Crystal Structure of Silkworm Bombyx mori JHBP in Complex With 2-Methyl-2,4-Pentanediol: Plasticity of JH-Binding Pocket and Ligand-Induced Conformational Change of the Second Cavity in JHBP

Juvenile hormones (JHs) control a diversity of crucial life events in insects. In Lepidoptera which major agricultural pests belong to, JH signaling is critically controlled by a species-specific high-affinity, low molecular weight JH-binding protein (JHBP) in hemolymph, which transports JH from the site of its synthesis to target tissues. Hence, JHBP is expected to be an excellent target for the development of novel specific insect growth regulators (IGRs) and insecticides. A better understanding of the structural biology of JHBP should pave the way for the structure-based drug design of such compounds. Here, we report the crystal structure of the silkworm Bombyx mori JHBP in complex with two molecules of 2-methyl-2,4-pentanediol (MPD), one molecule (MPD1) bound in the JH-binding pocket while the other (MPD2) in a second cavity. Detailed comparison with the apo-JHBP and JHBP-JH II complex structures previously reported by us led to a number of intriguing findings. First, the JH-binding pocket changes its size in a ligand-dependent manner due to flexibility of the gate α1 helix. Second, MPD1 mimics interactions of the epoxide moiety of JH previously observed in the JHBP-JH complex, and MPD can compete with JH in binding to the JH-binding pocket. We also confirmed that methoprene, which has an MPD-like structure, inhibits the complex formation between JHBP and JH while the unepoxydated JH III (methyl farnesoate) does not. These findings may open the door to the development of novel IGRs targeted against JHBP. Third, binding of MPD to the second cavity of JHBP induces significant conformational changes accompanied with a cavity expansion. This finding, together with MPD2-JHBP interaction mechanism identified in the JHBP-MPD complex, should provide important guidance in the search for the natural ligand of the second cavity.


Introduction
Juvenile hormones (JHs) are acyclic sesquiterpenoids which contain an a,b-unsaturated methyl ester and a terpenoid backbone with an epoxide distal to the ester. Both the ester and epoxide groups are required for hormone regulatory functions. JH regulates diverse processes including the growth, development, metamorphosis and reproduction of insects [1,2]. The JH levels control the full cycle of insect development from the immature larval stage to the adult form. Hence, insect growth regulators (IGRs) with JH-agonistic or -antagonistic activity have become attractive candidates for insect pest control since the identification of the structure of JH in 1967 [3]. Several JH analogues (JHAs) such as methoprene [4], fenoxycarb [5] and pyriproxyfen [6] have been proven to be effective IGRs and useful as insecticides against household and agricultural pests that cause damage worth billions of pounds to global agriculture each year.
The diversity of JH-mediated physiological effects suggests that target cells may respond to the hormone directly by gene expression and/or via a second messenger [7,8]. Since JH actions for the individual processes occur in tissue-and stage-specific manner, it is generally assumed that multiple numbers of JH receptors exist at the membranes and in cytosols and nuclei. Upon release from the corpora allata where JH is synthesized, the hormone is dispersed to the hemolymph to act at distant peripheral sites. In Lepidoptera almost every molecule of JH in the insect hemolymph appears in a complex with a specific 30 kDa JH-binding protein (JHBP) which serves as a carrier supplying the hormone to target cells [9][10][11][12]. Complex formation provides protection of the chemically labile JH against nonspecific enzymatic degradation and/or sequestration, and is crucial for effective signaling by the low amounts of the hormone. In this sense, JHBP is one of the most important proteins that regulate the JH signaling.
