMR analysis. Second, this structural information is combined with existing knowledge of the receptor primary sequence to construct a molecular model of the \u003cem\u003eC. morosus\u003c/em\u003e AKH receptor using molecular dynamics (MD) simulations. Third, we analyse the ligand\u0026ndash;receptor complex in detail to identify key interactions governing binding and activation. Comparable studies have been reported for AKH systems in several arthropods, including the mosquitoes \u003cem\u003eAnopheles gambiae\u003c/em\u003e and \u003cem\u003eAedes aegypti\u003c/em\u003e (including the ACP system), the water flea \u003cem\u003eDaphnia pulex\u003c/em\u003e, the vinegar fly \u003cem\u003eDrosophila melanogaster\u003c/em\u003e, and the desert locust \u003cem\u003eSchistocerca gregaria\u003c/em\u003e. (\u003cspan additionalcitationids=\"CR9 CR10\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e) Previous modelling work on the stick insect AKH system has also been published, allowing direct comparison with the results presented here. (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e)\u003c/p\u003e \u003cp\u003eUnderstanding ligand\u0026ndash;receptor interactions in the AKH system is of particular interest for the rational design of non-peptidic superagonists or antagonists that could serve as future \u0026ldquo;green insecticides\u0026rdquo;. (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e) Such compounds would ideally be species- or group-specific, biodegradable, and environmentally benign, with minimal effects on non-target organisms. Notably, earlier work on the AKH system of the desert locust identified a non-peptidic compound that not only exhibited favourable binding energies in modelling studies but also acted as a competitive inhibitor of the endogenous AKH peptide in an \u003cem\u003ein vivo\u003c/em\u003e bioassay. (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e)\u003c/p\u003e"},{"header":"Material and Method","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eSolution-state NMR Spectroscopy\u003c/h2\u003e \u003cp\u003eThe synthetic peptide Carmo-HrTH-II (pELTFTPNWGT-amide, Synpeptide Co. Ltd., Shanghai, China) was dissolved in perdeuterated d₃₈-dodecylphosphocholine (DPC) micelles (10 mM phosphate buffer, pH 5.0, containing 0.05 mM sodium trimethylsilylpropanesulfonate (DSS) and 10% v/v D₂O) to a final peptide concentration of 2 mM and a DPC-to-peptide molar ratio of 60:1.\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eNMR spectra were acquired at 298 K on a 700 MHz Bruker Neo equipped with a 5 mm TCI cryoprobe. Two-dimensional \u003csup\u003e1\u003c/sup\u003eH-\u003csup\u003e1\u003c/sup\u003eH TOCSY (τ\u003csub\u003emix\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;80 ms) and NOESY (τ\u003csub\u003emix\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;120 and 200 ms) spectra were recorded with 512 and 1024 complex points in the F₁ dimension, respectively, and 2048 points in F₂. Between 8 and 32 transients were accumulated using a 1.5 s recycle delay. Spectra were apodized with a squared sine-bell window shifted by 90\u0026deg; and a \u003csup\u003e1\u003c/sup\u003eH spectral width of 9090 Hz.\u003c/p\u003e\u003cp\u003e \u003csup\u003e13\u003c/sup\u003eC-\u003csup\u003e1\u003c/sup\u003eH HSQC spectra were collected with 256 \u0026times; 2048 complex points, 256 transients, and a 2 s recycle delay. The \u003csup\u003e13\u003c/sup\u003eC spectral window was set to 29050 Hz. \u003csup\u003e15\u003c/sup\u003eN-\u003csup\u003e1\u003c/sup\u003eH HSQC spectra were recorded with 128 \u0026times; 2048 points, 256 transients, a 1.5 s recycle delay, and a \u003csup\u003e15\u003c/sup\u003eN spectral width of 5675 Hz.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e \u003cp\u003eAll data were zero-filled to twice the number of acquired points. \u003csup\u003e1\u003c/sup\u003eH chemical shifts were referenced to DSS (0 ppm), and \u003csup\u003e13\u003c/sup\u003eC and \u003csup\u003e15\u003c/sup\u003eN shifts were indirectly referenced to the \u003csup\u003e1\u003c/sup\u003eH frequency. Data were processed in \u003cem\u003eTopSpin 4.0\u003c/em\u003e (Bruker) and analyzed using \u003cem\u003eCCPNmr Analysis\u003c/em\u003e (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e). Backbone and side-chain assignments were completed using the combined datasets.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eStructure Calculations\u003c/h3\u003e\n\u003cp\u003eNOESY cross-peaks (τ\u003csub\u003emix\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;120 ms) were used to derive interproton distance restraints, supplemented with dihedral angle restraints predicted by \u003cem\u003eTALOS-N\u003c/em\u003e (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e) from H\u003csub\u003eα\u003c/sub\u003e, \u003csup\u003eN\u003c/sup\u003eH, C\u003csub\u003eα\u003c/sub\u003e, C\u003csub\u003eβ\u003c/sub\u003e and \u003csup\u003eH\u003c/sup\u003eN chemical shifts. Structure calculations were performed using \u003cem\u003eXplor-NIH\u003c/em\u003e with standard protocols (16, 17) accessed via the \u003cem\u003eCCPNmr\u003c/em\u003e interface. The averaged structure of the ten lowest-energy conformers was selected for molecular dynamics (MD) simulations and validated with \u003cem\u003eMolProbity\u003c/em\u003e (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e18\u003c/span\u003e).\u003c/p\u003e\n\u003ch3\u003eComputational modelling\u003c/h3\u003e\n\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eLigand preparation and micelle simulation\u003c/h2\u003e \u003cp\u003eCarmo-HrTH-II was built and optimized using the \u003cem\u003eBuilder\u003c/em\u003e module in \u003cem\u003eMaestro\u003c/em\u003e (\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e), exported as a PDB file, and imported into \u003cem\u003eGROMACS\u003c/em\u003e (17). The peptide was placed in a simulation box containing a pre-equilibrated DPC micelle (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e18\u003c/span\u003e). The micelle was positioned at the bottom of the box, with Carmo-HrTH-II centered so that, under periodic boundary conditions, the micelle spanned both top and bottom boundaries. The micelle was taken from a previous study on adipokinetic hormones in water and DPC micelle solution (\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e)\u003c/p\u003e \u003cp\u003eSimulations were performed using the OPLS-AA force field (\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e). Following energy minimization, a 500 ns NVT simulation was conducted, saving trajectory frames every 100 ps. Cluster analysis was performed using the \u003cem\u003eg_cluster\u003c/em\u003e algorithm in \u003cem\u003eGROMACS\u003c/em\u003e with a 0.2 nm cutoff.\u003c/p\u003e \u003cp\u003eCarmo-HrTH-I, the second native AKH peptide of \u003cem\u003eCarausius morosus\u003c/em\u003e, was generated computationally by adding a C-linked mannose to the Trp residue of the Carmo-HrTH-II model.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eReceptor homology modelling and docking\u003c/h3\u003e\n\u003cp\u003eThe primary sequence of the \u003cem\u003eC. morosus\u003c/em\u003e adipokinetic hormone receptor (Carmo-AKHR) (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e) was used for homology modelling (\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e). The β₂-adrenergic receptor (PDB ID: 5D5A) (\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e) was chosen as a template based on resolution (2.48 \u0026Aring;), sequence identity (21.6%), coverage (0.52), and GMQE score (0.27) (\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e). The resulting model was refined in \u003cem\u003eMaestro\u003c/em\u003e (\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e) using the Protein Preparation Wizard at pH 7.0.\u003c/p\u003e \u003cp\u003eThe receptor was inserted into a 1-palmitoyl-2-eleoyl-sn-glycero-3-phosphocholine (POPC) membrane using \u003cem\u003eCHARMM-GUI\u003c/em\u003e (24), and a 50 ns MD simulation performed using \u003cem\u003eGROMACS\u003c/em\u003e with CHARMM parameters (\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e25\u003c/span\u003e). The final frame from the simulation was used for docking.\u003c/p\u003e \u003cp\u003eA conformational search in \u003cem\u003eMaestro\u003c/em\u003e (\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e) generated 100 Carmo-HrTH-II conformers, which were docked into the receptor using \u003cem\u003eGlide\u003c/em\u003e (\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e) in SP-Peptide mode (scaling factor 0.8, partial charge cutoff 0.15 nm). The top-scoring poses were subjected to MD simulation in a POPC membrane using \u003cem\u003eDesmond\u003c/em\u003e (\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e). Systems were solvated with SPC water, neutralized, and adjusted to 0.15 M NaCl. Binding free energies were calculated using MM/GBSA (\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e), and ligand\u0026ndash;receptor interactions were calculated by \u003cem\u003eMaestro\u003c/em\u003e.