In-silico analysis of E-β-farnesene for its possible insect-repellent activity through interaction with Odorant Binding Proteins (OBPs)

In-silico analysis of E-β-farnesene for its possible insect-repellent activity through interaction with Odorant Binding Proteins (OBPs) | 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 Research Article In-silico analysis of E-β-farnesene for its possible insect-repellent activity through interaction with Odorant Binding Proteins (OBPs) Aqsa Parvaiz, Hafsa Abid, Anam Altaf, Wajeeha Kainat, Ghulam Mustafa, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5603153/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Contrary to humans, many animals, especially insects, rely heavily on the sense of smell to detect their foods, predators, mating partners and nesting sites. Odor molecules are chemicals, originating from a source. When such chemical compounds interact with odorant binding proteins (OBPs) and subsequently with the receptors associated with the insect nervous system, a cascade of reactions commences. In the present study, an in-silico appraisal of a broad range of aphid repellent molecules was carried out in order to propose their mechanism of action at molecular level. Initially 3-D protein structure prediction of odorant binding proteins was carried out using SWISS model, Phyre2 and Modeller. The aphid repellent molecule E-beta-farnesene (a chemical compound used as ligand) was retrieved from PubChem. Physiochemical analyses carried out by Protparam revealed that OBPs are basic in nature with 9.30 isoelectric point (pI) and based on aliphatic index OBPs were found to be thermostable. Protein-ligand interaction was carried out with the help of Auto Dock Vina (ADV) tool that revealed complex interactions among ligand and proteins showing binding affinities by different bonds including hydrogen bonding, hydrophobic bonding, and elastic bonds. However, Phe230 residue in OBP of Apis gossypii showed interaction with ligand’s C9 atom via hydrogen bond having bond length of 3.04 Å. The use of new analogous for a wide range of aphid pests should be carried out in future studies. Bioinformatics Biotechnology and Bioengineering Insect repellence Odorant binding proteins E-beta-farnesene docking analysis. Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 INTRODUCTION Controlling pest attacks is a critical factor in growing healthy crops with high yield. Pest attacks cause heavy infestation to growing crops. Apparently, 42% of the world’s food supply is all wasted only because of the pest attacks (Ali et al. 2023 ). The insect olfactory system performs a vast range of activities like detecting the food sources, egg laying and mating preferences (Sun et al. 2023 ). Chemoreception in insects is believed as they thrive in a chemical world where they convey and receive information in the form of chemical signal like pheromones, plant volatiles and predator odors. Olfactory receptors are generally located in maxillary palps, antennae and labial palps of insects (Schuman 2023 ). Odorant binding proteins (OBPs) are soluble proteins of high concentration in sensory lymph. These are regarded as carriers of semiochemical molecules that play a crucial role in olfactory perception (Rana et al. 2024 ). Odorant binding proteins (OBPs) are a sub class of lipocalins present in olfactory apparatus. These proteins are illustrated by their characteristics of irreversibly binding with volatile molecules that are usually called “odorants”(Chandrasekaran et al. 2024 ). Odorant binding proteins are amphipathic proteins that are present in the olfactory system. OBPs have been characterized from various species of economically important insects (Tian et al. 2024). OBPs play a significant role in olfactory organs helping phytophageous insects for seeking of host (Liu et al. 2024 ). Earlier functional and structural studies on OBPs of insects raised two questions that (1) OBPs particularly identify the specific ligands. (2) Which way of transfer of OBPs uses to convey the chemical signal to Odorant Receptors (ORs). There have been long debates on this discussion because of selectivity and extremely sensitive behavior of insect olfactory system ( Kohlmeier and Billeter 2023 ). The principal organ of insect olfactory system is antennae, which mediate several processes in communication with other insects. For instance, male moths use olfactory organ which is antennae to locate their matting partners (Chu and Ågmo, 2023 ). Earlier, in insect peripheral nerve system, two alternatives have been examined for eco-friendly strategies in integrated pest management system (IPM). These are odorant receptors (ORs) and odorant binding proteins (OBPs) (Zhou et al. 2024 ). E-beta-farnesene plays a significant role as plant volatile in transgenic plants which not only interrupt the insect feeding process at midrib of plant but also interfere with the feeding process of other aphids in their proximity (Li et al. 2024 ). E-β-farnesene synthase catalyzes the formation of E-β-farnesene, which is used by many pest aphids as main component of alarming situations and involved in chemical communication with each other. Transgenic plants releasing E-β-farnesene molecules also attract the natural enemies which use E-β-farnesene for foraging cues. Therefore, it can minimize the level of aphid infestation. Symptoms of aphid attack on plants may vary according to plant species and aphid species (Wang et al. 2024 ) Chemosensory organs of insects are capable of receiving and programming a variety of chemical signals to exhibit their response to plant volatiles. These chemosensory systems include odorant binding receptors (ORs) and various odorant binding proteins (OBPs) along with odorant degrading enzymes (ODEs) having the ability of abrupt degradation of odorants (Pei et al. 2023 ;Qin et al. 2023 ; Gouda and Subramanian 2024 ). Insect OBPS are categorized into 5 subclasses depending on no. on cysteine residues that are Plus-c OBPs (two additional cysteine residues and pro residues), Mius-C OBPs (4 conserved cysteine), classical OBPs (6 conserved cysteine), Atypical OBPs (9–10 cysteine having long C-terminus) dimer (two cysteine signature motifs) (Abendroth et al. 2023 ; Liu et al. 2023 ). OBPs play a role in the association between the outer environment and ORs. Odorants arrive at the entry site, where sensilla are present in sensillum lymph (step1). Odorants molecules bound with the solubulized OBPs (step2) and transport from sensillum lymph (step3) that fill up the cavity with dendrites. At the end activation of membrane bound ORs is performed (step4) (Raiser et al. 2023 )The presence of N-terminus except of C-terminus covered the site closing off the conventional OBP pocket. Results of fluorescent binding assays and molecular docking show that OBP3 from Megoura viciae can bind to each alarm pheromone molecules (Jiang et al. 2023 ). Olfactory organs respond to odorant chemicals by proceeding the action potential and forwarding the odor to odorant receptor neuron (ORNs) in maxillary palps in insects. Information is transported to antennal lobe (AL) which is the influential functional unit of olfactory organ glomeruli. All information is collected into ORNs and thus expressing their response profile within the local neuron inside the glomeruli. Output of neurons contains axons which exit the local neurons and transfer to the mushroom body (MBs) of the insects. Local neurons (LNs) have excitatory role and Projection neurons (PNs) which target only local neurons and exhibit the inhibitory role by inhibition or termination of odorant signal (Wolff et al. 2023 ). Insects identify and distinguish olfactory cues with hair like structures called sensilla which is located inside the antennae. These sensilla are present in sensillum lymph. Various types of membrane bounded olfactory receptors and soluble odorant binding proteins (OBPs) are also present in sensillum lymph ( Manikkaraja et al. 2022 ). OBPs are widely identified in tarsi, larval heads, antennae and even present in eggs. They are indicative for their structural and functional range of diversities. Antennal odorant binding proteins (ABPs) and general odorant binding proteins (GOBPs) abundantly located in antennae of adults for odorant driven behavior responses (Li et al. 2023 ) The behavioral responses in drosophila mutants and aphids exhibited that OBPs are correctly engaged in semiochemical approach. But until now the exact mode of action of these proteins is unrevealed (Jaffar et al. 2024 ; Yi et al. 2024 ). In present study, we