Drosophila nuclear receptor E75 is a thiolate hemoprotein
Drosophila nuclear receptor E75 is a thiolate hemoprotein
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DOI：
10.1021/bi060537a
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发表时间：
2006-08-15
期刊：
影响因子：
2.9
通讯作者：
Jouve, Helene M.
中科院分区：
文献类型：
--
作者：
de Rosny, Eve;de Groot, Arjan;Jouve, Helene M.
果蝇E75是核受体超家族的成员。这些真核生物转录因子参与几乎所有的生理过程。它们响应刚性疏水激素配体的结合而调节转录。与许多核受体一样，E75激素配体最初是未知的。然而，最近的研究表明，E75的配体结合结构域（LBD）含有紧密结合的血红素辅基，并且是气体响应性的。在这里，我们已经使用了定点诱变沿着与紫外可见和电子顺磁共振（EPR）光谱表征和分配血红素铁轴向配体在E75。F370Y突变和在大肠杆菌中表达蛋白质期间向生长培养基中添加氯化血红素对于产生良好产量的富含血红素的E75 LBD是必要的。EPR研究表明，存在几个物种含有强烈的铁结合硫醇盐。半胱氨酸396和468参与血红素结合，随后通过单突变和双突变显示。使用类似的方法，我们还确定了第六个铁配体的一个明确的协调构象，占总物种的大约一半，是组氨酸574。讨论了其他铁配位对。我们的结论是，E75是一个新的例子，硫醇血红素蛋白，它可能参与激素合成调节。
Drosophila E75 is a member of the nuclear receptor superfamily. These eukaryotic transcription factors are involved in almost all physiological processes. They regulate transcription in response to binding of rigid hydrophobic hormone ligands. As it is the case for many nuclear receptors, the E75 hormone ligand was originally unknown. Recently, however, it was shown that the ligand binding domain (LBD) of E75 contains a tightly bound heme prosthetic group and is gas responsive. Here we have used site-directed mutagenesis along with UV-visible and electron paramagnetic resonance (EPR) spectroscopies to characterize and assign the heme iron axial ligands in E75. The F370Y mutation and addition of hemin to the growth medium during expression of the protein in Escherichia coli were necessary to produce good yields of heme-enriched E75 LBD. EPR studies revealed the presence of several species containing a strongly iron bound thiolate. The involvement of cysteines 396 and 468 in heme binding was subsequently shown by single and double mutations. Using a similar approach, we have also established that the sixth iron ligand of a well-defined coordination conformation, which accounts for approximately half of the total species, is histidine 574. The other iron coordination pairs are discussed. We conclude that E75 is a new example of a thiolate hemoprotein and that it may be involved in hormone synthesis regulation.