Protein Engineering, Vol. 16, No. 4, 247-254,
April 2003
© 2003 Oxford University Press
NMR and ICP spectroscopic analysis of the DNA-binding domain of the Drosophila GCM protein reveals a novel Zn2+-binding motif
1 Biomolecular Engineering Research Institute, 6–2–3 Furue-dai, Suita, Osaka 560-0874, 2 Japan Science and Technology Corporation,4–1–8 Honcho, Kawaguchi, Saitama 332-0012, 3 Center for Gene Research, Ehime University, 3–5–7 Tarumi, Matsuyama, Ehime 790-8566, 4 Division of Biophysics, Graduate School of Integrated Sciences, Yokohama City University, 1–7–29 Suehiro-cho, Tsurumi-ku, Yokohama, Kanagawa 230-0045, 5 Division of Structural Biology, Medical Institute of Bioregulation, Kyushu University, 3–1–1 Maidashi, Higashi-ku, Fukuoka 812-8582, 6 National Institute of Genetics, Mishima, Shizuoka 411-8540, 7 Department of Physics, Graduate School of Science, University of Tokyo, 7–3–1 Bunkyo-ku, Tokyo 113-0033 and 8 Venture Business Laboratory, Ehime University, 3–5 Bunkyo-cho, Matsuyama, Ehime 790-8577, Japan
To whom correspondence should be addressed. E-mail: ehmorita{at}dpc.ehime-u.ac.jp; morikawa{at}beri.or.jp
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 Zn2+ 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 1H–15N, 1H–15N–13C and 113Cd2+ NMR spectra, with or without its specific DNA molecule. Considering the results, it was concluded that the minimal DNA-binding domain includes two Zn2+-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 Zn2+-binding site is indispensable for stabilization of the higher order structure of this DNA-binding domain, but that the other is not.