2008 Solution Structure of the C-terminal Dimerization Domain of SARS Coronavirus Nucleocapsid Protein Solved by the SAI

15 Pages·2008·2.85 MB·English

Solution Structure of the C-terminal Dimerization
Domain of SARS Coronavirus Nucleocapsid Protein
Solved by the SAIL-NMR Method

Mitsuhiro Takeda1†, Chung-ke Chang2†, Teppei Ikeya1,3,
Peter Güntert3,4, Yuan-hsiang Chang2, Yen-lan Hsu2,
Tai-huang Huang2,5,6⁎ and Masatsune Kainosho1,3⁎

1Graduate School of Science,
Nagoya University, Furo-cho,
Chikusa-ku, Nagoya 464-8602,
Japan

2Institute of Biomedical
Sciences, Academia Sinica,
Taipei 115, Taiwan, ROC

3Graduate School of Science,
Tokyo Metropolitan University,
1-1 Minami-ohsawa,
Hachioji 192-0397, Japan

4Institute of Biophysical
Chemistry, J.W. Goethe-
University Frankfurt am Main,
Max-von-Laue-Str. 9, 60438
Frankfurt am Main, Germany

5Department of Physics, National
Taiwan Normal University,
Taipei, Taiwan, ROC

6The Genomic Research Center,
Academia Sinica, Taipei, Taiwan,
ROC

Received 26 October 2007;
received in revised form
24 November 2007;
accepted 28 November 2007
Available online
5 December 2007

The C-terminal domain (CTD) of the severe acute respiratory syndrome
coronavirus (SARS-CoV) nucleocapsid protein (NP) contains a potential
RNA-binding region in its N-terminal portion and also serves as a
dimerization domain by forming a homodimer with a molecular mass of
28 kDa. So far, the structure determination of the SARS-CoV NP CTD in
solution has been impeded by the poor quality of NMR spectra, especially
for aromatic resonances. We have recently developed the stereo-array
isotope labeling (SAIL) method to overcome the size problem of NMR
structure determination by utilizing a protein exclusively composed of
stereo- and regio-specifically isotope-labeled amino acids. Here, we
employed the SAIL method to determine the high-quality solution structure
of the SARS-CoV NP CTD by NMR. The SAIL protein yielded less crowded
and better resolved spectra than uniform 13C and 15N labeling, and enabled
the homodimeric solution structure of this protein to be determined. The
NMR structure is almost identical with the previously solved crystal
structure, except for a disordered putative RNA-binding domain at the
N-terminus. Studies of the chemical shift perturbations caused by the
binding of single-stranded DNA and mutational analyses have identified
the disordered region at the N-termini as the prime site for nucleic acid
binding. In addition, residues in the β-sheet region also showed significant
perturbations. Mapping of the locations of these residues onto the helical
model observed in the crystal revealed that these two regions are parts of
the interior lining of the positively charged helical groove, supporting the
hypothesis that the helical oligomer may form in solution.
© 2008 Published by Elsevier Ltd.

Edited by M. F. Summers

Keywords: SAIL-NMR; SARS nucleocapsid protein; protein structure
determination; nucleocapsid packaging

*Corresponding authors. T. Huang is to be contacted at Institute of Biomedical Sciences, Academia Sinica, Taipei 115,
Taiwan, ROC. M. Kainosho, Graduate School of Science, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8602,
Japan. E-mail addresses: