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Many protein-protein interactions (PPIs) affect the ways in which small molecules bind to their constituent proteins, which can impact drug efficacy and regulatory mechanisms. While recent advances have improved our ability to independently predict both PPIs and ligand-protein interactions (LPIs), a comprehensive understanding of how PPIs affect LPIs is still lacking. Here, we examined 63 pairs of ligand-protein complexes in a benchmark dataset for protein-protein docking studies and quantified six typical effects of PPIs on LPIs. A multi-chain dynamics perturbation analysis method, called mcDPA, was developed to model these effects and used to predict small-molecule binding regions in protein-protein complexes. Our results illustrated that the mcDPA can capture the impact of PPI on LPI to varying degrees, with six similar changes in its predicted ligand-binding region. The calculations showed that 52% of the examined complexes had prediction accuracy at or above 50%, and 55% of the predictions had a recall of not less than 50%. When applied to 33 FDA-approved protein-protein-complex-targeting drugs, these numbers improved to 60% and 57% for the same accuracy and recall rates, respectively. The method developed in this study may help to design drug-target interactions in complex environments, such as in the case of protein-protein interactions.

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In vivo molecular imaging tools hold immense potential to drive transformative breakthroughs by enabling researchers to visualize cellular and molecular interactions in real-time and/or at high resolution. These advancements will facilitate a deeper understanding of fundamental biological processes and their dysregulation in disease states. Here, we develop and characterize a self-assembling protein nanomicelle called collagen type I binding - thermoresponsive assembled protein (Col1-TRAP) that binds tightly to type I collagen in vitro with nanomolar affinity. For ex vivo visualization, Col1-TRAP is labeled with a near-infrared fluorescent dye (NIR-Col1-TRAP). Both Col1-TRAP and NIR-Col1-TRAP display approximately a 3.8-fold greater binding to type I collagen compared to TRAP when measured by surface plasmon resonance (SPR). We present a proof-of-concept study using NIR-Col1-TRAP to detect fibrotic type I collagen deposition ex vivo in the livers of mice with non-alcoholic steatohepatitis (NASH). We show that NIR-Col1-TRAP demonstrates significantly decreased plasma recirculation time as well as increased liver accumulation in the NASH mice compared to mice without disease over 4 hours. As a result, NIR-Col1-TRAP shows potential as an imaging probe for NASH with in vivo targeting performance after injection in mice. STATEMENT OF SIGNIFICANCE: Direct molecular imaging of fibrosis in NASH patients enables the diagnosis and monitoring of disease progression with greater specificity and resolution than do elastography-based methods or blood tests. In addition, protein-based imaging probes are more advantageous than alternatives due to their biodegradability and scalable biosynthesis. With the aid of computational modeling, we have designed a self-assembled protein micelle that binds to fibrillar and monomeric collagen in vitro. After the protein was labeled with near-infrared fluorescent dye, we injected the compound into mice fed on a NASH diet. NIR-Col1-TRAP clears from the serum faster in these mice compared to control mice, and accumulates significantly more in fibrotic livers.This work advances the development of targeted protein probes for in vivo fibrosis imaging.

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Human cytochrome P450 enzymes are membrane-embedded monooxygenases responsible for xenobiotic metabolism, steroidogenesis, fatty acid metabolism, and vitamin metabolism. Their active sites can accommodate diverse small molecules and understanding these interactions is key to decoding enzymatic functionality and designing drugs. The most common method for characterizing small molecule binding is quantifying absorbance changes that typically occur when substrates enter the active site near the heme iron. Traditionally such titrations are monitored by a spectrophotometer, requiring significant manual time, protein, and increasing solvents. This assay was adapted for semi-automated high throughput screening, increasing throughput 50-fold while requiring less protein and keeping solvent concentrations constant. This 384-well assay was validated for both type I and II shifts typically observed for substrates and heme-coordinating inhibitors, respectively. This assay was used to screen a library of ∼100 diverse imidazole-containing compounds which can coordinate with the heme iron if compatible with the overall active site. Three human cytochrome P450 enzymes were screened: drug-metabolizing CYP2A6 and CYP2D6 and sterol-metabolizing CYP8B1. Each bound different sets of imidazole compounds with varying Kd values, providing a unique binding fingerprint. As a final validation, the Kd values were used to generate pharmacophores to compare to experimental structures. Applications for the high-throughput assay include 1) facilitating generation of pharmacophores for enzymes where structures are not available, 2) screening to identify ligands for P450 orphans, 3) screening for inhibitors of P450s drug targets, 4) screening potential new drugs to avoid and/or control P450 metabolism, and 5) efficient validation of computational predictions.

