Binding Mechanism of PsauPBP3 to Sex Pheromones in Peridroma saucia: Insights from Computational and Experimental Approaches

Simple Summary

Peridroma saucia Hübner is a newly emerged agricultural pest in the Huang-Huai River Basin, China. The female sex pheromones of P. saucia are Z11-16: Ac and Z9-14: Ac. Pheromone-binding proteins (PBPs) serve as a key component in the initial step of sex pheromone recognition. Functional research on PBPs is a major current focus, yet the precise mechanism underlying their binding remains poorly understood. An improved understanding of PBP-mediated sex pheromone binding mechanisms could lead to eco-friendly strategies for insect population control by scrambling mate detection. Herein, we conducted a series of structure–function analyses of pheromone binding protein 3 (PsauPBP3) in sex pheromone recognition of P. saucia. PsauPBP3, which is predominantly expressed in male antennae of P. saucia, exhibited high binding affinities for both Z11-16: Ac and Z9-14: Ac. Further molecular dynamics simulation and molecular docking indicated that five amino acid residues could steadily interact with these two sex pheromones. Finally, computational alanine scanning and site-directed mutagenesis demonstrated that four residues were driving forces for the binding affinities of PsauPBP3, especially Phe-13 and Ile-95, which significantly contribute to the binding for both sex pheromones. These findings may lead to novel approaches for managing this pest by hindering its mating behaviors.

Abstract

The variegated cutworm Peridroma saucia Hübner, a recently emerged polyphagous pest in China’s Huang-Huai River Basin, uses sex pheromones (Z)-11-hexadecenyl acetate (Z11-16: Ac) and (Z)-9-tetradecenyl acetate (Z9-14: Ac) for mate finding. Insect pheromone-binding proteins (PBPs) serve as the primary filter for detecting specific sex pheromones. Although comprehensive functional analyses of PBPs exist, their binding mechanisms remain poorly characterized. In this study, we elucidated the binding properties and mechanisms of PsauPBP3 in sex pheromone recognition by computational and experimental approaches. PsauPBP3, predominantly expressed in male P. saucia antennae, showed high binding affinity for both Z11-16: Ac and Z9-14: Ac, as demonstrated by binding-free-energy calculations and fluorescence binding assays. Molecular dynamics simulations and docking studies identified five key residues (Thr-10, Phe-13, Ile-53, Ile-95, and Phe-119) that consistently interact with these pheromones, indicating their critical role in ligand binding. Computational alanine scanning further demonstrated that all five residues act as binding determinants, with Phe-13 and Ile-95 making particularly significant contributions to ligand affinity. The results were further validated by site-directed mutagenesis and fluorescence binding assays. This work provides insights into the function and binding mechanisms of PBPs in sex pheromone recognition and supports the development of targeted mating disruption strategies for P. saucia control.

1. Introduction

The variegated cutworm Peridroma saucia Hübner (Lepidoptera, Noctuidae) is a polyphagous pest on a large number of crops, including potato, soybean, wheat, and corn [1]. P. saucia was first recorded in Europe in 1790 and remains a main agricultural pest in Canada, the United States, and Europe [2]. It has been spreading as an invasive pest throughout South Korea and Japan since the 1970s [3,4,5]. In China, the first outbreak of P. saucia was recorded in Sichuan Province in 1985 [6]. Over the following three decades, this pest has been reported in at least 13 provinces in China [7,8,9]. In 2016, the larvae of P. saucia were identified in the tobacco fields in Xuchang, Henan Province. In 2018, an outbreak of P. saucia occurred in the soybean fields in Luanchuan County (Luoyang, Henan Province). The outbreak affected more than 6000 hectares, with the severely damaged soybean fields experiencing yield reductions of 30–60%. Subsequent field investigations revealed that P. saucia was widely distributed across the farmlands in the Huang-Huai River Basin [10].

The sex pheromone detection system is vital for almost all moth species, where males locate mating partners through sex pheromones released by conspecific females [11,12]. As their compositions and proportions vary interspecifically, sex pheromones not only serve as important information for mate finding but also establish chemical bases for interspecific reproductive isolation [13,14]. The sex pheromones of P. saucia are (Z)-11-hexadecenyl acetate (Z11-16: Ac) and (Z)-9-tetradecenyl acetate (Z9-14: Ac) [4,5]. During the period when female P. saucia are calling and copulating, the ratio of Z11-16: Ac and Z9-14: Ac in the sex pheromone gland ranges from 2.1:1 to 2.4:1 [5]. Electroantennogram recordings with Z11-16: Ac or Z9-14: Ac showed that both compounds elicited strong responses from male antennae [4]. Lure trapping studies in vegetable/grain fields demonstrated that the mixtures of Z9-14: Ac and Z11-16: Ac (1:1~1:2.3) could catch a great many male P. saucia [4,5]. Nevertheless, the molecular mechanism responsible for the sensing of Z9-14: Ac and Z11-16: Ac in P. saucia remains unclear.