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eIn silico Insecticide Screening\u003c/h2\u003e \u003cp\u003eAn insecticide-like compound library comprising 295,011 structures was obtained from Otava Chemicals Ltd. A total of 415,677 tautomers and ionization states were generated using \u003cem\u003eLigPrep\u003c/em\u003e (Schr\u0026ouml;dinger Release 2021-1). Compounds were filtered using \u003cem\u003eQikProp\u003c/em\u003e (\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e) for pharmacokinetic and physicochemical properties, yielding 320,689 candidates.\u003c/p\u003e \u003cp\u003eHigh-throughput virtual screening (HTVS) was performed against the Carmo-AKHR model. From 24,941 initial poses, 1,970 were refined by extra-precision docking, yielding 197 final ligand\u0026ndash;receptor complexes. Free energies of binding were computed via MM/GBSA, and the highest-scoring candidates were embedded into POPC membranes for 50 ns MD simulations as described above.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results and Discussions","content":"\u003cdiv id=\"Sec10\"\u003e\n \u003ch2\u003eNMR and structural data of the ligand Carmo-HrTH-II\u003c/h2\u003e\n \u003cp\u003eThe \u003csup\u003e1\u003c/sup\u003eH, \u003csup\u003e15\u003c/sup\u003eN and \u003csup\u003e13\u003c/sup\u003eC chemical shifts and assignments in DPC micelle solution of Carmo-HrTH-II are summarized in Table\u0026nbsp;1. Structure-induced chemical shift deviations were calculated by subtracting the random coil values from the observed chemical shifts (Fig.\u0026nbsp;1a,b). Positive deviations in C\u003csub\u003e\u0026alpha;\u003c/sub\u003e chemical shifts are characteristic of \u0026beta;-strand formation, while \u0026alpha;-helices typically produce negative deviations (22). Figure\u0026nbsp;1a demonstrates that Carmo-HrTH-II adopts a coiled conformation in DPC micelle solution. Although proton chemical shifts are less sensitive to structural changes than \u003csup\u003e13\u003c/sup\u003eC chemical shifts, all \u003csup\u003e1\u003c/sup\u003eH deviations were below 0.3 ppm (26), consistent with a turn structure following the first two residues. Together, the \u003csup\u003e13\u003c/sup\u003eC and \u003csup\u003e1\u003c/sup\u003eH chemical shift data indicate that Carmo-HrTH-II adopts a turn conformation in DPC micelle solution. This observation aligns with previous findings that adipokinetic hormones (AKHs) typically form \u0026beta;-turn structures in micellar environments. Indeed, Tyndal et al. (23) reported that over 100 mammalian GPCR ligands exhibit \u0026beta;-turns, and Cusinato et al. (24) showed by CD spectroscopy that AKH/RPCH peptides form \u0026beta;-turns in 0.6% SDS solution.\u003c/p\u003e\n \u003cdiv\u003e\n \u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv\u003eTable 1\u003c/div\u003e\n \u003cdiv\u003e\n \u003cp\u003eNMR assignments of Carmo-HrTH-II in DPC micelle solution.\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\" colname=\"c1\"\u003e\n \u003cp\u003eResidue\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colname=\"c2\"\u003e\n \u003cp\u003e\u0026delta;(N)/ppm\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colname=\"c3\"\u003e\n \u003cp\u003e\u0026delta;(HN)/ppm\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colname=\"c4\"\u003e\n \u003cp\u003e\u0026delta;(H\u0026alpha;)/ppm\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colname=\"c5\"\u003e\n \u003cp\u003e\u0026delta;(H\u0026beta;)/ppm\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colname=\"c6\"\u003e\n \u003cp\u003e\u0026delta;(H\u0026gamma;)/ppm\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colname=\"c7\"\u003e\n \u003cp\u003e\u0026delta;(C\u0026alpha;)/ppm\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colname=\"c8\"\u003e\n \u003cp\u003e\u0026delta;(C\u0026beta;)/ppm\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colname=\"c9\"\u003e\n \u003cp\u003e\u0026delta;(C\u0026gamma;)/ppm\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colname=\"c10\"\u003e\n \u003cp\u003e\u0026delta;(Other)/ppm\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colname=\"c1\"\u003e\n \u003cp\u003e\u003cstrong\u003e1-pGlu\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c2\"\u003e\n \u003cp\u003e125.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c3\"\u003e\n \u003cp\u003e7.99\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c4\"\u003e\n \u003cp\u003e4.38\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c5\"\u003e\n \u003cp\u003e2.50, 1.98\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c6\"\u003e\n \u003cp\u003e2.35*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c7\"\u003e\n \u003cp\u003e58.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c8\"\u003e\n \u003cp\u003e27.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c9\"\u003e\n \u003cp\u003e31.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c10\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colname=\"c1\"\u003e\n \u003cp\u003e\u003cstrong\u003e2-Leu\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c2\"\u003e\n \u003cp\u003e121.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c3\"\u003e\n \u003cp\u003e8.45\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c4\"\u003e\n \u003cp\u003e4.39\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c5\"\u003e\n \u003cp\u003e1.61, 1.49\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c6\"\u003e\n \u003cp\u003e1.61\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c7\"\u003e\n \u003cp\u003e54.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c8\"\u003e\n \u003cp\u003e41.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c9\"\u003e\n \u003cp\u003e26.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c10\"\u003e\n \u003cp\u003eH\u003csup\u003e\u0026delta;1\u003c/sup\u003e 0.90, H\u003csup\u003e\u0026delta;2\u003c/sup\u003e 0.87, C\u003csup\u003e\u0026delta;1\u003c/sup\u003e 24.7, C\u003csup\u003e\u0026delta;2\u003c/sup\u003e 23.5\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colname=\"c1\"\u003e\n \u003cp\u003e\u003cstrong\u003e3-Thr\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c2\"\u003e\n \u003cp\u003e114.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c3\"\u003e\n \u003cp\u003e8.10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c4\"\u003e\n \u003cp\u003e4.30\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c5\"\u003e\n \u003cp\u003e4.15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c6\"\u003e\n \u003cp\u003e1.10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c7\"\u003e\n \u003cp\u003e61.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c8\"\u003e\n \u003cp\u003e69.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c9\"\u003e\n \u003cp\u003e20.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c10\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colname=\"c1\"\u003e\n \u003cp\u003e\u003cstrong\u003e4-Phe\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c2\"\u003e\n \u003cp\u003e121.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c3\"\u003e\n \u003cp\u003e8.09\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c4\"\u003e\n \u003cp\u003e4.67\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c5\"\u003e\n \u003cp\u003e3.04, 3.01\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c6\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c7\"\u003e\n \u003cp\u003e56.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c8\"\u003e\n \u003cp\u003e39.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c9\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c10\"\u003e\n \u003cp\u003eH\u003csup\u003e\u0026delta;\u003c/sup\u003e 7.16, H\u003csup\u003e\u0026epsilon;\u003c/sup\u003e 7.21, H\u003csup\u003e\u0026zeta;\u003c/sup\u003e 7.16, C\u003csup\u003e\u0026delta;\u003c/sup\u003e 131.5, C\u003csup\u003e\u0026epsilon;\u003c/sup\u003e 130.4, C\u003csup\u003e\u0026zeta;\u003c/sup\u003e 128.8\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colname=\"c1\"\u003e\n \u003cp\u003e\u003cstrong\u003e5-Thr\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c2\"\u003e\n \u003cp\u003e116.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c3\"\u003e\n \u003cp\u003e8.00\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c4\"\u003e\n \u003cp\u003e4.47\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c5\"\u003e\n \u003cp\u003e4.04\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c6\"\u003e\n \u003cp\u003e1.12\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c7\"\u003e\n \u003cp\u003e58.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c8\"\u003e\n \u003cp\u003e69.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c9\"\u003e\n \u003cp\u003e20.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c10\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colname=\"c1\"\u003e\n \u003cp\u003e\u003cstrong\u003e6-Pro\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c2\"\u003e\n \u003cp\u003e121.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c3\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c4\"\u003e\n \u003cp\u003e4.21\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c5\"\u003e\n \u003cp\u003e1.87, 1.41\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c6\"\u003e\n \u003cp\u003e1.76, 1.69\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c7\"\u003e\n \u003cp\u003e62.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c8\"\u003e\n \u003cp\u003e31.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c9\"\u003e\n \u003cp\u003e26.