Introduction

Nicotinic acetylcholine receptors (nAChRs) are ligand-gated ion channels that mediate fast-acting excitatory cholinergic neurotransmission in vertebrates and invertebrates. Each nAChR molecule consists of five subunits; acetylcholine (ACh) binds at extracellular ligand-binding domain (LBD) at the subunit interfaces, and a central, cation-permeable ion channel opens transiently in response to ACh and agonists (Karlin 2002; Sine and Engel 2006). Each subunit possesses an N-terminal extracellular domain with a conserved di-cysteine loop, four transmembrane regions (TM1–TM4 of which TM2 provides most of the channel-lining residues), and a large intracellular loop between TM3 and TM4 (Karlin 2002). Usually, nAChRs are made up of α and non-α subunits (Fig. 1a), the α subunits being defined by adjacent cysteines in loop C of LBD. The ACh binding site is located at the LBD interfaces and formed by loops A, B and C of the α subunit and loops D, E and F which are normally located in non-α subunit (Corringer et al. 2000; Karlin 2002; Lindstrom 2003). However, α7, α8 and α9 subunits can form functional homo-pentamers (Couturier et al. 1990; Elgoyhen et al. 1994; Gerzanich et al. 1994) (Fig. 1a), whereas α9 and α10 subunits form α hetero pentamers (Elgoyhen et al. 2001). When only α subunits are present, they donate not only loops A–C, but also loops D–F.

Fig. 1
figure 1

Nicotinic acetylcholine receptors (nAChRs) and the acetylcholine binding protein (AChBP). a Schematic representation of neuronal nicotinic acetylcholine receptors and acetylcholine binding protein. b Chemical structures of natural nicotinic ligands and neonicotinoids [imidacloprid (IMI) and clothianidin (CTD)] used in this study. c Multiple sequence alignment of AChBP with the ligand binding domain of nAChR subunits. Lymnaea stagnalis AChBP (Ls-AChBP) and N-terminus LBD sequences of insect (species names are colored magenta) and vertebrate (species names are colored black) nAChRs are aligned using ClustalW2 program (Larkin et al. 2007) with default parameters and the details are manually adjusted. Secondary structure elements from the Ls-AchBP–IMI complex are indicated above the multiple alignments. Amino acids involved in the interactions with neonicotinoids are highlighted in bold with yellow and cyan backgrounds in the principal (+)- and complementary (-)-sides, respectively. Amino acid residue number from methionine for each protein was indicated at the top of each sequence

Full size image

In mammals, nAChRs play central roles in neuromuscular and inter-neuronal cholinergic neurotransmission. Mutations in muscle nAChR subunits account for many cases of congenital myasthenia syndrome (Engel et al. 2003) and also multiple pterygium (Morgan et al. 2006), whereas mutations in neuronal nAChR subunits (α4,β2) can produce epilepsies (Aridon et al. 2006; Steinlein 2004). It has also been shown that cholinergic neurotransmission is reduced in Alzheimer’s (Marutle et al. 1999) and Parkinson’s diseases as well as in schizophrenia (Woodruff-Pak and Gould 2002). Further, in some cases of Alzheimer’s disease, loss of nAChRs has been reported (Court et al. 2001). Thus there is a growing interest in nAChRs as potential drug targets (Arneric et al. 2007; Dani and Bertrand 2007).

