An Arabidopsis SBP-domain fragment with a disrupted C-terminal zinc-binding site retains its tertiary structure.
SQUAMOSA promoter-binding proteins (SBPs) form a major family of plant-specific transcription factors, mainly related to flower development. SBPs share a highly conserved DNA-binding domain of approximately 80 amino acids (SBP domain), which contains two non-interleaved zinc-binding sites formed by eight conserved Cys or His residues. In the present study, an Arabidopsis SPL12 SBP-domain fragment that lacks a Cys residue involved in the C-terminal zinc-binding pocket was found to retain a folded structure, even though only a single Zn2+ ion binds to the fragment. Solution structure of this fragment determined by NMR is very similar to the previously determined structures of the full SBP domains of Arabidopsis SPL4 and SPL7. Considering the previous observations that chelating all the Zn2+ ions of SBPs resulted in the complete unfolding of the structure and that a mutation of the Cys residue equivalent to that described above impaired the DNA-binding activity, we propose that the Zn2+ ion at the N-terminal site is necessary to maintain the overall tertiary structure, while the Zn2+ ion at the C-terminal site is necessary for the DNA binding, mainly by guiding the basic C-terminal loop to correctly fit into the DNA groove.
Yamasaki
Kazuhiko
K
Age Dimension Research Center, National Institute of Advanced Industrial Science and Technology (AIST), 1-1-1 Higashi, Tsukuba 305-8566, Japan. k-yamasaki@aist.go.jp
Kigawa
Takanori
T
Inoue
Makoto
M
Yamasaki
Tomoko
T
Yabuki
Takashi
T
Aoki
Masaaki
M
Seki
Eiko
E
Matsuda
Takayoshi
T
Tomo
Yasuko
Y
Terada
Takaho
T
Shirouzu
Mikako
M
Tanaka
Akiko
A
Seki
Motoaki
M
Shinozaki
Kazuo
K
Yokoyama
Shigeyuki
S
D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000737:chemistry* / Q000378:metabolism*; D029681:Arabidopsis Proteins / Q000737:chemistry* / Q000378:metabolism*; D001665:Binding Sites; D002942:Circular Dichroism; D004247:DNA / Q000378:metabolism; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry*; D017434:Protein Structure, Tertiary; D016415:Sequence Alignment; D020349:Surface Plasmon Resonance; D014157:Transcription Factors / Q000737:chemistry* / Q000378:metabolism*; D015032:Zinc / Q000378:metabolism*
D029681:Arabidopsis Proteins; D010446:Peptide Fragments; C509995:Spl12 protein, Arabidopsis; D014157:Transcription Factors; D004247:DNA; D015032:Zinc