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c10\"\u003e\n \u003cp\u003eH\u003csup\u003e\u0026delta;\u003c/sup\u003e 3.51, C\u003csup\u003e\u0026delta;\u003c/sup\u003e 50.0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colname=\"c1\"\u003e\n \u003cp\u003e\u003cstrong\u003e7-Asn\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c2\"\u003e\n \u003cp\u003e118.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c3\"\u003e\n \u003cp\u003e8.41\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c4\"\u003e\n \u003cp\u003e4.64\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c5\"\u003e\n \u003cp\u003e2.78, 2.69\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c6\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c7\"\u003e\n \u003cp\u003e52.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c8\"\u003e\n \u003cp\u003e38.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c9\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c10\"\u003e\n \u003cp\u003eN\u003csup\u003e\u0026delta;2\u003c/sup\u003e 112.5, H\u003csup\u003e\u0026delta;1\u003c/sup\u003e 7.61, H\u003csup\u003e\u0026delta;2\u003c/sup\u003e 6.85\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colname=\"c1\"\u003e\n \u003cp\u003e\u003cstrong\u003e8-Trp\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c2\"\u003e\n \u003cp\u003e120.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c3\"\u003e\n \u003cp\u003e7.98\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c4\"\u003e\n \u003cp\u003e4.64\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c5\"\u003e\n \u003cp\u003e3.32, 3.21\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c6\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c7\"\u003e\n \u003cp\u003e56.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c8\"\u003e\n \u003cp\u003e29.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c9\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c10\"\u003e\n \u003cp\u003eN\u003csup\u003e\u0026epsilon;\u003c/sup\u003e 130.3, H\u003csup\u003e\u0026delta;1\u003c/sup\u003e 7.26, H\u003csup\u003e\u0026epsilon;1\u003c/sup\u003e 10.51, H\u003csup\u003e\u0026epsilon;3\u003c/sup\u003e 7.56, H\u003csup\u003e\u0026zeta;2\u003c/sup\u003e 7.43, H\u003csup\u003e\u0026zeta;3\u003c/sup\u003e 7.02, H\u003csup\u003e\u0026eta;2\u003c/sup\u003e 7.07, C\u003csup\u003e\u0026delta;1\u003c/sup\u003e 126.6, C\u003csup\u003e\u0026epsilon;3\u003c/sup\u003e 120.3, C\u003csup\u003e\u0026zeta;2\u003c/sup\u003e 113.8, C\u003csup\u003e\u0026zeta;3\u003c/sup\u003e 120.8, C\u003csup\u003e\u0026eta;2\u003c/sup\u003e 123.3\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colname=\"c1\"\u003e\n \u003cp\u003e\u003cstrong\u003e9-Gly\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c2\"\u003e\n \u003cp\u003e108.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c3\"\u003e\n \u003cp\u003e8.41\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c4\"\u003e\n \u003cp\u003e3.95, 3.90\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c5\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c6\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c7\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c8\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c9\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c10\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colname=\"c1\"\u003e\n \u003cp\u003e\u003cstrong\u003e10-Thr\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c2\"\u003e\n \u003cp\u003e112.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c3\"\u003e\n \u003cp\u003e7.96\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c4\"\u003e\n \u003cp\u003e4.26\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c5\"\u003e\n \u003cp\u003e4.26\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c6\"\u003e\n \u003cp\u003e1.18\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c7\"\u003e\n \u003cp\u003e61.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c8\"\u003e\n \u003cp\u003e69.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c9\"\u003e\n \u003cp\u003e21.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c10\"\u003e\n \u003cp\u003eN\u003csup\u003e\u0026eta;2\u003c/sup\u003e 109.1, H\u003csup\u003eN1\u003c/sup\u003e 7.67, H\u003csup\u003eN2\u003c/sup\u003e 7.16\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colspan=\"10\" nameend=\"c10\" namest=\"c1\"\u003e\n \u003cp\u003eChemical shift in ppm, referenced from DSS, experiments run at 298K in DPC micelles\u003c/p\u003e\n \u003cp\u003e- Not observed\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003c/div\u003e\n \u003cp\u003eMunte et al.(25) reported that Carmo-HrTH-II in D₂O does not adopt a random coil structure but rather exists as an ensemble of conformations containing transient ordered species. In our study, binding to the DPC micelle appears to stabilize these conformers. Munte et al., (25) also measured random coil chemical shifts for the tryptophan C-mannosylated analogue, Carmo-HrTH-I, finding them nearly identical to those of Carmo-HrTH-II, indicating that the sugar moiety does not substantially alter peptide conformation. A comparison of random coil shift differences between Carmo-HrTH-I and -II is shown in Figure S1 Both \u003csup\u003e1\u003c/sup\u003eH and \u003csup\u003e13\u003c/sup\u003eC shift trends were similar, although Carmo-HrTH-II in DPC micelles showed larger C\u003csub\u003e\u0026alpha;\u003c/sub\u003e deviations than Carmo-HrTH-I in water, consistent with greater conformational stabilization in the micellar environment.\u003c/p\u003e\n \u003cp\u003eUsing all chemical shift data, the model free order parameter, \u003cem\u003eS\u003c/em\u003e\u003csup\u003e2\u003c/sup\u003e (26), was calculated to assess peptide flexibility (Fig.\u0026nbsp;1c). An \u003cem\u003eS\u003c/em\u003e\u0026sup2; value of 1 indicates complete rigidity, whereas 0 represents full flexibility. Carmo-HrTH-II exhibited \u003cem\u003eS\u003c/em\u003e\u0026sup2; values of 0.5\u0026ndash;0.6, indicating a structured but flexible conformation. The peptide is more rigid than some other members of this superfamily such as Aedae-ACP (\u003cem\u003eS\u003c/em\u003e\u0026sup2; = 0\u0026ndash;0.36) (27), Aedae-AKH (\u003cem\u003eS\u003c/em\u003e\u0026sup2; = 0.1\u0026ndash;0.3) (27), Schgr-AKH-II (\u003cem\u003eS\u003c/em\u003e\u0026sup2; = 0.1\u0026ndash;0.4) (9), and Dappu-RPCH (\u003cem\u003eS\u003c/em\u003e\u0026sup2; = 0.1\u0026ndash;0.25) (10), but less ordered than Melme-CC (\u003cem\u003eS\u003c/em\u003e\u0026sup2; = 0.85) (20), Declu-CC (\u003cem\u003eS\u003c/em\u003e\u0026sup2; = 0.7\u0026ndash;0.9) (20), Locmi-AKH-I (\u003cem\u003eS\u003c/em\u003e\u0026sup2; = 0.9) (9), and Anoga-HrTH (\u003cem\u003eS\u003c/em\u003e\u0026sup2; = 0.7\u0026ndash;0.8) (8). The NMR-derived RMSD values (Fig.\u0026nbsp;1d) similarly indicate moderate flexibility, with deviations ranging from 1.5 \u0026Aring; in the central region to 2.5 \u0026Aring; at the termini. By contrast, Aedae-ACP and Dappu-RPCH display RMSDs of 3\u0026ndash;5 \u0026Aring; centrally and up to 8\u0026ndash;9 \u0026Aring; at the termini, whereas the more rigid Melme-CC, Declu-CC, and Anoga-HrTH have RMSDs between 0.3 \u0026Aring; and 1.7 \u0026Aring;. The biological significance of these differing flexibilities remains unclear.\u003c/p\u003e\n \u003cp\u003eThe \u003csup\u003e15\u003c/sup\u003eN HSQC spectrum of Carmo-HrTH-II in DPC micelle solution (Figure S2A) displayed all 12 \u003csup\u003e15\u003c/sup\u003eN resonances. Distance restraints derived from NOESY spectra (mixing time\u0026thinsp;=\u0026thinsp;120 ms), combined with dihedral restraints from TALOS-N analysis of \u003csup\u003e15\u003c/sup\u003eN and \u003csup\u003e13\u003c/sup\u003eC shifts (Figure S2B), were used to calculate the peptide structure using the \u003cem\u003eXplor-NIH\u003c/em\u003e simulated annealing protocol. Due to force-field limitations, pyroglutamate and C-amidated residues were modeled as glutamic acid and a free C-terminus, respectively. A total of 244 restraints were applied. The 10 lowest-energy structures refined in a high-dielectric environment exhibited a backbone RMSD of 0.54 \u0026Aring;, with only four violations exceeding 0.4 \u0026Aring;. The averaged structure revealed two turns at Thr3 and Asn7, and a central proline-induced kink (Figure S2C), in agreement with the \u003cem\u003eS\u003c/em\u003e\u0026sup2; and chemical shift data (Fig.\u0026nbsp;1).\u003c/p\u003e\n \u003cp\u003eComparison with Locmi-AKH-I, which differs from Carmo-HrTH-II only at position 3 (Asn \u0026rarr; Thr), shows a strong correlation between their C\u003csub\u003e\u0026alpha;\u003c/sub\u003e and H\u003csub\u003e\u0026alpha;\u003c/sub\u003e random coil shift deviations (9). Both peptides share similar \u003cem\u003eS\u003c/em\u003e\u0026sup2; profiles, though Locmi-AKH-I is slightly more rigid (S\u0026sup2; = 0.8\u0026ndash;0.9).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec11\"\u003e\n \u003ch2\u003eMolecular dynamics simulation of Carmo-HrTH-II\u003c/h2\u003e\n \u003cp\u003eThe solution-state NMR structure served as an initial configuration for molecular dynamics simulation, with pyroglutamate (pGlu) and C-amidated Thr10 residues implemented. The simulation was started with the ligand emersed in water, above the surfaces of a DPC micelle. During the 100 ns simulation the ligand rapidly diffused and made contact with the micelle surface. This is shown by the contact area between Carmo-HrTH-II and the DPC micelle (Figure S3). Initial contact with the DPC micelle is made by the sidechains of Leu2 and Phe4 (inset snapshot 1). Thereafter, the rest of Carmo-Hr-TH-II makes contact with the DPC micelle (inset snapshot 2). The contact between Carmo-HrTH-II and the DPC surface fluctuated between 10 and 14 \u0026Aring;\u003csup\u003e2\u003c/sup\u003e, which is more contact than was seen for Declu-CC, Melme-CC, Dappu-RPCH (20), Aedae-AKH-I,(27) and Schgr-AKH-II. (9) However, the contact is very similar to that of Locmi-AKH-I (9) and Phote-HrTH. (11) Both Carmo-HrTH-II and Locmi-AKH-I (9) lie in a cleft on the surface of the micelle. Contact between the ligand and the cell surface helps the ligand find the receptor.