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. 2009 Mar;83(5):2255-64.
doi: 10.1128/JVI.02001-08. Epub 2008 Dec 3.

Multiple nucleic acid binding sites and intrinsic disorder of severe acute respiratory syndrome coronavirus nucleocapsid protein: implications for ribonucleocapsid protein packaging

Chung-Ke Chang  1 , Yen-Lan Hsu, Yuan-Hsiang Chang, Fa-An Chao, Ming-Chya Wu, Yu-Shan Huang, Chin-Kun Hu, Tai-Huang Huang
Affiliations

Multiple nucleic acid binding sites and intrinsic disorder of severe acute respiratory syndrome coronavirus nucleocapsid protein: implications for ribonucleocapsid protein packaging

Chung-Ke Chang et al. J Virol. 2009 Mar.

Abstract

The nucleocapsid protein (N) of the severe acute respiratory syndrome coronavirus (SARS-CoV) packages the viral genomic RNA and is crucial for viability. However, the RNA-binding mechanism is poorly understood. We have shown previously that the N protein contains two structural domains--the N-terminal domain (NTD; residues 45 to 181) and the C-terminal dimerization domain (CTD; residues 248 to 365)--flanked by long stretches of disordered regions accounting for almost half of the entire sequence. Small-angle X-ray scattering data show that the protein is in an extended conformation and that the two structural domains of the SARS-CoV N protein are far apart. Both the NTD and the CTD have been shown to bind RNA. Here we show that all disordered regions are also capable of binding to RNA. Constructs containing multiple RNA-binding regions showed Hill coefficients greater than 1, suggesting that the N protein binds to RNA cooperatively. The effect can be explained by the "coupled-allostery" model, devised to explain the allosteric effect in a multidomain regulatory system. Although the N proteins of different coronaviruses share very low sequence homology, the physicochemical features described above may be conserved across different groups of Coronaviridae. The current results underscore the important roles of multisite nucleic acid binding and intrinsic disorder in N protein function and RNP packaging.