have tried to unveil the mode of action of OBPs at molecular level. MATERIAL AND METHODS Sequence retrieval: Full length protein sequences of OBPs from Acrythosiphon pisum (accession # CAX63068.1), Aphis gossypii (accession # AJP06027.1), Sitobion avenae (accession # ACW03675.2) and Apis mellifera (accession # ABD92653.1) were retrieved from freely available web sources like National Center for Biotechnology Information (NCBI) and UniProtKB. The sequence characterization and conserved domains identification was carried out using CDD, a sub tool in NCBI. Superimposition of protein structures: Superimposition of protein structures is crucial for determining structural homology between proteins. Protein function and evolution can be better understood by comparing their structures which helps in deciphering the phylogenies of various organisms. Predicted protein structures were superimposed using UCSF Chimera with each other to know the similarities among them and understand how different they behave when docked with the same ligand ( Lyu et al. 2024 ) Homology modelling of both insect repellent molecules and OBPs: Protein threading/structure prediction was performed by using the automated online servers. Different protein structure modelling servers like Swiss model, I-TASSER, Phyre2 server and offline tool MODDELR was used for the prediction of protein structural models. The visualization of predicted models for better performance was done by online tools Chimera and I-TASSER server. Refinement of predicted models: Refinement of predicted models is a significant step to refine and correlate the energy minimization of protein models. For this purpose, different tools like ModeRefiner were used. Evaluation and validation: Evaluation and validation of proteins is an important step in modelling and performance of protein-ligand docking analysis. Evaluation and validation were done using RAMPAGE (Ramachandran Plot Assessment) which gave results in the form of graphs and protein identity scores in favored regions. Physiochemical characteristics of OBP were accessed by using PROCHECK server. Protein-ligand docking analysis: The docking analysis of OBPs and insect repellent molecules was performed by using automated tools like AutoDock tool, PatchDock tool and ZDOCK server. Hydrophobic and hydrophilic interactions and interaction affinity of proteins can be visualized by using the offline tool LigPlus which works with the java interface. RESULTS Semiochemicals play a vital role in insect’s life as they are used to convey information to one another. The study aimed to use these chemical cues to explore insect and plant interactions and cope the insects for causing damage to plants with a non-invasive pest control strategy. Also, this approach could help in avoiding the health hazards caused due to excessive pesticide usage to farmers, wildlife and the environment. By using OBPs of insects and E-beta farnesene as ligand and developing new analogues will provide a way for insect repellency from plants. Sequence of target OBPs were retrieved from NCBI given in Table 1. Model Prediction of E-beta-farnesene The 3-D model of E-beta-farense was identified by PubChem. Model translation was done using the online tool Smile Translator. Primary Structure and Conserved Domain Prediction of OBPs The primary structure prediction of proteins was carried out using ProtParam tools that gave the physicochemical parameters of all proteins shown in table 2. These parameters are important for protein primary structure prediction i.e., molecular weight, Iso-electric point (pI), aliphatic index, instability index, estimated half-life of OBPs models. The instability index of all the proteins under study was calculated within the range of 40 that showed an overall structural stability of proteins. The conserved domains of proteins observed via CDD (conserved domain database) depicted that all the proteins had a conserved domain that belongs to superfamily PBP-GOBP having 26 -112 residues in it, showing the similarity of proteins. Homology modeling of OBPs and their selection Homology modelling of OBPs was performed to get 3 Dimensional structures. At first, 3-dimensional structure prediction of the OBPs of four different aphid species ( Aphis gosypii, Acrythosiphon pisum, Sitobion avenae, Apis mellifera ) was carried out using three different tools SWISS-MODEL, phyre2 and MODELLER. In this case, the best 3-dimensional structure was predicted by SWISS-MODEL server as given in Figure 2. The evaluation of models from RAMPAGE described these structures with 96 (97.0%) residues of the model falling in the favored region and 3 (3.0%) residues were falling in the allowed region for OBP11 ( Acrythosiphon pisum) given in Figure 2(A), and 116 (92.8%) residues of the model fall in favored region and 7 (5.6 %) residues were falling in the allowed region for OBP of Aphis gosypii Figure 2(B). Similarly, predicted model of OBP ( Sitobion avenae ) when evaluated using RAMPAGE, its Ramachandran plot described those 118 (92.2%) residues of the model fall in favored region and 6 (4.7%) residues fell in allowed region as shown in Figure 4.2(C). The 3-dimensional structure evaluation of OBP ( Apis mellifera ) with its Ramachandran plot described it as 113 (99.1%) residues of the model fall in favored region and 4 (2.9%) residues fell in allowed region Figure 2(D). Superimposition of protein models OBPs models were superimposed to other OBPs from different organisms, to check the structural similarity between them. For this purpose, UCSF Chimera (v1.11.2) ( http://www.egl.ucsf.edu/chimera ) was used. RMSD value and Q-score estimation were also estimated through Chimera. This program provides diverse functions including analysis of structures, generation of images, and comparison between structures. The structure of OBP11 (A. pisum) when superimposed with OBP of A. gossypii gave an overall RMSD value of as 2.183, SDM 57.300 and Q-score 0.166. The superimposition values for OBP11 and OBP S. avenae were 2.497, SDM 65.091, and Q-score 0.136. Also, the superimposition technique was performed to check the overall effect of structural similarity between OBP11 ( Acrythosiphon pisum ) and OBP21 ( Apis mellifera ). The overall RMSD value as 2.148, SDM 47.115, and Q-score 0.301 was obtained. Thus, the lower values of RMSD showed relative similarity of structures when superimposed also they share a conserved domain and family resulting in the similarities. However, it is crucial to note, that these RMSD distributions may not reflect genuine model accuracy because flexible and poorly defined areas such as C-termini and extracellular loops in proteins heavily influence them (Luo et al. 2024) Evaluation and validation of OBPs model by PROCHECK Evaluation and validation of each OBP model was done by PROCHECK tool. The results of the validation were visualized by this tool in the form of Ramachandran plot which is based on an investigation of 118 structures of resolution (min.2.0 Angstroms). The Ramachandran plotofevery OBP displayed the backbone conformational angles (psi and phi) for all the residues in each OBP. The “red” region displayed as approved core region that indicated favourable region of phi-psi angle values. Other residues are present in allowed and disallowed regions. In “ Figure 3(A) ” Evaluation and validation of “OBP11 ( Acythosiphon pisum )” was done by PROCHECK tool. The program’s Ramachandran plot demonstrated that 97.8% residues of the model fall in favoured region. 2.2% residues fall in additionally allowed region. Evaluation and validation of “OBP ( Aphis gossypii )” in “ Figure 3(B) ” demonstrated that 87.9% residues of the model fall in favoured region. 11.2% Residues fall in additionally allowed region andin Figure 3(C) OBP ( Sitobion avenae ) Ramachandran plot was demonstrated that 88.2% residues of the model fall in favoured region. 11.8% residues fall in additionally allowed region. In “ Figure 3(D) ” Evaluation and validation of “OBP21 ( Apis Mellifera )” done by PROCHECK represents that 97.2% residues of the model fall in favoured region. 