In previous reports [13,14], we have reported a gate-latch mechanism of JH delivery in hemolymph by JHBP based upon the crystal and solution structures of apo-and JH-bound forms of the recombinant JHBP from the silkworm, Bombyx mori. JHBP adopts a unique elongated b-barrel fold, consisting of a long spine helix (a3) wrapped in a highly curved b-sheet, which is suitable for a transport of the hydrophobic JH ( Figure 1). Nearly the same folds are shared by several other lipid binding proteins: Takeout, a potential ubiquinone binding protein [15]; a bactericidal permeability-increasing protein [16]; and a cholesteryl ester transfer protein [17]. JH binds to a hydrophobic JH-binding pocket located at the one end of the elongated structure near the Cterminus of a3. The uptake and release of JH are regulated by the opening and closing of the a1-helix over the JH-binding pocket that functions as a gate sensing the ligand entry. In the crystal structure of the apo-JHBP (gate-open conformation), the location of the a1-helix generates a wide open conformation which permits access of JH to the hormone-binding site ( Figure 1A). Binding of the JH molecule induces a dramatic change in the orientation of a1, which swings towards the pocket about 70u from the position of the corresponding helix in the apo-structure. In the resulting fully gate-closed JHBP-JH complex structure ( Figure 1B), the bound JH is completely buried inside the protein and is thus protected from unfavorable nonspecific absorption and enzymatic degradation during its transport in the hemolymph. In solution, the apo-JHBP assumes multiple conformations of the gate a1-helix ranging from the fully closed to open forms while the protein core is well maintained in all of these structures. JH binding silences conformational dynamics of the a1 gate and leads to the formation of the unique conformation seen in the JHBP-JH complex [13]. Our dynamic structural model visualizes the JH-induced conformational change of JHBP previously reported by a range of different techniques such as proteolysis and ultracentrifugation [18], UV-difference and CD spectroscopy [12,19], and electrochemical impedance spectroscopy [20].
Structural studies revealed that JHBP has an additional hydrophobic cavity (second cavity) on the side opposite from the JH-binding pocket [13,21]. In both the apo-and JH-bound structures of the recombinant B. mori JHBP, this second cavity remains empty [13]. In contrast, ligand binding to the second cavity is suggested by the crystal structure of the native JHBP from the wax moth Galleria mellonella in which the second cavity accommodates an undefined small molecule while the JH-binding pocket remains empty [21]. Although the nature of the second ligand is not known, it was speculated that the second ligand could be bound to the cell membrane to allow for hormone delivery site recognition by JHBP [21]. Further studies are necessary to validate the accurate functional roles of the second ligand-binding site. Lepidopteran insects are major agricultural pests. For example, the tobacco hornworm Manduca sexta and the tobacco budworm Heliothis virescens are the most destructive pests of tobacco [22], while the wax moth G. mellonella causes serious damage to beehives [23]. Since the hemolymph JHBP mediates the first step in the JH signal transduction cascade, and is highly specific to Lepidopteran insects, JHBP is expected to be an excellent target for the design of novel specific IGRs and insecticides. The first three-dimensional structures of the JHBP-JH II complex in the crystalline state and the JHBP-JH III complex in solution, together with subsequent biochemical assays, enabled us to elucidate the molecular mechanism of JH recognition by JHBP that clearly explains the ligand specificity and enantioselectivity [13,[24][25][26][27][28]. The structural information derived from the JHBP-JH complexes opens the way to the structure-based design of IGRs which inhibit complex formation between JH and JHBP, and thus disrupt the JH signaling in Lepidoptera. The development of such IGRs should be further accelerated by enhancement of our understanding of interactions of JHBP with any ligands structurally related and unrelated to the JH molecule.
Here we report the crystal structure of the recombinant B. mori JHBP in complex with 2-methyl-2,4-pentanediol (MPD). This is the first structure of JHBP in complex with a ligand which is structurally unrelated to JH. In this structure, we found two bound MPD molecules, one (MPD1) in the JH-binding pocket and the other (MPD2) in a second cavity. MPD1 is anchored in the same hydrophobic cage as the epoxide of the JHBP-bound JH.
