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

Mitsuhiro Takeda, Chung ke Chang, Teppei Ikeya, Peter Güntert, Yuan hsiang Chang, Yen lan Hsu, Tai huang Huang, Masatsune Kainosho

Research output: Contribution to journalArticle

44 Citations (Scopus)

Abstract

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.

Original languageEnglish
Pages (from-to)608-622
Number of pages15
JournalJournal of Molecular Biology
Volume380
Issue number4
DOIs
Publication statusPublished - 2008 Jul 18

Fingerprint

SARS Virus
Isotope Labeling
Dimerization
Severe Acute Respiratory Syndrome
Protein C
DNA Mutational Analysis
Proteins
Single-Stranded DNA
Isotopes
Nucleic Acids
RNA
Amino Acids
Coronavirus nucleocapsid protein

Keywords

  • SAIL-NMR
  • SARS nucleocapsid protein
  • nucleocapsid packaging
  • protein structure determination

ASJC Scopus subject areas

  • Structural Biology
  • Molecular Biology

Cite this

Solution Structure of the C-terminal Dimerization Domain of SARS Coronavirus Nucleocapsid Protein Solved by the SAIL-NMR Method. / Takeda, Mitsuhiro; Chang, Chung ke; Ikeya, Teppei; Güntert, Peter; Chang, Yuan hsiang; Hsu, Yen lan; Huang, Tai huang; Kainosho, Masatsune.

In: Journal of Molecular Biology, Vol. 380, No. 4, 18.07.2008, p. 608-622.

Research output: Contribution to journalArticle

Takeda, Mitsuhiro ; Chang, Chung ke ; Ikeya, Teppei ; Güntert, Peter ; Chang, Yuan hsiang ; Hsu, Yen lan ; Huang, Tai huang ; Kainosho, Masatsune. / Solution Structure of the C-terminal Dimerization Domain of SARS Coronavirus Nucleocapsid Protein Solved by the SAIL-NMR Method. In: Journal of Molecular Biology. 2008 ; Vol. 380, No. 4. pp. 608-622.
@article{f817deb83b3e425681f700f0ef72e892,
title = "Solution Structure of the C-terminal Dimerization Domain of SARS Coronavirus Nucleocapsid Protein Solved by the SAIL-NMR Method",
abstract = "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.",
keywords = "SAIL-NMR, SARS nucleocapsid protein, nucleocapsid packaging, protein structure determination",
author = "Mitsuhiro Takeda and Chang, {Chung ke} and Teppei Ikeya and Peter G{\"u}ntert and Chang, {Yuan hsiang} and Hsu, {Yen lan} and Huang, {Tai huang} and Masatsune Kainosho",
year = "2008",
month = "7",
day = "18",
doi = "10.1016/j.jmb.2007.11.093",
language = "English",
volume = "380",
pages = "608--622",
journal = "Journal of Molecular Biology",
issn = "0022-2836",
publisher = "Academic Press Inc.",
number = "4",

}

TY - JOUR

T1 - Solution Structure of the C-terminal Dimerization Domain of SARS Coronavirus Nucleocapsid Protein Solved by the SAIL-NMR Method

AU - Takeda, Mitsuhiro

AU - Chang, Chung ke

AU - Ikeya, Teppei

AU - Güntert, Peter

AU - Chang, Yuan hsiang

AU - Hsu, Yen lan

AU - Huang, Tai huang

AU - Kainosho, Masatsune

PY - 2008/7/18

Y1 - 2008/7/18

N2 - 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.

AB - 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.

KW - SAIL-NMR

KW - SARS nucleocapsid protein

KW - nucleocapsid packaging

KW - protein structure determination

UR - http://www.scopus.com/inward/record.url?scp=45649085737&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=45649085737&partnerID=8YFLogxK

U2 - 10.1016/j.jmb.2007.11.093

DO - 10.1016/j.jmb.2007.11.093

M3 - Article

C2 - 18561946

AN - SCOPUS:45649085737

VL - 380

SP - 608

EP - 622

JO - Journal of Molecular Biology

JF - Journal of Molecular Biology

SN - 0022-2836

IS - 4

ER -