Insect Molecular Biology (2015) 24(5), 528–538

doi: 10.1111/imb.12179

Three amino acid residues of an odorant-binding protein are involved in binding odours in Loxostege sticticalis L.

J. Yin*, X. Zhuang*, Q. Wang*, Y. Cao*, S. Zhang*, C. Xiao† and K. Li* *State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing, China; and †College of Plant Protection, Yunnan Agricultural University, Kunming, China Abstract Odorant-binding proteins (OBPs) play an important role in insect olfactory processes and are thought to be responsible for the transport of pheromones and other semiochemicals across the sensillum lymph to the olfactory receptors within the antennal sensilla. As an important general odorant binding protein in the process of olfactory recognition, LstiGOBP1 of Loxostege sticticalis L. has been shown to have good affinity to various plant volatiles. However, the binding specificity of LstiGOBP1 should be further explored in order to better understand the olfactory recognition mechanism of L. sticticalis. In this study, real-time PCR experiments indicated that LstiGOBP1 was expressed primarily in adult antennae. Homology modelling and molecular docking were then conducted on the interactions between LstiGOBP1 and 1-heptanol to understand the interactions between LstiGOBP1 and their ligands. Hydrogen bonds formed by amino acid residues might be crucial for the ligand-binding specificity on molecular docking, a hypothesis that was tested by site-directed mutagenesis. As predicted binding sites for LstiGOBP1, Thr15, Trp43 and Val14 were replaced by alanine to determine the changes in binding affinity. Finally, fluorescence assays revealed that the First published online 8 July 2015. Correspondence: Professor Kebin Li, State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, 2 West Yuanmingyuan Road, Beijing 100193, China. Tel./fax: 1 86 106281 5619; e-mail: [email protected]

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mutants Thr15 and Trp43 had significantly decreased binding affinity to most odours; in mutants that had two-site mutations, the binding to the six odours that were tested was completely abolished. This result indicates that Thr15 and Trp43 were involved in binding these compounds, possibly by forming multiple hydrogen bonds with the functional groups of the ligands. These results provide new insights into the detailed chemistry of odours’ interactions with proteins. Keywords: Loxostege sticticalis, odorant binding protein, bioinformatics, mutants, fluorescence assays.

Introduction Olfaction in insects plays important roles in chemical communication. Odorant binding proteins (OBPs) are the first protein family found to function in the olfactory system (Vogt & Riddiford, 1981; Vogt et al., 1991; Pelosi & Maida, 1995; Prestwich et al., 1995; Steinbrecht, 1998). These are small, acidic and soluble proteins that are most likely involved in the binding and transport of odours through the sensillum lymph to specific odorant receptors (ORs) localized in the membrane of olfactory neurones. In the last few years, more than 400 OBPs have been isolated and cloned from more than 40 insect species, belonging to eight different orders (Pelosi et al., 2006; Leal, 2013). Insect OBPs are perhaps best known in the order Lepidoptera. Although lepidopteran OBPs represent a group of closely related proteins, they appear to be divergent compared with those of other insect orders. Three subfamilies of lepidopteran OBPs are classified primarily on the basis of the homology of their amino acid sequences and their expression patterns. These subfamilies are the pheromone-binding proteins (PBPs), the general odorant-binding proteins (GOBPs) and the antennalbinding X homologue proteins (ABPXs) (Breer et al., 1994; Robertson et al., 1999; Krieger et al., 1996, 2005; C 2015 The Royal Entomological Society V

