Abstract
Phototropin (phot), a blue light (BL) receptor in plants, has two photoreceptive domains named LOV1 and LOV2 as well as a Ser/Thr kinase domain (KD) and acts as a BL-regulated protein kinase. A LOV domain harbors a flavin mononucleotide that undergoes a cyclic photoreaction upon BL excitation via a signaling state in which the inhibition of the kinase activity by LOV2 is negated. To understand the molecular mechanism underlying the BL-dependent activation of the kinase, the photochemistry, kinase activity, and molecular structure were studied with the phot of Chlamydomonas reinhardtii. Full-length and LOV2-KD samples of C. reinhardtii phot showed cyclic photoreaction characteristics with the activation of LOV- and BL-dependent kinase. Truncation of LOV1 decreased the photosensitivity of the kinase activation, which was well explained by the fact that the signaling state lasted for a shorter period of time compared with that of the phot. Small angle x-ray scattering revealed monomeric forms of the proteins in solution and detected BL-dependent conformational changes, suggesting an extension of the global molecular shapes of both samples. Constructed molecular model of full-length phot based on the small angle x-ray scattering data proved the arrangement of LOV1, LOV2, and KD for the first time that showed a tandem arrangement both in the dark and under BL irradiation. The models suggest that LOV1 alters its position relative to LOV2-KD under BL irradiation. This finding demonstrates that LOV1 may interact with LOV2 and modify the photosensitivity of the kinase activation through alteration of the duration of the signaling state in LOV2.
Highlights
The plant photoreceptor “phototropin” is a light-regulated kinase containing two photosensory domains named LOV
When comparing the t1⁄2 in LOV2 of CrPFul (Fig. 3, dashed line) with that of the major component of CrPL2K, it was observed that the reversion of S390 in LOV2 is accelerated by ϳ2.5 times in the presence of light-oxygenvoltage 1 (LOV1)
The results showed that AtP1Nt serves as a substrate for the kinase of Chlamydomonas reinhardtii (Cr) phot as well as Arabidopsis thaliana (At) phots, suggesting that Cr phot has similar substrate recognition ability and kinase activity as At phots
Summary
The plant photoreceptor “phototropin” is a light-regulated kinase containing two photosensory domains named LOV. Because the LOV2-KDs have BL-regulated kinase activity, structural study of these molecules provides useful information regarding the molecular basis for the BL-dependent regulation of the kinase In this regard we previously measured the SAXS of a D720N substitute of At phot LOV-KD and reported its molecular models, which revealed the topological organization of LOV2 and KD and its BL-induced alteration [27]. The results revealed distinct functional roles of LOV1 in the regulation of kinase activity and, for the first time, the organization of LOV1, LOV2, and KD in a full-length phot molecule Based on these results, we discuss the regulatory mechanism of Cr phot kinase in comparison with those of higher plants
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