Specific ligand binding domain residues confer low dioxin responsiveness to AHR1β of Xenopus laevis.
Specific ligand binding domain residues confer low dioxin responsiveness to AHR1β of Xenopus laevis.
复制标题
特定的配体结合域残基赋予非洲爪蟾 AHR1β 的低二恶英反应性。
DOI：
10.1021/bi301722k
复制
发表时间：
2013
期刊：
影响因子：
2.9
通讯作者：
Powell,WadeH
中科院分区：
文献类型：
--
作者：
Odio,Camila;Holzman,SarahA;Denison,MichaelS;Fraccalvieri,Domenico;Bonati,Laura;Franks,DianaG;Hahn,MarkE;Powell,WadeH
芳烃受体 (AHR) 是一种 Per-ARNT-Sim (PAS) 家族蛋白，可介导 2,3,7,8-四氯二苯并-对二恶英 (TCDD) 对脊椎动物的毒性。青蛙对 TCDD 非常不敏感，非洲爪蟾的 AHR 与 TCDD 的亲和力较低。我们试图确定 X 的结构特征。 laevisAHR1β 与 TCDD 敏感性低相关。用小鼠 AHRb-1 的相应序列替换整个配体结合域 (LBD) 可显着提高反式激活测定中的 TCDD 响应性。为了确定负责的氨基酸残基，我们使用 PAS 蛋白 HIF2α 和 ARNT 的同源结构域构建了 AHR1β LBD 的比较模型。该模型揭示了一个内部空腔，其尺寸与假定的小鼠 AHRb-1 结合空腔的尺寸相似，这表明侧链相互作用对于空腔尺寸的重要性。在侧链明显指向空腔的残基中，只有两个与小鼠序列不同。当位于保守 β 链内的 A354 变为丝氨酸（相应的小鼠残基）时，TCDD 的 EC50 降低了 15 倍以上。当N325改为丝氨酸时，EC50降低了3倍。当突变组合时，EC50从18.6下降到0.8 nM，该值几乎与小鼠AHR的TCDD敏感性相匹配。速度沉降分析证实突变青蛙 AHR 的 TCDD 结合水平相应增加。我们还分析了突变 AHR 对候选内源配体 6-甲酰基吲哚并[3,2-b]咔唑 (FICZ) 的反应性。增加对 TCDD 敏感性的突变也增加了对 FICZ 的敏感性。这项比较研究代表了一种新颖的方法来辨别有关 AHR 结构及其与生物学上重要的激动剂相互作用的基本信息。
The aryl hydrocarbon receptor (AHR) is a Per-ARNT-Sim (PAS) family protein that mediates the toxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in vertebrates. Frogs are remarkably insensitive to TCDD, and AHRs fromXenopus laevisbind TCDD with low affinity. We sought to identify structural features ofX. laevisAHR1β associated with low TCDD sensitivity. Substitution of the entire ligand binding domain (LBD) with the corresponding sequence from mouse AHRb-1dramatically increased TCDD responsiveness in transactivation assays. To identify the amino acid residues responsible, we constructed a comparative model of the AHR1β LBD using homologous domains of PAS proteins HIF2α and ARNT. The model revealed an internal cavity with dimensions similar to those of the putative binding cavity of mouse AHRb-1, suggesting the importance of side chain interactions over cavity size. Of residues with side chains clearly pointing into the cavity, only two differed from the mouse sequence. When A354, located within a conserved β-strand, was changed to serine, the corresponding mouse residue, the EC50for TCDD decreased more than 15-fold. When N325 was changed to serine, the EC50decreased 3-fold. When the mutations were combined, the EC50decreased from 18.6 to 0.8 nM, the value nearly matching the TCDD sensitivity of mouse AHR. Velocity sedimentation analysis confirmed that mutant frog AHRs exhibited correspondingly increased levels of TCDD binding. We also assayed mutant AHRs for responsiveness to a candidate endogenous ligand, 6-formylindolo[3,2-b]carbazole (FICZ). Mutations that increased sensitivity to TCDD also increased sensitivity to FICZ. This comparative study represents a novel approach to discerning fundamental information about the structure of AHR and its interactions with biologically important agonists.