Covalent attachment of heme to the protein moiety in an insect E75 nitric oxide sensor.
Covalent attachment of heme to the protein moiety in an insect E75 nitric oxide sensor.
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DOI：
10.1021/bi300848x
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发表时间：
2012-09-18
期刊：
影响因子：
2.9
通讯作者：
Rodriguez-Crespo I
中科院分区：
文献类型：
--
作者：
Aicart-Ramos C;Valhondo Falcón M;Ortiz de Montellano PR;Rodriguez-Crespo I
我们重组表达并纯化了E75家族四种昆虫核受体的配体结合域。果蝇和家蚕核受体被纯化为铁血红素蛋白，在424 nm处具有Soret最大值，而它们的亚铁形式在425 nm处具有Soret最大值，其响应于·NO和CO结合。与此相反，纯化的LBD的Oncopeltus fasciatus显示Soret最大值在415 nm的铁蛋白，转移到425 nm的亚铁状态。·NO与D的血红素部分的结合。melanogaster和B. mori E75 LBD在385 nm处出现一个峰，而O. Fasciatus hemoprotein，类似于其人类同系物Rev-erbβ所显示的行为。HPLC分析表明，与D. melanogaster和B. mori对应物，O. Fasciatus通过两个氨基酸的侧链共价连接到蛋白质上。与O. Fasciatus E75的克隆和表达，证实了德国小蠊LBD的光谱特性与O. Fasciatus，并且它还具有与蛋白质共价结合的血红素基团。因此，·NO/CO调节这些核受体的转录活性可能在不同的昆虫物种之间受到不同的控制。此外，共价血红素结合提供了强有力的证据表明，至少有一些这些核受体的功能作为双原子气体传感器，而不是血红素传感器。最后，我们的研究结果扩展了血红素蛋白类，其中血红素基团通常共价连接到多肽链。
We have recombinantly expressed and purified the ligand binding domains (LBDs) of four insect nuclear receptors of the E75 family. The Drosophila melanogaster and Bombyx mori nuclear receptors were purified as ferric hemoproteins with Soret maxima at 424 nm, whereas their ferrous form had a Soret maximum at 425 nm that responds to ·NO and CO binding. In contrast, the purified LBD of Oncopeltus fasciatus displayed a Soret maximum at 415 nm for the ferric protein that shifted to 425 nm in its ferrous state. Binding of ·NO to the heme moiety of D. melanogaster and B. mori E75 LBD resulted in the appearance of a peak at 385 nm, whereas this peak appeared at 416 nm in the case of the O. fasciatus hemoprotein, resembling the behaviour displayed by its human homolog Rev-erbβ. HPLC analysis revealed that, unlike the D. melanogaster and B. mori counterparts, the heme group of O. fasciatus is covalently attached to the protein through the side-chains of two amino acids. The large sequence homology with O. fasciatus E75 led us to clone and express the LBD of Blattella germanica, which established that its spectral properties closely resemble those of O. fasciatus and that it also has the heme group covalently bound to the protein. Hence, ·NO/CO regulation of the transcriptional activity of these nuclear receptors might be differently controlled among various insect species. In addition, covalent heme binding provides strong evidence that at least some of these nuclear receptors function as diatomic gas sensors rather than heme sensors. Finally, our findings expand the classes of hemoproteins in which the heme group is normally covalently attached to the polypeptide chain.
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