Furthermore, interactions of MPD1 with JHBP are nearly the same as those observed for the epoxide moiety of JH in the JHBP-JH complex. We confirm that MPD and methoprene containing a MPD-like structural element, but not the unepoxydated JH III (methyl farnesoate), inhibit the binding of JH to JHBP using the ligand competitive binding assay. These findings strongly demonstrate that the epoxy group and its mimic structures are critically important for the ligand binding to the JH-binding pocket of JHBP, and should provide useful information for IGR design targeted for JHBP. We also found that MPD2 binding induces a significant conformational change in the second cavity. The MPD2-JHBP interactions reported here should provide important guidance in the search for the natural ligand of the second cavity.

Results and Discussion
Overall Structure of the JHBP-MPD Complex The crystal structure of B. mori JHBP in complex with MPD was solved at a resolution of 2.6 Å by a single-wavelength anomalous dispersion (SAD) method using selenomethionine (SeMet)-substituted crystals ( Figure 2 and Table 1). As shown in Figure 2A, the MPD-bound JHBP adopts a gate-closed conformation which is similar to the structure of the JH-bound JHBP [13]. The refined model contains four JHBP monomers (A, B, C and D) in the crystal asymmetric unit, which are related by 222 point group symmetry ( Figure 2B). No stable dimerization could be seen in any two pairs of the four NCS-related molecules. Consistent with this, gel filtration and ultracentrifugation experiments as well as NMR spectroscopy have shown unambiguously that JHBP is monomeric in solution [13,14,18,29,30].
We could trace the electron density of residues 7-174 and 183-222 for molecule A, of 7-175 and 180-222 for molecule B, of 5-175 and 183-223 for molecule C, and of 7-174 and 182-222 for molecule D. The four molecules overlay well with one another, giving root mean square difference (RMSD) values of less than 0.70 Å for C a atoms between any two pairs of them. For all the four JHBP molecules, we revealed two bound MPD molecules, one (MPD1) in the JH-binding pocket and the other (MPD2) in the second cavity at each end of the elongated structure (Figures 2A  and C). These MPD molecules were most likely incorporated from the precipitant solution during crystallization of the apoprotein.

Structural Plasticity of the JH Binding Pocket in JHBP
Comparison of the MPD-bound and JH II-bound JHBP structures reveals that the core of the protein assumes nearly the same structure with RMSD values ranging from 0.69 to 1.05 Å for C a atoms of residues 32-172 and 190-210 depending on the pair of chains superposed ( Figure 3A). However, pronounced displacements are observed at both ends of the elongated structure. The largest displacement involves the gate a1 helix which covers the JH-binding pocket ( Figure 3B). The structural rearrangement of a1 is correlated with the difference in the molecular size between JH and MPD. Comparing with the JH II-bound structure, the a1 helix of the MPD-bound structure is folded back into the hormone binding pocket about 11u and generates contacts of the Leu17 and Thr21 side chains in helix a1 with MPD, which is smaller in size than JH. Concomitantly, the N-terminal arm and the C-terminal tail that serve as a latch for the JH-binding pocket are pushed out toward solvent. When compared with the unliganded apo-JHBP structure [13], the movement of the a1 helix is 81u ( Figure 3B).
Reflecting differences in the orientation of the gate a1 helix, the sizes of the JH-binding pockets are different between the MPDbound and JH II-bound forms. According to the Swiss-PbdViewer version 4.1 (http://www.expasy.org/spdbv) [31], the JH-binding pocket of the JH II-bound form is a huge continuous cavity. The volume was calculated to be 874 and 903 Å 3 for the two JHcomplexes A and B in the crystal asymmetric unit, respectively. The cavity is completely closed, and matches almost accurately the van der Waals surface of the bound JH II molecule as shown in Figure 4A (drawn for JH-complex A). The cavity is sealed by two hydrogen bonds, Cys9 NH-Phe220 CO and Glu222 NH-Cys9 CO, between the N-terminal arm and the C-terminal tail ( Figure 4B). In contrast, the JH-binding pocket in the MPDbound form is separated into two smaller cavities, a large one with a volume of 507 Å 3 at the upper part which accommodates the MPD1 molecule and a tiny one with a volume of 56 Å 3 at the bottom wall as shown in Figure 4C (drawn for MPD-complex C). The latter tiny cavity corresponds to the open space below the methyl ester of the bound JH in the JHBP-JH complex. The former large cavity is slightly expanded upward compared with that of the JH-bound form, and has an open hole due to the structural rearrangements of the a1 helix, N-terminal arm, and Cterminal tail as mentioned above. It is noteworthy, however, that the Cys9 NH-Phe220 CO and Glu222 NH-Cys9 CO hydrogen bonds are still maintained in the MPD-bound structure ( Figure 4D). These observations suggest a high adaptability of the JH binding pocket which could change its size and shape in a ligand-dependent manner due to flexibility of the gate a1 helix.

