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

  • Koiwai, Kotaro1
  • Inaba, Kazue1, 2
  • Morohashi, Kana2
  • Enya, Sora2
  • Arai, Reina2
  • Kojima, Hirotatsu3
  • Okabe, Takayoshi3
  • Fujikawa, Yuuta4
  • Inoue, Hideshi4
  • Yoshino, Ryunosuke5
  • Hirokawa, Takatsugu6, 7, 8
  • Kato, Koichiro9
  • Fukuzawa, Kaori10
  • Shimada-Niwa, Yuko11
  • Nakamura, Akira12
  • Yumoto, Fumiaki1
  • Senda, Toshiya1, 13, 14
  • Niwa, Ryusuke1, 11
  • 1 Structural Biology Research Center, Photon Factory, Institute of Materials Structure Science, High Energy Accelerator Research Organization, 1-1 Oho, Tsukuba, Ibaraki 305-0801, Japan
  • 2 Graduate School of Life and Environmental Sciences, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8572, Japan
  • 3 Drug Discovery Initiative, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan
  • 4 School of Life Sciences, Tokyo University of Pharmacy and Life Sciences, 1432-1 Horinouchi, Hachioji, Tokyo 192-0392, Japan
  • 5 Graduate School of Comprehensive Human Sciences Majors of Medical Sciences, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8575, Japan
  • 6 Transborder Medical Research Center, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8575, Japan
  • 7 Division of Biomedical Science, Faculty of Medicine, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8575, Japan
  • 8 Aomi, Koto-ku, Tokyo 135-0064, Japan
  • 9 Mizuho Information & Research Institute, Inc., 2-3 Kanda Nishiki-cho, Chiyoda-ku, Tokyo 101-8443, Japan
  • 10 School of Pharmacy and Pharmaceutical Sciences, Hoshi University, 2-4-41 Ebara, Shinagawa-ku, Tokyo 142-8501, Japan
  • 11 Life Science Center for Survival Dynamics, Tsukuba Advanced Research Alliance (TARA), University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8577, Japan
  • 12 Institute of Molecular Embryology and Genetics, Kumamoto University, 2-2-1 Honjo, Chuo-ku, Kumamoto 860-0811, Japan
  • 13 School of High Energy Accelerator Science, Sokendai University, 1-1 Oho, Tsukuba, Ibaraki 305-0801, Japan
  • 14 Faculty of Pure and Applied Sciences, University of Tsukuba, 1-1-1 Tennodai, Ibaraki 305-8571, Japan
Published Article
Journal of Biological Chemistry
American Society for Biochemistry and Molecular Biology
Publication Date
Apr 02, 2020
DOI: 10.1074/jbc.RA119.011463
PMID: 32241910
PMCID: PMC7242711
PubMed Central


Ecdysteroids are the principal steroid hormones essential for insect development and physiology. In the last 18 years, several enzymes responsible for ecdysteroid biosynthesis encoded by Halloween genes were identified and genetically and biochemically characterized. However, the tertiary structures of these proteins have not yet been characterized. Here, we report the results of an integrated series of in silico , in vitro , and in vivo analyses of the Halloween GST protein Noppera-bo (Nobo). We determined crystal structures of Drosophila melanogaster Nobo (DmNobo) complexed with GSH and 17β-estradiol, a DmNobo inhibitor. 17β-Estradiol almost fully occupied the putative ligand-binding pocket and a prominent hydrogen bond formed between 17β-estradiol and Asp-113 of DmNobo. We found that Asp-113 is essential for 17β-estradiol–mediated inhibition of DmNobo enzymatic activity, as 17β-estradiol did not inhibit and physically interacted less with the D113A DmNobo variant. Asp-113 is highly conserved among Nobo proteins, but not among other GSTs, implying that this residue is important for endogenous Nobo function. Indeed, a homozygous nobo allele with the D113A substitution exhibited embryonic lethality and an 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 that appears to be essential for binding an endogenous sterol substrate to regulate ecdysteroid biosynthesis. To the best of our knowledge, ours is the first study describing the structural characteristics of insect steroidogenic Halloween proteins. Our findings provide insights relevant for applied entomology to develop insecticides that specifically inhibit ecdysteroid biosynthesis.

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