An integrated approach unravels a crucial structural property for the function of the insect steroidogenic Halloween protein Noppera-bo

Ecdysteroids are the principal insect steroid hormones essential for insect development and physiology. In the last 18 years, several enzymes responsible for ecdysteroid biosynthesis, encoded by Halloween genes, have been identified and well characterized, both genetically and biochemically. However, none of these proteins have yet been characterized at the tertiary structure level. Here, we report an integrated in silico, in vitro, and in vivo analyses of the Halloween glutathione S-transferase (GST) protein, Noppera-bo (Nobo). We determine crystal structures of Drosophila melanogaster Nobo (DmNobo) complexed with glutathione and 17β-estradiol, a DmNobo inhibitor. 17β-estradiol almost fully occupied the putative ligand-binding pocket, and a prominent hydrogen bond formed between Asp113 of DmNobo and 17β-estradiol. Asp113 is essential for inhibiting DmNobo enzymatic activity by 17β-estradiol, as 17β-estradiol does not inhibit and physically interacts less with the Asp113Ala DmNobo point mutant. Asp113 is highly conserved among Nobo proteins, but not among other GSTs, implying that Asp113 is important for endogenous Nobo function. Indeed, a homozygous nobo allele possessing the Asp113Ala point mutation exhibits embryonic lethality with undifferentiated cuticle structure, a phenocopy of complete loss-of-function nobo homozygotes. These results suggest that the nobo family of GST proteins has acquired a unique amino acid residue, which seems to be essential for binding an endogenous sterol substrate to regulate ecdysteroid biosynthesis. This is the first study to reveal the structural characteristics of insect steroidogenic Halloween proteins. This study also provides basic insight into applied entomology for developing a new type of insecticides that specifically inhibit ecdysteroid biosynthesis. Significance Statement Insect molting and metamorphosis are drastic and dynamic biological processes and, therefore, have fascinated many scientists. Ecdysteroids represent one class of insect hormones that are indispensable for inducing molting and metamorphosis. It is well known that proteins responsible for catalyzing ecdysteroid biosynthesis reactions are encoded by “Halloween” genes, most of which have names of ghosts and phantoms. However, no studies have focused on the structural properties of these biosynthetic proteins. In this study, we addressed this unsolved issue and successfully unraveled a structural property that is crucial for the function of the fruit fly Halloween protein, Noppera-bo (a Japanese faceless ghost). This is the first study to reveal the structural characteristics of an insect steroidogenic Halloween protein.

results suggest that Asp113 is critical for interaction with EST.

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We also employed MD simulations to confirm the contribution of Asp113 to the 2 2 8 interaction with EST using DmNobo [WT] and DmNobo[Asp113Ala] as models. In these MD for 100 nano seconds (ns), we found that the distance between Oδ of Asp113 and the in DmNobo [WT] was less than ~6.60 Å (SI Appendix, Fig. S6A, Fig. S6B). In contrast, with 2 3 7 the MD simulation of DmNobo[Asp113Ala], the maximum RMSD value was less than ~9.54 2 3 8 9 Å (SI Appendix, Fig. S6A, Fig. S6B). These simulation results also support the possibility that 2 3 9 hydrogen bonding between Asp113 and EST is required for stable binding of EST to the H- Previous reports have demonstrated that the nobo family of GSTs is found in Diptera and 2 4 4 Lepidoptera (18,30,31). Amino acid-sequence analysis revealed that all Nobo proteins from 2 4 5 6 dipteran and 13 lepidopteran species have Asp at the position corresponding to Asp113 of 2 4 6 DmNobo ( Fig. 3A, Fig. 3B, Fig. 3D). An exception is found in Nobo of the yellow fever residue was found at the corresponding position of the DmGSTD/E/T proteins, other than inhibit the enzymatic activity of the DmGSTE6 or DmGSTE9 recombinant proteins (Fig. 3G).