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Human phosphoglycerate kinase 1(hPGK1) is a key glycolytic enzyme that regulates the balance between ADP and ATP concentrations inside the cell. Phosphorylation of hPGK1 at S203 and S256 has been associated with enzyme import from the cytosol to the mitochondria and the nucleus respectively. These changes in subcellular locations drive tumorigenesis and are likely associated with site-specific changes in protein stability. In this work, we investigate the effects of site-specific phosphorylation on thermal and kinetic stability and protein structural dynamics by hydrogen-deuterium exchange (HDX) and molecular dynamics (MD) simulations. We also investigate the binding of 3-phosphoglycerate and Mg-ADP using these approaches. We show that the phosphomimetic mutation S256D reduces hPGK1 kinetic stability by 50-fold, with no effect of the mutation S203D. Calorimetric studies of ligand binding show a large decrease in affinity for Mg-ADP in the S256D variant, whereas Mg-ADP binding to the WT and S203D can be accurately investigated using protein kinetic stability and binding thermodynamic models. HDX and MD simulations confirmed the destabilization caused by the mutation S256D (with some long-range effects on stability) and its reduced affinity for Mg-ADP due to the strong destabilization of its binding site (particularly in the apo-state). Our research provides evidence suggesting that modifications in protein stability could potentially enhance the translocation of hPGK1 to the nucleus in cancer. While the structural and energetic basis of its mitochondrial import remain unknown.

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Retinoid X receptors (RXRs) belong to a retinoid-binding subgroup of the nuclear receptor family, and their synthetic agonists have been developed as therapeutics for glucose and lipid metabolism, inflammation, and inflammatory bowel disease, although RXR agonists could cause side effects such as hypothyroidism, hypertriglyceridemia, and hepatomegaly. We previously reported novel full and partial agonists, NEt-3IB and NEt-4IB, which reduce the side effects, but the molecular basis of their different activity was not clear. In this study, we report the crystal structures of the ligand-binding domain of human RXRα complexed with NEt-3IB and NEt-4IB. Detailed comparisons of the two structures showed that the full agonist, NEt-3IB, is more stably accommodated in the ligand-binding pocket due to the interactions of the bulky iso-butoxy group with helices 5 and 7. The stabilization of these helices led to the stabilization of helix 12, which is important for formation of the coactivator-binding site. The structures shed light on the novel mechanism of the regulation of RXR activity through the interaction between the bound agonist and helix 7, an interaction that was not previously considered important.

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Predicting protein-ligand binding sites is an integral part of structural biology and drug design. A comprehensive understanding of these binding sites is essential for advancing drug innovation, elucidating mechanisms of biological function, and exploring the nature of disease. However, accurately identifying protein-ligand binding sites remains a challenging task. To address this, we propose PGpocket, a geometric deep learning-based framework to improve protein-ligand binding site prediction. Initially, the protein surface is converted into a point cloud, and then the geometric and chemical properties of each point are calculated. Subsequently, the point cloud graph is constructed based on the inter-point distances, and the point cloud graph neural network (GNN) is applied to extract and analyze the protein surface information to predict potential binding sites. PGpocket is trained on the scPDB dataset, and its performance is verified on two independent test sets, Coach420 and HOLO4K. The results show that PGpocket achieves a 58% success rate on the Coach420 dataset and a 56% success rate on the HOLO4K dataset. These results surpass competing algorithms, demonstrating PGpocket's advancement and practicality for protein-ligand binding site prediction.