Three amino acid residues are involved in binding odours Pelosi et al., 2006; Kaissling, 2009), and these OBP subfamilies have different odorant-binding specificities. The PBPs are mostly male-specific, whereas two distinct classes of GOBPs are expressed in both males and females (Vogt et al., 1991; Pelosi et al., 2006). The lepidopteran GOBPs are thought to bind a wide range of odorants with broad specificity; however, several GOBPs have high binding activity to major pheromones in several insect species (Feng & Prestwich, 1997; MaibecheCoisne et al., 1998; Gong et al., 2009; He et al., 2010). ABPXs play a important role in insect olfactory, but until now, there is no detail report about its three-dimensional structure and function. Although many studies have demonstrated that OBPs are indispensable in the perception of olfaction in insects, the molecular mechanism of ligand–OBP interactions remains largely unknown. Structural studies at the molecular level have shed some light on the mode of action and binding specificity of OBPs. The 3D structure of the Bombyx mori PBP (BmorPBP) complexed with a molecule of bombykol, the sex pheromone of this species, was the first to be studied. The protein forms a pocket with helices a1, a4, a5 and a6, and at pHs below 6 the C-terminus acquires a a-helix and folds back into the binding pocket, whereas at pH 7.0, the helix withdraws from the binding pocket and makes it available for the pheromones (Wojtasek & Leal, 1999; Damberger et al., 2000; Sandler et al., 2000; Horst et al., 2001). Other OBPs with short length of their C-terminus, cannot form an additional helix to fill the binding cavity, but cover the binding pocket as a lid, which possibly affects the binding ability to specific ligands (Lartigue et al., 2003; Tegoni et al., 2004; Leite et al., 2009). The Culex quinquefasciatus provides another example. Although the CquiOBP1 binds to the oviposition pheromone MOP in a pH-dependent fashion, it lacks the C-terminus required for the pH-dependent binding/releasing model. CquiOBP binds to MOP in an brand new fashion using both a small central cavity for the lactone head group and a long hydrophobic channel for the tail (Mao et al., 2010). Therefore, different OBPs probably exhibit different ligand binding/releasing mechanisms. In recent years, some amino acid residues have been identified as the most critical residues for binding ligands (Sandler et al., 2000; Thode et al., 2008; Zhou et al., 2009; Jiang et al., 2009; Wang et al., 2013; Ahmed et al., 2014; Zhuang et al., 2014). The crystalline structure of BmorPBP bound to bombykol indicated that bombykol interacts with the binding cavity through numerous hydrophobic interactions (Sandler et al., 2000) and Ser56 in the binding pocket forms a specific hydrogen bond with bombykol (Lautenschlager et al., 2007); however, when bombykol is bound to BmorGOBP2, a hydrogen bond is formed with Arg110 rather C 2015 The Royal Entomological Society, 24, 528–538 V

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than with Ser56 (Zhou et al., 2009). The structure of the the pheromone-binding protein (LUSH) and the Drosophila pheromone (11-cis vaccenyl acetate, cVA) of Drosophila melanogaster complex (LUSHcVA) showed that cVA forms two polar interactions with Thr57 and Ser52 in the binding pocket (Laughlin et al., 2008). Lys123 in the C-terminus of HarmOBP7 formed hydrogen bonds with the functional groups of the ligands and is probably involved in binding these compounds (Sun et al., 2013). Loxostege sticticalis L. is one of the most important agricultural insect pests in the north of China, yet to date the characteristics and functions of only two OBPs have been reported for this species (Sun et al., 2011; Yin et al., 2012). The results from fluorescence binding assays with 50 related compounds clearly showed that the two LstiGOBPs had high binding affinities for plant volatiles, which suggests that LstiGOBPs might play a key role in olfactory chemoreception. Four odours, 1heptanol, cinnamic aldehyde, 1-hexanol and camphene, at 20 mM, replaced 50% N-phenyl-1-naphthylamine (1-NPN) from LstiGOBP1 (Sun et al., 2011; Yin et al., 2012). However, these results do not provide sufficient evidence for a better understanding of the olfactory recognition mechanism of L. sticticalis. The mode of action and the binding specificity of LstiGOBP1 should be further explored and would most likely offer some insight into the LstiGOBP1–ligand interaction. In this study, homology modelling and molecular docking were conducted on LstiGOBP1 and 1-heptanol, the highest affinity ligand in the 50 tested volatiles, to first predict the binding sites. Based on these results, we combined sitedirected mutagenesis and fluorescence binding assays to describe the binding sites and explore the ligandbinding mechanism. Results Expression pattern analysis of LstiGOBP1 We examined the expression pattern of LstiGOBP1 mRNA in different tissues with real-time quantitative PCR (qPCR). The desired product was largely amplified from cDNA templates that were reverse-transcribed from total RNA in male and female antennae with only a few derived from other tissues, indicating that GOBP1 was expressed specifically in the antennae (Fig. 1). In general, the levels of transcripts were very low in all tissues except the antennae, where LstiGOBP1 was highly expressed. 3D model of LstiGOBP1

From the BLAST search in the Protein Data Bank (PDB), five structurally determined OBPs, Bombyx mori OBP (BmorGOBP2), Amyelois transitella OBP (AtraPBP1),

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Figure 1. qPCR analysis of Loxostege sticticalis general odorant binding protein 1 expressed in different tissues. cDNAs were amplified with specific primers from antennae, thoraxes, abdomens, wings, legs and heads(without antennae).