2.8% residues fall in additionally allowed region. A summary of the results obtained from Ramachandran plot of all proteins is given in Table 3. Modification of OBP Models by Auto Dock Vina Modification of 3-D models of odorant binding proteins was performed by Auto Dock Vina program. For which, water molecules were eliminated and addition of hydrogen ions to the protein was done. The refinement was carried out for all structures in order to prepare them for protein-ligand interaction analysis. The refinement was done using the protonate3D function of ADV. Different parameters including Temperature (T):300, pH: 7, Solvent: 80 and van Waals: 800R3 and salt 0.1 were used. Energy minimization of OBPs model were performed by providing the Force Field: MMFF94X and gradient: 0.05. This improved the energies of OBPs models and along with eliminated the clashes. Figure 4 shows the modified models of OBPs. Protein-ligand docking analysis of OBPs and E-beta-farnesene molecule and their evaluation Molecular-docking protocols are progressively used to foresee binding affinities for a large number of ligands (27,28). The complex form between OBPs and E-beta-farnesene is due to hydrophobic interactions and hydrogen bonding. The generated complex from PatchDock server, displayed hydrophobic and hydrogen bonding interactions. OBP11 “( Acrythosiphon pisum )” Phe25 and E-beta-farnesene residue C7 shows interaction. The length of hydrogen bonding is 3.2 in “Figure 5 (A)” . Phe230 of OBP “( Apis gossypii )”and E-beta-farnesene residue C9 shows hydrogen bond interaction. The length of hydrogen bonding is 3.04 in “Figure 5 (B)” . OBP “( Sitobion avenae) ” and E-beta-farnesene residues Lys33, Lys32, Ala29 and C3, C13, C14 shows interaction. OBP“( Sitobion avenae) ” Ala29and E-beta-farnesene residue C3 shows hydrogen bond interaction. The length of hydrogen bonding is 3.14 in “Figure 5 (C)”. OBP21 “( Aphis mellifera )”and E-beta-farnesene residue Asp45, C14 hydrogen bond interaction. The length of hydrogen bonding is 2.70 in “Figure 5 (D)” . Visualization of these interactions was performed by LigPlus software. Discussion OBPs are major proteins in olfactory system of insects for communication with other insects (Kohlmeier and Billeter 2023 ). When the chemical signal arrives at the entry site of insects’ antennae these odorants molecules bound with the solubilized OBPs. Odorant molecules transported from sensillum lymph that fill the cavity with dendrites. At the end, activation of membrane-bound ORs is performed. It is persisted as a major challenge to discover olfactory genes that identify odorants, plant attractants, repellents and pheromones. Moreover, receptors are deorphanized by using heterologous systems such as Xenopus oocyte recording systems to identify specific silent receptors. Recently, it has been discovered that disclosure of an odorant activates alteration in the transcripts of those genes that are involved in the reception of that odorant. Here we have tried to unveil the mode of action of OBPs at molecular level using docking and other computational tools insects (Mounadi et al. 2024 ; Chennai et al. 2024 ) This study opened the doors for the identification of ORs and odorants they detect (Xiao et al. 2018 ; deMarch et al. 2024 ) Whereas the ligand spectrum of classical OBPs has been characterized and little is known about the Plus-C subgroup of OBPs. By focusing on AlinOBP14 OBP from the subgroup of Plus-C in Hemipteran mired bug pest Adelphocoris lineolatus . Q-PCR experiments suggested that AlinOBP14 expressed at various development stages ubiquitously, but extensively expressed in adult head the non-chemosensory organ. Fluorescence based competitive binding assays expressed that β-ionone, farnesol and nerolidol strongly bind to AlinOBP14. No significant internal binding pocket was predicted by homology modelling, but long N-terminal and C-terminal regions form a cupped cavity to dock ligands. Molecular docking exhibited that the four potential ligands have distinct binding orientations. These orientations involved dissimilar roles in the extension of N-terminal in the ligand recognition (Tian et al. 2021 ) E-beta-farnesene as a ligand play a significant role as plant volatile in transgenic plants that not only interrupt the insects feeding process at the midrib of plants also interfere the feeding process of other aphids in their proximity. Following the earlier work, this study was designed to create new analogous proteins and examine their affinity with their ligand by using in-silico techniques. In this research new analogous proteins were developed by performing the protein structure prediction, refinement of protein model and primary structure prediction including theoretical isoelectric point(pI) (the pH at which protein has no net charge and where pH is greater than pI than protein has a net negative charge while at the point where pH is less than pI than protein has a net positive charge), the molecular weight of protein, instability index values different other steps through various servers. For this purpose, Phyre2 Protein fold recognition server [v2.0], SWISS-MODEL, ADV, and ProtParam tools were used and the best model of each protein was selected based on their Ramachandran plot and PROCHECK values. Best structures were predicted with SWISS Model server as their Rmachandran values were significant in all other predicted structures. Model of E-beta-farnesene were created through PubChem tool and 3-D structure of E-beta-farnesene was translated by using Online Smiles Translator and structure file were generated in PDB format. Automated hydrogen bonds were added to the structure according to standard valences in the E-beta-farnesene molecule. Primary structure prediction of each OBP was performed through online ProtParam tool to govern the different physiochemical properties of these OBPs. Different physicochemical properties were analyzed and all values OBPs fell into the category of acceptable range. This tool identified the physiochemical properties of these proteins that these OBPs were well stable. Superimposition of OBPs was done by UCSF Chimera software for identification of structural similarity of OBPs. The superimposition technique was performed to check the overall effect of structural similarity between OBP11 ( rythosiphon pisum ) and OBP ( Aphis gossypii ). The overall SDM 57.300, RMSD value as 2.183, and Q-score 0.166 were obtained through superimposition performed by Chimera. Validation of OBPs models was performed by using four different tools such as verify 3D, ERRAT, QMEAN Server, and PROCHECK tool. PROCHECK values showed that predicted models of OBPs were highly reliable. Validation of “OBP ( Aphis gossypii )” by PROCHECK tool showed Ramachandran plot demonstrated that 87.9% residues of the model fall in favored region and 11.2% residues fall in additionally allowed region. The Q-Mean of “OBP ( Apis gossypii )” depicted that protein structure quality is good because this protein has a (-2.04) QMEANZ-score. “OBP (( tobion avenae )” structure evaluation which was also performed by ERRAT tool resulted 94.9153% evaluated overall quality factor value that indicated that the model of OBP is stable and reliable. OBPs models were modified and prepared for docking. Before docking of OBPs and E-beta farensene refinement and energy minimization of these OBPs were performed through the ADV tool. ADV tool is a highly extensive tool for energy minimization and protonation. Energy minimization of OBP “( Apis gossypii )” through the ADV model was performed by providing the Force Field: MMFF94X and gradient: 0.05. The PatchDock tool for protein and ligand interaction was performed and an evaluation of docking results was performed through the LigPlus tool. Evaluation of docking results showed the hydrogen bonding, double bond interaction, and hydrophobic and elastic bonding interaction between different atoms and residues. The generated complex from PatchDock server, and evaluation through LigPlus server displayed binding interactions of OBP “( Apis gossypii )”. The OBP residue Phe230 and ligand atom C9 shows hydrogen bond interaction and non-ligand residues showed hydrophobic interactions. The length of hydrogen bonding is 3.04. The dark brown interaction are depiction double bonding between different atoms. Structure facilitates the interactions between Phe230, Leu170, Leu169, Phe189 that showed the elastic bonding between ligand and OBP. The interaction showed that these proteins should be used for the repellency strategy of aphid pests from damaging the crop plants and preservation of food sources in future use. CONCLUSION There are many Odorant molecules which may interact with insect OBPs and serve as insect repellents however, the molecular basis of their repellent activity is not known. In the present study, an in-silico appraisal of a broad range of aphid repellent molecules was carried out in order to propose their mechanism of action at molecular level. Odorant binding protein (OBP) interaction with E-β-farnesene. Phe230 residue in OBP of Apis gossypii showed interaction with ligand’s C9 atom with hydrogen bond having bond length of 3.04 Å. Physicochemical analyses revealed that OBPs is basic in nature with 9.30 isoelectric point (pI), thermostable based on aliphatic index. Declarations Acknowledgements: None. Author contributions AQ and HA: data collection/entry, AA and WK: data analysis/ statistics, GM, MSK and FAJ: preparation of the manuscript, MMH : made a literature analysis/search, FAJ conceived and designed the study. All authors read and approved the final manuscript. 