In the insect nervous system, where ACh is the primary excitatory neurotransmitter, nAChRs are present at densities comparable to those in the electric organ of the electric fish, Electrophorus electricus (Sattelle 1980), and several classes of insecticides (cartap, bensultap, thiocyclam, spinosad and neonicotinoids) target insect nAChRs (Narahashi 2000; Raymond-Delpech et al. 2005; Millar and Denholm 2007). Of these, neonicotinoids (Fig. 1a) are increasingly used not only for controlling crop pests worldwide, but also for animal health application such as flea and louse controls. Imidacloprid (IMI; Fig. 1b), the first insecticide with billion dollars per annum sales, is a partial agonist of native (Deglise et al. 2002; Ihara et al. 2006; Tan et al. 2007) as well as of recombinant (Ihara et al. 2003) nAChRs expressed in Xenopus laevis oocytes. On the other hand, clothianidin (CTD; Fig. 1b) and its analog evoke supra-maximal responses (with reference to ACh responses) not only in the Drosophila Dα2/chicken β2 hybrid nAChRs (Ihara et al. 2004) expressed in Xenopus oocytes, but also in native Drosophila central cholinergic neurons (Brown et al. 2006). Single channel recording has shown that a CTD analog induces the opening of the Drosophila nAChR channels at the largest conductance state more frequently than ACh, offering a possible explanation for its super-agonist action (Brown et al. 2006). Contrasting with these actions, some neonicotinoids (Ihara et al. 2006; Salgado and Saar 2004), and bis-neonicotinoids containing two neonicotinoid units joined together by an alkyl chain (Ihara et al. 2007a), antagonize the acetylcholine-induced responses of native insect neurons.

Neonicotinoids show higher affinity for insect nAChRs, accounting, at least in part, for their selective toxicity to insects over vertebrates (Matsuda et al. 2001, 2005; Tomizawa and Casida 2005). Our studies using site-directed mutagenesis combined with two-electrode voltage-clamp electrophysiology have shown that the X residue in the YXCC motif of loop C (Shimomura et al. 2004), the region upstream of loop B (Shimomura et al. 2005) and basic residues in loop D (Shimomura et al. 2002, 2003, 2006) contribute to the high neonicotinoid sensitivity of insect nAChRs (see Fig. 1c for loop positions).

The acetylcholine binding protein (AChBP) from snail, Lymnaea stagnalis (Ls-AChBP) was discovered in glial cells as a water soluble protein modulating synaptic ACh concentration (Smit et al. 2001), and the X-ray crystal structure of the protein was described at the same time (Brejc et al. 2001). Subsequently, X-ray crystal structures of Ls-AChBP homologs from Aplysia californica (Ac-AChBP) (Bourne et al. 2005) and Bulinus truncatus (Bt-AChBP) (Celie et al. 2005b) were reported. Both Ls- and Ac-AChBPs show similarities to the extracellular domain of neuronal α7 nAChRs and form homopentamers in solution (Smit et al. 2001). Notably, all the six regions (loops A–F) that make up the ligand binding site are conserved in AChBPs. Thus AChBPs are considered as surrogates of the LBD in nAChRs and therefore have been employed to elucidate the mechanism for interactions with low molecular weight ligands as well as peptide toxin antagonists (Bourne et al. 2005; Celie et al. 2004, 2005; Dutertre et al. 2007; Hansen et al. 2005; Hansen and Taylor 2007; Ulens et al. 2006). Employing the crystal structures of AChBPs, we have previously modeled LBDs in complex with IMI to show that basic residues in loop D may interact electrostatically with the nitro group of neonicotinoids (Shimomura et al. 2006). Yet, details of neonicotinoid–nAChR interactions are poorly understood. Therefore, we have crystallized the Ls-AChBP in complex with two commercial neonicotinoids, IMI and CTD.

Materials and methods

Materials

Lymnaea stagnalis derived from the stocks of Vrije Universeit (Amsterdam) was provided from Professor Sakakibara at Tokai University. IMI and CTD were gifts from BayerCropscience Co. in Japan. 1-(6-Bromopyridylmethyl)-2-nitroiminoimidazolidine (bromine-substituted imidacloprid; Br-IMI) and N-(2-bromo-thiazol-5-ylmethyl)-N′-methyl-N′′-nitroguanidine (bromine-substituted clothianidin; Br-CTD) were synthesized according to the published methods (Maienfisch et al. 2000; Nishiwaki et al. 2000).