(28)\u003c/p\u003e\n \u003cp\u003eCluster analysis of the simulation of Carmo-HrTH-II in DPC micelle solution gave several clusters (1\u0026ndash;3 members) but only one main cluster of 49 members (Figure S4b). This cluster has a \u0026beta;-turn at proline, very similar to Locmi-AKH-I. The U-shaped structure is stabilised by intra- and inter-residue H-bonds. Comparison of the two conformations of Carmo-HrTH-II from NMR and MD simulation (Figures S4a and b) shows that they are very similar; both having a turn structure commonly found in G-protein coupled receptor ligands and both have the sidechain of Trp8 projecting inside the turn. The two conformers do differ in the orientation of the C-terminus, but this is expected as structure S4a has a free acid, while structure S4b is amidated. Another difference is that S4a has the Phe4 sidechain outside the turn, while S4b has the Phe4 sidechain inside the turn. Again there is a great similarity between the structure of Carmo-HrTH-II and Locmi-AKH-I (Figure S5). (9) The predominant conformations of both peptides show bends at position 3, Asn7, and Trp8, and a central proline kink. The principal difference lies in the tightness of the bend at residue 3: in Carmo-HrTH-II, the bend is sharper and extends to Phe4. Additionally, Locmi-AKH-I exhibits a subtle backbone twist, altering Trp8 orientation relative to Carmo-HrTH-II. Nevertheless, in both peptides, the aromatic rings of Trp8 and Phe4 are positioned to enable \u0026pi;-stacking interactions.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec12\"\u003e\n \u003ch2\u003eModelling of the Carausius AKH receptor\u003c/h2\u003e\n \u003cp\u003eThe primary sequence of Carmo-AKHR, provided by Paluzzi (6) has 528 amino acid residues. During homology modelling the 189 residues of the N- and C-termini were automatically excluded in the generation of the trans-membrane helix structures as they are unstructured and not resolved in the crystal structure. However, after generating the model, the N- and C-termini were added. The two models of the receptor, with and without the termini are shown in Fig.\u0026nbsp;2. A Ramachandran plot of the results had no torsion angles in a disallowed region, which means that the model is computationally acceptable.\u003c/p\u003e\n \u003cp\u003eThe homology model of Carmo-AKHR has long N- and C-termini, seven transmembrane helices, with the third helix tilted relative to the others, and an eighth helix, running parallel to the inner membrane surface, all of which are typical of GPCRs (Fig.\u0026nbsp;2). The constructed model has all the conserved residues, typical of the rhodopsin superfamily of GPCRs, namely (using the Ballesteros and Weinstein numbering system (29)): N\u003csup\u003e1.50\u003c/sup\u003e ; D\u003csup\u003e2.50\u003c/sup\u003e, P\u003csup\u003e2.59\u003c/sup\u003e ; C\u003csup\u003e3.25\u003c/sup\u003e, the DR\u003csup\u003e3.50\u003c/sup\u003eY motif, W\u003csup\u003e4.50\u003c/sup\u003e; P\u003csup\u003e5.50\u003c/sup\u003e ; F\u003csup\u003e6.44\u003c/sup\u003e, the CWxPY motif (CWTP\u003csup\u003e6.50\u003c/sup\u003e Y), the NPxxY (NP\u003csup\u003e7.50\u003c/sup\u003eIVY) motif, and a disulfide bridge between Cys262\u003csup\u003e3.25\u003c/sup\u003e on helix 3 and Cys338 on ECL2. These conserved residues are essential for maintaining the structure of the receptors and for their activation. When viewed from the extracellular side, the seven transmembrane helices have an anticlockwise arrangement, typical of class A GPCRs.(30)\u003c/p\u003e\n \u003cp\u003e\u003cem\u003eCarmo-AKH-II docked to Carmo-AKHR.\u003c/em\u003e\u003c/p\u003e\n \u003cp\u003eAttempts to dock Carmo-HrTH-II to the Carmo-AKHR homology model using \u003cem\u003eGlide\u003c/em\u003e were initially unsuccessful, showing only surface binding or limited entry into the binding site. Examination of the putative pocket indicated that the decapeptide ligand could not fit within the binding site. Consequently, molecular dynamics (MD) simulations of the receptor alone were performed in a POPC membrane for 500 ns. During the simulation, the extracellular helices moved apart, producing a more open structure (Figure S6a).\u003c/p\u003e\n \u003cp\u003eUsing this \u0026lsquo;open\u0026rsquo; model for \u003cem\u003eGlide\u003c/em\u003e docking yielded several successful poses with scores ranging from \u0026minus;\u0026thinsp;10.3 to \u0026minus;\u0026thinsp;6.4. Only peptide conformers containing a \u0026beta;-turn docked successfully, consistent with the hypothesis that a \u0026beta;-turn is essential for GPCR binding (23). The highest-scoring pose was subjected to a further 50 ns of MD simulation in a POPC membrane, giving a binding free energy of \u0026minus;\u0026thinsp;132 (\u0026plusmn;\u0026thinsp;13) kJ mol⁻\u0026sup1;. In the resulting complex (Fig.\u0026nbsp;3), the ligand spans the entrance of the binding pocket, with \u003cem\u003ep\u003c/em\u003eGlu located deep within the pocket and the N-terminus oriented toward the receptor surface. This orientation contrasts with that reported by Iyison \u003cem\u003eet al.\u003c/em\u003e (12) for Carmo-HrTH-I, where only the N-terminus entered the binding pocket. Because their model was based on the active-state \u0026beta;₂-adrenergic receptor structure, the pocket was likely too small to accommodate the full peptide. Similar behaviour was observed when docking to the closed conformation of our receptor model.\u003c/p\u003e\n \u003cp\u003eThe bound peptide structure (Figure S4c) closely resembles its predominant conformation in DPC micelles, differing mainly in a slightly more extended N-terminus. The free and bound conformations of Carmo-HrTH-II both exhibit a \u0026beta;-turn between Pro6 and Asn7, with Phe4 and Trp8 positioned within the turn, similar to the conformation reported for Locmi-AKH-I bound to its receptor.(9)\u003c/p\u003e\n \u003cp\u003eDuring MD simulation of the docked complex, the receptor transitioned from an inactive to an active state. In the inactive \u0026lsquo;open\u0026rsquo; form (Fig.\u0026nbsp;4b), ECL2 lies to the side of the helical bundle, permitting ligand entry, while H6 is displaced outward and ICL3 occludes the G-protein binding site Fig.\u0026nbsp;4d. Activation involves inward movement of H6 (extracellularly) and outward displacement (intracellularly) in a scissors-like motion, accompanied by ECL2 folding over the ligand and ICL3 shifting to expose the intracellular binding surface (Figs.\u0026nbsp;4a and c). This conformational rearrangement is consistent with activation mechanisms described for Aedae-ACPR and Aedes-AKH-I. (27)\u003c/p\u003e\n \u003cp\u003eRMSD analysis (Figure S7a) shows that the protein shifted by ~\u0026thinsp;3 \u0026Aring; over the simulation, mainly due to ECL2 closure, while the ligand reoriented without major conformational change. Ligand RMSF data (Figure S7b) indicate flexibility at the N-terminus and in the sidechains of Leu2, Phe4, and Asn7. Protein RMSF plots (Figs.\u0026nbsp;5a) reveal high mobility in loop regions and rigidity in transmembrane helices. ECL2 undergoes substantial movement, whereas ECL3, which interacts directly with the ligand, remains comparatively stable. The interacting residues, identified by green vertical lines, (Figs.\u0026nbsp;5a) are consistent with previous AKH/ACP receptor studies (7).\u003c/p\u003e\n \u003cp\u003eKey receptor\u0026ndash;ligand interactions (Figs.\u0026nbsp;5b) include persistent hydrogen bonds (\u0026gt;\u0026thinsp;75% of the simulation) with Glu246\u0026sup2;\u0026middot;\u003csup\u003e61\u003c/sup\u003e, Asn353⁵\u0026middot;\u0026sup3;⁹, Asn356⁵\u0026middot;⁴\u0026sup2;, Ser436 (ECL3), and Phe341 (ECL2). Bridging water is important in stabilizing the ligand in the binding site (31) and we see bridging waters with many of the protein residues but particularly with Val339 of ECL2. Hydrophobic contacts involve Trp249 and Phe341, and a conserved salt bridge links Glu246\u0026sup2;\u0026middot;⁶\u0026sup1;, Arg269\u0026sup3;\u0026middot;\u0026sup3;\u0026sup2;, and Lys446⁷\u0026middot;\u0026sup3;⁶, consistent with the model of Iyison \u003cem\u003eet al.\u003c/em\u003e. Additional interactions among Asn⁵\u0026middot;⁴\u0026sup2;, Arg\u0026sup3;\u0026middot;\u0026sup3;\u0026sup2;, Tyr⁶\u0026middot;⁵\u0026sup2;, and Phe⁷\u0026middot;\u0026sup3;⁹ correspond to those proposed for \u0026beta;₂-adrenergic receptor binding (32). The Arg\u0026sup3;\u0026middot;\u0026sup3;\u0026sup2;\u0026ndash;Trp⁸ interaction observed here also parallels that seen in the Aedae-ACPR/Aedae-ACP complex (27).\u003c/p\u003e\n \u003cp\u003eThe interaction diagram (Figs.