Olfactory perception is an important capability for insects, helping them to locate reproductive partners and food sources [15,16]. Insect olfactory organs have numerous sensilla that act as functional units in sensing of infochemicals [17,18,19]. Infochemicals enter the sensilla via pores that are distributed on the cuticular surface [17]. Because these chemicals are hydrophobic, they cannot pass through the sensillum lymph and get to the odorant receptors (ORs) on olfactory receptor neurons (ORNs) without assistance. These molecules are believed to be bound and transferred by odorant-binding proteins (OBPs) [20]. The first insect OBP was reported in the giant moth Antheraea polyphemus at the beginning of the 1980s. This protein, which is plentifully expressed in the antennal sensillum lymph and binds to the 3H-labeled sex pheromone (E)-6, (Z)-11-hexadecenyl acetate (E6, Z11-16: Ac), has since been termed pheromone-binding protein (PBP) [21]. Among moth species, PBPs constitute a subfamily of OBPs that are primarily involved in the detection of sex pheromones [22,23,24,25]. For instance, functional analyses in Helicoverpa armigera revealed that HarmPBP1 and HarmPBP2 bind to sex pheromone components (Z)-11-hexadecenal (Z11-16: Ald) and (Z)-9-hexadecenal (Z9-16: Ald). HarmPBP3 exhibits high affinity for acetate compounds (Z)-9-hexadecenyl acetate (Z9-16: Ac) and Z11-16: Ac [26]. In Plutella xylostella, PxylPBP1 and PxylPBP2 show high affinities to the sex pheromone components Z11-16: Ald and (Z)-11-hexadecenol (Z11-16: OH) [27], while PxylPBP3 exhibits high affinity to Z11-16: Ac [28]. CRISPR/Cas9-mediated knockout of SlitPBP1, SlitPBP2, or SlitPBP3 in male Spodoptera litura resulted in varying degrees of electrophysiological and/or behavioral response loss to sex pheromone components (Z)-9, (E)-11-tetradecenyl acetate (Z9, E11-14: Ac), and Z9-14: Ac [29,30]. Furthermore, the presence of PBPs could increase both sensitivity and selectivity of pheromone receptors ectopically expressed in Xenopus oocytes or Drosophila T1 sensilla [31,32]. A recent binding mode study of PBP3 from P. xylostella revealed that two key amino acid sites are essential for PxylPBP3 to distinguish between Z11-16: Ac and Z11-16: Ald [28]. However, compared with comprehensive functional studies on PBPs, research on their binding mechanisms remains limited.

Although applying chemical insecticides is still the main way to control P. saucia, the resulting environmental pollution problems cannot be neglected. One alternative method to control this pest is via olfaction regulation. Extensive research on PBPs will help us to develop novel strategies to control the pest population by scrambling mate detection. In this study, we selected PsauPBP3 as the research target and committed to characterizing its binding properties and mechanisms in sex pheromone recognition of P. saucia. We first analyzed the expression profile of PsauPBP3 by real-time quantitative PCR (RT-qPCR) and confirmed that this gene is predominantly expressed in the antennae and shows a male-biased profile. The binding properties of PsauPBP3 to a variety of moth sex pheromones and host plant volatiles were analyzed with binding free energy calculation and fluorescence binding assays. Molecular dynamics (MD) simulation was then performed to analyze the binding modes of PsauPBP3 with candidate ligands. Thereafter, a series of computational approaches, including per-residue free energy decomposition, molecular docking, and computational alanine scanning (CAS), were conducted to screen out amino acid residues associated with the binding affinities of PsauPBP3. Finally, site-directed mutagenesis combined with fluorescence binding assays was performed to validate the simulation results experimentally. This research enhances the functional and structural elucidations of moth PBPs, which may yield a more effective application of pheromone-based techniques for managing P. saucia.

2. Materials and Methods

2.1. Insect Rearing and Tissue Collection

A colony of larval P. saucia was collected from the potato fields in Xinxiang, Henan Province, China. The larvae were reared on an artificial diet in a climatic cabinet (25 ± 2 °C, with a 16 h L/8 h D cycle and 60% ± 5% relative humidity) as previously reported [10]. Male and female pupae were placed separately in square cages (40 cm in side length) for eclosion. Twenty adults (sex ratio 1:1) were kept in a cylinder cage (24 cm in diameter, 28 cm in length) for mating and oviposition. Adults were given a 15% (Q:V) sugar solution.

Antennae, proboscises, tarsi, wings, ovipositors, and hairbrushes were collected from 2 to 3-day-old adult P. saucia of both sexes for PCR and RT-qPCR analyses. Consistently, insects at this uniform developmental stage were used throughout the study. The collected samples were immediately transferred into 1.5 mL RNase-free tubes and stored at −70 °C for further study.

