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.

中文翻译：

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一种综合方法来揭示昆虫类固醇生成万圣节蛋白Noppera-bo的功能所需的关键结构特性。

蜕皮类固醇是昆虫发育和生理必不可少的主要类固醇激素。在过去的18年中，万圣节基因编码的几种负责蜕皮甾类生物合成的酶被鉴定出来，并进行了遗传和生物化学表征。然而，这些蛋白质的三级结构尚未被表征。在这里，我们报告了万圣节GST蛋白Noppera-bo（Nobo）的一系列计算机模拟，体外和体内分析的结果。我们确定了果蝇果蝇（DmNobo）与GSH和17β-雌二醇（一种DmNobo抑制剂）复合的晶体结构。17β-雌二醇几乎完全占据了假定的配体结合口袋，并且在17β-雌二醇和DmNobo的Asp-113之间形成了显着的氢键。我们发现Asp-113对于17β-雌二醇介导的DmNobo酶活性的抑制是必不可少的，因为17β-雌二醇不抑制D113A DmNobo变体并且与D113A DmNobo变体的相互作用较少。Asp-113在Nobo蛋白中是高度保守的，但在其他GST中却不是，这表明该残基对于内源性Nobo功能很重要。确实，具有D113A取代的纯合的nobo等位基因表现出胚胎致死性和未分化的表皮结构，即完全丧失功能的nobo纯合子的表型。这些结果表明，GST蛋白的nobo家族已经获得了独特的氨基酸残基，这似乎对于结合内源性固醇底物来调节蜕皮甾类生物合成至关重要。据我们所知，我们的研究是描述昆虫类固醇激素万圣节蛋白的结构特征的第一项研究。我们的发现提供了与应用昆虫学相关的见解，以开发能特异性抑制蜕皮甾类生物合成的杀虫剂。