Departments of Biophysics and Biochemistry, University of Texas Southwestern Medical Center, Dallas, TX 75390-8816, USA.
Biochemistry. 2012 Dec 18;51(50):10024-34. doi: 10.1021/bi301306t. Epub 2012 Dec 10.
Light-oxygen-voltage (LOV) domains serve as the photosensory modules for a wide range of plant and bacterial proteins, conferring blue light-dependent regulation to effector activities as diverse as enzymes and DNA binding. LOV domains can also be engineered into a variety of exogenous targets, allowing similar regulation for new protein-based reagents. Common to these proteins is the ability for LOV domains to reversibly form a photochemical adduct between an internal flavin chromophore and the surrounding protein, using this to trigger conformational changes that affect output activity. Using the Erythrobacter litoralis protein EL222 model system that links LOV regulation to a helix-turn-helix (HTH) DNA binding domain, we demonstrated that the LOV domain binds and inhibits the HTH domain in the dark, releasing these interactions upon illumination [Nash, A. I., et al. (2011) Proc. Natl. Acad. Sci. U.S.A. 108, 9449-9454]. Here we combine genomic and in vitro selection approaches to identify optimal DNA binding sites for EL222. Within the bacterial host, we observe binding at several genomic sites using a 12 bp sequence consensus that is also found by in vitro selection methods. Sequence-specific alterations in the DNA consensus reduce EL222 binding affinity in a manner consistent with the expected binding mode, a protein dimer binding to two repeats. Finally, we demonstrate the light-dependent activation of transcription of two genes adjacent to an EL222 binding site. Taken together, these results shed light on the native function of EL222 and provide useful reagents for further basic and applications research of this versatile protein.
光氧电压 (LOV) 结构域是广泛存在于植物和细菌蛋白中的光感觉模块，为各种效应器活性（如酶和 DNA 结合）提供蓝光依赖性调节。LOV 结构域也可以被工程改造到各种外源靶标中，为新的基于蛋白质的试剂提供类似的调节。这些蛋白质的共同点是 LOV 结构域能够在内部黄素发色团和周围蛋白质之间可逆地形成光化学反应加合物，利用这一反应来触发影响输出活性的构象变化。我们使用将 LOV 调节与螺旋-转角-螺旋 (HTH) DNA 结合域联系起来的 Erythrobacter litoralis 蛋白 EL222 模型系统，证明 LOV 结构域在黑暗中结合并抑制 HTH 结构域，在光照下释放这些相互作用 [Nash, A. I., et al. (2011) Proc. Natl. Acad. Sci. U.S.A. 108, 9449-9454]。在这里，我们结合基因组和体外选择方法来确定 EL222 的最佳 DNA 结合位点。在细菌宿主中，我们使用通过体外选择方法也发现的 12 个碱基对序列共识来观察几个基因组位点的结合。DNA 共识序列的特异性改变以与预期结合模式一致的方式降低 EL222 的结合亲和力，即蛋白质二聚体与两个重复结合。最后，我们证明了与 EL222 结合位点相邻的两个基因的转录在光依赖性下被激活。总之，这些结果揭示了 EL222 的天然功能，并为进一步研究这种多功能蛋白质的基础和应用提供了有用的试剂。