2.2. RNA Extraction, cDNA Synthesis, and Gene Cloning

Total RNA was isolated following the manufacturer’s instructions for the Total RNA Extraction Kit (ACMEC, Shanghai, China). The quantity of the RNA was checked by 2.0% agarose gel electrophoresis and a Nano-Drop 2000 spectrophotometer (Nano-Drop Products, Wilmington, DE, USA). The RNA was then treated with DNase I (HARVEYBIO, Beijing, China) to remove residual genomic DNA. The cDNA was reverse transcribed using the TIANScript II RT Kit (TIANGEN, Beijing, China). The newly synthesized cDNA was then applied as a template for PCR and RT-qPCR analyses.

Full-length PsauPBP3 was acquired by PCR (gene-specific primers are shown in Table S1) with Taq Plus DNA Polymerase (TIANGEN) under the following conditions: 94 °C for 3 min; 34 cycles of 94 °C for 30 s, 60 °C for 30 s, and 72 °C for 1 min, followed by 72 °C for 5 min. The PCR products were ligated into the pLB-T vector using the Fast Ligation Kit (TIANGEN). The products were transformed into competent cells (E. coli Top10, TIANGEN). Positive colonies were selected by PCR using gene-specific primers and custom sequenced at Sangon Biotech, Shanghai, China. The signal peptide of PsauPBP3 was predicted with SignalP-6.0.

2.3. Real-Time Quantitative PCR

RT-qPCR was performed to evaluate the transcript levels of PsauPBP3 in chemosensory organs of P. saucia. Operations were carried out following the manufacturer’s instructions for the SuperReal PreMix Plus kit (TIANGEN) using the CFX384 Touch Real-Time PCR Detection System (Bio-Rad, United States). The reaction conditions were set as: one cycle of 95 °C for 15 min and 40 cycles of 95 °C for 10 s and 62 °C for 25 s. The P. saucia β-actin gene (Psauactin, GenBank number: OQ472022.1) was selected as the control gene and used for normalizing target gene expression. Relative transcript levels of PsauPBP3 were calculated using the 2^−ΔΔCt method. Three biological replicates were set for each tissue, and each biological replicate was operated three times (three technical replicates). Tukey’s multiple comparison test after one-way ANOVA was used to ascertain statistical differences for transcript levels of the target gene in different tissues. All the primers are listed in Table S1.

2.4. Expression, Purification, and Fluorescence Binding Assay

For the expression of recombinant PsauPBP3, its coding region was amplified by PCR with specific primers preceded by NdeI (forward) and EcoRI (reverse) restriction sites. The PCR product was ligated into the pLB-T vector (TIANGEN) and transformed into E. coli Top10 cells. pLB-T plasmids containing the PsauPBP3 sequence were extracted with the TIANprep Mini Plasmid Kit (TIANGEN) and then digested with NdeI and EcoRI restriction enzymes for 2–3 h at 37 °C. The digestion product was purified from the agarose gel using the TIANquick MiDi Purification Kit (TIANGEN), then ligated into the expression vector pET28a (Novagen, Darmstadt, Germany). The ligated products were used to transform BL21 E. coli cells. PsauPBP3 expression was achieved by the addition of IPTG to a final concentration of 0.4 mM when OD₆₀₀ of the E. coli culture had reached 0.6–0.8. The cells were cultured for a further 4 h at 37 °C, then collected by centrifugation and sonication. Purification of the recombinant PsauPBP3 was accomplished along with standard protocols of His-tag purification using magnetic beads (BeaverBeads, IDA-Nickel) (Beaver, Suzhou, China). The protein was renatured through dissolving with urea solutions and centrifugation with ultrafiltration tubes (Amicon Ultra, Merck Millipore, Darmstadt, Germany).

For the fluorescence binding assay, the fluorescence spectra were recorded on a Hitachi F-4500 at 25 ± 2 °C. The protein was dissolved in Tris-HCl buffer (50 mM, pH 7.4), while the tested compounds were added as 1 mM methanol solutions. N-Phenyl-1-naphthylamine (1-NPN) is the most widely used fluorescent probe in binding experiments with insect OBPs. When excited at 337 nm in water, this compound exhibits weak emission with a peak at approximately 502 nm. The main binding force for 1-NPN arises from π-π stacking with aromatic residues in OBPs. Upon binding to an OBP, 1-NPN shows a several-fold increase in emission intensity accompanied by a spectral shift to 407–412 nm. Using this probe with different odorant compounds enables measurement of ligand binding affinities through competitive binding assays [17]. To determine the affinity of 1-NPN for PsauPBP3, a 2 µM protein solution was titrated with aliquots of 1 mM 1-NPN to a final probe concentration of 16 µM. The system was excited at 337 nm, and emission spectra were recorded between 380 and 450 nm. Binding parameters, including the dissociation constant for 1-NPN and the binding stoichiometry, were derived by processing the data with GraphPad Prism software. To analyze the binding affinities of PsauPBP3, a panel of seven moth sex pheromones and 21 host plant volatiles was used as competitors in the competitive binding assay. A solution of the protein and the probe, both at a concentration of 2 µM, was titrated with 1 mM methanol solutions of competitors at a concentration of 8 µM. Detailed information on these compounds is listed in Table S2. Dissociation constants of the tested compounds were calculated from the corresponding IC₅₀ values, using the equation Kd = [IC₅₀]/(1 + [1-NPN]/K_1-NPN). All the data were processed with GraphPad Prism 10.