Protein preparation

The cDNA of Ls-AChBP was amplified by RT-PCR from L. stagnalis and was cloned into the pPICZα B vector (Invitrogen) at the Pst I site, with an L1A mutation introduced to facilitate cloning. The Ls-AChBPs were expressed in yeast, Pichia pastris X-33 using EasySelect Pichia Expression Kit (Invitrogen). Secreted proteins were purified with Q-Sepharose column (GE Healthcare) immediately following concentration and buffer-exchange using Vivaflow200 (Zartorius), and then treated with 50 U/mg protein of peptide-N-glycosidase F (Wako Pure Chemical Industries) at 37°C for 24 h to remove the glycosyl-chain at Asn67. The protein samples were subsequently purified with Mono Q and Superdex 200 columns (GE Healthcare). Purified proteins were confirmed to be the samples of interest by N-terminal sequencing and mass spectrometry. Purified proteins were buffer-exchanged against 20 mM Tris–HCl buffer with 0.02% NaN3 (pH 8.0).

Evaluation of equilibrium binding of neonicotinoids

The equilibrium binding of neonicotinoids with Ls-AChBP was determined by quenching of intrinsic tryptophan fluorescence. Ls-AChBP at 600 nM in binding site concentration in 20 mM Tris–HCl buffer with 0.02% NaN3 (pH 8.0) was incubated with each ligand at various concentrations on a 96-well plate. Ls-AChBP was excited at 280 nm, and emission intensity was recorded at 342 nm using Varioskan microplate reader (Thermo Fisher Scientific) at room temperature. Data were fitted by non-linear regression according to the following equation using Prism Software version 4.03 (GraphPad Software):

$$ \frac{{\Delta F}} {{F_{0} }} = \frac{{\Delta F_{{\max }} }} {{F_{0} }} \cdot \frac{{K_{{\text{d}}} + [{\text{X}}] + [{\text{Protein}}] - {\sqrt {{\left( {K_{{\text{d}}} + [{\text{X}}] + [{\text{Protein}}]} \right)}^{2} - 4 \cdot [{\text{X}}] \cdot [{\text{Protein}}]} }}} {{2 \cdot [{\text{Protein}}]}}, $$

where ΔF and ΔF max represent quenching of fluorescence at a ligand concentration X and maximum quenching of fluorescence at saturation, respectively. F 0 and [Protein] are fluorescence measured in the absence of ligands and the concentration of Ls-AChBP, respectively.

Crystallization and X-ray data collection

Purified Ls-AChBPs (6.0 mg/mL) were incubated with 0.5 mM neonicotinoids at 4°C for 1 h prior to crystallization. Ls-AChBP-neonicotinoid complex crystals were obtained by vapor diffusion method at 20°C with 1:1 ratio of protein to reservoir solution containing 0.2 M Na citrate pH 5.7, 15–22% PEG3350 and about 0.5 mM of either IMI, CTD, Br-IMI or Br-CTD. The crystals were flash-cooled in liquid nitrogen after soaking into the cryoprotectant solutions containing 0.2 M Na citrate pH 5.7, 25% PEG3350, 20% glycerol and about 0.5 mM of each neonicotinoid. X-ray diffraction data sets were collected at 90 K using either Bruker AXS DIP6040 detector at BL44XU (Yoshimura et al. 2007) or ADSC QUANTUM 210 detector at BL44B2 (Adachi et al. 2001) beamlines in SPring-8, and processed with Mosflm (Leslie 1992) or HKL2000 (Otwinowski and Minor 1997). In order to identify the positions of the sulfur atom at the thiazole ring of CTD as well as the bromine atom of Br-IMI and Br-CTD, anomalous data from the crystals complexed with these neonicotinoids were collected at wavelength of 1.75 Å and 0.919 Å (Table 1), respectively.

