NMR and ICP spectroscopic analysis of the DNA-binding domain of the Drosophila GCM protein reveals a novel Zn2+ -binding motif.
Drosophila GCM (glial cell missing) is a novel DNA-binding protein that determines the fate of glial precursors from the neural default to glia. The GCM protein contains the functional domain that is essential for recognition of the upstream sequence of the repo gene. In the DNA-binding region of this GCM protein, there is a cysteine-rich region with which divalent metal ions such as Zn(2+) must bind and other proteins belonging to the GCM family have a corresponding region. To obtain a more detailed insight into the structural and functional features of this DNA-binding region, we have determined the minimal DNA-binding domain and obtained inductively coupled plasma atomic emission spectra and (1)H-(15)N, (1)H-(15)N-(13)C and (113)Cd(2+) NMR spectra, with or without its specific DNA molecule. Considering the results, it was concluded that the minimal DNA-binding domain includes two Zn(2+)-binding sites, one of which is adjacent to the interface for DNA binding. Systematic mutational analyses of the conserved cysteine residues in the minimal DNA-binding domain revealed that one Zn(2+)-binding site is indispensable for stabilization of the higher order structure of this DNA-binding domain, but that the other is not.
Biomolecular Engineering Research Institute, 6-2-3 Furue-dai, Suita, Osaka 560-0874, Japan.
Masato
M
Shimizu
Hidekazu
H
Hiroaki
Daisuke
D
Kohda
Toshihiko
T
Hosoya
Yasuko
Y
Akiyama-Oda
Yoshiki
Y
Hotta
Eugene Hayato
EH
Morita
Kosuke
K
Morikawa
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002413:Cations, Divalent; D003545:Cysteine / Q000737:chemistry; D004268:DNA-Binding Proteins / Q000737:chemistry*; D004330:Drosophila / Q000235:genetics / Q000378:metabolism*; D029721:Drosophila Proteins; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D009479:Neuropeptides / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics; D016415:Sequence Alignment; D013054:Spectrophotometry, Atomic; D015534:Trans-Activators / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics; D014157:Transcription Factors; D015032:Zinc / Q000737:chemistry
D002413:Cations, Divalent; D004268:DNA-Binding Proteins; D029721:Drosophila Proteins; C102805:GCM protein, Drosophila; D009479:Neuropeptides; D015534:Trans-Activators; D014157:Transcription Factors; D015032:Zinc; D003545:Cysteine