insects Article The Crystal Structure of the Spodoptera litura Chemosensory Protein CSP8 Qian Jia 1 , Hui Zeng 1, Jinbing Zhang 1, Shangfang Gao 1 , Nan Xiao 1, Jing Tang 1, Xiaolin Dong 2,* and Wei Xie 1,* 1 MOE Key Laboratory of Gene Function and Regulation, State Key Laboratory for Biocontrol, School of Life Sciences, Sun Yat-Sen University, Guangzhou 510006, China; [email protected] (Q.J.); [email protected] (H.Z.); [email protected] (J.Z.); [email protected] (S.G.); [email protected] (N.X.); [email protected] (J.T.) 2 Forewarning and Management of Agricultural and Forestry Pests, Hubei Engineering Technology Center, Yangtze University, Jingzhou 434025, China * Correspondence: [email protected] (X.D.); [email protected] (W.X.); Tel.: +86-716-806-6314 (X.D.); +86-203-933-2943 (W.X.) Simple Summary: Worldwide, pest control involves extensive use of insecticides, which results in serious environmental pollution problems. On the other hand, insecticides can be recognized by proteins named CSPs in insects, which allow them to accurately respond to these environmental chemical signals for their survival, but the mechanism is poorly studied. Here, we report the crystal structure of the CSP8 protein from the tobacco cutworm Spodoptera litura, a major plant pest in Asia. We also studied its binding properties to compounds like rhodojaponin III, a non-volatile plant metabolite. Our studies showed that the protein binds to these molecules with different affinities and provided important insight into the molecular recognition mechanism of the sensory protein SlCSP8 and the CSP protein family in general. Citation: Jia, Q.; Zeng, H.; Zhang, J.; Gao, S.; Xiao, N.; Tang, J.; Dong, X.; Abstract: Spodoptera litura F. is a generalist herbivore and one of the most important economic pests Xie, W. The Crystal Structure of the feeding on about 300 host plants in many Asian countries. Specific insect behaviors can be stimulated Spodoptera litura Chemosensory after recognizing chemicals in the external environment through conserved chemosensory proteins Protein CSP8. Insects 2021, 12, 602. (CSPs) in chemoreceptive organs, which are critical components of the olfactory systems. To explore https://doi.org/10.3390/ its structural basis for ligand-recognizing capability, we solved the 2.3 Å crystal structure of the insects12070602 apoprotein of S. litura CSP8 (SlCSP8). The SlCSP8 protein displays a conserved spherical shape with Academic Editor: Ian M. Scott a negatively charged surface. Our binding assays showed that SlCSP8 bound several candidate ligands with differential affinities, with rhodojaponin III being the most tightly bound ligand. Our Received: 18 May 2021 crystallographic and biochemical studies provide important insight into the molecular recognition Accepted: 28 June 2021 mechanism of the sensory protein SlCSP8 and the CSP family in general, and they suggest that CSP8 Published: 1 July 2021 is critical for insects to identify rhodojaponin III, which may aid in the CSP-based rational drug design in the future. Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in Keywords: crystal structure; CSP; rhodojaponin III; structure-function relationship; ligand-binding published maps and institutional affil- specificity iations. 1. Introduction Copyright: © 2021 by the authors. Worldwide, pest control involves the extensive use of insecticides. The last three Licensee MDPI, Basel, Switzerland. decades have witnessed great advantages and successes in food production, but the nega- This article is an open access article tive consequences of these chemicals used at large scales can never be underestimated. The distributed under the terms and extensive use of synthetic, broad-spectrum pesticides has the potential hazardous effects conditions of the Creative Commons on the environment, human health, and so on [1]. Attribution (CC BY) license (https:// CSP creativecommons.org/licenses/by/ The gene was first discovered in 1994 and named factory-specific protein D 4.0/). (OS-D) [2]. Later, Angeli et al. found a class of proteins mediating chemical pheromone Insects 2021, 12, 602. https://doi.org/10.3390/insects12070602 https://www.mdpi.com/journal/insects Insects 2021, 12, 602 2 of 12 recognition in Schistocerca gregaria, which they renamed chemosensory proteins (CSPs) [3]. CSPs are present in chemoreceptive organs and can bind a range of aliphatic compounds, esters, and other long-chain compounds [2,4] to stimulate specific insect behaviors. CSPs consist of 100–115 amino acids with molecular weights of ~13 kDa. They are highly stable and are evolutionarily conserved except for their C-termini, suggesting that they play essential roles in insects’ lives. For example, the expression levels of some CSP proteins were significantly upregulated in the abdomen of Bombyx mori when it was treated with sublethal concentrations of avermectin [5]. Similar observations were obtained when Terthron albovittata was treated with thiamethoxam [5]. Furthermore, the expression levels of three CSP proteins (CSP4, CSP8, and CSP3) were upregulated in the head of Plutella xylostella in the presence of pyrethroids [6]. In addition, the expression of CSP2 in Lepidoptera was significantly increased in the presence of rhodojaponin III, a non-volatile secondary metabolite of plants [7]. Conversely, after blocking the CSP2 expression by RNAi, insects mistakenly laid eggs on rhodojaponin III-treated azalea, suggesting that CSP2 is implicated in identifying non-volatile metabolites in plants [8]. The approach to understanding the role of CSPs in the chemoreception process relies on a detailed knowledge of their three-dimensional structures and their binding modes, and possible conformational changes induced by the formation of complexes with their ligands [9]. The first structure of CSP (MbCSPA6) was reported from Mamestra brassi- cae (moth) [10,11]. Overall, MbCSPA6 is a globular protein and consists of six α-helices named helices A-F. A hydrophobic slit is formed in the center of the helices. Four con- served cysteines (Cys29-Cys36 and Cys55-Cys58) form two disulfide bonds and maintain a stable tertiary structure. Following the success on the apoprotein, the crystal struc- ture of MbCSPA6 in complex with 12-bromododecanol was determined by Campanacci et al. in 2003 (PDB 1N8V) [12]. Significant conformational changes were found when 12-bromododecanol was bound. In order to accommodate the large ligand molecule, the binding cavity increased its volume and the position of each helix changed to various degrees. Of note, helix C displayed the most dramatic structural rearrangements and was pushed out by ~5 Å. Then the structures of Bombyx mori CSP1 and Schistocerca gre- garia CSP4 were solved, respectively [9,13]. No further structures of CSPs were described and documented. Although several structures of CSPs have been reported, our understanding of the ligand recognition and the binding modes by CSPs is still limited, given the large variety of CSPs. In addition, we are unclear about the molecular mechanism behind their physio- logical functions and therefore unable to carry out rational designs of pesticides due to a lack of structural information. Our previous study showed that rhodojaponin III might be a potential ligand of CSP [7,8]. To further study the structure–activity relationships of CSP, here we report the structure of CSP8 from S. litura (SlCSP8) and intended to provide evi- dence of its potential ligands. By gaining a better understanding of the olfactory system of S. litura, we expect to provide a new theoretical basis for the development and applications of novel insecticides. 2. Materials and Methods 2.1. Expression and Purification of SlCSP8 Total RNA of S. litura was isolated from twenty individual adults using Trizol ac- cording to the manufacturer’s specifications (Invitrogen, Carlsbad, CA, USA). First-strand cDNA was synthesized with the first-strand synthesis kit using Reverse transcriptase M-MLV (RNase H) (TaKaRa, Shiga, Japan). Briefly, 1 µg of total RNA, 1µL of Oligo ◦ (dT) primer (50 mM), and RNase-free deionized H2O were mixed, incubated at 70 C for 10 min, and chilled on ice for 2 min immediately. Then, 0.5 µL of RTase M-MLV (RNase H), 2 µL of 5× M-MLV buffer, 0.5 µL of dNTP mixture (10 mM each), 0.25 µL RNase inhibitor were added to a final volume of 10 µL. The reaction mix was incubated at 42 ◦C for 60 min, 70 ◦C for 15 min, and cooled on ice. Then, the cDNA was stored at −20 ◦C. The gene encoding SlCSP8 was amplified by PCR from the cDNA of Spodoptera Insects 2021, 12, 602 3 of 12 litura using the primers 50-GCGGCAGCGGATCCGATGAAATTCGTACTAGTATTGTG-30 and 50-TTGCACTTCTCGAGTTCTGGGATGACGATGCCGTT-30, respectively. The primer sequences contain the protective bases and restriction enzyme sites. After the double diges- tion by the restriction enzymes BamHI and XhoI, the PCR product was inserted into the pET-20b (+) (MerckMillipore) vector. The proteins expressed would possess a C-terminal 6× His tag. The D18-E128 fragment (SF) was subcloned in the same manner. The primers of the SF version are 50-GCGGCAGCGGATCCGGATGAAAAGTACCCTAGCAAGTA-30 and 50-TTGCACTTCTCGAGTTCTGGGATGACGATGCCGTT-30, respectively. The plasmid was transformed into the Escherichia coli strain BL21 (DE3) cells for overexpression. When OD600 reached 0.6, expression of the target protein was induced with 0.2 mM IPTG, and the temperature was decreased to 25 ◦C. Cells were harvested after 16-h induction. The E. coli cells were pelleted by centrifugation at 3500× g for 20 min. Then, the cells were resuspended in a solution containing 30 mM Tris-HCl pH 8.0, 20% sucrose, and 1 mm EDTA. The precipitation was obtained by centrifugation at 23,500× g for 10 min at 4 ◦C, while the supernatant was removed. Repeat this step once. The precipitation was completely redissolved in 5 mM MgSO4 solution and slowly stirred on ice for 10 min. At this time, periplasmic proteins were released.