Table 1 X-ray diffraction data collection and refinement statistics for Ls-AChBP–neonicotinoid complexes
Full size table

Structure determination and refinement

The structure of the IMI complex was solved by molecular replacement with Phaser (McCoy 2007) using the coordinates of an Ls-AChBP in complex with nicotine (PDB entry: 1UW6) (Celie et al. 2004) as a search model. Subsequently, the refined coordinates of the IMI complex were used to solve the structure of the CTD complex. Refinements and manual model building were performed with CNS version 1.2 (Brünger et al. 1998) and Coot (Emsley and Cowtan 2004), respectively. The non-crystallographic-symmetry restraints between the subunits were initially applied for the refinements and removed at later steps. The details of data collection and refinement statistics are provided in Table 1. Figures are generated by PyMOL (DeLano Scientific LLC), Bobscript (Esnouf 1999) and Raster3D (Merritt and Bacon 1997).

Results and discussion

Structure analysis

In this study, we have crystallized the Ls-AChBP in complex with IMI and CTD to elucidate the mechanism underpinning their selective and diverse actions on nAChRs. Both complexes were crystallized in the same space group with one pentamer in an asymmetric unit. Ligand-omit and anomalous electron density maps clearly indicated AChBP in the crystals bound neonicotinoids (see Fig. 3a, b). The positions and orientations of the 2-chloropyridine ring in IMI and 2-chloro-1,3-thiazole ring in CTD were modeled to satisfy the observation of Br- and S-anomalous peak positions. The X-ray crystal structures of the Ls-AChBP complexed with IMI and CTD were refined at 2.58 and 2.70 Å resolutions, respectively (Table 1).

The neonicotinoid-bound Ls-AChBPs form the same homopentameric structure (Fig. 2a, b) as those determined for the 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES)-bound apo form (Brejc et al. 2001) and the other ligand-bound forms (Celie et al. 2004, 2005; Hansen et al. 2005; Ulens et al. 2006). Typically, each subunit has a secondary structure composed of an N-terminal 12-residue α-helix followed by β-strand-rich parts as shown previously for other Ls-AChBPs. Root mean square differences (rmsd) of Cα atoms between the IMI- or CTD-complex and the nicotine-complex (Celie et al. 2004) were observed as 0.975 and 0.758 Å, respectively. In two structures determined in the present study, neonicotinoids occupied all five ligand-binding sites that are formed between the two adjacent subunits (Fig. 2a, b). The neonicotinoids occupy broadly the same position as HEPES and (-)-nicotine within the pocket formed by “A-F loop” regions. Although “loops A, B, D and E” are mainly composed of β-strands and only loops C and F have no canonical secondary structure, we use these designations to facilitate comparison with previous studies on nAChRs (Corringer et al. 2000; Matsuda et al. 2001, 2005). The principal side [(+)-chain] of the ligand-binding site possesses residues from loops A, B and C which are located in α subunits, whereas the complementary side [(-)-chain] contains loops D, E and F, which are donated by non-α subunits in α/non-α heteropentamers. Overall, the structures of the IMI and CTD complexes were very similar with Cα rmsd of 0.597 Å. However, the loop C region has a specific conformation in each complex. For example, compared with the IMI complex, loop C of the CTD complex takes a “closed conformation”, showing approximately 4 Å shift of the positions of the Cα atoms (Fig. 2c). The variations in loop C conformation were often observed between different chains within the same Ls-AChBP pentamer in complex with neonicotinoids (e.g. subunits A and C of the CTD complex, data not shown). Conformational change in loop C is evident when comparing structures with the apo form Ls-AChBP complexed with HEPES (PDB entry code: 1UX2) (Fig. 2c). The Thr155-Asp160 loop region of the (-)-chain upstream of loop F also indicates a conformational change in the ligand-binding site. These results suggest that induced-fit movement of loop regions is essential for recognition of ligands including neonicotinoids. The detailed binding interactions of neonicotinoids will be considered in relation to their key components, the aromatic ring and the guanidine/related moieties.

