Spectroscopic and DNA-binding characterization of the isolated heme-bound basic helix-loop-helix-PAS-A domain of neuronal PAS protein 2 (NPAS2), a transcription activator protein associated with circadian rhythms
Spectroscopic and DNA-binding characterization of the isolated heme-bound basic helix-loop-helix-PAS-A domain of neuronal PAS protein 2 (NPAS2), a transcription activator protein associated with circadian rhythms
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DOI：
10.1111/j.1742-4658.2006.05259.x
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发表时间：
2006-06-01
期刊：
影响因子：
5.4
通讯作者：
Shimizu, Toru
中科院分区：
文献类型：
--
作者：
Mukaiyama, Yuji;Uchida, Takeshi;Shimizu, Toru
神经元PAS结构域蛋白2(NPAS2)是一种昼夜节律相关的转录因子，在两个PAS结构域上有两个血红素结合位点。在本研究中，我们比较了含有NPAS2第一个PAS(PAS-A)结构域的N-末端碱性螺旋-环-螺旋(BHLH)的分离片段和单独的PAS-A结构域的光吸收光谱、共振拉曼光谱、血红素结合动力学和DNA结合特性。我们发现，血红素结合的bHLH-PAS-A结构域在溶液中主要以二聚体形式存在。铁(III)络合物与bHLH-PAS-A的Soret吸收峰(421 Nm)比单独的PAS-A(412 Nm)高9 nm。根据共振拉曼光谱，bHLH-PAS-A中反式为CO的轴向配体似乎是His。此外，血红素与apo-bHLH-PAS结合的速率常数(3.3x10~(-7)摩尔~(-1))中心点S(~(-1))比与apo-PAS-A(~10(5)摩尔~(-1))中心点S(~(-1))结合的速率常数高两个数量级以上。这些结果表明，bHLH域有助于血红素与NPAS2稳定的结合。光学和共振拉曼光谱表明，Fe(II)-NO-HO络合物是五配位的。利用石英晶体微天平方法，我们发现bHLH-PAS-A结构域在有血红素存在的情况下与E-box DNA序列特异结合，而在没有血红素的情况下没有。在这些结果的基础上，我们讨论了bHLH-PAS-A与血红素结合的方式及其在调节DNA结合中的潜在作用。
Neuronal PAS domain protein 2 (NPAS2) is a circadian rhythm-associated transcription factor with two heme-binding sites on two PAS domains. In the present study, we compared the optical absorption spectra, resonance Raman spectra, heme-binding kinetics and DNA-binding characteristics of the isolated fragment containing the N-terminal basic helix-loop-helix (bHLH) of the first PAS (PAS-A) domain of NPAS2 with those of the PAS-A domain alone. We found that the heme-bound bHLH-PAS-A domain mainly exists as a dimer in solution. The Soret absorption peak of the Fe(III) complex for bHLH-PAS-A (421 nm) was located at a wavelength 9 nm higher than for isolated PAS-A (412 nm). The axial ligand trans to CO in bHLH-PAS-A appears to be His, based on the resonance Raman spectra. In addition, the rate constant for heme association with apo-bHLH-PAS (3.3 x 10(7) Mol(-1)center dot s(-1)) was more than two orders of magnitude higher than for association with apo-PAS-A (< 10(5) mol(-1)center dot s(-1)). These results suggest that the bHLH domain assists in stable heme binding to NPAS2. Both optical and resonance Raman spectra indicated that the Fe(II)-NO heme complex is five-coordinated. Using the quartz-crystal microbalance method, we found that the bHLH-PAS-A domain binds specifically to the E-box DNA sequence in the presence, but not in the absence, of heme. On the basis of these results, we discuss the mode of heme binding by bHLH-PAS-A and its potential role in regulating DNA binding.