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BACKGROUND: Metastatic castration-resistant prostate cancer (mCRPC) that progresses on androgen receptor pathway inhibitors (ARPIs) may continue to be driven by AR signaling. BMS-986365 is an orally administered ligand-directed degrader targeting the AR via a first-in-class dual mechanism of AR degradation and antagonism. CC-94676-PCA-001 (NCT04428788) is a phase 1 multicenter study of BMS-986365 in patients with progressive mCRPC. PATIENTS AND METHODS: Patients who progressed on androgen deprivation therapy, ≥ 1 ARPI, and taxane chemotherapy (unless declined/ineligible) were enrolled. The study included dose escalation (Part A) and expansion (Part B) of BMS-986365 up to 900 mg twice daily (BID). Primary objectives were safety, tolerability, and to define maximum tolerated dose (MTD) and/or recommended phase 2 dose (RP2D). Key secondary endpoints included decline in prostate-specific antigen ≥50% (PSA50) and radiographic progression-free survival (rPFS). RESULTS: Parts A and B enrolled 27 and 68 patients, respectively. In Part B, the median number of prior therapies was 4 (range 2-11). The most common treatment-related adverse events (TRAEs) were asymptomatic prolonged corrected QT interval (47%) and bradycardia (34%). Part A MTD was not reached and RP2D selection is ongoing. Across Part B three highest doses (400-900 mg BID, n = 60), PSA50 was 32% (n = 19), including 50% (n = 10/20) at 900 mg; median rPFS (95% CI) was 6.3 months (5.3-12.6), including 8.3 months (3.8-16.6) at 900 mg; and rPFS was longer in patients without versus with prior chemotherapy: 16.5 months (5.5-not evaluable) versus 5.5 months (2.7-8.3), respectively. Efficacy was observed in patients with AR ligand binding domain (LBD) WT or with AR LBD mutations. CONCLUSIONS: BMS-986365 was well tolerated, with a manageable safety profile, and demonstrated activity in heavily pretreated patients with potentially higher benefit in chemotherapy-naïve patients. These data show BMS-986365's potential to overcome resistance to current ARPIs, regardless of AR LBD mutation status.

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Propofol is a widely used anesthetic and sedative that acts as a positive allosteric modulator (PAM) of gamma-aminobutyric acid type A (GABAA) receptors. Several potential propofol binding sites that may mediate this effect have been identified using propofol-analogue photoaffinity labeling. o-PD labels ß-H267, a pore-lining residue, whereas AziPm labels residues ß-M286, ß-M227 and α-I239 in the two membrane-facing interfaces (ß(+)/α(-) and α(+)/ß(-)) between α and ß subunits. This study used photoaffinity labeling of α1ß3 GABAA receptors to reconcile the apparently conflicting results obtained with AziPm and o-PD labeling, focusing on whether ß3-H267 identifies specific propofol binding site(s). The results show that propofol, but not AziPm protects ß3-H267 from labeling by o-PD, whereas both propofol and o-PD protect against AziPm labeling of ß3-M286, ß3-M227 and α1I239. These data indicate that there are three distinct classes of propofol binding sites, with AziPm binding to two of the classes and o-PD to all three. Analysis of binding stoichiometry using native mass spectrometry in ß3 homomeric receptors, demonstrated a minimum of five AziPm labeled residues and three o-PD labeled residues per pentamer, suggesting that there are two distinct propofol binding sites per ß-subunit. The native MS data, coupled with photolabeling performed in the presence of zinc, indicate that the binding site(s) identified by o-PD are adjacent to, but not within the channel pore, since the pore at the 17' H267 residue can accommodate only one propofol molecule. These data validate the existence of three classes of specific propofol binding sites on α1ß3 GABAA receptors.