Bombyx mori OBP (BmorPBP), Antheraea polyphemus (ApolPBP) and Drosophila melanogaster OBP (LUSH), were found with similar sequences to LstiGOBP1. With homology modelling, models are generated for a homologous sequence (target) that shares either a significant sequence (30% or more) or a structural similarity with the template (Guex & Peitsch, 1997; Schwede et al., 2003; Cavasotto & Phatak, 2009). In this study, the total sequence identity between the target protein (LstiGOBP1) and the template protein (BmorGOBP2) was 53% (Fig. 2A), and the root mean square deviation

(RMSD) was calculated with the formula: delta (A˚) 5 0.40 e1.87H. As described by Chothial & Lesk ˚ (0.2 with VERIFY-3D, whereas the verification score of the final LstiGOBP1 model with PROFILES-3D was 65.19, which was close to the expected high score of 60.13. Therefore, the predicted model was reasonable and reliable for LstiGOBP1. The superposition of the model and the template is shown in Fig. 2C. It indicates that the overall conformation of the modelling target was very similar to that of the template. The predicted 3D structure of LstiGOBP1 consisted of six a-helices located between residues 8– 29 (a1), 52–63 (a2), 76–84 (a3), 90–105 (a4), 113–131 (a5) and 138–141 (a6) (Fig. 2A). Five antiparallel helices (a1, a2, a3, a4 and a5) converged to form the hydrophobic binding pocket. The converging ends of the helices formed the narrow end of the pocket, and the opposite end of the pocket was capped by another helix, a6

Figure 2. 3D model of Loxostege sticticalis general odorant binding protein 1. (A) Sequence alignment between LstiGOBP1 and BmorGOBP2. (B) Threedimensional structure of LstiGOBP1. The N and C termini and the six helices are labeled and the three disulfide linkages are red stick representations. (C) Superimposed structures of BmorGOBP2 and LstiGOBP1. The model of LstiGOBP1 and crystal structures of BmorGOBP2 are shown in grass green and kermesinus, respectively. (D) An analysis of LstiGOBP1 by Pro-CHECK. C 2015 The Royal Entomological Society, 24, 528–538 V

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Figure 3. The binding pocket of Loxostege sticticalis general odorant binding protein 1 and the docking result with 1-heptanol. 1-Heptanol shown as a stick model with the carboxyl oxygen in red. (A) Tertiary structure of 1-heptanol. (B) The binding pocket of LstiGOBP1 and 1-heptanol docked into the active-site of LstiGOBP1 receptor. (C) Diagram (LIGPLOT) of the van der Waals interactions and hydrophobic interactions of 1-heptanol with key binding site residues. Residues shown as labeled drawings have a distance to 1-heptanol of less than 4 A˚, Val14 are shown as orange stick. (D) Hydrogen bonds in the Protein active area.

(Fig. 2B). Disulphide bonds and helix–helix packing reinforced the organization of the helices. Three pairs of disulphide bridges connected Cys25 in a1 and Cys60 in a2, Cys56 in a2 and Cys114 in a5, and Cys103 in a4 and Cys123 in a5 (Fig. 2B). In this model, most of the residues (methionine, tryptophan, phenylalanine, leucine and valine) that formed the pocket were hydrophobic. Only one hydrophilic residue (threonine) was present in the binding site. Molecular docking To further explore the characteristics of the LstiGOBP1 binding site, 1-heptanol (Fig. 3A) was chosen to dock with the predicted LstiGOBP1 model because of its strong binding affinity with LstiGOBP1 (Sun et al., 2011). AutoDock (http://autodock.scripps.edu/resources/ adt) molecular docking produced nine likely conformations. The best conformation of complex was determined by the docking score and the binding mode. The docking result is shown in Fig. 3B, and the orientations and conformations of 1-heptanol in the binding site were clear. Based on the docking study, hydrogen bonds were the C 2015 The Royal Entomological Society, 24, 528–538 V