\u0026nbsp;3b) summarizes ligand residues maintaining receptor contact for \u0026gt;\u0026thinsp;30% of the simulation. Trp8 is involved in a stable hydrogen bond (84%) with Glu246\u0026sup2;\u0026middot;⁶\u0026sup1; and a \u0026pi;\u0026ndash;\u0026pi; interactions with Trp249\u0026sup2;\u0026middot;⁶⁴. Experimental data confirm that Trp8 is essential for hypertrehalosaemic activity (6, 7). There is a hydrogen bond (75%) between Phe341\u0026sup2;\u0026middot;⁵⁶ and the amide CO of Asn7 and a water bridge a water bridge with Val339 for 59% of the simulation, although \u003cem\u003ein vitro\u003c/em\u003e results suggest this residue is not critical for activation. Leu2 shows no significant receptor contact, consistent with our \u003cem\u003ein vitro\u003c/em\u003e results (6) and the \u003cem\u003ein vivo\u003c/em\u003e findings of Katali \u003cem\u003eet al.\u003c/em\u003e (7). This is reminiscent of the situation in B. \u003cem\u003ediscoidalis\u003c/em\u003e where Ala2 substitution for Val2 is well tolerated (33). Substitution of Leu2 with its \u0026beta;-branched isomer, isoleucine, was not tested here but was tested using the natural peptide of the cockroach \u003cem\u003ePolyphaga aegyptiaca\u003c/em\u003e, Polae-HrTH (see later).\u003c/p\u003e\n \u003cp\u003eCarmo-HrTH-II forms several internal hydrogen bonds that stabilize its \u0026beta;-turn conformation, though fewer than observed in Aedae-ACP,(27) likely because the Pro6 residue intrinsically induces a turn without requiring additional hydrogen bonding.\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eComparison of Carmo-HrTH-I and\u003c/strong\u003e \u003cem\u003eII\u003c/em\u003e\u003c/p\u003e\n \u003cp\u003eSince Iyison \u003cem\u003eet al\u003c/em\u003e (12) investigated the docking of Carmo-HrTH-I to their model of Carmo-AKHR, it was of interest to compare the binding of Carmo-HrTH-I to our inactive receptor model. Munte \u003cem\u003eet al.\u003c/em\u003e (25) reported that addition of a mannose moiety to Trp8 does not significantly alter peptide conformation. Accordingly, the NMR-derived structure of Carmo-HrTH-II was used as a template, and a C-linked mannose group was added to Trp8 to generate the Carmo-HrTH-I model. It should be noted that the mannose used by Iyison \u003cem\u003eet al.\u003c/em\u003e (12) lacked the CH₂OH substituent at C5 and was therefore not a true sugar.\u003c/p\u003e\n \u003cp\u003eDocking and MD simulation of Carmo-HrTH-I bound to Carmo-AKHR in a POPC membrane yielded a binding free energy (\u0026Delta;G\u003csub\u003ebinding\u003c/sub\u003e) of \u0026minus;\u0026thinsp;146\u0026thinsp;\u0026plusmn;\u0026thinsp;12 kJ mol⁻\u0026sup1;, compared with \u0026minus;\u0026thinsp;159.7 kJ mol⁻\u0026sup1; reported by Iyison \u003cem\u003eet al.\u003c/em\u003e (12) An overlay of the Carmo-HrTH-I and Carmo-HrTH-II complexes revealed that both peptides adopt essentially the same conformation within the binding site. Minor adjustments in receptor helices were observed to accommodate the bulkier Carmo-HrTH-I ligand, but both peptides occupy the same binding pocket (Figures S8). The ligand interaction diagram (Figures S9) shows that the mannose moiety resides in a hydrophilic pocket and forms several interactions mediated by water molecules.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec13\"\u003e\n \u003ch2\u003eAlanine-Scanning Mutagenesis and Computational Correlation\u003c/h2\u003e\n \u003cp\u003eIn an \u003cem\u003ein vivo\u003c/em\u003e study, Katali \u003cem\u003eet al.\u003c/em\u003e (7) used a calcium bioluminescence reporter assay to measure the activity of several alanine-substituted analogues of Carmo-HrTH-II. To interpret these \u003cem\u003ein vitro\u003c/em\u003e (6) and \u003cem\u003ein vivo\u003c/em\u003e (7) results, each residue of Carmo-HrTH-II was systematically replaced with alanine, and the docking recalculated. The computational results were compared with the \u003cem\u003ein vitro\u003c/em\u003e data in Figs.\u0026nbsp;6a, where the computed change in ligand affinity (relative to the native peptide) is plotted alongside the experimentally determined EC₅₀ values. A positive change in affinity indicates that the mutated ligand binds less strongly than the native AKH.\u003c/p\u003e\n \u003cp\u003eOverall, the correlation between calculated and experimental data is strong, though not perfect. EC₅₀ values for the Phe4 and Trp8, Ala mutants were not determined because both peptides exhibited minimal activity even at the highest concentration tested. Consistently, these two substitutions produced the largest decreases in computed binding affinity, with Trp8 showing a 21.7 kJ mol⁻\u0026sup1; reduction relative to the native peptide. Mutations at Thr3, Thr5, Pro6, and Asn7 resulted in moderately reduced affinities and correspondingly higher EC₅₀ values. Substitution of Leu2 decreased affinity by 9 kJ mol⁻\u0026sup1;, an unexpected result given that Leu2 forms no direct receptor interactions; this suggests an entropic contribution to the binding free energy. Ligand RMSF data (Figures S7b) show that the Leu2 side chain fluctuates by ~\u0026thinsp;2.5 \u0026Aring; within the binding pocket, which may contribute to this effect. Experimentally, the Ala2 analogue showed a moderate decrease in activity.(6)\u003c/p\u003e\n \u003cp\u003eIn contrast, substitution of Gly9 by alanine was predicted to slightly increase affinity (by 2.3 kJ mol⁻\u0026sup1;), whereas \u003cem\u003ein vitro\u003c/em\u003e results showed that Ala9 was approximately two orders of magnitude less active than the native peptide. (6)\u003c/p\u003e\n \u003cp\u003eBecause residue-scanning calculations estimate binding free energy changes without accounting for conformational flexibility, each mutant was subsequently subjected to 50 ns of MD simulation in a POPC membrane. Binding free energies were extracted from the trajectories (Figs.\u0026nbsp;6b). The resulting trend in \u0026Delta;G\u003csub\u003ebinding\u003c/sub\u003e differed somewhat from that of \u0026Delta;Affinity, though both showed good overall agreement with experimental activity. In this case, a higher (less negative) \u0026Delta;G\u003csub\u003ebinding\u003c/sub\u003e corresponds to a lower EC₅₀ and thus greater activity, meaning that the two plots should be approximately mirror images. This relationship holds for Trp8, which has a low (strong) binding energy and is inactive, and for Thr5, Pro6, and Asn7, which exhibit higher \u0026Delta;G\u003csub\u003ebinding\u003c/sub\u003e values and moderate activity. However, the Phe4 mutant showed a \u0026Delta;G\u003csub\u003ebinding\u003c/sub\u003e similar to that of the native peptide despite being inactive.(6, 7) As Phe4 is known to be essential for receptor activation, this discrepancy suggests that binding affinity alone cannot fully account for activity. Similarly, Carmo-HrTH-II itself does not have the highest \u0026Delta;G\u003csub\u003ebinding\u003c/sub\u003e value, yet it is the most active ligand. These results highlight that receptor activation involves additional steps beyond initial ligand binding, of which only the binding event was modeled here.\u003c/p\u003e\n \u003cp\u003eG\u0026auml;de \u003cem\u003eet al.\u003c/em\u003e (6) and Katali \u003cem\u003eet al.\u003c/em\u003e (7) also measured the activity of several endogenous AKH peptides, allowing comparison with our computational results (Table\u0026nbsp;2). While the agreement between calculated and experimental data is not exact, the overall correlation is strong, supporting the validity of the docking and MD models.\u003c/p\u003e\n \u003cdiv\u003e\n \u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv\u003eTable 2\u003c/div\u003e\n \u003cdiv\u003e\n \u003cp\u003eCalculated DG\u003csub\u003ebinding\u003c/sub\u003e of AKH peptides bound to Carmo-AKHR, and EC\u003csub\u003e50\u003c/sub\u003e values from \u003cem\u003ein vitro\u003c/em\u003e bioluminescence assays.\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\" colname=\"c1\"\u003e\n \u003cp\u003ePeptide name\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colname=\"c2\"\u003e\n \u003cp\u003eNative source\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colname=\"c3\"\u003e\n \u003cp\u003ePeptide sequence \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colname=\"c4\"\u003e\n \u003cp\u003eEC\u003csub\u003e50\u003c/sub\u003e value (mol dm\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003e ) \u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colname=\"c5\"\u003e\n \u003cp\u003eDG\u003csub\u003ebinding\u003c/sub\u003e (kJ/mol)\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colname=\"c1\"\u003e\n \u003cp\u003eCarmo-HrTH-II\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c2\"\u003e\n \u003cp\u003e\u003cem\u003eCarausius morosus\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c3\"\u003e\n \u003cp\u003epELTFTPNWGT amide\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c4\"\u003e\n \u003cp\u003e7.36E-08\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c5\"\u003e\n \u003cp\u003e-132 (7)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colname=\"c1\"\u003e\n \u003cp\u003eTriin-AKH\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c2\"\u003e\n \u003cp\u003e\u003cem\u003eTriatoma infestans\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c3\"\u003e\n \u003cp\u003epELTFTPNWG\u003cstrong\u003e-\u003c/strong\u003e amide\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c4\"\u003e\n \u003cp\u003e5.48E-07\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c5\"\u003e\n \u003cp\u003e-101 (9)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colname=\"c1\"\u003e\n \u003cp\u003ePeram-CAH-II\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c2\"\u003e\n \u003cp\u003e\u003cem\u003ePeriplaneta americana\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c3\"\u003e\n \u003cp\u003epELTFTPNW\u0026ndash; amide\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c4\"\u003e\n \u003cp\u003e3.88E-07\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c5\"\u003e\n \u003cp\u003e-118 (9)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colname=\"c1\"\u003e\n \u003cp\u003ePhyle-CC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c2\"\u003e\n \u003cp\u003e\u003cem\u003ePhymateus leprosus\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c3\"\u003e\n \u003cp\u003epELTFTPNWG\u003cstrong\u003eS\u003c/strong\u003e amide\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c4\"\u003e\n \u003cp\u003e1.80E-08\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c5\"\u003e\n \u003cp\u003e-136 (8)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colname=\"c1\"\u003e\n \u003cp\u003eLocmi-AKH-I\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c2\"\u003e\n \u003cp\u003e\u003cem\u003eLocusta migratoria\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c3\"\u003e\n \u003cp\u003epEL\u003cstrong\u003eN\u003c/strong\u003eFTPNWGT amide\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c4\"\u003e\n \u003cp\u003e4.14E-06\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c5\"\u003e\n \u003cp\u003e-112 (14)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colname=\"c1\"\u003e\n \u003cp\u003ePhymo-AKH\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c2\"\u003e\n \u003cp\u003e\u003cem\u003ePhymateus morbillosus\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c3\"\u003e\n \u003cp\u003epEL\u003cstrong\u003eN\u003c/strong\u003eFTPNWG\u003cstrong\u003eS\u003c/strong\u003e amide\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c4\"\u003e\n \u003cp\u003e5.05E-06\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c5\"\u003e\n \u003cp\u003e-114 (14)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colname=\"c1\"\u003e\n \u003cp\u003ePolae-HrTH\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c2\"\u003e\n \u003cp\u003e\u003cem\u003ePolyphaga aegyptiaca\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c3\"\u003e\n \u003cp\u003epE\u003cstrong\u003eI\u003c/strong\u003eTFTPNW\u0026ndash; amide\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c4\"\u003e\n \u003cp\u003e8.76E-06\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c5\"\u003e\n \u003cp\u003e-119 (15)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colname=\"c1\"\u003e\n \u003cp\u003eAedae-AKH\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c2\"\u003e\n \u003cp\u003e\u003cem\u003eAedes aegypti\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c3\"\u003e\n \u003cp\u003epELTFTP\u003cstrong\u003eS\u003c/strong\u003eW\u0026ndash; amide\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c4\"\u003e\n \u003cp\u003e1.03E-06\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c5\"\u003e\n \u003cp\u003e-118 (7)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colname=\"c1\"\u003e\n \u003cp\u003ePeram-CAH-I\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c2\"\u003e\n \u003cp\u003e\u003cem\u003ePeriplaneta americana\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c3\"\u003e\n \u003cp\u003epE\u003cstrong\u003eVN\u003c/strong\u003eF\u003cstrong\u003eS\u003c/strong\u003ePNW\u0026ndash; amide\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c4\"\u003e\n \u003cp\u003eND*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c5\"\u003e\n \u003cp\u003e-94 (9)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colname=\"c1\"\u003e\n \u003cp\u003ePanbo-RPCH\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c2\"\u003e\n \u003cp\u003e\u003cem\u003ePandalus borealis\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c3\"\u003e\n \u003cp\u003epEL\u003cstrong\u003eN\u003c/strong\u003eF\u003cstrong\u003eS\u003c/strong\u003eP\u003cstrong\u003eG\u003c/strong\u003eW\u0026ndash; amide\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c4\"\u003e\n \u003cp\u003e3.35E-05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c5\"\u003e\n \u003cp\u003e-122 (8)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colname=\"c1\"\u003e\n \u003cp\u003eRommi-CC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c3\"\u003e\n \u003cp\u003epE\u003cstrong\u003eVN\u003c/strong\u003eFTPNWGT amide\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c4\"\u003e\n \u003cp\u003e1.5E-7\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c5\"\u003e\n \u003cp\u003e-107 (14)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003c/div\u003e\n \u003cp\u003e\u003csup\u003e\u0026nbsp;\u003cstrong\u003ea\u003c/strong\u003e\u0026nbsp;\u003c/sup\u003e Amino acid residues in bold text indicate a substitution relative to Carmo-HrTH-II.\u003c/p\u003e\n \u003cp\u003e\u003csup\u003e\u0026nbsp;\u003cstrong\u003eb\u003c/strong\u003e\u0026nbsp;\u003c/sup\u003e Data from (6)\u003c/p\u003e\n \u003cp\u003e\u003csup\u003e\u0026nbsp;\u003cstrong\u003ec\u003c/strong\u003e\u0026nbsp;\u003c/sup\u003e Standard deviation is given in parentheses.\u003c/p\u003e\n \u003cp\u003e\u003csup\u003ed\u003c/sup\u003e Estimated from carbohydrate bioassay (7)\u003c/p\u003e\n \u003cp\u003ePhyle-CC differs from Carmo-HrTH-II only by substitution of Thr10 with Ser. As both residues are hydrophilic, this change was expected to have little effect on receptor binding or activity\u0026mdash;consistent with our results. Phyle-CC had the same \u0026Delta;G\u003csub\u003ebinding\u003c/sub\u003e and essentially the same bioactivity as Carmo-HrTH-II. Similarly, replacement of Thr10 with the more hydrophobic Ala10 produced no measurable change in binding or activity, indicating that position 10 is not critical for receptor activation. Comparable biological activity results were reported for \u003cem\u003eL. migratoria\u003c/em\u003e (34) and \u003cem\u003eB. discoidalis\u003c/em\u003e (33).\u003c/p\u003e\n \u003cp\u003eThe importance of residue 3 is illustrated by comparison of Carmo-HrTH-II and Locmi-AKH-I, where Thr3 is replaced by Asn3. This substitution reduces the binding free energy by 20 kJ mol⁻\u0026sup1; and increases the EC₅₀ from 7.36 \u0026times; 10⁻⁸ to 4.14 \u0026times; 10⁻⁶ mol dm⁻\u0026sup3;, indicating a substantial loss of potency. Also, the tolerance at position 10 is further demonstrated by comparison of Locmi-AKH-I and Phymo-AKH, which differ only at this position but have nearly identical binding energies and activities. Together, these data indicate that Thr10 does not contribute significantly to receptor activation, whereas position 3 is critical, requiring threonine for Carmo-AKHR, but asparagine for Locmi-AKHR.\u003c/p\u003e\n \u003cp\u003eAccording to the \u003cem\u003ein vitro\u003c/em\u003e activation data (6) position 2 plays a limited role in receptor activation. Consistent with this result, the residue-scanning experiment shows that substituting the bulky Leu2 with alanine had minimal effect on binding as well. However, in the bioassay data set (7) it was measured that replacement of Leu2 by valine in Locmi-AKH-I (yielding Rommi-CC) increased hypertrehalosaemic activity from 19% to 48% relative to Carmo-HrTH-II, suggesting that steric effects at this position may be receptor- or species-specific.\u003c/p\u003e\n \u003cp\u003eCarmo-HrTH-II is a decapeptide; deletion of one residue, as in Triin-AKH, reduced the binding free energy by 31 kJ mol⁻\u0026sup1; and caused a one-order-of-magnitude loss in activity. Removal of two terminal residues, as in Peram-CAH-II, produced a smaller decrease in \u0026Delta;G\u003csub\u003ebinding\u003c/sub\u003e (14 kJ mol⁻\u0026sup1;) yet a similar reduction in activity.(6) Structural comparison revealed that both peptides occupy the same binding pocket as Carmo-HrTH-II but adopt different orientations. In Peram-CAH-II, the N-terminus lies deep within the binding pocket and the C-terminus faces the receptor surface, resulting in minimal interaction between Gly9 and Thr10, consistent with the small effect of their removal. By contrast, Triin-AKH binds in the reverse orientation, with the C-terminus buried in the pocket. This reversal, along with the lower binding energy, may explain its reduced but not abolished activity.\u003c/p\u003e\n \u003cp\u003eThe influence of position 2 was examined by comparing Peram-CAH-II and Polae-HrTH, which differ only at this site: Leu2 versus Ile2. Both peptides exhibit similar binding energies and activities. Examination of the receptor surface (Figure S10) shows that Leu2 resides in a hydrophobic pocket capable of accommodating isoleucine, explaining the tolerance at this position.\u003c/p\u003e\n \u003cp\u003eA comparable pattern is observed for position 7. Peram-CAH-II and Aedae-AKH differ only by Asn7 versus Ser7 yet have nearly identical \u0026Delta;G\u003csub\u003ebinding\u003c/sub\u003e and bioactivity. These data suggest that residue 7 is not critical for receptor activation.