2.5. Three-Dimensional (3D) Modeling and Molecular Docking

The 3D structure of PsauPBP3 was predicted with AlphaFold3. The 3D structures of Z9-14: Ac and Z11-16: Ac were downloaded from the PubChem database, and their energies were minimized under the MMFF94 force field. Molecular docking was performed using AutoDock Vina 1.1.2 software. Prior to docking, the targeted protein was hydrogenated with PyMOL 2.5. Docking boxes were subsequently set up using PyMOL to encapsulate active pockets. All processed small molecules and the protein were then converted to AutoDock Vina 1.1.2-compatible PDBQT format using ADFRsuite 1.0.

2.6. MD Simulation

The MD simulations were conducted using the AMBER 18 software package [33]. The charges of sex pheromone molecules were calculated with the antechamber module and Gaussian 09 software. The sex pheromone molecules and the protein were modeled using the GAFF2 small molecule force field and the ff14SB protein force field, respectively. All systems were hydrogen-atomized using the LEaP module, with a truncated octahedral TIP3P solvent box added 10 Å away from the system. Na⁺/Cl⁻ was introduced to balance the system’s charge, followed by outputting topology and parameter files for the simulations. Prior to simulation, energy optimization was performed through 2500 steps of steepest descent and 2500 steps of conjugate gradient optimization. After optimization, the system underwent a 200 ps heating process at a fixed volume and constant heating rate, gradually raising the temperature from 0 K to 298.15 K. A 500 ps NVT (isothermal-isobaric) system state was performed to homogenize solvent distribution. Finally, a 500 ps NPT (isothermal–isobaric–temperature) equilibrium was conducted for the entire system. Two composite systems underwent 200 ns of NPT MD simulations under periodic boundary conditions. During simulation, the non-bonded cutoff distance was set to 10 Å. The Particle Mesh Ewald method was employed to compute long-range electrostatic interactions, the SHAKE method for hydrogen bond length constraints, and the Langevin algorithm for temperature control. The system pressure was maintained at 1 atm, using an integration time step of 2 fs. Trajectories were saved every 10 ps for subsequent analyses.

2.7. Per-Residue Free Energy Decomposition and Computational Alanine Scanning

Binding free energies between PsauPBP3 and the candidate ligands were calculated using the MM/GBSA method. In this study, 200 ns molecular dynamics trajectories were applied for calculations with the following formula:

∆G_bind = ∆G_complex − (∆G_receptor + ∆G_ligand)

=ΔE_vdW + ΔE_elec + ΔG_GB + ΔG_SA

In the equation, ΔE_vdW represents van der Waals interactions, and ΔE_elec indicates electrostatic interactions. ΔG_GB and ΔG_SA refer to solvation free energies. ΔG_GB stands for polar solvation free energy, while ΔG_SA denotes non-polar solvation free energy. ΔG_GB was calculated based on the GB model [34]. ΔG_SA was calculated as ΔG_SA = 0.0072 × ΔSASA, based on the surface tension and solubility surface area (SA).

For computational alanine scanning (CAS), the calculation was repeated based on the same principle of the aforementioned MM/GBSA method to obtain the difference in binding energy before and after mutation. Mutation energy was calculated with the formula ∆∆G_mutation = ∆G_{bind (mutant)} − ∆G_{bind (WT)}, where ∆∆G_mutation means mutation energy, and ∆G_{bind (mutant)} and ∆G_{bind (WT)} are the binding free energies in mutated and wild type systems, respectively. If ∆∆G_mutation > 0.5, this mutation will cause a weakened interaction and decreased affinities; if ∆∆G_mutation < −0.5, the mutation will give rise to an increase in interactions and affinities; if the value of ∆∆G_mutation is between −0.5 and 0.5, the mutation has no effect on the affinities.

2.8. Site-Directed Mutagenesis and Fluorescence Binding Assay

According to the CAS result, five PsauPBP3 mutants (T10A, F13A, I53A, I95A, and F119A) were prepared following the manufacturer’s instructions of the Hieff Mut Site-Directed Mutagenesis Kit (Yeasen, Shanghai, China). Expression, purification, and fluorescence binding assays of the mutants were performed as described for the wild-type PsauPBP3. The primers are listed in Table S1.