main linkage between LstiGOBP1 and 1-heptanol (Fig. 3D). 1-heptanol formed hydrogen bonds separately with Thr15 and Trp43, and a hydrogen bond was formed between Thr15 and Trp43. These three hydrogen bonds acted as a holder that strongly fixed 1-heptanol in the binding site (Fig. 3D). However, van der Waals and hydrophobic interactions were the important linkages between LstiGOBP1 and 1-heptanol. All the residues with less than 4.0 A˚ distances to 1-heptanol are represented in Fig. 3C, namely, methionine11 (Met11), valine14 (Val14), threonine15 (Thr15), phenylalanine18 (Phe18), phenylalanine39 (Phe39), phenylalanine42 (Phe42), tryptophan43 (Trp43), leucine67 (Leu67), phenylalanine82 (Phe82) and phenylalanine124 (Phe124). The distance from Val14 to 1-heptanol was 2.09 A˚, which was the shortest distance over which van der Waals interactions were formed (Fig. 3C). Binding specificities of mutants Amino acids were chosen for site-directed mutation based on two criteria: (1) the sequence alignment of LstiGOBP1 to the insect OBPs with validated key

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Figure 4. Purified Loxostege sticticalis general odorant binding protein 1 and mutants after rEK cleavaged the his-tag. M: Protein Marker; 1: Repurified LstiGOBP1 original protein; 2: Repurified LstiGOBP1-Val14A mutant; 3: Repurified LstiGOBP1-Thr15A mutant; 4: Repurified LstiGOBP1-Trp43A mutant. 5: Repurified LstiGOBP1-Thr15A &Trp43A two site mutant.

binding sites, and (2) the molecular docking results of LstiGOBP1 bound to 1-heptanol. The sequence alignment of several insect OBPs showed that Val14 of LstiGOBP1 was similar with the important binding site of OBPs, such as Ile80 to HoblOBP1 (Zhuang et al., 2014) and V87A to LmigOBP1 (Jiang et al., 2009), and formed the greatest van der Waals interactions with 1-heptanol. The docking experiments showed that Thr15 and Trp43 formed hydrogen bonds with 1-heptanol to keep it within the binding sites of the protein (Fig. 3D). Thus, we predicted that three residues (Thr15, Trp43 and Val14) were most probably the important sites for protein binding. Using site-directed mutagenesis, these three residues (Thr15, Trp43 and Val14) were replaced with alanine, which generated four mutants, Thr15A, Trp43A, Val14A and the two-site mutant (Thr15A and Trp43A). After mutagenesis, the recombinant LstiGOBP1 and mutants were expressed in Escherichia coli as completely soluble proteins with high yields (more than 20 mg/l), and the expression levels of the mutated proteins were apparently the same. The His-tag of the recombinant protein was removed by recombinant enterokinase (rEK). The

protein was purified by two rounds of Ni ion affinity chromatography: the first round was to separate and purify the recombinant protein from total protein, and the second round was to divide the His-tag and the uncleaved His-tagged proteins. After purification by column chromatography, the proteins were treated with acidic buffer to remove potential endogenous ligands by dialysis. Then the purity of the proteins was examined by sodium dodecyl sulfate polyacrylamide gel electropheresis (SDS-PAGE). The single bands between 16 and 24 kDa had greater than 95% purity (Fig. 4), which met the expectation for a monomer of LstiGOBP1. The fluorescence binding assays showed that maximum emission wavelengths of the four mutants were in the range of 410–430 nm after being probed using 1-NPN, similar to LstiGOBP1 (420 nm). In the first instance, the binding curve and Kdiss between mutants of LstiGOBP1 and 1-NPN were determined (Fig. 5). These results indicated that the interaction between LstiGOBP1 and 1-NPN was affected by the mutations, especially for the two-site mutant (Fig. 5). The binding ability of the four mutants was then evaluated with six compounds, namely, 1-heptanol, 1-hexanol, cinnamic aldehyde, benzaldehyde, trans-2-hexenal and trans-11-tetradecen-1-yl acetate (Table 1). Compared with the original protein (Fig. 6A), all of the mutants showed low binding affinity to the tested compounds (Fig. 6B–E). The two-site mutant lost almost all of its binding ability to the six compounds. None of these compounds could compete with the 50% 1-NPN, even when the ligand concentrations reached 40 lM (Fig. 6E). The mutants Thr15A and Trp43A showed a slightly lower affinity for cinnamic aldehyde and benzaldehyde compared with the wild-type protein, but 1-heptanol, 1hexanol and trans-11-tetradecen-1-yl acetate could not compete for binding with 1-NPN in the mutants over the concentration range that was tested (Fig. 6B, C). By contrast, the mutant Val14A had only a weak effect on the binding affinities of the tested compounds compared with the wild-type protein (Fig. 6D).

