The SARS-CoV nucleocapsid (N) protein is a major antigen in severe acute respiratory syndrome. It binds to the viral RNA genome and forms the ribonucleoprotein core. The SARS-CoV N protein has also been suggested to be involved in other important functions in the viral life cycle. Here we show that the N protein consists of two non-interacting structural domains, the N-terminal RNA-binding domain (RBD) (residues 45-181) and the C-terminal dimerization domain (residues 248-365) (DD), surrounded by flexible linkers. The C-terminal domain exists exclusively as a dimer in solution. The flexible linkers are intrinsically disordered and represent potential interaction sites with other protein and protein-RNA partners. Bioinformatics reveal that other coronavirus N proteins could share the same modular organization. This study provides information on the domain structure partition of SARS-CoV N protein and insights into the differing roles of structured and disordered regions in coronavirus nucleocapsid proteins.

We have employed NMR to investigate the structure of SARS coronavirus nucleocapsid protein dimer. We found that the secondary structure of the dimerization domain consists of five a helices and a b-hairpin. The dimer interface consists of a continuous four-stranded b-sheet superposed by two long a helices, reminiscent of that found in the nucleocapsid protein of porcine respiratory and reproductive syndrome virus. Extensive hydrogen bond formation between the two hairpins and hydrophobic interactions between the b-sheet and the a helices render the interface highly stable. Sequence alignment suggests that other coronavirus may share the same structural topology.

Onconase (rONC), otherwise known as ranpirnase or P-30 protein, which was initially purified from extracts of Rana pipiens oocytes and early embryos, exhibits anticancer activity both in vitro and in vivo and is in phase III clinical trials for tumor therapy. We have determined the solution NMR structure of a recombinant onconase with Met ؊1 , Gln 1 , and Leu 23 residues (M؊1, Q1, M23L)rONC. The 20 best solution structures had a backbone root mean square deviation of 0.41 ؎ 0.09 Å with respect to the average structure. The energy-minimized average NMR structure had a backbone root mean square deviation of 0.72 Å from the x-ray crystallographic structure of native onconase; however, the orientation of the N-terminal residue in the two structures was very different. Comparison of the 15 N HSQC spectrum of (M؊1, Q1, M23L)rONC with that of a mutant E1S-rONC, which is identical to the nONC except with the N-terminal pyroglutamyl residue replaced by Ser, showed that N-terminal and residue 23 mutations induced structural changes in regions beyond the mutation sites. Model-free analysis of the backbone amide 15 N-T 1 , 15 N-T 2 , and 15 N-1 H NOE relaxation data for (M؊1, Q1, M23L)rONC and E1S-rONC revealed that the E1S-rONC molecule showed very little flexibility, whereas (M؊1, Q1, M23L)rONC exhibited substantial flexibility, which may account for the previously observed reduced stability and increased protease susceptibility. The ␣ 1 helix and ␤-sheets of (M؊1, Q1, M23L)rONC displayed bending motions. These data provided strong evidence for the presence of an N-terminal hydrogen bond network in E1S-rONC, but not in (M؊1, Q1, M23L)rONC.