3. Results

3.1. Expression Profiles of PsauPBP3

The open reading frame of PsauPBP3 (GenBank: WKF45277.1) was obtained through reverse transcription-PCR with gene-specific primers (Table S1). Sequence analyses showed that the open reading frame of PsauPBP3 is 495 bp, encoding 164 amino acids, and the predicted signal peptide contains 22 amino acid residues (Figure S1A). The predicted molecular weight of the mature PsauPBP3 is 16.4 kDa with an isoelectric point of 5.32. The mature PsauPBP3 has a predicted molecular weight of 16.4 kDa and an isoelectric point of 5.32. Multiple sequence alignments further revealed that moth PBP3s (including PsauPBP3) exhibit the six-cysteine signature, forming the typical motif: C1–X₂₅–₄₀–C2–X₃–C3–X₃₅–₄₂–C4–X₈–₁₅–C5–X₈–C6 (Figure S1B). PsauPBP3 shares an identity of 57.76–88.41% with other moth PBP3s, and the highest is 88.41% with AipsPBP3 of Agrotis ipsilon.

To investigate the expression profile of PsauPBP3 in P. saucia, we measured its transcript levels in different chemosensory tissues, including antennae, proboscises, tarsi, wings, ovipositors, and hairbrushes. RT-qPCR results showed that PsauPBP3 was expressed specifically in the antennae. Additionally, transcript levels of PsauPBP3 were significantly higher in male antennae than female antennae (p < 0.0001) (Figure 1).

3.2. Virtual Screening Based on Binding Free Energy Calculation

To determine the binding properties of PsauPBP3, an odorant library comprising 28 compounds was assembled. This library included host plant volatiles [35,36,37], sex pheromone components from P. saucia [4,5], and sex pheromones from several other noctuid species [38,39,40], which were pooled for affinity assays (Table S2). The binding properties of PsauPBP3 to the 28 odorants were first measured based on their binding free energies (ΔG_bind) with the MM/GBSA method. The results indicated that four odorants, i.e., Z9-14: Ac, Z11-16: Ac, Z9-16: Ac, and Z11-16: OH, act as potential ligands with ΔG_bind < −20.00 kcal·mol⁻¹. Notably, the strongest binding affinities were observed with Z9-14: Ac (−41.74 kcal·mol⁻¹) and Z11-16: Ac (−47.68 kcal·mol⁻¹). Energy component analysis reveals that van der Waals interactions (ΔE_vdW) predominantly contribute to the binding energies, with respective values of −43.61 kcal·mol⁻¹ for Z9-14:Ac and −50.95 kcal·mol⁻¹ for Z11-16:Ac. Non-polar solvation free energy (ΔG_SA) constitutes the secondary contribution, followed by electrostatic interactions (ΔE_elec) and polar solvation free energy (ΔG_GB). Similar energy contribution profiles were observed in all protein-ligand complexes involving the 28 compounds (Table S3).

3.3. In Vitro Binding Assays

Fluorescence binding assays were further performed to better clarify affinities of the candidate odorants with PsauPBP3. PsauPBP3 was recombinantly expressed in a bacterial system and purified (Figure 2A). To assess the binding ability of PsauPBP3, we first measured its affinity to the fluorescent probe 1-NPN. The results showed that 1-NPN bound PsauPBP3 with an isotherm indicating a single binding site with a dissociation constant of 4.32 µM. Affinities of other ligands were then evaluated in competitive-binding experiments. Among the tested odorants, PsauPBP3 displayed the highest affinities (Kd < 5 μM) with Z11-16: Ac (Kd = 3.36 μM) and Z9-14: Ac (Kd = 3.55 μM). It also exhibited minor affinities to alcohol and aldehyde pheromones from other moth species, i.e., Z9-16: Ac, Z11-16: OH, Z11-16: Ald, Z9-16: Ald, and Z9-16: OH (Kd > 5 μM) (Figure 2B,C, Table S4).

3.4. Structural Dynamics Analysis

A 200 ns MD simulation was performed to determine the structural dynamics in the binding of PsauPBP3 to Z9-14: Ac and Z11-16: Ac. Molecular dynamics simulations revealed that the PsauPBP3 maintained structural stability, with RMSD values consistently ranging between 1.0 and 1.5 Å throughout the simulation trajectory. While the Z9-14: Ac molecule exhibited fluctuations, with the highest RMSD value reaching up to 3.0 Å. The PsauPBP3/Z9-14: Ac complex showed a rapid RMSD increase to 2.0–2.5 Å at the initial phase and then stabilized. For PsauPBP3/Z11-16: Ac, the protein maintains stable RMSD values between 1.0 and 1.5 Å. The Z11-16: Ac molecule shows an RMSD range within 1.0–1.5 Å, exhibiting less fluctuation than Z9-14: Ac. The PsauPBP3/Z11-16: Ac complex shows a gradual RMSD increase during the first 50 ns before stabilizing at 2.0 Å, with smaller fluctuations compared to the PsauPBP3/Z9-14: Ac complex (Figure 3A,B).