Figure 5. The binding curve and Kd of 1-NPN of mutant and wild-type Loxostege sticticalis general odorant binding protein 1. C 2015 The Royal Entomological Society, 24, 528–538 V

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Figure 6. Competitive binding curves of selected ligands to the mutants of Loxostege sticticalis general odorant binding protein 1. The ligands are shown on the right. (A) Original protein. (B) LstiGOBP1-Thr15A mutant. (C) LstiGOBP1-Trp43A mutant. (D) LstiGOBP1-Val14A mutant. (E) LstiGOBP1- Thr15A & Trp43A mutant.

Discussion OBPs play a crucial role in transporting odorant molecules from the sensillum lymph to the olfactory receptors to initiate behavioural responses. A previous study conducted in our laboratory showed that LstiGOBP1 was capable of binding efficiently to various plant volatiles (Sun et al., 2011). In the present study, the long-chain chemical trans-11-tetradecen-1-yl acetate, a sex pheromone component of L. sticticalis (Struble & Lilly, 1977), also showed a high binding affinity to LstiGOBP1 (Fig. 6A), which was very similar to the result with LstiGOBP2 C 2015 The Royal Entomological Society, 24, 528–538 V

(Yin et al., 2012). However, another important piece of information towards the understanding of the physiological function of a protein is its expression in different tissues. Quantitative examination of transcript levels showed that LstiGOBP1 was expressed in male and female antennae, and if any expression occurred in other tissues, it was very low (Fig. 1). These results were consistent with the OBPs of other insects (Gu et al., 2010, 2011; Zhang et al., 2010; Yin et al., 2012; Ahmed et al., 2014). Our results indicate that LstiGOBP1 plays a major role in olfactory chemoreception.

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Studies of the interactions between protein receptors and ligands were necessary to better understand the function and binding mechanism of LstiGOBP1. Although no experimental figures of nuclear magnetic resonance (NMR) or X-ray diffraction crystal structures were available for L. sticticalis, fortunately, structures of several OBPs in Lepidoptera were. From a BLAST search in the PDB, BmorGOBP2 was the most similar OBP (53% identity) to LstiGOBP1 with sequence similarity of more than 30%, which was qualified for 3D-homology modelling. Therefore, the crystalline structure of BmorGOBP2 was selected as the template to build a 3D-homology structure of LstiGOBP1. PRO-CHECK, PROFILES-3D and VERIFY-3D confirmed that the 3D model was accurate and could be used for molecular docking studies. As the ligand with the highest binding affinity to LstiGOBP1 (Sun et al, 2011), 1-heptanol was selected as the compound to dock with the 3D model of LstiGOBP1. The docking experiments suggested that three hydrogen bonds were formed by the amino acids Thr15 and Trp43 at the entrance of the binding cavity. Hydrogen bonds have been confirmed as the primary linkage between proteins and ligands in several insect OBPs (Thode et al., 2008; Jiang et al., 2009; Zhou et al., 2009; Sun et al., 2013; Ahmed et al., 2014). The distances of amino acids to 1-heptanol and the location of amino acids in the 3D structure of the protein were also analysed. The hydrophobic amino acid Val14 was the nearest amino acid, which might have affected the formation of van der Waals interactions and hydrophobic interactions, as occurred with V87A in LmigOBP1 (Jiang et al., 2009) and Ile80A in HoblOBP1 (Zhuang et al., 2014). This might be the other linkage found between LstiGOBP1 and the ligands. After site-directed mutagenesis, the four mutants of the LstiGOBP1 protein were analysed to characterize their ligand-binding mechanism. Fluorescence binding assays revealed that the single amino acid mutants Thr15A and Trp43A could not efficiently bind to 1heptanol, 1-hexanol or trans-11-tetradecen-1-yl acetate. Furthermore, binding affinity was completely lost in the two-site mutant, which could not bind with the six tested odours. A possible explanation is that mutants arose from Thr15 and/or Trp43 to alanine avoided the formation of hydrogen bonds between LstiGOBP1 and the ligand odours. These compounds could not stay in the binding cavity because of the loss of hydrogen bonding and were not bound by the mutants. The mutant Val14A showed a slight decrease in binding to most compounds, including 1-heptanol, 1-hexanol, cinnamic aldehyde, trans-2-hexenal and trans-11-tetradecen-1-yl acetate, perhaps because of the change of van der Waals and hydrophobic interactions between the mutant and the