Fig. 2
figure 2

Overall view of crystal structure of Ls-AChBP complexed with imidacloprid (IMI). The pentameric structure of Ls-AChBP is viewed from the top (a) corresponding to an extracellular surface of nAChRs and from the side (b). IMI molecules bound to Ls-AChBP are shown in space-filling model, whereas carbons, nitrogens and oxygens are colored green, blue and red, respectively. Two subunits, A and B chains, are colored by pink and blue, respectively. c The structure of A–B domain boundary of the Ls-AChBP complexed with IMI (red) is compared with that of the Ls-AChBP complexed with CTD (blue) and the apo form (light brown) complexed with 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) (PDB entry code: 1UX2) (Celie et al. 2004). Ligands are presented by stick models

Full size image

Interactions with aromatic rings of insecticides

When complexed with Ls-AChBP, the pyridine ring of IMI was buried deep in the binding site, interacting with loop E segments from the (-)-chain (Fig. 3c). Recently, Tomizawa et al. (2008) has reported that two azido-neonicotinoid probes containing the photolabile group in the pyridine ring labeled Tyr164 in loop F of the (-)-chain and Tyr192 in loop C of the (+)-chain in the Ls-AChBP. However, these two distinct binding modes were not observed in our models, neither in the IMI complex, nor in the CTD complex of the Ls-AChBP. The nitrogen atom of the pyridine ring forms a hydrogen bond with the amide group of Met114 and the carbonyl group of Leu102 in loop E via a water molecule, resembling the observations for both the Ls-AChBP-nicotine complex (Celie et al. 2004) and Ac-AChBP-epibatidine complex (Hansen et al. 2005).

Fig. 3
figure 3

Electron density maps of bound ligands and their interactions with the loop E region. The annealed F o –F c omit maps (blue) for imidacloprid (IMI) (a) and clothianidin (CTD) (b) bound to the Ls-AChBP are contoured at 4σ and drawn with the final models of the native and non-Br-neonicotinoids. In addition, the anomalous maps of bromine (purple) from the Br-neonicotinoid-complexes and sulfur atoms (yellow) from the native-neonicotinoid-complexes for ligands, which are contoured at 5 and 1.5σ, respectively, are overlaid. Interactions of the loop E region with pyridine moiety of IMI (c) and thiazole moiety of CTD (d) are shown in the binding site of the Ls-AChBP. In each ligand and amino acid, chlorine, nitrogen, oxygen and sulfur atoms are colored purple, blue, red and yellow, respectively. The hydrogen bonds are presented by orange broken lines

Full size image

The chlorine atom was located in the vicinity of a hydrogen bond bridge between the backbone C = O of Leu112 and the backbone amide NH of Arg104. As a result, it made van der Waals contact the peptide backbones of these two amino acids. The aromatic ring of CTD also contacts with the loop E segment in a manner similar to IMI (Fig. 3d). The nitrogen atom of the thiazole ring of CTD, in a similar fashion to the nitrogen atom of the pyridine ring of IMI, formed hydrogen bond with the backbone carbonyl of Leu102 and the backbone amide group of Met114 via water molecule (Fig. 3d).

Nicotine, which is active not only on insect, but also on vertebrate nAChRs, has also been shown to interact with the backbone of loop E in a similar manner to neonicotinoids (Celie et al. 2004). Therefore interactions with the backbone are insufficient to explain the selective actions of neonicotinoids. On the other hand, Met114 in loop E was located in the vicinity of the nitroimine group (Fig. 3c, d) which plays a key role in selective insect nAChR–neonicotinoid interactions (Ihara et al. 2003). Some insect α subunits possess either basic or hydrogen bondable residues favorable for interactions with the nitro group such as those corresponding to Met114 (Fig. 1c). Hence, if two adjacent α subunits form a ligand-binding site, such residues may contribute to the neonicotinoid sensitivity of insect nAChRs.