\u003c/p\u003e\n \u003cp\u003eFinally, Peram-CAH-I, an octapeptide that differs from Carmo-HrTH-II by three residues and the shorter length, showed no \u003cem\u003ein vitro\u003c/em\u003e activity and the lowest \u0026Delta;G\u003csub\u003ebinding\u003c/sub\u003e of all peptides examined. Conversely, Panbo-RPCH displayed a slightly higher \u0026Delta;G\u003csub\u003ebinding\u003c/sub\u003e (comparable to Carmo-HrTH-II and Phyle-CC) but lower activity. This underscores that strong binding is necessary but not sufficient for receptor activation. In Peram-CAH-I, Phe4 and Trp8, both essential for activity, show weak interactions with the receptor and move continuously within the binding pocket. By contrast, in Panbo-RPCH, Phe4 and Trp8 align closely with the corresponding residues in Carmo-HrTH-II and interact with the same receptor residues (Figure S11), explaining the residual activity despite the overall sequence divergence.\u003c/p\u003e\n \u003cp\u003eBecause all known endogenous AKH peptides retain Phe4 and Trp8, direct experimental testing of these positions was not possible. However, residue-scanning analysis confirmed that both residues are indispensable for receptor activation and reversing the order of these two essential amino acids in a peptide such as Peram-CAH-I (thus pEVN\u003cstrong\u003eW\u003c/strong\u003eSPN\u003cstrong\u003eF\u003c/strong\u003e amide instead of pEVN\u003cstrong\u003eF\u003c/strong\u003eSPN\u003cstrong\u003eW\u003c/strong\u003e amide) resulted in no hypertrehalosaemic activity in the American cockroach. (35)\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec14\"\u003e\n \u003ch2\u003eInsecticide Screening\u003c/h2\u003e\n \u003cp\u003eTo identify potential antagonists capable of blocking receptor activation by endogenous ligands, \u003cem\u003ein silico\u003c/em\u003e screening was conducted using the Octavia database of commercially available insecticide-like molecules. From an initial library of 295,011 compounds, 415,677 conformers were generated and docked to the Carmo-AKHR model. Of these, 197 met the screening criteria, yielding Glide scores from \u0026minus;\u0026thinsp;10.7 to \u0026minus;\u0026thinsp;1.4 and \u0026Delta;G\u003csub\u003ebinding\u003c/sub\u003e binding energies between \u0026minus;\u0026thinsp;118 and \u0026minus;\u0026thinsp;3 kJ mol⁻\u0026sup1;. The top-scoring candidates were subsequently subjected to 50 ns MD simulations in a POPC membrane. Results are summarized in Table\u0026nbsp;3, which also includes reference data from the study of Iyison (12) for comparison. All but one of the compounds has an amide bond and the best two are carboxylic acids. Hydrophobic \u0026pi;-\u0026pi; stacking is an important contributor to the binding of Carmo-HrTH-II to Carmo-AKHR and all the compounds have multiple aromatic rings, which are able to form \u0026pi;-\u0026pi; interactions. All compounds bound within the Carmo-HrTH-II binding pocket of the receptor. Among these, compound \u003cstrong\u003e2429\u003c/strong\u003e achieved the highest Glide docking score (\u0026ndash;10.75), while compound \u003cstrong\u003e9072\u003c/strong\u003e exhibited the most favourable free energy of binding (\u0026ndash;98 kJ mol⁻\u0026sup1;). These two ligands are discussed in detail below; results for the remaining compounds are provided in the Supplementary Information.\u003c/p\u003e\n \u003cdiv\u003e\n \u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\" class=\"fr-table-selection-hover\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv\u003eTable 3\u003c/div\u003e\n \u003cdiv\u003e\n \u003cp\u003eBest hit compounds found after after in silico screening of Otava database of insecticide-like molecules using our model of Carmo-AKHR. For comparison top hits from Iyison (12) are included.\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\" colname=\"c1\"\u003e\n \u003cp\u003eCode\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colname=\"c2\"\u003e\n \u003cp\u003eStructure\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colname=\"c3\"\u003e\n \u003cp\u003eGlide score kcal/mol\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colname=\"c4\"\u003e\n \u003cp\u003eDG\u003csub\u003ebinding\u003c/sub\u003e kJ/mol\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colname=\"c1\"\u003e\n \u003cp\u003e2429\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003cimg src=\"https://myfiles.space/user_files/69519_bce2c0439cd956a6/69519_custom_files/img1774468125.png\"\u003e\u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c3\"\u003e\n \u003cp\u003e-10.75\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c4\"\u003e\n \u003cp\u003e-91\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colname=\"c1\"\u003e\n \u003cp\u003e7831\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003cimg src=\"https://myfiles.space/user_files/69519_bce2c0439cd956a6/69519_custom_files/img1774468133.png\"\u003e\u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c3\"\u003e\n \u003cp\u003e-9.82\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c4\"\u003e\n \u003cp\u003e-83\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colname=\"c1\"\u003e\n \u003cp\u003e9072\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003cimg src=\"https://myfiles.space/user_files/69519_bce2c0439cd956a6/69519_custom_files/img1774468139.png\"\u003e\u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c3\"\u003e\n \u003cp\u003e-9.64\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c4\"\u003e\n \u003cp\u003e-98\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colname=\"c1\"\u003e\n \u003cp\u003e7727\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003cimg src=\"https://myfiles.space/user_files/69519_bce2c0439cd956a6/69519_custom_files/img1774468143.png\"\u003e\u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c3\"\u003e\n \u003cp\u003e-9.56\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c4\"\u003e\n \u003cp\u003e-73\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colname=\"c1\"\u003e\n \u003cp\u003e6026\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c2\"\u003e\u003cimg src=\"https://myfiles.space/user_files/69519_bce2c0439cd956a6/69519_custom_files/img1774468146.png\"\u003e\u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c3\"\u003e\n \u003cp\u003e-8.75\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c4\"\u003e\n \u003cp\u003e-118\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colname=\"c1\"\u003e\n \u003cp\u003eP7715460398\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003cimg src=\"https://myfiles.space/user_files/69519_bce2c0439cd956a6/69519_custom_files/img1774468150.png\"\u003e\u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c3\"\u003e\n \u003cp\u003e-8.44\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c4\"\u003e\n \u003cp\u003e-83\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colname=\"c1\"\u003e\n \u003cp\u003eP771556530\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003cimg src=\"https://myfiles.space/user_files/69519_bce2c0439cd956a6/69519_custom_files/img1774468154.png\"\u003e\u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c3\"\u003e\n \u003cp\u003e-7.31\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c4\"\u003e\n \u003cp\u003e-85\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colname=\"c1\"\u003e\n \u003cp\u003eP2187686\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c2\"\u003e\u003cimg src=\"https://myfiles.space/user_files/69519_bce2c0439cd956a6/69519_custom_files/img1774468159.png\"\u003e\u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c3\"\u003e\n \u003cp\u003e-7.77\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c4\"\u003e\n \u003cp\u003e-75\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec15\"\u003e\n \u003ch2\u003eCompound 2429\u003c/h2\u003e\n \u003cp\u003eThe RMSF plot for compound 2429 (Figure S12a) shows that the aromatic portions of the molecule are tightly bound (RMSF\u0026thinsp;\u0026asymp;\u0026thinsp;0.8 \u0026Aring;), whereas the aliphatic butane segment fluctuates by ~\u0026thinsp;3 \u0026Aring;, consistent with limited receptor contacts. Throughout the simulation, the ligand forms persistent hydrogen-bond and ionic interactions with Lys446 (Figure S12b). Additional interactions include an H-bond and water bridge with Phe344, and hydrophobic contacts with Phe449, Trp420, Tyr423, and Phe273. The ligand interaction diagram (Figure S13) reveals that the phenyl group of compound 2429 occupies a hydrophobic pocket, engaging in \u0026pi;\u0026ndash;\u0026pi; stacking with Trp420, Phe449, and Phe273. Arg346 forms a \u0026pi;\u0026ndash;cation interaction with the benzene ring, while the ligand\u0026rsquo;s carboxylic acid and amide carbonyl groups hydrogen-bond to Lys446, Gln337, and Trp249.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec16\"\u003e\n \u003ch2\u003eCompound 9072\u003c/h2\u003e\n \u003cp\u003eCompound 9072 contains two phenyl rings linked by a thiadiazole and amide bridge, with one hydrophobic (alkyl-substituted) and one hydrophilic (ether-substituted) aromatic ring. The ligand exhibits limited mobility in the binding pocket, with all atoms fluctuating by approximately 1 \u0026Aring; (Figure S14). Its key receptor interactions largely overlap with those of compound 2429, including hydrogen bonding to Lys446 and hydrophobic contacts with Phe273, Trp420, Tyr423, and Phe449. In addition, compound 9072 interacts with Arg269, Arg346, and Tyr424, further stabilizing the complex (Figure S13).