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Menin is an essential oncogenic co-factor of MLL1 fusion proteins in acute leukemias and inhibitors of the menin-MLL1 interaction are under evaluation in clinical trials. Recent studies found emerging resistance to menin inhibitor treatment in leukemia patients as a result of somatic mutations in menin. To understand how patient mutations in menin affect the interaction with MLL1, we performed systematic characterization of the binding affinity of these menin mutants (T349M, M327I, G331R and G331D) and the N-terminal fragment of MLL1. We also determined the crystal structures of menin patient mutants and their complexes with MLL1-derived peptides. We found that drug-resistant mutations in menin occur at a site adjacent to the MLL1 binding site, but they do not affect MLL1 binding to menin. On the contrary, our structural analysis shows that all these point mutations in menin generate steric clash with menin inhibitors. We also found that mutation G331D results in a very slow dissociation of MLL1 from menin and this mutant might be particularly difficult to inhibit with small molecule drugs. This work provides structural information to support the development of a new generation of small molecule inhibitors that overcome resistance caused by menin mutations.

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Sol g 2, a major protein found in the venom of the tropical fire ant (Solenopsis geminata), is well-known for its ability to bind various hydrophobic molecules. In this study, we investigate the binding activity of recombinant Sol g 2.1 protein (rSol g 2.1) with potential molecules, including (E)-ß-Farnesene, α-Caryophyllene, and 1-Octen-3-ol at different pH levels (pH 7.4 and 5.5) using fluorescence competitive binding assays (FCBA). Our results revealed that Sol g 2.1 protein has higher affinity binding with these ligands at neutral pH. Relevance to molecular docking and molecular dynamics simulations were utilized to provide insights into the stability and conformational dynamics of Sol g 2.1 and its ligand complexes. After simulation, we found that Sol g 2.1 protein has higher affinity binding with these ligands as well as high structural stability at pH 7.4 than at an acidic pH level, indicating by RMSD, RMSF, Rg, SASA, and principal component analysis (PCA). Additionally, the Sol g 2.1 protein complexes at pH 7.4 showed significantly lower binding free energy (∆Gbind) and higher total residue contributions, particularly from key non-polar amino acids such as Trp36, Met40, Cys62, and Ile104, compared to the lower pH environment. These explain why they exhibited higher binding affinity than the lower pH. Therefore, we suggested that Sol g 2.1 protein is a pH-responsive carrier protein. These findings also expand our understanding of protein-ligand interactions and offer potential avenues for the development of innovative drug delivery strategies targeting Sol g 2.1 protein.

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Aim: Oligonucleotide therapeutics can be quantified using various bioanalytical methods, and these methods have been compared extensively. However, few comparisons exist where the same analyte is evaluated by multiple assay platforms.Materials & methods: Hybrid LC-MS, SPE-LC-MS, HELISA and SL-RT-qPCR methods were developed for an siRNA analyte, and samples from a pharmacokinetic study were analyzed by all four methods.Results: All assay platforms provided comparable data, though higher concentrations were observed using the non-LC-MS assays. Hybrid LC-MS and SL-RT-qPCR were the most sensitive methodologies, and SL-RT-qPCR and HELISA demonstrated the highest throughput.Conclusion: Each assay platform is suitable for oligonucleotide bioanalysis, and the ultimate choice of methodology will depend on the prioritization of needs such as sensitivity, specificity and throughput.

[Box: see text].