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Xiao Y, Sun L, Wang Q, Zhang Q, Gu S hua, Khashaveh A. (2018) Entomology Molecular characterization and expression analysis of putative odorant carrier proteins in Adelphocoris lineolatus . J Asia Pac Entomol. 21(3):958–70. Yi S, Chen X, Wu Y, Wu J, Wang J, Wang M. (2024) Identification of odorant‐binding proteins and functional analysis of antenna‐specific BhorOBP28 in Batocera horsfieldi (Hope). Pest Manag Sci. 80(8):40558–4068. Zhou Y, Huang C, Fu G, Tang R, Yang N, Liu W, et al. (2024) Molecular and Functional Characterization of Three General Odorant-Binding Protein 2 Genes in Cydia pomonella (Lepidoptera: Tortricidae). Int J Mol Sci. 25(3):1746. Tables Tables 1 to 3 are available in the Supplementary Files section Additional Declarations The authors declare no competing interests. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5603153","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":387614095,"identity":"353cbeb1-7cc0-458a-b232-46043f56f4e7","order_by":0,"name":"Aqsa Parvaiz","email":"","orcid":"https://orcid.org/0000-0001-8180-2168","institution":"Department of Biochemistry and Biotechnology, The Women University Multan; Multan, Pakistan.","correspondingAuthor":false,"prefix":"","firstName":"Aqsa","middleName":"","lastName":"Parvaiz","suffix":""},{"id":387614096,"identity":"a550355e-d1b9-451e-ade1-7590c154d8b4","order_by":1,"name":"Hafsa Abid","email":"","orcid":"https://orcid.org/0000-0002-6704-8231","institution":"Centre of Agricultural Biochemistry and Biotechnology, University of Agriculture Faisalabad, Faisalabad, Pakistan","correspondingAuthor":false,"prefix":"","firstName":"Hafsa","middleName":"","lastName":"Abid","suffix":""},{"id":387614097,"identity":"ef5a7b12-a509-4b98-b0b2-0387d59d8be1","order_by":2,"name":"Anam Altaf","email":"","orcid":"https://orcid.org/0000-0002-6202-8776","institution":"Centre of Agricultural Biochemistry and Biotechnology, University of Agriculture Faisalabad, Faisalabad, Pakistan","correspondingAuthor":false,"prefix":"","firstName":"Anam","middleName":"","lastName":"Altaf","suffix":""},{"id":387614098,"identity":"536c264f-9f57-43e6-9ca8-a18a601a11b5","order_by":3,"name":"Wajeeha Kainat","email":"","orcid":"","institution":"Centre of Agricultural Biochemistry and Biotechnology, University of Agriculture Faisalabad, Faisalabad, Pakistan","correspondingAuthor":false,"prefix":"","firstName":"Wajeeha","middleName":"","lastName":"Kainat","suffix":""},{"id":387614099,"identity":"90341c4b-5c52-4cc3-89a9-783a83ebda7c","order_by":4,"name":"Ghulam Mustafa","email":"","orcid":"https://orcid.org/0000-0003-1879-2053","institution":"Centre of Agricultural Biochemistry and Biotechnology, University of Agriculture Faisalabad, Faisalabad, Pakistan","correspondingAuthor":false,"prefix":"","firstName":"Ghulam","middleName":"","lastName":"Mustafa","suffix":""},{"id":387614100,"identity":"c12e793c-6cc6-4ae5-863a-b24f4255bb97","order_by":5,"name":"Muhammad Sarwar Khan","email":"","orcid":"https://orcid.org/0000-0001-7194-8622","institution":"Centre of Agricultural Biochemistry and Biotechnology, University of Agriculture Faisalabad, Faisalabad, Pakistan","correspondingAuthor":false,"prefix":"","firstName":"Muhammad","middleName":"Sarwar","lastName":"Khan","suffix":""},{"id":387614101,"identity":"5ee39b51-8218-4b7c-9bac-aabb98c90999","order_by":6,"name":"Muhammad Mudassir Hussain","email":"","orcid":"","institution":"Potato Research Institute, Sahiwal, Pakistan","correspondingAuthor":false,"prefix":"","firstName":"Muhammad","middleName":"Mudassir","lastName":"Hussain","suffix":""},{"id":387614102,"identity":"47454cbc-8e0d-4dfe-aee8-163e99449dd1","order_by":7,"name":"Faiz Ahmad Joyia","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABBUlEQVRIiWNgGAWjYBAC9gbGBgYgYmA4zHzwQUIFkMHM3IBXC88BmJbjbMkGH86AtDAS0gIkwGrO85hJzmxjgHHxaGE/3Pzh545t8nyHeYyNeefVRvO3A7X8qNiGWwtPYoNh75nbhjMPsxU+5t12PHfGYcYGxp4zt3FqsWdIbEjgbbvNuOEw82Zj3m3HchuAWpgZ23Br4eF/2HDwb9tt+w2HGcykeeccy51PUItEYmMz0JbEDYdZgN5vqMndQFjLw2Zm2bbbyUC/AAP52IHcjUAtB/H5hYc//fHHt223bfvOHwZGZU1d7jwQ40cFbi3o4DCYPEC0eiCoI0XxKBgFo2AUjBAAAHfGZkrcSZDuAAAAAElFTkSuQmCC","orcid":"https://orcid.org/0000-0002-9540-6972","institution":"Centre of Agricultural Biochemistry and Biotechnology, University of Agriculture Faisalabad, Faisalabad, Pakistan","correspondingAuthor":true,"prefix":"","firstName":"Faiz","middleName":"Ahmad","lastName":"Joyia","suffix":""}],"badges":[],"createdAt":"2024-12-08 13:33:38","currentVersionCode":1,"declarations":{"humanSubjects":false,"vertebrateSubjects":false,"conflictsOfInterestStatement":false,"humanSubjectEthicalGuidelines":false,"humanSubjectConsent":false,"humanSubjectClinicalTrial":false,"humanSubjectCaseReport":false,"vertebrateSubjectEthicalGuidelines":false},"doi":"10.21203/rs.3.rs-5603153/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5603153/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":71002586,"identity":"2f4ee76c-ec0d-4393-87c7-e46f0df2f3a3","added_by":"auto","created_at":"2024-12-10 05:59:50","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":78123,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMolecular structure of E beta farnesene\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-5603153/v1/437e4693ac9c03fab6046b6a.png"},{"id":71002584,"identity":"0fda7d89-42c6-4e8f-9852-dc3893a80672","added_by":"auto","created_at":"2024-12-10 05:59:50","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":247925,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003e3-D Models of odorant binding proteins: A) \u003c/strong\u003eOBP11 (\u003cem\u003eAcrythosiphon pisum)\u003c/em\u003e \u003cstrong\u003eB) \u003c/strong\u003eOBP of \u003cem\u003eAphis gosypii \u003c/em\u003e\u003cstrong\u003eC) \u003c/strong\u003eOBP (\u003cem\u003eSitobion avenae\u003c/em\u003e) \u003cstrong\u003eD) \u003c/strong\u003eOBP (\u003cem\u003eApis mellifera\u003c/em\u003e).\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-5603153/v1/728821149d0347f17235ee1a.png"},{"id":71005113,"identity":"31e21a1d-669e-4036-8ad4-e07244850f15","added_by":"auto","created_at":"2024-12-10 06:15:50","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":210364,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eRamachandran Plot of OBPs created through PROCHECK; A) \u003c/strong\u003eOBP11 of \u003cem\u003eAcythosiphon pisum\u003c/em\u003e, \u003cstrong\u003eB) \u003c/strong\u003eOBP of \u003cem\u003eAphis gossypi,\u003c/em\u003e\u003cstrong\u003e C) \u003c/strong\u003e\u003cem\u003eSitobion avenae\u003c/em\u003eOBP,\u003cstrong\u003e D) \u003c/strong\u003eOBP21 of\u003cstrong\u003e \u003c/strong\u003e\u003cem\u003eApis mellifera.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-5603153/v1/72a66b38e85578a3b943e907.png"},{"id":71003823,"identity":"1fcd8423-3d9d-42d2-b64d-712e26d750a3","added_by":"auto","created_at":"2024-12-10 06:07:50","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":265390,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eModified model of OBP\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-5603153/v1/5b449fb0100dad5885ea3fe6.png"},{"id":71002599,"identity":"b51aa2e3-e32c-4be2-8661-93afe3d7e7da","added_by":"auto","created_at":"2024-12-10 05:59:50","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":176741,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMolecular interaction of OBPs and E-Beta Farnesene; A)\u003c/strong\u003e OBP11 (Acrythosiphon pisum) and E-Beta Farnesene \u003cstrong\u003eB) \u003c/strong\u003eOBP (Apis gossypii) and E-beta-farnesene, \u003cstrong\u003eC) \u003c/strong\u003eOBP (Sitobion avenae) and E-beta-farnesene, \u003cstrong\u003eD) \u003c/strong\u003eOBP21 “(Aphis mellifera)”\u003cstrong\u003e \u003c/strong\u003eand E-beta-farnesene.