odours. Hence, we concluded that the amino acids Thr15 and Trp43 were the critical residues for LstiGOBP1 binding with these compounds and that Val14 also affected the initial ligand recognition. Interestingly, when LstiGOBP1 was aligned with other lepidopteran GOBPs (Zhong et al., 2008), both Thr15 and Trp43 were found to be conserved in all lepidopteran GOBPs; thus, these two residues may play a role that is common to all lepidopteran GOBPs, rather than a role in binding a ligand specific for LstiGOBP1. However, the functions of these two residues in other lepidopteran GOBPs should be studied further. Different OBPs in the same insect are capable of binding to several of the same ligands, although the sequences are completely different (Sun et al., 2011; Deng et al. 2012; Gu et al., 2012; Yin et al., 2012; Zhong et al., 2012; Zhuang et al., 2013, 2014). From the molecular docking model, we found that the same ligand recognition mechanism could be formed by special binding sites in different full-length sequences (Zhuang et al., 2013, 2014), which led to the hypothesis that the binding site is more important than the entire sequence in the process of ligand recognition. Our work revealed that two amino acids, Thr15 and Trp43, at the opening of the OBP binding cavity were involved simultaneously in the process of ligand binding. Comparing our results with other reports, we confirmed that the hydrophilic amino acids at the opening of the OBP binding cavity contribute to the initial ligand recognition. For instance, Ser56 of BmorPBP (Sandler et al., 2000) and Thr57 of LUSH (Thode et al., 2008), which are located at the opening of the binding pocket and form hydrogen bonds with compounds, determine the binding specificity of OBPs. More importantly, our results show that multiple hydrogen bonding plays a key role in the binding specificity of LstiGOBP1 and provide new insight into the detailed chemistry of odours’ interactions with proteins. Although these computational simulations need to be confirmed with NMR tests and X-ray diffraction of the protein–ligand complexes, both the molecular docking and the mutant binding assay provide new directions for further study on the molecular mechanisms of ligand– OBP interactions. Moreover, it is also hoped that these results may have a far-reaching impact on insect control, through inhibiting the olfactory recognition to plant volatiles. Experimental procedures Material Loxostege sticticalis were fed with Chenopodium glaucum in the laboratory of the Institute of Plant Protection, Chinese Academy of Agricultural Sciences. Real-time PCR was performed using reverse-transcribed RNA isolated from the antennae, heads C 2015 The Royal Entomological Society, 24, 528–538 V

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Table 1. Binding affinities of six odours to Loxostege sticticalis general odorant-binding protein 1 original protein and mutants Kd (lM) Ligand

Original protein

Thr15A

Trp43A

Val14A

Thr15A&Trp43A

1-Heptanol 1-Hexanol Cinnamic aldehyde Benzaldehyde, Trans-2-hexenal Trans-11-tetradecen-1-yl acetate

7.73 13.44 10.82 18.75 16.06 13.58

– – 20.45 21.70 – –

– – 16.22 18.93 26.14 –

16.42 20.11 19.70 18.06 24.62 23.39

– – – – – –

Values are means of three independent experiments. Ligand concentrations >40 lM for half-maximal relative fluorescence intensity are represented as ‘–’.