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The nuclear receptor Nur77 is a transcription factor belonging to the NR4A subfamily. Upon activation, it regulates a wide array of biological and pathophysiological processes by modulating the expression of its target genes. Previous findings have classified Nur77 as an orphan receptor because of the discovery of a structurally atypical ligand-binding domain and the lack of identification of an endogenous ligand. Nevertheless, recent studies have uncovered several endogenous, natural, and small synthetic molecules that can bind to and activate Nur77. However, developing selective and potent Nur77 activators remains a significant challenge. The discovery of novel and potential small synthetic molecules that modulate Nur77 activity will facilitate therapeutic interventions of Nur77 against several human diseases. In this study, we have reported the development of a novel and effective Nur77 ligand. Our data show that (1E,4E)-1,5-di(pyrazin-2-yl)penta-1,4-dien-3-one (PB) induces Nur77 transcriptional activity by interacting with a putative Nur77 ligand binding site by forming solid hydrogen bonding. Calculated chemical parameters denote that PB has sophisticated chemical features that will enhance its interaction with the Nur77 ligand-binding domain. Molecular docking simulations showed that PB fits in the Nur77 putative ligand binding site with solid hydrogen bonding, and molecular dynamics simulations indicate that PB binding would stabilize the Nur77 ligand binding domain. Further, in vitro studies revealed that PB increases Nur77 nuclear expression and activity, inhibits cigarette smoke-induced inflammatory phenotype of airway epithelial cells, and protects against apoptosis. These findings provide insights into developing an effective Nur77 small-molecule activator which could be developed into a therapeutic agent against inflammatory diseases.

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Glyphodes pyloalis Walker (Lepidoptera: Pyralidae) is the most destructive pest, causing severe damage to mulberry production in China's sericulture industry. The insecticide application in mulberry orchards poses a significant risk of poisoning to Bombyx mori. Shifting from insecticides to odor attractants is a beneficial alternative, but not much data is available on the olfactory system of G. pyloalis. We identified 114 chemosensory genes from the antennal transcriptome database of G. pyloalis, with 18 odorant-binding protein (OBP) and 17 chemosensory protein (CSP) genes significantly expressed in the antennae. Ligand-binding assays for two antennae-biased expressed general odorant-binding proteins (GOBPs) showed high binding affinities of GOBP1 to hexadecanal, ß-ionone, and 2-ethylhexyl acrylate, while GOBP2 exhibited binding to 4-tert-octylphenol, benzyl benzoate, ß-ionone, and farnesol. Computational simulations indicated that van der Waal forces predominantly contributed to the binding free energy in the binding processes of complexes. Among them, Phe12 of GOBP1 and Phe19 of GOBP2 were demonstrated to play crucial roles in their bindings to plant volatiles using site-directed mutagenesis experiments. Moreover, hexadecanal and ß-ionone attracted G. pyloalis male moths in the behavioral assays, while none of the candidate plant volatiles significantly affected female moths. Our findings provide a comprehensive understanding of the molecular mechanisms underlying olfactory recognition in G. pyloalis, setting the groundwork for novel mulberry pests control strategies based on insect olfaction.

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P2X receptors are ATP-activated, non-specific cation channels involved in sensory signalling, inflammation, and certain forms of pain. Investigations of agonist binding and activation are essential for comprehending the fundamental mechanisms of receptor function. This encompasses the ligand recognition by the receptor, conformational changes following binding, and subsequent cellular signalling. The ATP-induced activation of P2X receptors is further influenced by the concentration of Mg2+ that forms a complex with ATP. To explore these intricate mechanisms, two new fluorescently labelled ATP derivatives have become commercially available: 2-[DY-547P1]-AHT-ATP (fATP) and 2-[DY-547P1]-AHT-α,ßMe-ATP (α,ßMe-fATP). We demonstrate a subtype-specific pattern of ligand potency and efficacy on human P2X2, P2X3, and P2X2/3 receptors with distinct relations between binding and gaiting. Given the high in vivo concentrations of Mg2+, the complex formed by Mg2+ and ATP emerges as an adequate ligand for P2X receptors. Utilising fluorescent ligands, we observed a Mg2+-dependent reduction in P2X2 receptor activation, while binding remained surprisingly robust. In contrast, P2X3 receptors initially exhibited decreased activation at high Mg2+ concentrations, concomitant with increased binding, while the P2X2/3 heteromer showed a hybrid effect. Hence, our new fluorescent ATP derivatives are powerful tools for further unravelling the mechanism underlying ligand binding and activation gating in P2X receptors.