MPD Mimics Epoxide of JH
The MPD molecule (MPD1) bound to the JH-binding pocket is confined in a hydrophobic cage formed by Phe78 and Met80 in b2b, Val87 and Leu89 in b3, Tyr128 and Tyr130 in b4, Phe142 and Val144 in b5a, and Phe220 and Phe221 in the C-terminal tail as shown in Figure 5A (drawn for MPD-complex C). The same cage accommodates the epoxide group of JH in the JHBP-JH II complex [13] as shown in Figure 5B (drawn for JH-complex A). In fact, MPD1 overlaps the epoxide group of JH II in the complex with JHBP as shown in Figure 5C. Furthermore, the recognition mode of MPD1 is essentially the same as that of the epoxide group of JH ( Figures 5A and B). MPD1 is anchored in the cage by a direct hydrogen bond of the O 2 H group of MPD with the hydroxyl group of Tyr128 which forms a direct hydrogen bond with the epoxy oxygen of JH in the JHBP-JH II complex. In addition, MPD1 is further stabilized by CH-p stacking interactions between two methyl groups attached to C 2 of MPD and the aromatic side chains of Tyr130 and Phe142. These two aromatic residues make similar CH-p stacking interactions with the methyl and ethyl groups attached to the epoxide of JH II in the JHBP-JH II complex.
Similarity in the binding mode between MPD1 and the epoxide moiety of JH raises the question if MPD competes with the binding of JH to JHBP. To answer this question, we performed a ligand competition binding assay by using a described method [32].
Binding of 3 H-JH III to JHBP was measured in the absence and presence of MPD. For comparison, we also tested methyl farnesoate (MF) which is the unepoxidated form of JH III and JH-agonist methoprene which can be used as an insecticide. Figure 5D shows chemical structures of the tested ligands as well as JH II. Since the local structure of methoprene around the methoxy group resembles the MPD structure, the methoxy oxygen is expected to act as the hydrogen-bond acceptor from the Tyr128 of JHBP. It has been reported by electrochemical impedance spectroscopy that methoprene binds to the recombinant G. mellonella JHBP with the dissociation constant (K d ) of 33.3 nM which is comparable to the K d values for JH I (28.6 nM) and JH III (43.5 nM) [20]. As shown in Figure 5E, MPD inhibits the binding of 3 H-labeled JH III to JHBP in a dose-dependent manner. The IC 50 value was determined to be 160 mM by least-square-fitting to the experimental data using Eq. 1 described in Materials & Methods. Methoprene displayed 5-fold stronger inhibitory activity (IC 50 = 35 mM) than MPD likely due to additional interactions with JHBP. The curve-fitting also provided the maximum reduction in the radioactivity, DF max in Eq. 1, 66% for MPD and 76% for methoprene, which are comparable to the value for JH III (68%) estimated by use of the same binding assay [32]. In contrast with MPD and methoprene, MF inhibited only 30% of the 3 H-JH III binding to JHBP even at the concentration of 2 mM ( Figure 5E). The weak inhibitory activity of MF could be attributed to the lack of the epoxy group because the structure of the remaining part is the same as JH. Hence the results of our binding assay strongly demonstrate that the epoxy group and its mimic structures, particularly the existence of the hydrogen-bond acceptor from the Tyr128 O g H, are critically important for the ligand binding to the JH-binding pocket of JHBP.
