Blue light-induced dimerization of monomeric aureochrome-1 enhances its affinity for the target sequence.
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.
From the Department of Earth and Space Science, Graduate School of Science, Osaka University, Toyonaka, Osaka 560-0043, hisatomi@ess.sci.osaka-u.ac.jp.
Osamu
O
Hisatomi
From the Department of Earth and Space Science, Graduate School of Science, Osaka University, Toyonaka, Osaka 560-0043.
Yoichi
Y
Nakatani
From the Department of Earth and Space Science, Graduate School of Science, Osaka University, Toyonaka, Osaka 560-0043.
Ken
K
Takeuchi
the Department of Biotechnology, College of Life Sciences, Ritsumeikan University, Kusatsu, Shiga 525-8577, PRESTO, Japan Science and Technology Agency, 4-1-8 Honcho Kawaguchi, Saitama 332-0012, and.
Fumio
F
Takahashi
the Botanical Gardens, Tohoku University, 12-2 Kawauchi, Aoba-ku, Sendai 980-0862, Japan.
Hironao
H
Kataoka
D020418:Algal Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004247:DNA / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004220:Disulfides / Q000737:chemistry / Q000378:metabolism; D008027:Light; D017434:Protein Structure, Tertiary; D058009:Stramenopiles / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D014157:Transcription Factors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
D020418:Algal Proteins; D004220:Disulfides; D014157:Transcription Factors; D004247:DNA
Biophysics; DNA-Protein Interaction; Flavin Mononucleotide (FMN); Molecular Biology; Oxidation-Reduction (Redox); Transcription Factor