From the Department of Physics, Faculty of Science and Technology, Keio University, 3-14-1 Hiyoshi, Kohoko-ku, Yokohama, Kanagawa 223-8522, Japan.
RIKEN SPring-8 Center, 1-1-1 Kouto, Sayo-cho, Sayo-gun, Hyogo 679-5148, Japan.
J Biol Chem. 2018 Jan 19;293(3):963-972. doi: 10.1074/jbc.RA117.000324. Epub 2017 Dec 1.
Phototropin2 (phot2) is a blue-light (BL) receptor that regulates BL-dependent activities for efficient photosynthesis in plants. phot2 comprises two BL-receiving light-oxygen-voltage-sensing domains (LOV1 and LOV2) and a kinase domain. BL-excited LOV2 is thought to be primarily responsible for the BL-dependent activation of the kinase. However, the molecular mechanisms by which small BL-induced conformational changes in the LOV2 domain are transmitted to the kinase remain unclear. Here, we used full-length wild-type and mutant phot2 proteins from to study their molecular properties in the dark and under BL irradiation. Phosphorylation assays and absorption measurements indicated that the LOV1 domain assists the thermal relaxation of BL-excited LOV2 and vice versa. Using small-angle X-ray scattering and electron microscopy, we observed that phot2 forms a dimer and has a rod shape with a maximum length of 188 Å and a radius of gyration of 44 Å. Under BL, phot2 displayed large conformational changes that bent the rod shape. By superimposing the crystal structures of the LOV1 dimer, LOV2, and a homology model of the kinase to the observed changes, we inferred that the BL-dependent change consisted of positional shifts of both LOV2 and the kinase relative to LOV1. Furthermore, phot2 mutants lacking the photocycle in LOV1 or LOV2 still exhibited conformational changes under BL, suggesting that LOV1 and LOV2 cooperatively contribute to the conformational changes that activate the kinase. These results suggest that BL-activated LOV1 contributes to the kinase activity of phot2. We discuss the possible intramolecular interactions and signaling mechanisms in phot2.
光受体 2（phot2）是一种蓝光（BL）受体，可调节植物中 BL 依赖的活性，以实现有效的光合作用。phot2 由两个 BL 接收光氧电压感应结构域（LOV1 和 LOV2）和一个激酶结构域组成。BL 激发的 LOV2 被认为主要负责激酶的 BL 依赖性激活。然而，小分子 BL 诱导的 LOV2 结构域构象变化如何传递到激酶的分子机制尚不清楚。在这里，我们使用来自 全长野生型和突变型 phot2 蛋白研究它们在黑暗中和 BL 照射下的分子特性。磷酸化测定和吸收测量表明，LOV1 结构域有助于 BL 激发的 LOV2 的热弛豫，反之亦然。使用小角 X 射线散射和电子显微镜，我们观察到 phot2 形成二聚体，具有 188 Å 的最大长度和 44 Å 的回转半径的棒状形状。在 BL 下，phot2 显示出较大的构象变化，使棒状形状弯曲。通过将 LOV1 二聚体、LOV2 和激酶的同源模型的晶体结构叠加到观察到的变化上，我们推断 BL 依赖性变化包括 LOV2 和激酶相对于 LOV1 的位置移动。此外，缺失 LOV1 或 LOV2 光循环的 phot2 突变体在 BL 下仍表现出构象变化，表明 LOV1 和 LOV2 共同促进激活激酶的构象变化。这些结果表明 BL 激活的 LOV1 有助于 phot2 的激酶活性。我们讨论了 phot2 中可能的分子内相互作用和信号机制。