The residue fluctuation of these two complexes is shown in Figure 3C,D. For Z9-14: Ac, the overall root mean square fluctuation (RMSF) fluctuates between 0.5 and 1.0 Å, with most regions showing good conformational stabilities. Notably, the N-terminal and C-terminal regions, especially the C-terminal region (from approximately > 140 aa), exhibit larger fluctuations with an RMSF value exceeding 2.0 Å. The core residues in the ligand binding region (roughly distributed in the range of 10–120 amino acids) exhibit smaller fluctuations, indicating their involvement in forming a stable binding pocket. For Z11-16: Ac, the overall fluctuation trend mirrors that of Z9-14: Ac, with RMSF values concentrated between 0.5 and 1.0 Å. Also, the C-terminal region (>140 aa) demonstrates flexibility with RMSF approaching 2.0 Å.

3.5. Prediction of Key Amino Acid Residues

Molecular docking was conducted to predict key amino acid residues in the ligand binding of PsauPBP3. The docking score for Z9-14: Ac and Z11-16: Ac is −6.903 kcal·mol⁻¹ and −7.327 kcal·mol⁻¹, respectively. For Z9-14: Ac, the ligand forms hydrophobic interactions with Phe-13, Leu-34, Trp-38, Ile-53, Leu-63, Ile-95, Val-112, Ala-116, and Phe-119. Additionally, Z9-14: Ac forms hydrogen bonds with Ser-57, enhancing its positioning accuracy and stability within the binding pocket. Similarly, Z11-16: Ac forms multiple hydrophobic interactions with PsauPBP3, involving key amino acid residues including Ile-9, Phe-13, Trp-38, Ile-53, Phe-77, Ile-95, Val-115, Ala-116, and Phe-119 (Figure 4 and Figure S2). No hydrogen bonds were detected between Z11-16: Ac and the protein. For both ligands, although the two uncharged polar amino acids Thr-10 and Tyr-37 were predicted to be key residues in the binding site, the binding stability of PsauPBP3 is primarily maintained through hydrophobic interactions, indicating a hydrophobic-driven binding mode.

To ensure prediction accuracy, another computational approach, i.e., per-residue energy distribution, was also employed to identify key amino acid residues contributing to binding affinities. Analysis of the top ten amino acid residues contributing to ΔG_bind revealed consistently negative binding energy values in both PsauPBP3/Z9-14: Ac and PsauPBP3/Z11-16: Ac complexes. Subsequently, residues Phe-13, Ile-53, Met-69, Ile-95, and Phe-119 contributed binding energies < −1.0 kcal·mol⁻¹ in both complexes, with Phe-13 and Ile-95 exhibiting the most significant contributions (

Table 1. Prediction of key amino acid residues in the binding of PsauPBP3 to Z9-14: Ac and Z11-16: Ac with molecular docking and MD simulation.

Ligand	Key Amino Acid Residues *
	Molecular docking	MD simulation
Z9-14: Ac	Thr-10, Phe-13, Leu-34, Tyr-37, Trp-38, Ile-53, Ser-57, Leu-63, Ile-95, Val-112, Ala-116, Phe-119	Ile-9, Thr-10, Phe-13, Ile-53, Leu-62, Met-69, Ile-95, Leu-110, Val-112, Phe-119
Z11-16: Ac	Ile-9, Thr-10, Phe-13, Tyr-37, Trp-38, Ile-53, Phe-77, Ile-95, Val-115, Ala-116, Phe-119	Ile-9, Thr-10, Phe-13, Ile-53, Ile-54, Met-69, Ile-95, Val-112, Ala-116, Phe-119

* Identical residues predicted in both methods are underlined.

, Figure 5).

3.6. CAS and Site-Directed Mutagenesis

Five identical amino acid residues (Thr-10, Phe-13, Ile-53, Ile-95, and Phe-119) were screened out based on comparison of molecular docking and MD simulation results. We inferred that these residues may play a pivotal role in the binding of PsauPBP3 to both sex pheromones (Table 1, Figure 4 and Figure 5). Therefore, we selected these residues as the target for CAS analysis. The result showed that alanine substitution of these residues resulted in a mutation energy (∆∆G_mutation) of >0.5 kcal·mol⁻¹, with ∆∆G_mutation values of 0.98 (Thr-10 > Ala), 2.98 (Phe-13 > Ala), 1.59 (Ile-53 > Ala), 1.85 (Ile-95 > Ala), and 3.96 (Phe-119 > Ala) kcal·mol⁻¹ for PsauPBP3/Z9-14: Ac; and 0.68 (Thr-10 > Ala), 2.10 (Phe-13 > Ala), 1.41 (Ile-53 > Ala), 2.16 (Ile-95 > Ala), and 3.55 (Phe-119 > Ala) kcal·mol⁻¹ for PsauABPX/Z11-16: Ac (

Table 2. Computational alanine scanning for the selected residues based on molecular docking and MD simulation.

Complex	Mutation	Mutation Energy (kcal·cal−1)
PsauPBP3/Z9-14: Ac	Thr-10 > Ala	0.98
	Phe-13 > Ala	2.98
	Ile-53 > Ala	1.59
	Ile-95 > Ala	1.85
	Phe-119 > Ala	3.96
PsauPBP3/Z11-16: Ac	Thr-10 > Ala	0.68
	Phe-13 > Ala	2.10
	Ile-53 > Ala	1.41
	Ile-95 > Ala	2.16
	Phe-119 > Ala	3.55

).