Interactions with guanidine and related moieties

The crystal structures of the Ls-AChBP bound with nicotine or carbamylcholine (Celie et al. 2004) and Ac-AChBP in the presence of epibatidine (Hansen et al. 2005), as well as quantum chemical calculations (Cashin et al. 2005), suggested that the protonated nitrogen atom in nicotinic agonists undergoes cation–π interactions with Trp143 in loop B. In line with this notion, it was earlier demonstrated that the electron-deficient guanidine moiety of neonicotinoids is likely to contact with Trp143 (Matsuda et al. 2005; Tomizawa and Casida 2005). However, the guanidine moiety of IMI and CTD was found to stack with the aromatic ring of Tyr185 in the crystal structures of CTD- as well as IMI-Ls-AChBP complexes (Fig. 4a, d). Since Tyr185 is conserved throughout vertebrate and invertebrate nAChR α subunits (Fig. 1c), this residue itself is not involved directly in determining the selective interactions with neonicotinoids. Yet the interactions with Tyr185 contribute to the affinity for neonicotinoids. The stacking level for CTD appears to be weaker than that for IMI (Fig. 4a, b). In addition, the stacking interaction resulted in CH–π contacts with Trp143 of the methylene (CH2–CH2) bridge in IMI, which was not seen in CTD, accounting, at least in part, for the higher binding potency of IMI (K d = 1.57 ± 0.21 μM, n = 3) compared to that of CTD (K d = 7.26 ± 1.13 μM, n = 3) on the Ls-AChBP as evaluated by quenching of the protein fluorescence.

Fig. 4
figure 4

Imidacloprid (IMI) and clothianidin (CTD) binding to Lymnaea stagnalis AChBP (Ls-AChBP). a IMI–Ls-AChBP complex. b CTD–Ls-AChBP complex. The binding site is located at the interface between two adjacent subunits, as drawn in green for (+)-chains and in cyan for (-)-chain (see text). The subunits EA and CD interfaces are shown for the binding sites for IMI (a) and CTD (b), respectively. Orange broken lines depict hydrogen bonds. In each ligand and amino acid, chlorine, nitrogen, oxygen and sulfur atoms are colored purple, blue, red and yellow, respectively

Full size image

Another difference between CTD and IMI is that the former exposes a hydrogen bond donor NH at position 1, whereas the latter lacks it. We suggested earlier that this NH may form a hydrogen bond with the backbone carbonyl of Trp143 in loop B (Ihara et al. 2007b). As suggested, the NH of CTD made a hydrogen bond with the backbone carbonyl of Trp143 from the (+)-chain in the crystal structures of the Ls-AChBP (Fig. 4b), although it was observed at only 2 of 5 interfaces. Such hydrogen bond formation may assist CTD in keeping contact with nAChRs when large conformational changes occur in LBD to open the central channel (Miyazawa et al. 2003; Unwin 2005), accounting, at least in part, for higher agonist efficacy of CTD compared to IMI and ACh in certain native and recombinant nAChRs (Brown et al. 2006; Ihara et al. 2004).

Interactions with loop D and selectivity of neonicotinoids

We have previously shown that substitution of a glutamate for the glutamine corresponding to Gln55 of Ls-AChBP (Fig. 1c) markedly reduced the maximum response to IMI and nitenpyram of the chicken α7 nAChR, whereas mutation of the same glutamine to arginine enhanced it, slightly shifting the concentration–response curve to the left (lower concentrations). In complete contrast, the same mutation reduced the (-)-nicotine sensitivity of the α7, whereas substitution with a glutamate enhanced it (Shimomura et al. 2002). Therefore, it was postulated that this glutamine residue is likely to be located in close proximity to the nitro group of neonicotinoids, and that marked changes in the α7 response to neonicotinoids as well as (-)-nicotine are the results of electrostatic interactions between the added residues and the nitro group. Consistent with this hypothesis, Gln55 faced the nitro groups of IMI and CTD in the crystal structure of the Ls-AChBP within a hydrogen bondable distance in the AChBP–IMI complex (<3.3 Å in 2 of 5 interfaces in the crystal structure of the Ls-AChBP) (Fig. 4). For CTD, however, the distance between the Gln55 and the nitro group was longer than 3.5 Å, excluding or minimizing a contribution of hydrogen bonding to the binding potency of CTD.