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-5603153/v1/c4fa5d352f1547704a1629fe.png"},{"id":71005119,"identity":"539d7857-6a8e-42c4-9766-04ddcc19d27d","added_by":"auto","created_at":"2024-12-10 06:15:55","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1375745,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5603153/v1/bf6e39a2-64d9-4fce-b513-8e2e424117a9.pdf"},{"id":71002583,"identity":"3d746624-67b3-422b-9c6c-b9eb319c6947","added_by":"auto","created_at":"2024-12-10 05:59:50","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":17526,"visible":true,"origin":"","legend":"","description":"","filename":"TABLES.docx","url":"https://assets-eu.researchsquare.com/files/rs-5603153/v1/2ea849814cbbf0e282a40c13.docx"}],"financialInterests":"The authors declare no competing interests.","formattedTitle":"\u003cp\u003eIn-silico analysis of E-β-farnesene for its possible insect-repellent activity through interaction with Odorant Binding Proteins (OBPs)\u003c/p\u003e","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eControlling pest attacks is a critical factor in growing healthy crops with high yield. Pest attacks cause heavy infestation to growing crops. Apparently, 42% of the world\u0026rsquo;s food supply is all wasted only because of the pest attacks (Ali et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). The insect olfactory system performs a vast range of activities like detecting the food sources, egg laying and mating preferences (Sun et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Chemoreception in insects is believed as they thrive in a chemical world where they convey and receive information in the form of chemical signal like pheromones, plant volatiles and predator odors. Olfactory receptors are generally located in maxillary palps, antennae and labial palps of insects (Schuman \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Odorant binding proteins (OBPs) are soluble proteins of high concentration in sensory lymph. These are regarded as carriers of semiochemical molecules that play a crucial role in olfactory perception (Rana et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Odorant binding proteins (OBPs) are a sub class of lipocalins present in olfactory apparatus. These proteins are illustrated by their characteristics of irreversibly binding with volatile molecules that are usually called \u0026ldquo;odorants\u0026rdquo;(Chandrasekaran et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eOdorant binding proteins are amphipathic proteins that are present in the olfactory system. OBPs have been characterized from various species of economically important insects (Tian et al. 2024). OBPs play a significant role in olfactory organs helping phytophageous insects for seeking of host (Liu et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Earlier functional and structural studies on OBPs of insects raised two questions that (1) OBPs particularly identify the specific ligands. (2) Which way of transfer of OBPs uses to convey the chemical signal to Odorant Receptors (ORs). There have been long debates on this discussion because of selectivity and extremely sensitive behavior of insect olfactory system ( Kohlmeier and Billeter \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe principal organ of insect olfactory system is antennae, which mediate several processes in communication with other insects. For instance, male moths use olfactory organ which is antennae to locate their matting partners (Chu and \u0026Aring;gmo, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Earlier, in insect peripheral nerve system, two alternatives have been examined for eco-friendly strategies in integrated pest management system (IPM). These are odorant receptors (ORs) and odorant binding proteins (OBPs) (Zhou et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eE-beta-farnesene plays a significant role as plant volatile in transgenic plants which not only interrupt the insect feeding process at midrib of plant but also interfere with the feeding process of other aphids in their proximity (Li et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). E-β-farnesene synthase catalyzes the formation of E-β-farnesene, which is used by many pest aphids as main component of alarming situations and involved in chemical communication with each other. Transgenic plants releasing E-β-farnesene molecules also attract the natural enemies which use E-β-farnesene for foraging cues. Therefore, it can minimize the level of aphid infestation. Symptoms of aphid attack on plants may vary according to plant species and aphid species (Wang et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2024\u003c/span\u003e)\u003c/p\u003e \u003cp\u003eChemosensory organs of insects are capable of receiving and programming a variety of chemical signals to exhibit their response to plant volatiles. These chemosensory systems include odorant binding receptors (ORs) and various odorant binding proteins (OBPs) along with odorant degrading enzymes (ODEs) having the ability of abrupt degradation of odorants (Pei et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2023\u003c/span\u003e;Qin et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Gouda and Subramanian \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Insect OBPS are categorized into 5 subclasses depending on no. on cysteine residues that are Plus-c OBPs (two additional cysteine residues and pro residues), Mius-C OBPs (4 conserved cysteine), classical OBPs (6 conserved cysteine), Atypical OBPs (9\u0026ndash;10 cysteine having long C-terminus) dimer (two cysteine signature motifs) (Abendroth et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Liu et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). OBPs play a role in the association between the outer environment and ORs. Odorants arrive at the entry site, where sensilla are present in sensillum lymph (step1). Odorants molecules bound with the solubulized OBPs (step2) and transport from sensillum lymph (step3) that fill up the cavity with dendrites. At the end activation of membrane bound ORs is performed (step4) (Raiser et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2023\u003c/span\u003e)The presence of N-terminus except of C-terminus covered the site closing off the conventional OBP pocket. Results of fluorescent binding assays and molecular docking show that OBP3 from \u003cem\u003eMegoura viciae\u003c/em\u003e can bind to each alarm pheromone molecules (Jiang et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Olfactory organs respond to odorant chemicals by proceeding the action potential and forwarding the odor to odorant receptor neuron (ORNs) in maxillary palps in insects. Information is transported to antennal lobe (AL) which is the influential functional unit of olfactory organ glomeruli. All information is collected into ORNs and thus expressing their response profile within the local neuron inside the glomeruli. Output of neurons contains axons which exit the local neurons and transfer to the mushroom body (MBs) of the insects. Local neurons (LNs) have excitatory role and Projection neurons (PNs) which target only local neurons and exhibit the inhibitory role by inhibition or termination of odorant signal (Wolff et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eInsects identify and distinguish olfactory cues with hair like structures called sensilla which is located inside the antennae. These sensilla are present in sensillum lymph. Various types of membrane bounded olfactory receptors and soluble odorant binding proteins (OBPs) are also present in sensillum lymph ( Manikkaraja et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eOBPs are widely identified in tarsi, larval heads, antennae and even present in eggs. They are indicative for their structural and functional range of diversities. Antennal odorant binding proteins (ABPs) and general odorant binding proteins (GOBPs) abundantly located in antennae of adults for odorant driven behavior responses (Li et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2023\u003c/span\u003e)\u003c/p\u003e \u003cp\u003eThe behavioral responses in drosophila mutants and aphids exhibited that OBPs are correctly engaged in semiochemical approach. But until now the exact mode of action of these proteins is unrevealed (Jaffar et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Yi et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). In present study, we have tried to unveil the mode of action of OBPs at molecular level.