Abstract
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 Arabidopsis 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.
Highlights
Phototropin2 is a blue-light (BL) receptor that regulates BL-dependent activities for efficient photosynthesis in plants. phot2 comprises two BL-receiving light-oxygenvoltage–sensing domains (LOV1 and LOV2) and a kinase domain
The biochemical, spectroscopic, and structural studies on purified full-length phot2 and its mutants suggested a contribution of LOV1, as well as LOV2, to the kinase activity and BL-dependent conformational changes of Atphot2
We discuss the implications of the results for the illustration of the molecular organization and intramolecular signaling to induce cellular signaling cascades in the BL response by phot2
Summary
Phototropin (phot2) is a blue-light (BL) receptor that regulates BL-dependent activities for efficient photosynthesis in plants. The molecular mechanisms by which small BL-induced conformational changes in the LOV2 domain are transmitted to the kinase remain unclear. Upon blue-light excitation, each LOV domain in the dark state (D450 state) undergoes a characteristic photocycle, and the FMN transiently forms a covalent bond with a wellconserved cysteine residue in LOV (S390 state) (14 –16). In phot proteins of land plants, LOV1 contributes to the regulation of LOV2 and the kinase activity [19, 31]. We established a large-scale preparation system to provide full-length Atphot, which comprised all residues in the amino acid sequence, and characterized the molecular properties of the wild-type protein and its mutants in terms of their kinase activities, dark recovery kinetics, and molecular structures. We report the results of biochemical, spectroscopic, and structural studies using small-angle X-ray scattering (SAXS) and electron microscopy (EM) and discuss the regulatory mechanism of Atphot
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