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These results suggest that Nobo proteins utilize Asp113 to recognize their target compounds 2 5 3 as a common feature and that Asp113 serves a biological role. Finally, we examined whether Asp113 is essential for any in vivo biological function of 2 5 7 DmNobo. We utilized a CRISPR-Cas9-based knock-in strategy to generate a nobo allele 2 5 8 encoding an Asp113Ala point mutation (nobo 3×FLAG-HA-D113A ). We found that no trans- nobo 3×FLAG-HA-D113A /nobo KO genotype is embryonic lethal. We also found that nobo 3×FLAG-HA-2 6 4 D113A /nobo KO embryos exhibit an undifferentiated cuticle phenotype (Fig. 5A, Fig. 5B) and a 2 6 5 failure of head involution (Fig. 5C, Fig. 5D). These phenotypic characteristics were very Nobo 3×FLAG-HA-WT (Fig. 5E, Fig. 5F), suggesting that the phenotypes were due to loss of 2 6 9 protein function, but not impaired gene expression. Taken together, these results show that GSTs have been shown to exhibit "ligandin" function to carry and transport specific ligands The activities of insect ecdysteroids can be disrupted in vivo using chemical agonists 3 0 9 and antagonists of the ecdysone receptor, some of which are also utilized as insecticides (33).

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However, chemical compounds that specifically inhibit ecdysteroid biosynthesis are not 3 1 1 available. This study provides the first structural information for guiding the development of practical compound that can be utilized as an actual insecticide must display no-estrogenic 3 1 7 activity. To consider this problem, it is important to note a difference in the EST-recognition of ERα, and DmNobo utilizes a Cys residue of GSH for an SH/π interaction with the A-ring 3 2 6 of EST. However, no interaction was found between O17 of EST and residues of the H-site of 3 2 7 DmNobo (SI Appendix, Fig. S8). Given this difference, we expect that a Nobo-specific, non- computational calculations to select chemical compounds that satisfy those conditions and an 3 3 0 in vitro enzymatic assay to examine DmNobo inhibition.

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We emphasize that this report is the first to describe the physical interactions between 3 3 2 a Halloween protein and a potent inhibitor at the atomic level. Our interdisciplinary approach 3 3 3 will also be applicable for Nobo proteins other than D. melanogaster, such as disease vector insecticides useful for human societies. Kd values were determined by SPR using a Biacore T200 instrument and a CM5 sensorchip CT+mix, and DI components) using paired interaction-energy decomposition analysis 3 8 5 (PIEDA) (28, 29). suitable for pH 7.0 ± 2.0 were predicted using Epik (52), and H-bond optimization was 3 9 1 conducted using PROPKA (53). Energy minimization was performed in Maestro using the 3 9 2 OPLS3 force field (54). Preparation for MD simulation was conducted using the Molecular DmNobo[Asp113Ala]_EST-GSH, which were subjected to energy minimization, were placed 3 9 5 in an orthorhombic box with a buffer distance of 10 Å to create a hydration model, using the 3 9 6 TIP3P water model (55). NaCl (0.15 M) was added as the counterion to neutralize the system. The MD simulations were performed using Desmond ver. 2.3 (Schrödinger) (56). The cut-off 3 9 8 radii for the van der Waals and electrostatic interactions, and the time step, initial temperature, and pressure of the system were set to 9 Å, 2.0 fs, 300 K, and 1.01325 bar, respectively. The  For the phylogenetic analysis of insect GSTD/E/T proteins, previously described  Grishin-sequence difference of 0.9, which included 151 GSTDs, 178 GSTEs, and 42 GSTTs. The GSTEs included 21 Nobo proteins. To calculate the amino acid frequencies, the obtained 4 1 6 alignment was manually edited based on the known crystal structures, using Jalview (63).

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The amino acid frequencies were calculated and illustrated using WebLOGO version 3.7.4 4 1 8 (64), and colored using the "Chemistry (AA)" scheme. The generation of D. melanogaster knock-in flies was performed as described in the SI 4 2 2 materials. We found that nobo 3×FLAG-HA-WThomozygous flies were fully viable, whereas HA-D113A -heterozygous and -homozygous embryos for cuticle preparation and immunostaining.

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To formally rule out the possibility that the embryonic lethality was due to anonymous 4 2 6 deleterious mutations other than nobo 3×FLAG-HA-D113A , we counted the number of trans-1 5 Embryonic cuticle preparation was performed as previously described (65). Immunostaining 4 3 8 for whole-mount embryos was conducted as previously described (18) immunostaining of the brain-ring gland complex in third-instar larvae, we first crossed  We thank Teruki Honma and Chiduru Watanabe at RIKEN for discussions regarding the Iowa) for providing us with antibodies. We are also grateful to Yoshiaki Nakagawa and Private University Research Branding Project. In addition, this work was supported by the hydroxylation of ecdysone to the steroid insect molting hormone 20-hydroxyecdysone.