The nucleocapsid (N) phosphoprotein of the severe acute respiratory syndrome coronavirus (SARS-CoV) packages the viral genome into a helical ribonucleocapsid and plays a fundamental role during viral self-assembly. We showed that the N protein consists of two structural domains interspersed between intrinsically disordered regions and dimerizes through the C-terminal structural domain (CTD). The crystal structure of CTD revealed the formation of an octamer in an asymmetric unit. Packing of the octamers in the crystal forms two parallel, basic helical grooves, which was proposed to be the oligonucleotide attachment sites, and suggests a mechanism for helical RNA packaging in the virus. Solution structure and the nucleic acid binding site of CTD identified by SAIL-NMR are in good agreements with the RNP model. EMS experiments further showed that nucleic acid binds to N at multiple sites cooperatively in a “coupled-allostery” manner, reminiscent of the allosteric effect in a multi-domain regulatory system and underscore the importance of intrinsic disorder in nucleocapsid packaging and function. Transient formation of N oligomers was confirmed by disulfide trapping technique and a systematic study of the oligomerization behavior revealed that altering the intermolecular electrostatic repulsion through changes in solution salt concentration or phosphorylation- mimicking mutations affects oligomerization propensity. Thus, electrostatic repulsion may act as a switch to regulate N protein oligomerization. A SARS-CoV RNP packaging model based on structural-biophysical principles is proposed.
Chen, C.Y. et al. (2006) J. Mol. Biol. 368, 1075-1086. Takeda, M. et al. (2008) J. Mol. Biol. 380, 608-622. Chang, C.K. et al. (2009) J. Virol. 83: 2255-2264. Chang, C.K. et al. (2013) PLoS ONE 8 (5) e65045.