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 , , and analyses of the Halloween glutathione -transferase (GST) protein, Noppera-bo (Nobo). We determine crystal structures of 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 allele possessing the Asp113Ala point mutation exhibits embryonic lethality with undifferentiated cuticle structure, a phenocopy of complete loss-of-function homozygotes. These results suggest that the 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.