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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
Author(s) -
Nagata Takashi,
Niyada Emi,
Fujimoto Natsuki,
Nagasaki Yuuya,
Noto Kazuaki,
Miyanoiri Youhei,
Murata Jun,
Hiratsuka Kazuyuki,
Katahira Masato
Publication year - 2010
Publication title -
proteins: structure, function, and bioinformatics
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.699
H-Index - 191
eISSN - 1097-0134
pISSN - 0887-3585
DOI - 10.1002/prot.22827
Subject(s) - phosphorylation , mutant , dna , dna binding domain , dephosphorylation , hmg box , transcription factor , binding site , biology , biochemistry , dna binding protein , microbiology and biotechnology , biophysics , gene , phosphatase
Abstract 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. Proteins 2010. © 2010 Wiley‐Liss, Inc.