(without antennae), thoraxes, abdomens, legs and wings. All tissues were stored at 2708C prior to use. 3D modelling and molecular docking The sequence of LstiGOBP1 was retrieved from GenBank (EU413989), and a BLAST search (Altschul et al., 1990) of the amino acid sequence of LstiGOBP1 was conducted in the current PDB (http://www.rcsb.org) to find structural templates. The target and the template sequences were aligned using the CLUSTALW program (http://www.ebi.ac.uk/Tools/msa/clustalw2/). Finally, the 3D model of LstiGOBP1 was built by homology modelling using the crystalline structure of an odorant binding protein from Bombyx mori (BmorGOBP2) as the template at a ˚ . The resulting alignment was subsequently resolution of 1.5 A submitted to the SWISS-MODEL server (http://swissmodel. expasy.org/) for comparative structural modelling (Schwede et al., 2003). The optimum alignment, as determined by the lowest Anolea score and the QMEAN4 score, was selected, and the modelling rationality was further estimated using PROCHECK (http://nihserver.mbi.ucla.edu/SAVS/; Zhuang et al., 2014), VERIFY-3D (Bowie et al., 1991; Luethy et al., 1992), and PROFILES-3D (Luethy et al., 1992). Based on the established homology model, a docking program, AutoDock Vina, was used to find the potential binding mode between LstiGOBP1 and the ligand 1-heptanol. The 3D structure of 1-heptanol was obtained from ChemOffice (http:// www.cambridgesoft.com/Ensemble_for_Chemistry/ChemOffice/ ChemOfficeProfessional/) and was further refined by the CHARMm force field (http://www.charmm.org/). First, the LstiGOBP1 structure was modified by adding polar hydrogen, and was then kept flexible in the docking process, whereas all of the torsional ligand bonds were set free by the ligand module in AUTODOCK Tools-ADT. The AUTODOCK Tools were used to generate both the grid and the docking parameter files. The default AUTODOCK force field was applied (Huey et al., 2007), and the RMSD tolerance of the resulting docked structures was 2 A˚. The binding energy included van der Waals energy, hydrogen bonding energy, desolvation energy, electrostatic energy, total internal energy and torsional free energy. Site-directed mutagenesis The substitution in LstiGOBP1 was designed to disrupt the binding affinity of the protein to six compounds. The LstiGOBP1 C 2015 The Royal Entomological Society, 24, 528–538 V

coding sequence was mutated to yield the four mutants: LstiGOBP1-Thr15A (threonine to alanine at position 15), LstiGOBP1-Trp43A (tryptophane to alanine at position 43), LstiGOBP1-Val14A (valine to alanine at position 14) and the two-site mutant LstiGOBP1-Thr15A&Trp43A (threonine to alanine at position 15 and tryptophane to alanine at position 43). The mutations were generated using a Fast Mutagenesis System kit (Beijing TransGen Biotech Co., Lid, Beijing, China) with FastPfu DNA polymerase (Beijing TransGen Biotech Co., Lid, Beijing, China). The expression vector pET30/LstiGOBP1 cDNA served as a template. The primers were designed according to the directions of the Fast Mutagenesis System kit using PRIMER5 (http://www.premierbiosoft.com/services/bioinformatics.html). Expression and purification of mutants The correct insertion of the mutations was verified with DNA sequencing. Rosetta (DE3) E. coli competent cells were transformed with the plasmids pET30/LstiGOBP1-Thr15A, pET30/ LstiGOBP1-Trp43A, pET30/LstiGOBP1-Val14A and pET30/LstiGOBP1-Thr15A&Trp43A, and protein synthesis was induced with 0.5 mM Isopropyl b-D-1-thiogalactopyranoside (IPTG) when the absorbance values of E. coli at 600 nm wavelength (OD600) is 1.0. All expressed proteins were found in the supernatant, and the recombinant proteins were purified by Ni ion affinity chromatography (GE-Healthcare, Sangon Biotech Co., Ltd., Shanghai, China). SDS-PAGE was used to monitor protein expression and purification. The purified proteins were stored at 2208C in 50 mM Tris-HCl buffer with a pH of 7.4. Protein concentration was determined by the standard bicinchoninic acid (BCA) method (Sangon Biotech Co., Ltd.). To prevent the Histag from affecting the protein functional studies, the His-tag was removed by rEK (Bio. Basic Inc.). Any remaining uncleaved Histag proteins were removed with a second round of Ni ion affinity chromatography. The purified original protein and four mutants were treated with 50 mM Tris-HCl (pH 5.4) to remove potential endogenous ligands by dialysis. The corrected proteins were then dissolved in 50 mM Tris-HCl (pH 7.4) by dialysis again and used in fluorescence binding assays as described below. Fluorescence binding assays Fluorescence spectra were recorded in a right-angle configuration on a Lengguang 970CRT spectrofluorimeter (Shanghai Jingmi, Shanghai, China) at room temperature using a 1-cm light path fluorimeter quartz cuvette. Slit widths of 10 nm were