According to the CAS result, site-directed mutagenesis of Thr-10, Phe-13, Ile-53, Ile-95, and Phe-119 was conducted to verify their involvement in the binding of PsauPBP3 to Z9-14: Ac and Z11-16: Ac. We substituted these amino acid residues with alanine to delete the active group on the side chain, considering the methyl group of alanine is relatively small and exerts negligible influence on the protein conformation. Each PsauPBP3 mutant was expressed and purified (Figure 6A,B; Table S1) for subsequent fluorescence binding assays. However, recombinant expression of the Ile-53 mutant (I53A) failed despite testing multiple plasmids and induction conditions. For the successfully expressed mutants, the dissociation constants (Kd) with fluorescent probe 1-NPN were: 4.9 μM (T10A), 6.5 μM (F13A), 6.4 μM (I95A), and 5.5 μM (F119A) (Figure 6C). According to our molecular docking results, the key residues in the protein binding cavity for 1-NPN are Phe-13, Tyr-37, Ile-53, Ile-95, and Ala-116 (Figure S3). Compared with the key residues involved in sex pheromone binding, two residues (Phe-13 and Ile-95) are shared between the two binding pockets. The lack of significant impact from the F13A or I95A mutation on 1-NPN binding affinity may be due to the fact that binding is maintained by the other three residues. Alternatively, other aromatic amino acids (such as Tyr-37) could compensate for the loss of Phe-13 or Ile-95 to preserve affinity. Finally, the binding affinities of each mutant to Z9-14: Ac and Z11-16: Ac were analyzed. The competitive binding curves showed that the initial fluorescence values (100%) of 1-NPN dropped by only about 30% for mutants T10A and F119A and showed no decrease for mutants F13A and I95A, even when the concentration of the sex pheromones reached 8 μM (Figure 6D), demonstrating a marked loss in binding capacity of these mutants for both sex pheromones.

4. Discussion

Despite the indefinite history and major economic importance of P. saucia, biological control strategies for this pest remain limited. Functional and binding dynamics analyses of its PBPs could pave the way for novel management approaches by disrupting mating behaviors.

In expression profile analyses across different chemosensory tissues, PsauPBP3 was exclusively detected in antennae, indicating its involvement in olfaction. This finding aligns with observations of PBP3s in other moth species [41,42]. For instance, HcunPBP3 was exclusively expressed in the antennae of Hyphantria cunea [42]. In contrast, CsasPBP3 was also detected in the tarsi and wings of Carposina sasakii beyond its antennal expression [43]. Similarly, PxylPBP3 in Plutella xylostella showed expression not only in antennae but also in female reproductive organs and male legs [27]. HarmPBP3 in H. armigera also exhibits expression in wings as detected by immunostaining experiments [26]. These discrepancies may reflect functional differentiation of PBP3s during the evolution of distinct moth species.

As carriers transporting odorant molecules to ORN membranes, OBPs rely critically on ligand-binding properties for olfactory function [17,20]. To date, PBP3s have been functionally characterized in diverse moth species. Competitive binding assays showed that PxylPBP3 in P. xylostella could discriminate Z11-16: Ac from Z11-16: Ald [28]. However, in vitro binding assays found that CsupPBP3 in Chilo suppressalis had high affinities for the female sex pheromones Z11-16: Ald, (Z)-13-octadecenal (Z13-18: Ald), and Z9-16: Ald and showed no obvious selectivity among the three components [44]. Such non-selectivity was also reported in ligand binding assays of other moths, such as Spodoptera exigua [45] and Ostrinia furnacalis [46]. Our findings demonstrate that PsauPBP3 exhibits high binding affinity for both sex pheromone components of P. saucia, aligning with previous reports on PBP3s. Thus, moth PBP3s, at least in certain species, lack precise discriminatory capacity for distinct pheromone components. The task of distinguishing sex pheromone components is suggested to be accomplished by specific ORs [47,48,49]. Except for the female sex pheromones Z11-16: Ac and Z9-14: Ac, PsauPBP3 also showed moderate binding affinities for the alcohol and aldehyde pheromones Z11-16: Ald, Z9-16: Ald, Z11-16: OH, and Z9-16: OH, which were identified as sex pheromones from other moth species [38,39,40]. Similar binding characteristics have been documented in H. armigera and H. assulta PBP3s. Both HarmPBP3 and HassPBP3 exhibit high binding affinity not only to their respective sex pheromones (Z11-16: Ald and Z9-16: Ald) but also to Z11-16: Ac and Z9-16: Ac [26]. This functional versatility suggests that PBP3s facilitate detection of interspecific sex pheromones in addition to conspecific signals, potentially enabling interspecies differentiation. Nevertheless, additional future studies are needed to substantiate this theory.