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Cytochrome c (CytC), a one-electron carrier, transfers electrons from complex bc1 to cytochrome c oxidase (CcO) in the electron-transport chain. Electrostatic interaction with the partners, complex bc1 and CcO, is ensured by a lysine cluster near the heme forming the Universal Binding Site (UBS). We constructed three mutant variants of mitochondrial CytC with one (2Mut), four (5Mut), and five (8Mut) Lys->Glu substitutions in the UBS and some compensating Glu->Lys substitutions at the periphery of the UBS for charge compensation. All mutants showed a 4-6 times increased peroxidase activity and accelerated binding of cyanide to the ferric heme of CytC. In contrast, decomposition of the cyanide complex with ferrous CytC, as monitored by magnetic circular dichroism spectroscopy, was slower in mutants compared to WT. Molecular dynamic simulations revealed the increase in the fluctuations of Cα atoms of individual residues of mutant CytC compared to WT, especially in the Ω-loop (70-85), which can cause destabilization of the Fe…S(Met80) coordination link, facilitation of the binding of exogenous ligands cyanide and peroxide, and an increase in peroxidase activity. It was found that only one substitution K72E is enough to induce all these changes, indicating the significance of K72 and the Ω-loop (70-85) for the structure and physiology of mitochondrial CytC. In this work, we also propose using a ferro-ferricyanide buffer as a substrate to monitor the peroxidase activity of CytC. This new approach allows us to determine the rate of peroxidase activity at moderate (200 µM) concentrations of H2O2 and avoid complications of radical formation during the reaction.

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Odorant binding proteins (OBPs) are involved in odorant discrimination and act as the first filter in the peripheral olfactory system. Previous studies have shown that BhorOBP29 is potentially involved in olfactory perception in an important wood-boring pest Batocera horsfieldi (Hope) (Coleoptera: Cerambycidae), however, its function remains unclear. Here, we investigated the ligand-binding profiles of recombinant BhorOBP29 with 22 compounds from its host plant using fluorescence competitive binding assays and fluorescence quenching assays. The results showed that BhorOBP29 could bind to five ligands relying mainly on hydrophobic interactions. Molecular docking analysis indicated that residues Ile48, Leu51, Met52, Trp57, Asn105, and Val119 were extensively involved in the interactions between BhorOBP29 and the five ligands. Furthermore, the site-directed mutagenesis analysis revealed that Leu51 and Met52 residues were indispensable for BhorOBP29-ligands binding. Finally, electroantennogram (EAG) assays confirmed that hexanal, (-)-limonene, and 2-methylbutyraldehyde elicited a concentration-dependent EAG response with a maximum at the concentration of 1/10 v/v. These findings suggest that BhorOBP29 may play a significant role in the perception of host plant volatiles by B. horsfieldi. This study may help to discover novel behavioral regulation and environmentally friendly strategies for controlling B. horsfieldi in the future.

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Fluorescent ligands have proved to be powerful tools in the study of G protein-coupled receptors in living cells. Here we have characterized a new fluorescent ligand PSB603-BY630 that has high selectivity for the human adenosine A2B receptor (A2BR). The A2BR appears to play an important role in regulating immune responses in the tumor microenvironment. Here we have used PSB603-BY630 to monitor specific binding to A2BRs in M1- and M2-like macrophages derived from CD14+ human monocytes. PSB603-BY630 bound with high affinity (18.3 nM) to nanoluciferase-tagged A2BRs stably expressed in HEK293G cells. The ligand exhibited very high selectivity for the A2BR with negligible specific-binding detected at NLuc-A2AR, NLuc-A1R, or NLuc-A3R receptors at concentrations up to 500 nM. Competition binding studies showed the expected pharmacology at A2BR with the A2BR-selective ligands PSB603 and MRS-1706 demonstrating potent inhibition of the specific binding of 50 nM PSB603-BY630 to A2BR. Functional studies in HEK293G cells using Glosensor to monitor Gs-coupled cyclic AMP responses indicated that PSB603-BY630 acted as a negative allosteric regular of the agonist responses to BAY 60-6583. Furthermore, flow cytometry analysis confirmed that PSB603-BY630 could be used to selectively label endogenous A2BRs expressed on human macrophages. This ligand should be an important addition to the library of fluorescent ligands which are selective for the different adenosine receptor subtypes, and will enable study of the role of A2BRs on immune cells in the tumor microenvironment.

