Helix 11 dynamics is critical for constitutive androstane receptor activity.
Helix 11 dynamics is critical for constitutive androstane receptor activity.
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Helix 11 动力学对于组成型雄甾烷受体活性至关重要。
DOI：
10.1016/j.str.2010.11.008
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发表时间：
2011
期刊：
影响因子：
--
通讯作者：
Fernandez,EliasJ
中科院分区：
文献类型：
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作者：
Wright,Edward;Busby,ScottA;Wisecarver,Sarah;Vincent,Jeremy;Griffin,PatrickR;Fernandez,EliasJ
组成型雄烷受体（CAR）反式激活可在不存在外源性配体的情况下发生，并且该活性通过激动剂TCPOBOP和美克洛嗪增强。我们使用生物物理和基于细胞的测定来显示CAR（TCPOBOP）相对于CAR（美克洛嗪）的增加的活性对应于CAR（TCPOBOP）对类固醇受体辅激活因子-1的更高亲和力。此外，稳态荧光光谱表明CAR（TCPOBOP）：RXR和CAR（美克洛嗪）：RXR之间的构象差异。氢/氘交换（HDX）数据表明CAR活化功能2（AF-2）在CAR（TCPOBOP）：RXR和CAR（美克洛嗪）：RXR中比在CAR：RXR中更稳定。HDX动力学还显示CAR（TCPOBOP）：RXR和CAR（美克洛嗪）：RXR之间的显著差异。与CAR（美克洛嗪）：RXR不同，CAR（TCPOBOP）：RXR显示出延伸到RXR的更高的整体稳定性。我们将CAR中的残基339-345鉴定为别构调节位点，其在CAR（TCPOBOP）：RXR中的交换动力学降低幅度大于CAR（美克洛嗪）：RXR。因此，在CAR上亮氨酸-340和亮氨酸-343处具有突变的测定证实了该区域是CAR活性的重要决定因素。
The constitutive androstane receptor (CAR) transactivation can occur in the absence of exogenous ligand and this activity is enhanced by agonists TCPOBOP and meclizine. We use biophysical and cell-based assays to show that increased activity of CAR(TCPOBOP) relative to CAR(meclizine) corresponds to a higher affinity of CAR(TCPOBOP) for the steroid receptor coactivator-1. Additionally, steady-state fluorescence spectra suggest conformational differences between CAR(TCPOBOP):RXR and CAR(meclizine):RXR. Hydrogen/deuterium exchange (HDX) data indicate that the CAR activation function 2 (AF-2) is more stable in CAR(TCPOBOP):RXR and CAR(meclizine):RXR than in CAR:RXR. HDX kinetics also show significant differences between CAR(TCPOBOP):RXR and CAR(meclizine):RXR. Unlike CAR(meclizine):RXR, CAR(TCPOBOP):RXR shows a higher overall stabilization that extends into RXR. We identify residues 339–345 in CAR as an allosteric regulatory site with a greater magnitude reduction in exchange kinetics in CAR(TCPOBOP):RXR than CAR(meclizine):RXR. Accordingly, assays with mutations on CAR at leucine-340 and leucine-343 confirm this region as an important determinant of CAR activity.