Heme-binding characteristics of the isolated PAS-A domain of mouse Per2, a transcriptional regulatory factor associated with circadian rhythms
Heme-binding characteristics of the isolated PAS-A domain of mouse Per2, a transcriptional regulatory factor associated with circadian rhythms
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DOI：
10.1021/bi7023892
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发表时间：
2008-06-10
期刊：
影响因子：
2.9
通讯作者：
Shimizu, Toru
中科院分区：
文献类型：
--
作者：
Kitanishi, Kenichi;Igarashi, Jotaro;Shimizu, Toru
神经元PAS蛋白2（NPAS 2）是一种血红素结合转录调节因子，参与昼夜节律。周期同源物（Period homologue，Per）是与隐花色素（cryptochrome，Cry）结合的另一个重要的转录调控因子。所得Per/Cry异二聚体与NPAS 2/BMAL 1异二聚体相互作用以抑制Per和Cry的转录。先前的细胞生物学实验表明小鼠Per 2（mPef 2）也是血红素结合蛋白，并且血红素穿梭于mPer 2和NPAS 2之间可以调节转录。在本研究中，我们表明，分离的PAS-A结构域的mPer 2（PAS-A-mPer 2）结合铁（III）原卟啉IX复合物（氯化血红素）与血红素：蛋白质化学计量为1：1。光学吸收和EPR光谱研究结果表明，Fe（III）结合的PAS-A-mPer 2是一种六配位低自旋复合物，具有Cys和未知的轴向配体。Hg 2+结合研究支持Cys是Fe（III）结合PAS-A-mPer 2的轴向配体之一的理论。PAS-A-mPer 2的Fe（III）络合物的解离速率常数（6.3 x 10（-4）s（-1））与血红素调节抑制剂（HRI）（一种血红素传感器酶）的解离速率常数（1.5 x 10（-3）s（-1））相当，但明显高于高铁肌红蛋白的解离速率常数（8.4 x 10（-7）s（-1））。如Soret吸收光谱位移所证实的，血红素从NPAS 2的全碱性螺旋-环-螺旋PAS-A转移到apoPAS-A-mPer 2。C215 A突变体PAS-A-mPer 2蛋白的Soret CD谱与野生型蛋白的Soret CD谱显著不同。根据这些数据，我们提出PAS-A-mPer 2是一个血红素传感器蛋白，其中Cys 215是血红素轴向配体。
Neuronal PAS protein 2 (NPAS2), a heme-binding transcriptional regulatory factor, is involved in circadian rhythms. Period homologue (Per) is another important transcriptional regulatory factor that binds to cryptochrome (Cry). The resultant Per/Cry heterodimer interacts with the NPAS2/BMAL1 heterodimer to inhibit the transcription of Per and Cry. Previous cell biology experiments indicate that mouse Per2 (mPef2) is also a heme-binding protein, and heme shuttling between mPer2 and NPAS2 may regulate transcription. In the present study, we show that the isolated PAS-A domain of mPer2 (PAS-A-mPer2) binds the Fe(III) protoporphyrin IX complex (hemin) with a heme:protein stoichiometry of 1:1. Optical absorption and EPR spectroscopic findings suggest that the Fe(III)-bound PAS-A-mPer2 is a six-coordinated low-spin complex with Cys and an unknown axial ligand. A Hg2+ binding study supports the theory that Cys is one of the axial ligands for Fe(III)-bound PAS-A-mPer2. The dissociation rate constant of the Fe(III) complex from PAS-A-mPer2 (6.3 x 10(-4) s(-1)) was comparable to that of the heme-regulated inhibitor (HRI), a heme-sensor enzyme (1.5 x 10(-3) s(-1)), but markedly higher than that of metmyoglobin (8.4 x 10(-7) s(-1)). As confirmed by a Soret absorption spectral shift, heme transferred from the holo basic helix-loop-helix PAS-A of NPAS2 to apoPAS-A-mPer2. The Soret CD spectrum of the C215A mutant PAS-A-mPer2 protein was markedly different from that of the wild-type protein. On the basis of the data, we propose that PAS-A-mPer2 is a heme-sensor protein in which Cys215 is the heme axial ligand.