\u003c/p\u003e"},{"header":"MATERIAL AND METHODS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eSequence retrieval:\u003c/h2\u003e \u003cp\u003eFull length protein sequences of OBPs from \u003cem\u003eAcrythosiphon pisum\u003c/em\u003e (accession # CAX63068.1), \u003cem\u003eAphis gossypii\u003c/em\u003e (accession # AJP06027.1), \u003cem\u003eSitobion avenae\u003c/em\u003e (accession # ACW03675.2) and \u003cem\u003eApis mellifera\u003c/em\u003e (accession # ABD92653.1) were retrieved from freely available web sources like National Center for Biotechnology Information (NCBI) and UniProtKB. The sequence characterization and conserved domains identification was carried out using CDD, a sub tool in NCBI.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eSuperimposition of protein structures:\u003c/h3\u003e\n\u003cp\u003eSuperimposition of protein structures is crucial for determining structural homology between proteins. Protein function and evolution can be better understood by comparing their structures which helps in deciphering the phylogenies of various organisms. Predicted protein structures were superimposed using UCSF Chimera with each other to know the similarities among them and understand how different they behave when docked with the same ligand ( Lyu et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2024\u003c/span\u003e)\u003c/p\u003e\n\u003ch3\u003eHomology modelling of both insect repellent molecules and OBPs:\u003c/h3\u003e\n\u003cp\u003eProtein threading/structure prediction was performed by using the automated online servers. Different protein structure modelling servers like Swiss model, I-TASSER, Phyre2 server and offline tool MODDELR was used for the prediction of protein structural models. The visualization of predicted models for better performance was done by online tools Chimera and I-TASSER server.\u003c/p\u003e\n\u003ch3\u003eRefinement of predicted models:\u003c/h3\u003e\n\u003cp\u003eRefinement of predicted models is a significant step to refine and correlate the energy minimization of protein models. For this purpose, different tools like ModeRefiner were used.\u003c/p\u003e\n\u003ch3\u003eEvaluation and validation:\u003c/h3\u003e\n\u003cp\u003eEvaluation and validation of proteins is an important step in modelling and performance of protein-ligand docking analysis. Evaluation and validation were done using RAMPAGE (Ramachandran Plot Assessment) which gave results in the form of graphs and protein identity scores in favored regions. Physiochemical characteristics of OBP were accessed by using PROCHECK server.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eProtein-ligand docking analysis:\u003c/h2\u003e \u003cp\u003eThe docking analysis of OBPs and insect repellent molecules was performed by using automated tools like AutoDock tool, PatchDock tool and ZDOCK server. Hydrophobic and hydrophilic interactions and interaction affinity of proteins can be visualized by using the offline tool LigPlus which works with the java interface.\u003c/p\u003e \u003c/div\u003e"},{"header":"RESULTS","content":"\u003cp\u003eSemiochemicals play a vital role in insect’s life as they are used to convey information to one another. The study aimed to use these chemical cues to explore insect and plant interactions and cope the insects for causing damage to plants with a non-invasive pest control strategy. Also, this approach could help in avoiding the health hazards caused due to excessive pesticide usage to farmers, wildlife and the environment. By using OBPs of insects and E-beta farnesene as ligand and developing new analogues will provide a way for insect repellency from plants. Sequence of target OBPs were retrieved from NCBI given in Table 1.\u003c/p\u003e\n\u003cp\u003eModel Prediction of E-beta-farnesene\u003c/p\u003e\n\u003cp\u003eThe 3-D model of E-beta-farense was identified by PubChem. Model translation was done using the online tool Smile Translator.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003ePrimary Structure and Conserved Domain Prediction of OBPs\u003c/p\u003e\n\u003cp\u003eThe primary structure prediction of proteins was carried out using ProtParam tools that gave the physicochemical parameters of all proteins shown in table 2. These parameters are important for protein primary structure prediction i.e., molecular weight, Iso-electric point (pI), aliphatic index, instability index, estimated half-life of OBPs models. The instability index of all the proteins under study was calculated within the range of 40 that showed an overall structural stability of proteins.\u003c/p\u003e\n\u003cp\u003eThe conserved domains of proteins observed via CDD (conserved domain database) depicted that all the proteins had a conserved domain that belongs to superfamily PBP-GOBP having 26 -112 residues in it, showing the similarity of proteins.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eHomology modeling of OBPs and their selection\u003c/p\u003e\n\u003cp\u003eHomology modelling of OBPs was performed to get 3 Dimensional structures. At first, 3-dimensional structure prediction of the OBPs of four different aphid species (\u003cem\u003eAphis gosypii, Acrythosiphon pisum, Sitobion avenae, Apis mellifera\u003c/em\u003e) was carried out using three different tools SWISS-MODEL, phyre2 and MODELLER. In this case, the best 3-dimensional structure was predicted by SWISS-MODEL server as given in Figure 2. The evaluation of models from RAMPAGE described these structures with 96 (97.0%) residues of the model falling in the favored region and 3 (3.0%) residues were falling in the allowed region for OBP11 (\u003cem\u003eAcrythosiphon pisum)\u003c/em\u003e given in Figure 2(A), and 116 (92.8%) residues of the model fall in favored region and 7 (5.6 %) residues were falling in the allowed region for OBP of \u003cem\u003eAphis gosypii\u0026nbsp;\u003c/em\u003eFigure 2(B). Similarly, predicted model of OBP (\u003cem\u003eSitobion avenae\u003c/em\u003e) when evaluated using RAMPAGE, its Ramachandran plot described those 118 (92.2%) residues of the model fall in favored region and 6 (4.7%) residues fell in allowed region as shown in Figure 4.2(C). \u0026nbsp;The 3-dimensional structure evaluation of OBP (\u003cem\u003eApis mellifera\u003c/em\u003e) with its Ramachandran plot described it as 113 (99.1%) residues of the model fall in favored region and 4 (2.9%) residues fell in allowed region Figure 2(D).\u003c/p\u003e\n\u003cp\u003eSuperimposition of protein models\u003c/p\u003e\n\u003cp\u003eOBPs models were superimposed to other OBPs from different organisms, to check the structural similarity between them. For this purpose, UCSF Chimera (v1.11.2) (\u003ca href=\"http://www.egl.ucsf.edu/chimera\"\u003ehttp://www.egl.ucsf.edu/chimera\u003c/a\u003e) was used. RMSD value and Q-score estimation were also estimated through Chimera. This program provides diverse functions including analysis of structures, generation of images, and comparison between structures. The structure of OBP11 \u003cem\u003e(A. pisum)\u0026nbsp;\u003c/em\u003ewhen superimposed with OBP of \u003cem\u003eA. gossypii\u0026nbsp;\u003c/em\u003egave an overall RMSD value of as 2.183, SDM 57.300 and Q-score 0.166. The superimposition values for OBP11 and OBP \u003cem\u003eS. avenae\u0026nbsp;\u003c/em\u003ewere 2.497, SDM 65.091, and Q-score 0.136. Also, the superimposition technique was performed to check the overall effect of structural similarity between OBP11 (\u003cem\u003eAcrythosiphon pisum\u003c/em\u003e) and OBP21 (\u003cem\u003eApis mellifera\u003c/em\u003e). The overall RMSD value as 2.148, SDM 47.115, and Q-score 0.301 was obtained. Thus, the lower values of RMSD showed relative similarity of structures when superimposed also they share a conserved domain and family resulting in the similarities. However, it is crucial to note, that these RMSD distributions may not reflect genuine model accuracy because flexible and poorly defined areas such as C-termini and extracellular loops in proteins heavily influence them (Luo et al. 2024)\u003c/p\u003e\n\u003cp\u003eEvaluation and validation of OBPs model by PROCHECK\u003c/p\u003e\n\u003cp\u003eEvaluation and validation of each OBP model was done by PROCHECK tool. The results of the validation were visualized by this tool in the form of Ramachandran plot which is based on an investigation of 118 structures of resolution (min.2.0 Angstroms). The Ramachandran plotofevery OBP displayed the backbone conformational angles (psi and phi) for all the residues in each OBP. The “red” region displayed as approved core region that indicated favourable region of phi-psi angle values. Other residues are present in allowed and disallowed regions. In “\u003cstrong\u003eFigure 3(A)\u003c/strong\u003e” Evaluation and validation of “OBP11 (\u003cem\u003eAcythosiphon pisum\u003c/em\u003e)” was done by PROCHECK tool. The program’s Ramachandran plot demonstrated that 97.8% residues of the model fall in favoured region. 