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A fundamental mistake in receptor theory has led to an enduring misunderstanding of how to estimate the affinity and efficacy of an agonist. These properties are inextricably linked and cannot be easily separated in any case where the binding of a ligand induces a conformation change in its receptor. Consequently, binding curves and concentration-response relationships for receptor agonists have no straightforward interpretation. This problem-the affinity-efficacy problem-remains overlooked and misunderstood despite it being recognized in 1987. To avoid the further propagation of this misunderstanding, we propose in this review that the affinity-efficacy problem should be included in the core curricula for pharmacology undergraduates proposed by the British Pharmacological Society and the International Union of Basic and Clinical Pharmacology (IUPHAR).

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Apriona germari (Hope) presents a significant threat as a dangerous wood-boring pest, inflicting substantial harm to forest trees. Investigating the olfactory sensory system of A. germari holds substantial theoretical promise for developing eco-friendly control strategies. To date, however, the olfactory perception mechanism in A. germari remains largely unknown. Therefore, we performed transcriptome sequencing of A. germari across four distinct body parts: antennae, foreleg tarsal segments, mouthparts (maxillary and labial palps), and abdomen terminals, pinpointing the odorant binding protein (OBP) genes and analyzing their expression. We found eight AgerOBPs (5, 19, 23, 25, 29, 59, 63, 70) highly expressed in the antennae. In our competitive binding experiments, AgerOBP23 showed strong binding abilities to the pheromone component fuscumol acetate, eight plant volatiles (farnesol, cis-3-hexenal, nerolidol, myristol acetate, cis-3-hexenyl benzoate, (-)-α-cedrene, 3-ethylacetophenone, and decane), and four insecticides (chlorpyrifos, phoxim, indoxacarb, and cypermethrin). However, AgerOBP29 and AgerOBP63 did not show prominent binding activities to these tested chemicals. Through homology modeling and molecular docking, we identified the key amino acid sites involved in the binding process of AgerOBP23 to these ligands, which shed light on the molecular interactions underlying its binding specificity. Our study suggests that AgerOBP23 may serve as a potential target for future investigations of AgerOBP ligand binding. This approach is consistent with the reverse chemical ecology principle, establishing the groundwork for future studies focusing on attractant or repellent development by exploring further the molecular interactions between OBP and various compounds.

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Odorant receptors (ORs) play a crucial role in insect chemoreception. Here, a female-biased odorant receptor MmedOR48 in parasitoid Microplitis mediator was fully functionally characterized. The qPCR analysis suggested that the expression level of MmedOR48 increased significantly after adult emergence and was expressed much more in the antennae. Moreover, an in situ hybridization assay showed MmedOR48 was extensively located in the olfactory sensory neurons. In two-electrode voltage clamp recordings, recombinant MmedOR48 was broadly tuned to 23 kinds of volatiles, among which five plant aldehyde volatiles excited the strongest current recording values. Subsequent molecular docking analysis coupled with site-directed mutagenesis demonstrated that key amino acid residues Thr142, Gln80, Gln282, and Thr312 together formed the binding site in the active pocket for the typical aldehyde ligands. Furthermore, ligands of MmedOR48 could stimulate electrophysiological activities in female adults of the M. mediator. The main aldehyde ligand, nonanal, aroused significant behavioral preference of M. mediator in females than in males. These findings suggest that MmedOR48 may be involved in the recognition of plant volatiles in M. mediator, which provides valuable insight into understanding the olfactory mechanisms of parasitoids.

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