Abstract
Aureochrome-1 (AUREO1) is a blue light (BL) receptor that mediates the branching response in stramenopile alga, Vaucheria frigida. AUREO1 contains a basic leucine zipper (bZIP) domain in the central region and a light-oxygen-voltage sensing (LOV) domain at the C terminus, and has been suggested to function as a light-regulated transcription factor. We have previously reported that preparations of recombinant AUREO1 contained the complete coding sequence (full-length, FL) and N-terminal truncated protein (ZL) containing bZIP and LOV domains, and suggested that wild-type ZL (ZLwt2) was in a dimer form with intermolecular disulfide linkages at Cys(162) and Cys(182) (Hisatomi, O., Takeuchi, K., Zikihara, K., Ookubo, Y., Nakatani, Y., Takahashi, F., Tokutomi, S., and Kataoka, H. (2013) Plant Cell Physiol. 54, 93-106). In the present study, we report the photoreactions, oligomeric structures, and DNA binding of monomeric cysteine to serine-mutated ZL (ZLC2S), DTT-treated ZL (DTT-ZL), and FL (DTT-FL). Recombinant AUREO1 showed similar spectral properties and dark regeneration kinetics to those of dimeric ZLwt2. Dynamic light scattering and size exclusion chromatography revealed that ZLC2S and DTT-ZL were monomeric in the dark state. Dissociation of intermolecular disulfide bonds of ZLwt2 was in equilibrium with a midpoint oxidation-redox potential of approximately -245 ± 15 mV. BL induced the dimerization of monomeric ZL, which subsequently increased its affinity for the target sequence. Also, DTT-FL was monomeric in the dark state and underwent BL-induced dimerization, which led to formation of the FL2·DNA complex. Taken together, our results suggest that monomeric AUREO1 is present in vivo, with dimerization playing a key role in its role as a BL-regulated transcription factor.
Highlights
Aureochromes in stramenopiles are thought to function as light-regulated transcription factors, the molecular mechanism is unknown
We have previously reported that preparations of recombinant AUREO1 contained the complete coding sequence and N-terminal truncated protein (ZL) containing basic leucine zipper (bZIP) and light-oxygen-voltage sensing (LOV) domains, and suggested that wild-type ZL (ZLwt2) was in a dimer form with intermolecular disulfide linkages at Cys162 and Cys182 (Hisatomi, O., Takeuchi, K., Zikihara, K., Ookubo, Y., Nakatani, Y., Takahashi, F., Tokutomi, S., and Kataoka, H. (2013) Plant Cell Physiol. 54, 93–106)
Our results suggest that monomeric AUREO1 is present in vivo, with dimerization playing a key role in its role as a blue light (BL)-regulated transcription factor
Summary
Aureochromes in stramenopiles are thought to function as light-regulated transcription factors, the molecular mechanism is unknown. To elucidate the molecular mechanism of AUREO1, we have previously reported the preparation of six recombinant AUREO1 proteins with a histidine tag at the N or C terminus, full-length AUREO1 (AUREO1-FL), and N terminally truncated AUREO1 (AUREO1-ZL), consisting of the bZIP and LOV domains, and AUREO1-LOV, consisting of the LOV domain, and have investigated global conformational changes in these recombinant proteins using the transient grating technique, size exclusion chromatography (SEC), circular dichroism (CD) spectropolarimetry, and dynamic light scattering (DLS) [20, 21]. Our results suggested that AUREO1 exists as a monomer in reducing conditions and BL induces dimerization of monomeric AUREO1, which subsequently increases affinity for the target DNA sequence. Consistent with these results, we propose a model describing the molecular mechanism of the BL-regulated transcription factor, AUREO1
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