Additional file 1 of Molecular action of larvicidal flavonoids on ecdysteroidogenic glutathione S-transferase Noppera-bo in Aedes aegypti
Creators: name: Inaba, Kazue
name: Ebihara, Kana
name: Senda, Miki
name: Yoshino, Ryunosuke
name: Sakuma, Chisako
name: Koiwai, Kotaro
name: Takaya, Daisuke
name: Watanabe, Chiduru
name: Watanabe, Akira
name: Kawashima, Yusuke
name: Fukuzawa, Kaori
name: Imamura, Riyo
name: Kojima, Hirotatsu
name: Okabe, Takayoshi
name: Uemura, Nozomi
name: Kasai, Shinji
name: Kanuka, Hirotaka
name: Nishimura, Takashi
name: Watanabe, Kodai
name: Inoue, Hideshi
name: Fujikawa, Yuuta
name: Honma, Teruki
name: Hirokawa, Takatsugu
name: Senda, Toshiya
name: Niwa, Ryusuke
Publisher: name: figshare
Year: 2022
Subjects: subject: Biochemistry
subject: Cell Biology
subject: Molecular Biology
subject: Pharmacology
subject: Biotechnology
subject: 39999 Chemical Sciences not elsewhere classified
subject: FOS: Chemical sciences
subject: Science Policy
subject: Developmental Biology
subject: Cancer

description: Additional file 1 : Figure S1. Glu-113 of AeNobo and Asp-113 of DmNobo. (A) Comparison of predicted amino acid sequences between Ae. aegypti LOC5569853 proteins encoded by the LOC5569853 gene and the wildtype DmNobo proteins. There are two predicted amino acid sequences in the GenBank database, EAT40301.1 and XM_001658698.3, whose amino acid (aa) lengths are 220 and 271, respectively. The 220-aa protein, rather than the 271-aa protein, is substantially similar to DmNobo. Therefore, we used the gene encoding the 220-aa protein for the transgenic rescue experiment (Additional file 2: Table S1) and all biochemical and structure biological analyses in this study. Glu-113 of AeNobo and Asp-113 of DmNobo are marked with a red box. (B) The hydrogen bonds between Asp-113 of DmNobo and 17β-estradiol, Glu-113 of AeNobo and luteolin, and Glu-113 of AeNobo and daidzein. Carbon atoms of 17β-estradiol and flavonoids are colored pink and green, respectively. Oxygen and nitrogen atoms are colored red and blue, respectively. Hydrogen bonds are illustrated by dashed yellow lines. As shown in the most right panel (Superposed), Asp-113 of DmNobo and Glu-113 of AeNobo are present at the similar location. Figure S2. 17β-estradiol inhibitory activity against AeNobo is less than that against DmNobo. (A) Chemical structure of 17β-estradiol. (B) Inhibition of the GSH conjugation activities of DmNobo (left) and AeNobo (right) determined using an artificial fluorescent substrate 3,4-DNADCF in the presence of 17β-estradiol. Each relative activity is defined as the ratio of activity compared between the respective proteins without 17β-estradiol. All the data points in triplicate (for DmNobo) or duplicate (for AeNobo) assays are indicated. Figure S3. Chemical structure of 14 flavonoids used in our initial chemical screen. 2′-hydroxyflavanone was used for the experiment shown in Fig. 1A. Thirteen other flavonoids, including the subclasses of flavonone, flavone, isoflavone, flavonol, isoflavan, and anthocyanidin, were used for the experiment shown in Fig. 1B. The IC50 values of the 14 flavonoids are shown in Table 1. Figure S4. Purification of AeNobo-WT. (A) AeNobo-WT was purified using size exclusion column chromatography. The UV charts of AeNobo-WT and AeNobo-E113A are shown using blue and orange lines, respectively. The peak points of marker proteins are indicated using black solid lines. The marker proteins used were: β-amylase from sweet potato (223.8 kDa; a), alcohol