Solution structures of the trihelix DNA-binding domains of the wild-type and a phosphomimetic mutant of Arabidopsis GT-1: mechanism for an increase in DNA-binding affinity through phosphorylation.
GT-1 is a plant transcription factor that binds to one of the cis-acting elements, BoxII, which resides within the upstream promoter region of light-responsive genes. GT-1 was assumed to act as a molecular switch modulated through Ca(2+)-dependent phosphorylation/dephosphorylation in response to light signals. It was shown previously that the phosphorylation of threonine 133 in the DNA-binding domain (DBD) of GT-1 results in enhancement of the BoxII-binding activity. Interestingly, point mutation of Thr133 to Asp also enhances the BoxII-binding activity. Here, we report the solution structures of hypothetical trihelix DBDs of the wild-type (WT) and a phosphomimetic mutant (T133D) of GT-1. First, we demonstrated that the isolated DBD of GT-1 alone has the ability to bind to DNA, and that the T133D mutation of the isolated DBD can enhance the DNA-binding affinity. The structures of these DBDs turned out to be almost identical. The structural topology resembles that of Myb DBDs, but all α-helices are longer in GT-1. Our NMR titration experiments suggested that these longer α-helices yield an enlarged DNA-binding surface. The phosphorylation site is located at the N-terminus of the third α-helix. We built a structural model of the T133D DBD:BoxII complex with the program HADDOCK. The model resembles the structure of the TRF1 DBD:telomeric DNA complex. Interestingly, the model implies that the phosphorylated side chain may directly interact with the bases of DNA. On the basis of our findings, we propose a mechanism by which the DNA-binding activity toward BoxII of the phosphorylated GT-1 could be enhanced.
Nagata
Takashi
T
Department of Supramolecular Biology, Graduate School of Nanobioscience, Yokohama City University, Tsurumi-ku, Yokohama 230-0045, Japan.
Niyada
Emi
E
Fujimoto
Natsuki
N
Nagasaki
Yuuya
Y
Noto
Kazuaki
K
Miyanoiri
Youhei
Y
Murata
Jun
J
Hiratsuka
Kazuyuki
K
Katahira
Masato
M
D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000235:genetics / Q000378:metabolism; D001665:Binding Sites; D018744:DNA, Plant / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D024202:Electrophoretic Mobility Shift Assay; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010766:Phosphorylation; D017433:Protein Structure, Secondary; D017386:Sequence Homology, Amino Acid; D012996:Solutions; D014157:Transcription Factors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
D018744:DNA, Plant; D004268:DNA-Binding Proteins; C077926:GT-1 transcription factor; D012996:Solutions; D014157:Transcription Factors