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec17\"\u003e\n \u003ch2\u003eComparison and Functional Implications\u003c/h2\u003e\n \u003cp\u003eAlthough both ligands occupy the same binding pocket as Carmo-HrTH-II, their receptor contacts differ markedly. Apart from Trp249 and Phe341, most peptide-specific interactions are absent. Notably, both compounds disrupt the conserved salt bridge between Glu246\u0026sup2;\u0026middot;⁶\u0026sup1;, Arg269\u0026sup3;\u0026middot;\u0026sup3;\u0026sup2;, and Lys446⁷\u0026middot;\u0026sup3;⁶ by forming persistent interactions with Lys446 and a water-mediated bridge to Arg269.\u003c/p\u003e\n \u003cp\u003eA critical requirement for antagonist design is that a compound binds to the receptor without triggering activation. None of the tested compounds induced the conformational change associated with receptor activation, as indicated by the distance between Arg287 and Leu406. In the active state this distance is ~\u0026thinsp;15 \u0026Aring;, whereas in the inactive state it is ~\u0026thinsp;7 \u0026Aring;; for all docked insecticides, the measured distance remained between 7.2 and 7.9 \u0026Aring;, consistent with the inactive conformation.\u003c/p\u003e\n \u003cp\u003eAll but one of the docked ligands contain an amide bond, and the two highest-affinity compounds are carboxylic acids. Consistent with the dominant role of hydrophobic \u0026pi;\u0026ndash;\u0026pi; stacking in the binding of Carmo-HrTH-II to Carmo-AKHR, all hit compounds possess multiple aromatic rings capable of such interactions.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn this study, the structural, dynamic, and functional properties of Carmo-HrTH-II were characterized using NMR, molecular dynamics, and docking studies, providing a comprehensive view of its interaction with the Carmo-AKH receptor. NMR and MD data indicate that Carmo-HrTH-II adopts a stable β-turn conformation, with flexibility primarily at the termini, consistent with other AKH peptides and suitable for GPCR recognition. Docking and MD simulations reveal that successful receptor binding requires this β-turn, positioning key residues Phe4 and Trp8 within the binding pocket, consistent with their essential roles in receptor activation. Alanine-scanning and computational free energy analyses demonstrate that positions 3 and 8 are critical for activity, whereas positions 2, 7, and 10 are more tolerant to substitutions, highlighting structural determinants of receptor specificity and potency. Comparison with Carmo-HrTH-I shows that glycosylation minimally affects peptide conformation and receptor binding which is consistent with the in vitro activation studies.(\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e)\u003c/p\u003e \u003cp\u003eIn silico screening of insecticide-like compounds identified several molecules that occupy the same binding pocket as Carmo-HrTH-II, with stable interactions primarily mediated by hydrophobic π\u0026ndash;π stacking and hydrogen bonding, without inducing receptor activation. These results validate the binding pocket as a target for antagonist design and provide lead candidates for the development of novel insecticides capable of modulating AKH receptor activity. Overall, this integrative approach links peptide structure, receptor dynamics, and ligand-receptor interactions, offering insights into AKH receptor activation and a framework for rational design of species-specific insecticides.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eInformed Consent\u003c/h2\u003e \u003cp\u003e \u003cb\u003eStatement\u003c/b\u003e: Not applicable.\u003c/p\u003e \u003c/p\u003e\u003cp\u003e \u003ch2\u003eConflicts of Interest:\u003c/h2\u003e \u003cp\u003eThe authors declare no conflict of interest.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding:\u003c/h2\u003e \u003cp\u003eThis research was funded by the National Research Foundation of South Africa (grant Nos 93450 and 85466 to Graham E. Jackson; grant numbers 85768 [IFR13020116790] to GG and 109204 [IFR170221223270] to HGM, and the University of Cape Town Research Committee.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eConceptualization, GG and GEJ; methodology, GEJ, MAS and FS; formal analysis, GEJ, GG, HGM and MAS; investigation, GEJ and MAS; resources, GEJ, GG, HGM, MAS and FS; data curation, GEJ; writing\u0026mdash;original draft preparation, GEJ and MAS.; writing\u0026mdash;review and editing, GEJ, GG, HGM, MAS and FS; funding acquisition, GEJ, GG, HGM, MAS and FS. All authors have read and agreed to the published version of the manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eMAS and FS acknowledge access to the Bio21 NMR Facility, University of Melbourne. The Centre for High-Performance Computing (CHPC), South Africa, provided computational resources for this research project.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eAll data supporting the findings of this study are available within the paper and its Supplementary Information. Raw data is available from the corresponding author upon request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eLiessem, S., Ragionieri, L., Neupert, S., B\u0026uuml;schges, A. \u0026amp; Predel, R. Transcriptomic and Neuropeptidomic Analysis of the Stick Insect, Carausius morosus. \u003cem\u003eJ. Proteome Res.\u003c/em\u003e \u003cb\u003e17\u003c/b\u003e (6), 2192\u0026ndash;2204 (2018).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eG\u0026auml;de, G. Adipokinetic and hyperglycaemic factors of different insect species: Separation with high performance liquid chromatography. \u003cem\u003eJ. 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Predicted novel hypertrehalosaemic peptides of cockroaches are verified by mass spectrometry. \u003cem\u003eAmino Acids\u003c/em\u003e. \u003cb\u003e55\u003c/b\u003e, 1641\u0026ndash;1654 (2023).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-9099089/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9099089/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThis study combines NMR spectroscopy, molecular dynamics (MD), and docking simulations to elucidate the structure, dynamics, and receptor interactions of the decapeptide Carmo-HrTH-II, from the stick insect, \u003cem\u003eCarausius morosus\u003c/em\u003e. NMR analysis revealed a flexible peptide adopting a β-turn between Pro⁶ and Asn⁷, a feature retained during MD simulations and critical for receptor recognition. Docking Carmo-HrTH-II to the Carmo-AKHR homology model identified a stable binding mode in which Phe⁴ and Trp⁸ lie within the binding pocket, consistent with their known importance for GPCR activation. Alanine-scanning mutagenesis and residue scanning demonstrated strong agreement between calculated binding energies and experimental EC₅₀ values, confirming the essential roles of Phe⁴, Pro⁶, Asn⁷, and Trp⁸ in receptor activation. Comparative studies of Carmo-HrTH-I and other AKH analogues showed that position 10 substitutions have minimal influence on binding, whereas position 3 substitutions significantly alter affinity.\u003c/p\u003e \u003cp\u003eIn silico screening of insecticide-like compounds identified several potential receptor antagonists that bind to the same site as Carmo-HrTH-II. These ligands interact predominantly through π\u0026ndash;π stacking and hydrogen bonding, disrupting the conserved salt bridge essential for receptor activation. Together, these results provide a molecular basis for AKH receptor activation and suggest a new route for the rational design of AKH-targeted insecticides.\u003c/p\u003e","manuscriptTitle":"Molecular modelling of the adipokinetic hormone receptor from the stick insect Carausius morosus, and its endogenous agonist","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-03-25 19:53:42","doi":"10.21203/rs.3.rs-9099089/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-04-09T11:34:25+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-04-09T08:07:29+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"174040150357861837987667662489357047777","date":"2026-03-24T12:01:00+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-03-24T07:09:44+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"247409129212177252505706934300172383517","date":"2026-03-23T01:50:47+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-03-20T11:09:11+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2026-03-20T10:24:58+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-03-12T11:45:17+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-03-12T11:44:47+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2026-03-12T01:58:16+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"512c9bf4-8996-451b-8066-b2234d5c60da","owner":[],"postedDate":"March 25th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":65047944,"name":"Biological sciences/Biochemistry"},{"id":65047945,"name":"Biological sciences/Chemical biology"},{"id":65047946,"name":"Physical sciences/Chemistry"},{"id":65047947,"name":"Biological sciences/Computational biology and bioinformatics"},{"id":65047948,"name":"Biological sciences/Drug discovery"},{"id":65047949,"name":"Biological sciences/Structural biology"}],"tags":[],"updatedAt":"2026-05-11T08:28:37+00:00","versionOfRecord":[],"versionCreatedAt":"2026-03-25 19:53:42","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9099089","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9099089","identity":"rs-9099089","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}