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selected for both excitation and emission. The spectra data were processed with 970CRT 2.0l software (Shanghai Jingmi, Shanghai, China). 1-NPN was dissolved in methanol to yield a 1.0 mM stock solution. The binding affinity for 1-NPN was titrated by adding aliquots into a 2 lM protein sample to final concentrations of 1 to 40 lM. The fluorescence of 1-NPN was excited at 337 nm and emission was recorded between 350 and 600 nm. Spectra were recorded with a high-speed scan. The competitive binding of ligands was measured using 1-NPN (2 lM) as the fluorescent reporter. The final concentration of each ligand dissolved in high-performance liquid chromatography (HPLC) purity grade methanol ranged from 2 to 40 lM. To determine the binding ability of the four mutants, different organic compounds were chosen that succeeded in displacing 1-NPN from the LstiGOBP1/1-NPN complex at low concentrations. The organic compounds that were used were 1-heptanol, 1-hexanol, cinnamic aldehyde, benzaldehyde, trans-2-hexenal and trans-11-tetradecen-1-yl acetate (Sun et al., 2011). The binding data were collected repeatedly by three independent measurements. The concentrations of competitors that caused a reduction in fluorescence to half-maximal intensity IC50 were used as a measure of the binding dissociation constants and were calculated from the corresponding IC50 values using the formula: Kdiss 5 [IC50]/(1 1 [1-NPN]/K1-NPN). In the equation, [1NPN] is the free concentration of 1-NPN, and K1-NPN is the dissociation constant of the complex LstiGOBP1/1-NPN. Expression pattern analysis of LstiGOBP1 Antennae, thoraxes, abdomens, wings, legs and heads (without antennae) were collected and immediately frozen in liquid nitrogen for storage at 2808C until use. Total RNA isolated from the different tissues using Trizol reagent (Invitrogen, Carlsbad, CA, USA) was prepared in triplicate and then treated with gDNA Eraser (TaKaRa Co., Dalian, China) to remove residual genomic DNA. The quality and concentration of the RNA were estimated by determining the ratio of Ultraviolet absorbance 260 and absorbance 280 (A260/A280 ratio) and then modified to the same concentration (0.1 lg/ll) using diethy pyrocarbonate (DEPC). For RT-PCR, the cDNA was synthesised with a firststrand cDNA synthesis kit (TaKaRa Co., Dalian, China). Taqman primers and probes were designed using PRIMER EXPRESS 3.0 (Applied Biosystems, Foster City, CA, USA) and are listed in Supporting Information Table S1. The Taqman probes were labelled with the 5-Carboxyfluorescein (FAM) reporter dye at the 50 ends and with the quencher dye 50 -Carboxytetramethylrhodamine (TAMRA) at the 30 ends. The 18S rRNA was used as an endogenous control to normalize the results of a variable target gene and to correct for sample-to-sample variation. Real-time qPCR was conducted on an ABI Prism 7500 Fast Detection System (Applied Biosystems, Inc. Carlsbad,CA, USA). Each amplification reaction was performed using a 20-ml reaction mixture under the following conditions: denaturation at 958C for 10 s, followed by 40 cycles at 958C for 5 s and at 608C for 34 s. The relative quantification was performed by using the comparative 2(2DDCT) method (Livak and Schmittgen, 2001). All data were normalized to endogenous 18S rRNA levels from the same tissue sample, and

the relative fold change in the different tissues was calculated with the transcript level of the abdomen as the calibrator. By comparing the expression level of LstiGOBP1 in other tissues to that in the abdomen, the relative fold change in different tissues was assessed.

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Supporting Information Additional Supporting Information may be found in the online version of this article at the publisher’s web-site: Table S1. Oligonucleotide primers used in real-time quantitative PCR.

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