The dissociation constant (K d ) for the tested ligand can be theoretically calculated from the IC 50 value if the amount of the unliganded JHBP is negligible. For the ligand competition assay, we employed the condition that JHBP exists much more than JH as in the insect hemolymph (800 nM JHBP and 10 nM 3 H-JH III in our case). It is, therefore, difficult to estimate the precise K d value from the IC 50 value obtained from our assay. Applying the reported K d values of JH III (43.5 nM) and methoprene (33.3 nM) [20] to the equation of K d = IC 50 /(1+ [JH]/K d,JH ), the IC 50 of methoprene is theoretically calculated to be 41 nM which is threeorder of magnitude smaller than the IC 50 value (35 mM) obtained by our assay. Assuming that this three-order difference between the theoretical and our observed IC 50 values is held in the case of MPD, the K d value is estimated to be 152 nM. This value might be the maximum because MPD can also bind to the second cavity of JHBP.

Ligand-induced Conformational Change of the Second Cavity in JHBP
JHBP has a second hydrophobic cavity located on the side opposite from the JH-binding pocket. The second cavity is formed by the a2-helix, the N-terminal portion of the a3-helix, and the inner side of the highly curved b-sheet. The entrance of the cavity is formed by the b5c strand, the a2-helix, the N-terminal portion of the a3-helix, and presumably the a2-a3 loop, which is not observable for the MPD-bound JHBP because of the flexibility of the loop or conformational heterogeneity. The end of the b5c strand and the whole a2-helix forms one side-wall while the Nterminal portion of the a3-helix forms the other side-wall. Seven residues, Val50, Phe62, Tyr116, Ile159, Arg189, Ala192 and Ile193, form the inside-wall which closes the cavity at the middle of the protein structure. In contrast, the outside-part at the end of the second cavity remains open.
Comparison of the MPD-bound JHBP structure with the crystal structures of the apo-and JH II-bound JHBP [13] suggests a ligand-induced structural rearrangement of the second cavity. This cavity accommodates one MPD molecule (MPD2) in the MPDbound form ( Figure 6A) but remains empty in the apo-and JHbound forms [13] (Figure 6B). Figures 6A and 6B are drawn for the MPD-complex A and the JH-complex A, respectively. There is essentially no difference in the structure of the unliganded second cavity between the apo-and JH II-bound forms. MPD2 is partially solvent-exposed, and is held in the cavity by a water-mediated hydrogen-bond network with Tyr116 O g H and Arg189 CO, and hydrophobic interactions with Ile60 and Phe62 in b2a, Ile101 in b3, Tyr116 in b4, Ile159 and Leu161 in b5c, and Leu185, Leu189, Arg189 and Ala192 at the N-terminus of helix a3 ( Figure 6A). The a3-helix has no kink in its conformation in the MPD-bound second cavity ( Figure 6A), but is kinked at position 190 near the N-terminus in the unliganded second cavity (Figure 6B), where the side chain of Leu188 occupies the same space as MPD2 in the MPD-bound structure. In addition, a loop connecting helices a2 and a3 is visible in the apo-and JH IIbound forms. As a result, the unliganded second cavity is shallow when compared to the liganded one.
Ligand binding to the second cavity is also suggested by the crystal structure of the native G. mellonella JHBP [21]. In this structure, the second cavity accommodates an undefined small molecule for which a 7 Å long patch of electron density starts from a point about 3 Å from the functional groups of Lys51, Tyr62 and Thr193 toward the main entrance. The equivalent residues in B. mori JHBP are hydrophobic: Val50, Phe62 and Ala192. It is worth mentioning that the starting point of this undefined molecule seems to match the location of MPD2 in our JHBP-MPD complex structure. Furthermore, in the G. mellonella JHBP structure long spine a4 helix, which corresponds to the a3 helix in the B. mori JHBP, has no kink in its conformation as the MPD-bound structure of B. mori JHBP reported here. Unlike the liganded and unliganded B. mori JHBP, the a2 and a4 helices of G. mellonella JHBP are connected by a clearly observable extra helix a3.
