Disruption of the LOV-Jα helix interaction activates phototropin kinase activity
Disruption of the LOV-Jα helix interaction activates phototropin kinase activity
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DOI：
10.1021/bi048092i
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发表时间：
2004-12-28
期刊：
影响因子：
2.9
通讯作者：
Gardner, KH
中科院分区：
文献类型：
--
作者：
Harper, SM;Christie, JM;Gardner, KH
光在激活光致蛋白中起着至关重要的作用，光致蛋白是一类对蓝色和UV-A波长敏感的植物光感受器。先前的研究表明，phototropin在其光-氧-电压（LOV）结构域中使用结合的黄素单核苷酸（FMN）在光照下产生蛋白质-黄素共价键。在A vena sativa phototropin 1的C-末端LOV 2结构域中，该键的形成触发构象变化，导致该结构域外部的称为J α的螺旋解折叠[哈珀，S. M.，等人（2003）Science 301，1541-1545]。虽然照明的结构效应的特点，这是未知的，这些变化是如何耦合到激酶激活。为了检验这一点，我们沿着Ja螺旋沿着进行了一系列点突变，以类似于光活化的方式破坏其与LOV结构域的相互作用。使用NMR光谱和有限的蛋白水解，我们证明，这些突变中的几个取代的JA螺旋从LOV域独立的照明。当置于全长光致蛋白质中时，这些点突变显示组成型激酶激活，而不照射样品。这些结果表明，Jalpha螺旋的解折叠是光致蛋白激酶信号调节的关键事件。
Light plays a crucial role in activating phototropins, a class of plant photoreceptors that are sensitive to blue and UV-A wavelengths. Previous studies indicated that phototropin uses a bound flavin mononucleotide (FMN) within its light-oxygen-voltage (LOV) domain to generate a protein-flavin covalent bond under illumination. In the C-terminal LOV2 domain of A vena sativa phototropin 1, formation of this bond triggers a conformational change that results in unfolding of a helix external to this domain called Jalpha [Harper, S. M., et al. (2003) Science 301, 1541-1545]. Though the structural effects of illumination were characterized, it was unknown how these changes are coupled to kinase activation. To examine this, we made a series of point mutations along the Ja helix to disrupt its interaction with the LOV domain in a manner analogous to light activation. Using NMR spectroscopy and limited proteolysis, we demonstrate that several of these mutations displace the Ja helix from the LOV domain independently of illumination. When placed into the full-length phototropin protein, these point mutations display constitutive kinase activation, without illumination of the sample. These results indicate that unfolding of the Jalpha helix is the critical event in regulation of kinase signaling for the phototropin proteins.