2.2% residues fall in additionally allowed region. Evaluation and validation of “OBP (\u003cem\u003eAphis gossypii\u003c/em\u003e)” in “\u003cstrong\u003eFigure 3(B)\u003c/strong\u003e” demonstrated that 87.9% residues of the model fall in favoured region. 11.2% \u0026nbsp; Residues fall in additionally allowed region andin\u003cstrong\u003e\u0026nbsp;Figure 3(C)\u0026nbsp;\u003c/strong\u003eOBP (\u003cem\u003eSitobion avenae\u003c/em\u003e) Ramachandran plot was demonstrated that 88.2% residues of the model fall in favoured region. 11.8% residues fall in additionally allowed region. In “\u003cstrong\u003eFigure 3(D)\u003c/strong\u003e” Evaluation and validation of “OBP21 (\u003cem\u003eApis Mellifera\u003c/em\u003e)” done by PROCHECK represents that 97.2% residues of the model fall in favoured region. 2.8% residues fall in additionally allowed region. A summary of the results obtained from Ramachandran plot of all proteins is given in Table 3.\u003c/p\u003e\n\u003cp\u003eModification of OBP Models by Auto Dock Vina\u003c/p\u003e\n\u003cp\u003eModification of 3-D models of odorant binding proteins was performed by Auto Dock Vina program. For which, water molecules were eliminated and addition of hydrogen ions to the protein was done. The refinement was carried out for all structures in order to prepare them for protein-ligand interaction analysis.\u003c/p\u003e\n\u003cp\u003eThe refinement was done using the protonate3D function of ADV. Different parameters including Temperature (T):300, pH: 7, Solvent: 80 and van Waals: 800R3 and salt 0.1 were used. Energy minimization of OBPs model were performed by providing the Force Field: MMFF94X and gradient: 0.05. This improved the energies of OBPs models and along with eliminated the clashes. Figure 4 shows the modified models of OBPs.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eProtein-ligand docking analysis of OBPs and E-beta-farnesene molecule and their evaluation\u003c/p\u003e\n\u003cp\u003eMolecular-docking protocols are progressively used to foresee binding affinities for a large number of ligands (27,28). The complex form between OBPs and E-beta-farnesene is due to hydrophobic interactions and hydrogen bonding. The generated complex from PatchDock server, displayed hydrophobic and hydrogen bonding interactions. OBP11 “(\u003cem\u003eAcrythosiphon pisum\u003c/em\u003e)” Phe25 and E-beta-farnesene residue C7 shows interaction. The length of hydrogen bonding is 3.2 in \u003cstrong\u003e“Figure 5 (A)”\u003c/strong\u003e. Phe230 of OBP “(\u003cem\u003eApis gossypii\u003c/em\u003e)”and E-beta-farnesene residue C9 shows hydrogen bond interaction. The length of hydrogen bonding is 3.04 in \u003cstrong\u003e“Figure 5 (B)”\u003c/strong\u003e. OBP “(\u003cem\u003eSitobion avenae)\u003c/em\u003e” and E-beta-farnesene residues Lys33, Lys32, Ala29 and C3, C13, C14 shows interaction. OBP“(\u003cem\u003eSitobion avenae)\u003c/em\u003e” Ala29and E-beta-farnesene residue C3 shows hydrogen bond interaction. The length of hydrogen bonding is 3.14 in \u003cstrong\u003e“Figure 5 (C)”.\u0026nbsp;\u003c/strong\u003eOBP21 “(\u003cem\u003eAphis mellifera\u003c/em\u003e)”and E-beta-farnesene residue Asp45, C14 hydrogen bond interaction. The length of hydrogen bonding is 2.70 in \u003cstrong\u003e“Figure 5 (D)”\u003c/strong\u003e. Visualization of these interactions was performed by LigPlus software.\u0026nbsp;\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eOBPs are major proteins in olfactory system of insects for communication with other insects (Kohlmeier and Billeter \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). When the chemical signal arrives at the entry site of insects\u0026rsquo; antennae these odorants molecules bound with the solubilized OBPs. Odorant molecules transported from sensillum lymph that fill the cavity with dendrites. At the end, activation of membrane-bound ORs is performed. It is persisted as a major challenge to discover olfactory genes that identify odorants, plant attractants, repellents and pheromones. Moreover, receptors are deorphanized by using heterologous systems such as \u003cem\u003eXenopus\u003c/em\u003e oocyte recording systems to identify specific silent receptors. Recently, it has been discovered that disclosure of an odorant activates alteration in the transcripts of those genes that are involved in the reception of that odorant. Here we have tried to unveil the mode of action of OBPs at molecular level using docking and other computational tools insects (Mounadi et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Chennai et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2024\u003c/span\u003e)\u003c/p\u003e \u003cp\u003eThis study opened the doors for the identification of ORs and odorants they detect (Xiao et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; deMarch et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2024\u003c/span\u003e) Whereas the ligand spectrum of classical OBPs has been characterized and little is known about the Plus-C subgroup of OBPs. By focusing on AlinOBP14 OBP from the subgroup of Plus-C in Hemipteran mired bug pest \u003cem\u003eAdelphocoris lineolatus\u003c/em\u003e. Q-PCR experiments suggested that AlinOBP14 expressed at various development stages ubiquitously, but extensively expressed in adult head the non-chemosensory organ. Fluorescence based competitive binding assays expressed that β-ionone, farnesol and nerolidol strongly bind to AlinOBP14. No significant internal binding pocket was predicted by homology modelling, but long N-terminal and C-terminal regions form a cupped cavity to dock ligands. Molecular docking exhibited that the four potential ligands have distinct binding orientations. These orientations involved dissimilar roles in the extension of N-terminal in the ligand recognition (Tian et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2021\u003c/span\u003e)\u003c/p\u003e \u003cp\u003eE-beta-farnesene as a ligand play a significant role as plant volatile in transgenic plants that not only interrupt the insects feeding process at the midrib of plants also interfere the feeding process of other aphids in their proximity. Following the earlier work, this study was designed to create new analogous proteins and examine their affinity with their ligand by using \u003cem\u003ein-silico\u003c/em\u003e techniques. In this research new analogous proteins were developed by performing the protein structure prediction, refinement of protein model and primary structure prediction including theoretical isoelectric point(pI) (the pH at which protein has no net charge and where pH is greater than pI than protein has a net negative charge while at the point where pH is less than pI than protein has a net positive charge), the molecular weight of protein, instability index values different other steps through various servers. For this purpose, Phyre2 Protein fold recognition server [v2.0], SWISS-MODEL, ADV, and ProtParam tools were used and the best model of each protein was selected based on their Ramachandran plot and PROCHECK values.\u003c/p\u003e \u003cp\u003eBest structures were predicted with SWISS Model server as their Rmachandran values were significant in all other predicted structures. Model of E-beta-farnesene were created through PubChem tool and 3-D structure of E-beta-farnesene was translated by using Online Smiles Translator and structure file were generated in PDB format. Automated hydrogen bonds were added to the structure according to standard valences in the E-beta-farnesene molecule.