All vertebrate nAChRs, perhaps apart from those containing either human or rat β4 subunit, are likely to show reduced sensitivity to neonicotinoids due to the lack of the basic residues in loop D. The human β4 subunit (Fig. 1c), as well as rat β4 subunit (sequence not shown), possesses a lysine as residue corresponding to Gln55 of Ls-AChBP. However, the vertebrate β4 subunits possess a glutamate residue as that corresponding to Thr57 in Ls-AChBP which is located in the vicinity of Gln55 (Fig. 5). This glutamate residue is postulated to interfere electrostatically with the neighboring basic residue–neonicotinoid interactions, thereby reducing the neonicotinoid sensitivity of nAChRs containing the β4 subunits.

Fig. 5
figure 5

Interactions between loops C, D and F. IMI-binding site of Ls-AChBP is presented by especially focusing on loops C, D and F. The binding site at the interface of the subunits C and D is drawn in green for (+)-chains and in cyan for (-)-chain (see text). In each ligand and amino acid, chlorine, nitrogen, oxygen and sulfur atoms are colored purple, blue, red and yellow, respectively. Orange broken lines depict hydrogen bonds

Full size image

Role of loops C and F in their interactions with neonicotinoids

We have found that replacement by glutamate of the proline corresponding to Ser186 in the YXCC motif in loop C of the Drosophila Dα2 subunit reduces the IMI sensitivity of the Dα2β2 hybrid nAChR, whereas its reverse mutation in the α4β2 nAChR enhanced sensitivity (Shimomura et al. 2004), suggesting a contribution of the loop C residue to the neonicotinoid sensitivity of nAChRs. However, another view has been proposed in which the serine or threonine residue corresponding to Ser186 from (+)-chain of Ls-AChBP in loop C of insect nAChRs (see Fig. 1c) forms a hydrogen bond with the nitro group of neonicotinoids, thereby enhancing the binding potency of neonicotinoids (Tomizawa et al. 2007). In the crystal structures of Ls-AChBP, Ser186 made hydrogen bonds with Glu163 and Tyr164 in loop F of (-)-chain but not with the nitro group of IMI (Fig. 5). This hydrogen bond web, as well as the basic residues in loop D, seems to play an important role in determining the neonicotinoid sensitivity of nAChRs. In most heteromeric vertebrate nAChRs, α subunits possess acidic residues as those corresponding to Ser186 in loop C, resulting in electrostatic repulsion of the acidic residues in loop F of β subunits corresponding to Glu163 in Ls-AChBP (Fig. 1c). On the other hand, insect nAChR α subunits do not possess such an acidic residue in loop C (Fig. 1c). Hence, it is conceivable that amino acids not only from α subunits, but also from non-α subunits of insect nAChRs, contact more effectively with neonicotinoids than those of vertebrate nAChRs. In this context, Tyr164 may also contribute indirectly to the neonicotinoid sensitivity of insect nAChRs because vertebrate non-α subunits possess phenylalanine residue incapable of assisting the formation of the hydrogen-bond-network at this position.

In conclusion, we have for the first time elucidated the crystal structures of the Ls-AChBP in complex with two commercial neonicotinoids IMI and CTD. Met114 in loop E and Gln55 in Loop D closest to the nitro group in neonicotinoids, suggesting that corresponding residues in insect nAChRs may play important roles in determining neonicotinoid sensitivity. Ser186 in loop C was found to form hydrogen bonds with Gln163 as well as Tyr164 in loop F, suggesting that these hydrogen bondable residues can also indirectly influence the selective interactions with neonicotinoids. CTD forms hydrogen bond with the backbone carbonyl of Trp143 in loop B and shows weaker hydrophobic contacts with Trp143 compared with other neonicotinoids tested, which may altogether lead to its unique actions on nAChRs. Although there are sequence and structural similarities between AChBPs and nAChRs, there are also important differences. Future structural studies on AChBP mutants engineered to resemble more closely insect or human nAChRs could help to enhance even further our understanding of neonicotinoid actions and selectivity. Nevertheless, we have provided new insights into the molecular basis of differential actions of neonicotinoid molecules. These results may be of value in the design of even safer new crop protection agents.