In this study, PsauPBP3 exhibited no binding affinity to host plant volatiles. Similar findings were reported in studies of other moth species, including H. armigera, H. assulta, C. sasakii, and P. xylostella, where PBP3s bound exclusively to sex pheromones but not to host plant volatiles [26,28,43]. While a study in H. cunea reported that HcunPBP3 has high binding affinities to (E)-2-hexenal and β-ocimene [42]. Such divergence indicated that PBP3s in different moth species may differ in their functions. Further intensive studies, such as in vivo gene knockout using the CRISPR/Cas9 system, are needed to demonstrate the functions of PBP3s. Furthermore, we cannot rule out the possibility that the odorant library constructed in our study may omit the odorant compounds potentially detected by PsauPBP3.

Molecular dynamics (MD) simulation is a powerful tool for revealing atomic-level structural mechanisms. Its reliability derives from the computational accuracy of force fields in describing interatomic interactions [33]. For example, computational investigations using MD simulations to uncover binding mechanisms have successfully characterized the OBPs of Grapholita molesta [50] and Athetis lepigone [51]. In this study, molecular dynamics analyses revealed conformationally stable complexes of PsauPBP3 with both Z11-16: Ac and Z9-14: Ac, as evidenced by RMSD and RMSF profiles. Comparative data indicated marginally enhanced structural stability in the PsauPBP3/Z11-16: Ac complex, consistent with competitive fluorescence binding assays showing slightly higher affinity for Z11-16: Ac than for Z9-14: Ac. Energy decomposition analysis identified van der Waals interactions as the primary binding energy contributor. Furthermore, the per-residue energy distribution reveals hydrophobic residues as key participants, indicating the ligand was embedded within a hydrophobic binding cavity of PsauPBP3. Overall, the two ligands exhibit high overlaps in key binding sites in PsauPBP3, particularly hydrophobic residues.

It is well established that genes are transcribed and translated into specific sequences of proteins, which are then folded and modified to bind with the corresponding ligands [52]. Structural changes in key amino acid residues during protein-ligand interactions usually give rise to structural alteration and induce functional destruction of proteins [53,54,55]. Investigating key amino acid residues’ contributions to OBP binding affinities is critically important. Molecular docking combined with site-directed mutagenesis has been employed in studies to identify residues involved in odorant ligand binding. Using this method, five amino acid residues were demonstrated to be associated with the binding affinities of AlepOBP6 to distinct ligands in Athetis lepigone [56]. Similarly, ten putatively crucial amino acid residues are involved in the binding of TabsPBP3 to the sex pheromone (3E, 8Z, 11Z)-tetradecatrien-1-yl acetate in Tuta absoluta [57]. Recently, a novel structure–function approach, i.e., per-residue energy distribution analysis combined with CAS and/or site-directed mutagenesis, has been successfully applied to identify key amino acid residues in insect OBP binding pockets. For example, per-residue energy distribution analysis and experimental studies identified Phe-12, Ile-52, Ile-94, and Phe-118A as critical residues for AlepGOBP2 binding to the insecticides chlorpyrifos and phoxim [58]. Using the same methodology, Phe-34, Tyr-37, Trp-38, and Arg-111 were identified as essential residues for Z11-16: Ac binding in P. xylostella PxylPBP3 [28]. To ensure prediction accuracy, we combined both methods to identify key ligand-binding residues in PsauPBP3. Five amino acid residues, i.e., Thr-10, Phe-13, Ile-53, Ile-95, and Phe-119, were predicted to be crucial in the ligand-binding of PsauPBP3 to Z11-16: Ac and Z9-14: Ac. This finding was further validated by the site-directed mutagenesis combined with fluorescence binding assays. Mutations of four residues into alanine caused a decrease (Thr-10 and Phe-119) or abolishment (Phe-13 and Ile-53) of the binding affinities of PsauPBP3 to both sex pheromones. The failed expression of the I53A mutant may result from the critical role of Ile-53 in maintaining the protein’s structural stability. A mutation at this site could disrupt folding, primarily as this residue presumably resides in the hydrophobic core region. Its hydrophobicity and specific side-chain structure are crucial for maintaining tight packing and overall core stability. Even conservative mutations, such as replacing leucine with alanine (another nonpolar amino acid), can weaken van der Waals interactions due to altered side-chain volume or shape. Additionally, Ile-53 may stabilize local structures or influence folding kinetics. Deciphering the binding mechanisms of PsauPBP3 with female sex pheromones provides a theoretical foundation for developing novel semiochemicals (e.g., pheromone-based mimetics targeting key binding sites) to disrupt P. saucia mating behaviors.

5. Conclusions

Data from this study demonstrate that PsauPBP3 exhibits male antennal-biased expression. Competitive fluorescence binding assays and molecular dynamics simulations confirmed its high binding affinity for female sex pheromone components. Structural analyses identified Phe-13 and Ile-95 as critical residues for binding specificity. These findings elucidate the binding mechanisms of moth PBP3s and provide a foundation for developing novel P. saucia control strategies.