\u003c/p\u003e \u003cp\u003ePrimary structure prediction of each OBP was performed through online ProtParam tool to govern the different physiochemical properties of these OBPs. Different physicochemical properties were analyzed and all values OBPs fell into the category of acceptable range. This tool identified the physiochemical properties of these proteins that these OBPs were well stable. Superimposition of OBPs was done by UCSF Chimera software for identification of structural similarity of OBPs. The superimposition technique was performed to check the overall effect of structural similarity between OBP11 (\u003cem\u003erythosiphon pisum\u003c/em\u003e) and OBP (\u003cem\u003eAphis gossypii\u003c/em\u003e). The overall SDM 57.300, RMSD value as 2.183, and Q-score 0.166 were obtained through superimposition performed by Chimera. Validation of OBPs models was performed by using four different tools such as verify 3D, ERRAT, QMEAN Server, and PROCHECK tool. PROCHECK values showed that predicted models of OBPs were highly reliable. Validation of \u0026ldquo;OBP (\u003cem\u003eAphis gossypii\u003c/em\u003e)\u0026rdquo; by PROCHECK tool showed Ramachandran plot demonstrated that 87.9% residues of the model fall in favored region and 11.2% residues fall in additionally allowed region. The Q-Mean of \u0026ldquo;OBP (\u003cem\u003eApis gossypii\u003c/em\u003e)\u0026rdquo; depicted that protein structure quality is good because this protein has a (-2.04) QMEANZ-score. \u0026ldquo;OBP ((\u003cem\u003etobion avenae\u003c/em\u003e)\u0026rdquo; structure evaluation which was also performed by ERRAT tool resulted 94.9153% evaluated overall quality factor value that indicated that the model of OBP is stable and reliable. OBPs models were modified and prepared for docking. Before docking of OBPs and E-beta farensene refinement and energy minimization of these OBPs were performed through the ADV tool. ADV tool is a highly extensive tool for energy minimization and protonation. Energy minimization of OBP \u0026ldquo;(\u003cem\u003eApis gossypii\u003c/em\u003e)\u0026rdquo; through the ADV model was performed by providing the Force Field: MMFF94X and gradient: 0.05. The PatchDock tool for protein and ligand interaction was performed and an evaluation of docking results was performed through the LigPlus tool. Evaluation of docking results showed the hydrogen bonding, double bond interaction, and hydrophobic and elastic bonding interaction between different atoms and residues. The generated complex from PatchDock server, and evaluation through LigPlus server displayed binding interactions of OBP \u0026ldquo;(\u003cem\u003eApis gossypii\u003c/em\u003e)\u0026rdquo;. The OBP residue Phe230 and ligand atom C9 shows hydrogen bond interaction and non-ligand residues showed hydrophobic interactions. The length of hydrogen bonding is 3.04. The dark brown interaction are depiction double bonding between different atoms. Structure facilitates the interactions between Phe230, Leu170, Leu169, Phe189 that showed the elastic bonding between ligand and OBP. The interaction showed that these proteins should be used for the repellency strategy of aphid pests from damaging the crop plants and preservation of food sources in future use.\u003c/p\u003e"},{"header":"CONCLUSION","content":"\u003cp\u003eThere are many Odorant molecules which may interact with insect OBPs and serve as insect repellents however, the molecular basis of their repellent activity is not known. In the present study, an \u003cem\u003ein-silico\u003c/em\u003e appraisal of a broad range of aphid repellent molecules was carried out in order to propose their mechanism of action at molecular level. Odorant binding protein (OBP) interaction with E-β-farnesene. Phe230 residue in OBP of \u003cem\u003eApis gossypii\u003c/em\u003e showed interaction with ligand\u0026rsquo;s C9 atom with hydrogen bond having bond length of 3.04 \u0026Aring;. Physicochemical analyses revealed that OBPs is basic in nature with 9.30 isoelectric point (pI), thermostable based on aliphatic index.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003eAcknowledgements:\u0026nbsp;None.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAuthor contributions\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAQ and HA: data collection/entry,\u0026nbsp;AA and WK: data analysis/ statistics,\u0026nbsp;GM,\u0026nbsp;MSK and FAJ: preparation of the manuscript, \u003cem\u003eMMH\u003c/em\u003e: made a literature analysis/search,\u0026nbsp;FAJ\u0026nbsp;conceived and designed the study. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003eCompliance with ethical standards:\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eResearch involving Human Participants and/or Animals:\u0026nbsp;N/A\u003c/p\u003e\n\u003cp\u003eInformed consent:\u0026nbsp;N/A\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eConflict of interest: The authors declare that they have no conflict of interest.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eAbendroth JA, Moural TW, Wei H, Zhu F. (2023) Roles of insect odorant binding proteins in communication and xenobiotic adaptation. Front Insect Sci. 3:1274197.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eAli MA, Abdellah IM, Eletmany MR. (2023) Towards sustainable management of insect pests: Protecting food security through Ecological Intensification. Int J Chem Biochem Sci. 24(4):386\u0026ndash;94.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eChandrasekaran P, Weiskirchen S, Weiskirchen R. 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(2023) Neuromodulation and differential learning across mosquito species. Proc R Soc B. 290(1990):20222118.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eXiao Y, Sun L, Wang Q, Zhang Q, Gu S hua, Khashaveh A. (2018) \u0026nbsp;Entomology Molecular characterization and expression analysis of putative odorant carrier proteins in \u003cem\u003eAdelphocoris lineolatus\u003c/em\u003e. J Asia Pac Entomol. 21(3):958\u0026ndash;70.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eYi S, Chen X, Wu Y, Wu J, Wang J, Wang M. (2024) Identification of odorant‐binding proteins and functional analysis of antenna‐specific BhorOBP28 in \u003cem\u003eBatocera horsfieldi\u003c/em\u003e (Hope). Pest Manag Sci. 80(8):40558\u0026ndash;4068.\u003c/li\u003e\n \u003cli\u003eZhou Y, Huang C, Fu G, Tang R, Yang N, Liu W, et al. (2024) Molecular and Functional Characterization of Three General Odorant-Binding Protein 2 Genes in \u003cem\u003eCydia pomonella\u003c/em\u003e (Lepidoptera: Tortricidae). Int J Mol Sci. 25(3):1746.\u0026nbsp;\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003eTables 1 to 3 are available in the Supplementary Files section\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"Centre of Agricultural Biochemistry and Biotechnology, University of Agriculture Faisalabad, Faisalabad, Pakistan ","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Insect repellence, Odorant binding proteins, E-beta-farnesene, docking analysis. ","lastPublishedDoi":"10.21203/rs.3.rs-5603153/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5603153/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eContrary to humans, many animals, especially insects, rely heavily on the sense of smell to detect their foods, predators, mating partners and nesting sites. Odor molecules are chemicals, originating from a source. When such chemical compounds interact with odorant binding proteins (OBPs) and subsequently with the receptors associated with the insect nervous system, a cascade of reactions commences. In the present study, an \u003cem\u003ein-silico\u003c/em\u003eappraisal of a broad range of aphid repellent molecules was carried out in order to propose their mechanism of action at molecular level. Initially 3-D protein structure prediction of odorant binding proteins was carried out using SWISS model, Phyre2 and Modeller. The aphid repellent molecule E-beta-farnesene (a chemical compound used as ligand) was retrieved from PubChem. Physiochemical analyses carried out by Protparam revealed that OBPs are basic in nature with 9.30 isoelectric point (pI) and based on aliphatic index OBPs were found to be thermostable. Protein-ligand interaction was carried out with the help of Auto Dock Vina (ADV) tool that revealed complex interactions among ligand and proteins showing binding affinities by different bonds including hydrogen bonding, hydrophobic bonding, and elastic bonds. However, Phe230 residue in OBP of \u003cem\u003eApis gossypii\u003c/em\u003e showed interaction with ligand’s C9 atom via hydrogen bond having bond length of 3.04 Å. The use of new analogous for a wide range of aphid pests should be carried out in future studies.\u003c/p\u003e","manuscriptTitle":"In-silico analysis of E-β-farnesene for its possible insect-repellent activity through interaction with Odorant Binding Proteins (OBPs)","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-12-10 05:59:45","doi":"10.21203/rs.3.rs-5603153/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"dd6e8bc0-c839-4b2b-8bf8-17a2809fa69e","owner":[],"postedDate":"December 10th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":41305831,"name":"Bioinformatics"},{"id":41305832,"name":"Biotechnology and Bioengineering"}],"tags":[],"updatedAt":"2024-12-10T05:59:45+00:00","versionOfRecord":[],"versionCreatedAt":"2024-12-10 05:59:45","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-5603153","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5603153","identity":"rs-5603153","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}