dehydrogenase from Saccharomyces cerevisiae (146.8 kDa; b), bovine serum albumin (66.5 kDa; c), human GSTP1-1 (46.7 kDa in solution; d), and RNase A (13.7 kDa; e). (B) The asymmetric unit of AeNobo is composed of four chains: A, B, C, and D. (C) Biological dimers with a symmetry-related subunit by a crystallographic two-fold axis: Chain A–Chain A´, Chain B–Chain B´´, Chain C–Chain D´´´, and Chain D–Chain C´´´´. (D) Overlaid structures of the 4 dimer pairs shown in C. Figure S5. Simulated annealing (SA) omit maps for GSH and flavonoids in all chains of AeNobo. mFo-DFc maps (green) (3.0 σ) are shown. Carbon atoms of AeNobo and GSH and flavonoids are colored deep teal and wheat, respectively. Carbon atoms of daidzein (B), luteolin (C), and desmethylglycitein (D) are colored orange, light violet, and gray, respectively. Oxygen, nitrogen, and sulfur atoms are colored red, blue, and yellow, respectively. Figure S6. Interaction between AeNobo and GSH. Carbon atoms of GHS, daidzein, luteolin, and desmethylglycitein (DMG) are colored wheat, orange, light violet, and gray, respectively. Oxygen, nitrogen, and sulfur atoms are colored red, blue, and yellow, respectively. Water molecules are shown as yellow spheres. Hydrogen bonds are illustrated by yellow dashed line. (A) Position of GSH and surrounding amino acids of the chain D of AeNobo complexed with GSH (left), with GSH and daidzein (middle), and with GSH and desmethylglycitein (DMG). (B) Position of GSH and surrounding amino acids of the chain B of AeNobo complexed with GSH (left), and with GSH and luteolin. Figure S7. Hydrogen bonds between all chains of AeNobo and daidzein, luteolin, and desmethylglycitein (DMG). Hydrogen bonds are illustrated by yellow dashed line. (A) Position of Glu-113 of AeNobo-GSH with (orange) or without (pink) daidzein. (B) Position of Glu-113 of AeNobo-GSH with (red) or without (pink) luteolin. (C) Position of Glu-113 of AeNobo-GSH with (gray) or without (pink) DMG. Note that the hydrogen bonds are observed in all 4 chains. Figure S8. Distribution of RMSD values of daidzein and desmethylglycitein (DMG) in AeNobo-WT and AeNobo-E113A. All RMSD values were calculated from three independent MD simulations for each complex (3 × 10000 snapshots). ** p&lt;0.01 Student’s t-test. Figure S9. Phe-39 of AeNobo. (A, B) A 3-dimentional position of desmethylglycitein (DMG) (A) and luteolin (B) with Glu-113 and Phe-39 of AeNobo. Carbon atoms of DMG and luteolin are colored gray and light violet, respectively. Oxygen, nitrogen, and sulfur atoms are colored red, blue, and yellow, respectively. (A) The hydrogen bonds (dashed yellow lines) between Glu-113 of AeNobo and DMG. Additionally, DMG forms a CH-π interaction (dashed black line) with Phe-39. (B) The hydrogen bonds (dashed yellow lines) between Glu-113 of AeNobo and luteolin. There is no obvious interaction between Phe-39 and luteolin. (C, D) Inhibition of the GSH conjugation activities of AeNobo-WT (blue) and AeNobo-F39L (red), determined using an artificial fluorescent substrate 3,4-DNADCF in the presence of DMG (C) or luteolin (D). Each relative activity is defined as the ratio of activity compared between the respective proteins without flavonoids. All the data points in triplicate (for DmNobo) and duplicate (for AeNobo) assays are indicated. Figure S10. Pupariation ratio of control and 10 ppm DMG-treated D. melanogaster. The pupariation timing was not changed in 10 ppm DMG-treated larvae (red, n=240) as compared with control larvae (grey, n=240).