The structural change of the a3 helix of the B. mori JHBP from the kinked conformation to the straight one after ligand binding to the second cavity results in a cavity expansion. We used the GHECOM program (http://strcomp.protein.osaka-u.ac.jp/ ghecom/) [33] to evaluate the volume of the second cavity instead of the Swiss-PbdViewer program [31] used for the JH-binding pocket because the former program provides more reasonable results for shallow pockets like the second cavity. The GHECOM analysis revealed that the volume of the MPD-bound second cavity of the B. mori JHBP is in a range of 741-929 Å 3 ( Figure 6C) while the unliganded second cavity of the same protein is separated into two smaller cavities with volumes of 86 (site 2a) and 233 (site 2b) Å 3 by the Leu185 side chain in the kinked a3 helix ( Figure 6D). It has been reported that the volume of the liganded second cavity of the G. mellonella JHBP is estimated to be 668 Å 3 by the CASTp analysis [21,34]. Our GHECOM [33] analysis of the same structure provided the volume of 706 Å 3 for the second cavity of the G. mellonella JHBP. Hence, we suggest that binding of a small molecule to the second cavity of JHBP straightens the long spine helix, a3 in B. mori JHBP or a4 in G. mellonella JHBP, and creates a proper space for the ligand. This finding, together with the MPD2-JHBP interaction mechanism, should assist in identification of the natural ligand(s) for the second cavity of JHBP.

Conclusion
In this paper, we present the crystal structure of the recombinant B. mori JHBP in complex with two molecules of MPD (MPD1 and MPD2), where MPD1 is bound in the JHbinding pocket and MPD2 in a second hydrophobic cavity. They are at different ends of the elongated protein structure. This is the first structure of JHBP in complex with a ligand which is structurally unrelated to JH. Detailed comparison with the apo-JHBP and JHBP-JH II complex structures previously reported by us [13] led to a number of intriguing findings. First, the JHbinding pocket changes its size and shape in a ligand-dependent manner due to flexibility of the gate a1 helix. Second, MPD1 mimics interactions of the epoxide moiety of JH previously observed in the JHBP-JH complex, and MPD can compete with JH in binding to the JH-binding pocket. We also confirmed that methoprene, with an MPD-like structural element, inhibits the complex formation between JH and JHBP. The existence of the hydrogen-bond acceptor from Tyr128 is critical for ligand binding to the JH-binding pocket, because methyl farnesoate, which is structurally similar to JH but lacks such acceptor, showed significantly weaker binding to JHBP. These findings could open the door to the structure-based design of novel Lepidopteraspecific IGRs which inhibit complex formation between JH and JHBP, and thus disrupt JH signaling. Third, binding of MPD to the second cavity of JHBP induces significant conformational changes accompanied with cavity expansion. This finding, together with the MPD2-JHBP interaction mechanism identified in the JHBP-MPD complex, should provide important guidance in the search for the natural ligand of the second cavity, which is presumably important for JH delivery site recognition by JHBP.

Crystallization, Data Collection and Structure Determination
Mature JHBP from Bombyx mori was expressed as a GST-fusion protein in Escherichia coli and purified as described previously [14,32]. SeMet-substituted JHBP was expressed in E. coli strain B834 (DE3). Crystals of JHBP in complex with MPD and SeMet derivative crystals were obtained by the hanging-drop vapordiffusion method at 20uC from solution containing 3 mL of apoprotein at 20 mg mL 21 in 2 mM Tris buffer, pH 8.0, and 1.5 mL of crystallant: 25% (6)-MPD (Hampton Research, Aliso Viejo, CA, USA), 0.05 M zinc acetate (Wako Pure Chemical Industry, Osaka, Japan) and 0.1 M sodium cacodylate buffer, pH 7.0 (Hampton Research). After a week, thin plate crystal clusters appeared and a typical crystal grew to the size of dimensions 500 6 20 6 10 mm.