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Figures

FIG. 1.
FIG. 1.
(A) Schematic of the domain architecture of the SARS-CoV N protein. Structured domains are shown as balls, and unstructured regions are shown as lines. (B) Protein constructs used in the current study. Numbers represent the amino acid residue range relative to the full-length N protein (NP). Sumo-1-FL contains a Sumo-1 tag (shown as an oval), followed by the flexible linker of the N protein between residues 181 and 246.
FIG. 2.
FIG. 2.
Sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel strips of the various SARS-CoV NP protein constructs after purification. Almost all constructs appear as a single band in the gel strips, and for the few exceptions, the purity of the main band exceeds 90%. Lanes are labeled in the following order: M, light molecular mass marker; 1, NP1-181; 2, NP45-181; 3, NP45-247; 4, NP181-365; 5, NP248-365; 6, NP248-422; 7, NP45-365; 8, Sumo-1-FL.
FIG. 3.
FIG. 3.
Effects of the ID regions (residues 1 to 44 and 182 to 247) on the RNA binding affinity of the NTD. (A through C) Fitting of the binding isotherms of NP45-181 (NTD) (A), NP1-181 (B), and NP45-247 (C), based on the EMSA results. Each binding isotherm represents the overall fitting against three independent experiments, taking into account the standard deviation of each data point. (D through F) Representative EMSA results for NP45-181 (D), NP1-181 (E), and NP45-247 (F).
FIG. 4.
FIG. 4.
Effects of the ID regions (residues 182 to 247 and 366 to 422) on the RNA binding activity of the CTD. (A through C) Fitting of the binding isotherms of NP248-365 (CTD) (A), NP248-422 (B), and NP182-365 (C), based on the EMSA results. Each binding isotherm represents the overall fitting against three independent experiments, taking into account the standard deviation of each data point. (D through F) Representative EMSA results for NP248-365 (D), NP248-422 (E), and NP182-365 (F).
FIG. 5.
FIG. 5.
Residues 182 to 247 are ID when attached to the NTD. (A) 15N-edited HSQC spectra of NP45-181 (NTD) (left) and NP45-247 (right) show additional resonances clustered in the middle of the spectrum of NP45-247. Axis units are ppm. (B) Size exclusion chromatogram of NP45-247. The corresponding apparent molecular weight was calculated from the equation log(MW) = 6.5404 − 0.1802 EV, where MW is the molecular weight in thousands and EV is the elution volume in milliliters.
FIG. 6.
FIG. 6.
Residues 182 to 247 are ID when attached to the CTD. (A) 15N-edited HSQC spectra of NP248-365 (CTD) (left) and NP182-365 (right) show additional resonances clustered in the middle of the spectrum of NP182-365. Axis units are ppm. (B) Size exclusion chromatogram of NP182-365. The corresponding apparent molecular weight was calculated from the equation log(MW) = 6.5404 − 0.1802 EV, where MW is the molecular weight in thousands and EV is the elution volume in milliliters.
FIG. 7.
FIG. 7.
SAXS results for the didomain construct NP45-365. (A) Scattering profile of NP45-365 (crosses) and normalization fitting with GNOM (dashed lines). J, scattering intensity; s, scattering angle vector. (B) Normalized results from GNOM showing the pairwise distance distribution [P(r)] and the maximum distance. The radius of gyration is fitted to 61 Å. “r” represents the pairwise distances. (C) Representative model of NP45-365 structure based on CRYSOL simulations of SAXS data. Only the alpha carbons are shown. Notice the difference in distance between the two NTDs and the CTD core.
FIG. 8.
FIG. 8.
Alignment of the flexible linker regions from different coronavirus N proteins. Residues that are predicted by JPred to form a helix are boxed. The arginines of the SR-rich regions are underlined. The names of the coronaviruses (with SwissProt accession numbers and phylogenetic groups in parentheses) are as follows: SARS-CoV (P59595; group 2b); NL63, human coronavirus NL63 (Q6Q1R8; group 1b); 229E, human coronavirus 229E (P15139; group 1b); TGEV, porcine transmissible gastroenteritis virus strain Purdue (P04134; group 1a); OC43, human coronavirus OC43 (P33469; group 2a); MHV-1, murine hepatitis virus 1 (P18446; group 2a); IBV, avian infectious bronchitis virus strain Beaudette (P69596; group 3).
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References

    1. Chang, C. K., S. C. Sue, T. H. Yu, C. M. Hsieh, C. K. Tsai, Y. C. Chiang, S. J. Lee, H. H. Hsiao, W. J. Wu, C. F. Chang, and T. H. Huang. 2005. The dimer interface of the SARS coronavirus nucleocapsid protein adapts a porcine respiratory and reproductive syndrome virus-like structure. FEBS Lett. 5795663-5668. - PMC - PubMed
    1. Chang, C. K., S. C. Sue, T. H. Yu, C. M. Hsieh, C. K. Tsai, Y. C. Chiang, S. J. Lee, H. H. Hsiao, W. J. Wu, W. L. Chang, C. H. Lin, and T. H. Huang. 2006. Modular organization of SARS coronavirus nucleocapsid protein. J. Biomed. Sci. 1359-72. - PMC - PubMed
    1. Chen, C. Y., C. K. Chang, Y. W. Chang, S. C. Sue, H. I. Bai, L. Riang, C. D. Hsiao, and T. H. Huang. 2007. Structure of the SARS coronavirus nucleocapsid protein RNA-binding dimerization domain suggests a mechanism for helical packaging of viral RNA. J. Mol. Biol. 3681075-1086. - PMC - PubMed
    1. Chiti, F., M. Stefani, N. Taddei, G. Ramponi, and C. M. Dobson. 2003. Rationalization of the effects of mutations on peptide and protein aggregation rates. Nature 424805-808. - PubMed
    1. Cuff, J. A., and G. J. Barton. 2000. Application of multiple sequence alignment profiles to improve protein secondary structure prediction. Proteins 40502-511. - PubMed

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