Nakatani Yoichi, Hisatomi Osamu
Department of Earth and Space Science, Graduate School of Science, Osaka University, Toyonaka, Osaka 560-0043, Japan.
Biophys Physicobiol. 2018 Jan 20;15:8-17. doi: 10.2142/biophysico.15.0_8. eCollection 2018.
Aureochrome1 is a blue-light-receptor protein identified in a stramenopile alga, . Photozipper (PZ) is an N-terminally truncated, monomeric, aureochrome1 fragment containing a basic leucine zipper (bZIP) domain and a light-oxygen-voltage (LOV)-sensing domain. PZ dimerizes upon photoexcitation and consequently increases its affinity for the target sequence. In the present study, to understand the equilibria among DNA complexes of PZ, DNA binding by PZ and mutational variants was quantitatively investigated by electrophoretic-mobility-shift assay and fluorescence-correlation spectroscopy in the dark and light states. DNA binding by PZ was sequence-specific and light-dependent. The half-maximal effective concentration of PZ for binding to the target DNA sequence was ~40 nM in the light, which was >10-fold less than the value in the dark. By contrast, the dimeric PZ-SC variant (with intermolecular disulfide bonds) had higher affinity for the target sequence, with dissociation constants of ~4 nM, irrespective of the light conditions. Substitutions of Glu159 and Lys164 in the leucine zipper region decreased the affinity of PZ for the target sequence, especially in the light, suggesting that these residues form inter-helical salt bridges between leucine zipper regions, stabilizing the dimer-DNA complex. Our quantitative analyses of the equilibria in PZ-DNA-complex formation suggest that the blue-light-induced dimerization of LOV domains and coiled-coil formation by leucine zipper regions are the primary determinants of the affinity of PZ for the target sequence.
金藻视蛋白1是在一种不等鞭毛藻中鉴定出的蓝光受体蛋白。光拉链（PZ）是一种N端截短的单体金藻视蛋白1片段，含有一个碱性亮氨酸拉链（bZIP）结构域和一个光-氧-电压（LOV）传感结构域。PZ在光激发时二聚化，从而增加其对靶序列的亲和力。在本研究中，为了了解PZ的DNA复合物之间的平衡，通过电泳迁移率变动分析和荧光相关光谱法，在黑暗和光照状态下对PZ及其突变变体与DNA的结合进行了定量研究。PZ与DNA的结合具有序列特异性且依赖于光。在光照下，PZ与靶DNA序列结合的半数最大有效浓度约为40 nM，比黑暗中的值低10倍以上。相比之下，二聚体PZ-SC变体（具有分子间二硫键）对靶序列具有更高的亲和力，无论光照条件如何，其解离常数约为4 nM。在亮氨酸拉链区域中替换Glu159和Lys164会降低PZ对靶序列的亲和力，尤其是在光照下，这表明这些残基在亮氨酸拉链区域之间形成了螺旋间盐桥，稳定了二聚体-DNA复合物。我们对PZ-DNA复合物形成平衡的定量分析表明，LOV结构域的蓝光诱导二聚化和亮氨酸拉链区域形成的卷曲螺旋是PZ对靶序列亲和力的主要决定因素。