Diffraction data were collected at beamlines of the Photon Factory (PF), High Energy Accelerator Research Organization, Tsukuba, Japan. A single crystal was scooped up in a nylon loop and directly flash-frozen in a nitrogen stream at 95 K. Native data were collected by CCD detectors (Area Detector Systems Corp., Poway, CA, USA). Data were integrated and scaled using the program DENZO and Scalepack in the HKL2000 program suite [35]. MPD complex and SeMet crystals belonged to the space group P2 1 2 1 2 1 and four molecules were present in the asymmetric unit with a Vm value of 3.0 Å 3 Da 21 and a solvent content of 59.6% [36]. Anomalous dispersion data sets for the SeMet crystal were collected near the selenium absorption edge.
Structure solution of JHBP was performed using the SAD method. The heavy atom search and initial phase calculation were conducted using the program SOLVE/RESOLVE [37,38]. A total of 16 heavy atom positions were determined, four of which were identified as bound zinc ions. An initial model produced by RESOLVE indicated that the crystal contains four JHBP molecules in the asymmetric unit. Successively, the structural model was refined using the MPD complex data of 2.6 Å resolution. Manual model rebuilding, introduction of water molecules, and molecular refinement were conducted using Coot [39] and Refmac5 [40]. A total of 20 zinc ions, eight MPD molecules and 121 water molecules were added into the final model. The stereochemistry of the models was analyzed with the program RAMPAGE [41]. Data collection and structure refinement statistics are summarized in Table 1. The atomic coordinates and structure factors have been deposited in the Protein Data Bank (3A1Z).
Figures of the proteins were generated by a combination of PyMOL (version 1.5: Schrödinger, LLC.) and PovRay (Version 3.7: Persistence of Vision Pty. Ltd.). Cavities on the protein surface were detected either by the Swiss-PbdViewer version 4.1 [31] or the program GHECOM [33]. The shapes of the cavities detected by GHECOM were visualized by use of OOSAWA (http://www. cfca.nao.ac.jp/,takedatk/COMPUTER/OOSAWA/oosawa. html) and PovRay.

Competitive Ligand Binding Assay
Competitive ligand binding assays were conducted based on the method used for the JH binding assay [32] with slight modifications. For the assay, the recombinant B. mori JHBP (800 nM) was dissolved in 20 mM Tri-HCl buffer, pH 7.9, supplemented with 5 mM magnesium acetate, 1 mM EDTA, 1 mM DTT, 1 mM PMSF (phenylmethylsulfonyl fluoride), 2 mg mL 21 Leupeptides, 1 mg mL 21 pepstatin A, 0.1 mM diisopropyl fluorophosphate, and 10 mM 3-octylthio-1,1,1-trifluoropropanone. The protein samples were incubated for 30 min at 4uC in a volume of 100 mL containing 10 nM 3 H-JH III (62.90 GBq mmol 21 ; New England Nuclear Chemicals) in the absence or the presence of the competitive ligand delivered in ethanol (1% v/v). Unbound JH was removed by the addition of dextran-coated charcoal solution (100 mL) and centrifugation for 2 min at 10,000 g. The radioactivity of a 50 mL aliquot of the supernatant was measured using Perkin-Elmer Liquid Scintillation Counter (Tri-Carb 2900TR) for quantitative determination of JHBP-bound 3 H-JH III. Data were fitted to a hyperbolic equation describing binding to a single site [42], where F obs is the observed radioactivity at any given competitive ligand concentration, F 0 is the radioactivity of the JHBP-bound 3 H-JH III in the absence of competitors, and DF max is the maximum reduction in the radioactivity. IC 50 and DF max are fitted as free parameters by non-linear squares regression analysis.