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The dimer interface of the SARS coronavirus nucleocapsid protein adapts a porcine respiratory and reproductive syndrome virus‐like structure

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 α helices and a β‐hairpin. The dimer interface consists of a continuous four‐stranded β‐sheet superposed by two long α 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 β‐sheet and the α helices render the interface highly stable. Sequence alignment suggests that other coronavirus may share the same structural topology.


Introduction
Severe acute respiratory syndrome (SARS) is the first emerging infectious disease in the 21st century with a fatality rate of ca. 8% and is caused by a novel coronavirus (CoV) [1]. The nucleocapsid protein is a key component of the virus and is essential for virus formation. It binds to the viral RNA to form a ribonucleoprotein core, which can enter the host cell and interact with cellular processes [2][3][4][5]. The free protein presumably exists as a dimer in solution, with the dimerization domain located at the C-terminus [6,7]. We have previously defined the structural domains of the SARS-CoV N protein [8]. The C-terminal structural domain coincides with the dimerization domain identified in previous studies, and our biochemical studies showed that it exists as a dimer in solution. Denaturation studies have shown that dissociation of the SARS-CoV N protein is coupled with loss of structure, implying a structure-dependent mechanism for self-association [9]. Understanding this mechanism would not only provide insights into the viral assembly process, but also identify additional targets for drugs to combat SARS through disruption of N protein self-association. However, there has been no 3D structure of the dimerization domain of coronavirus N protein published and the underlining principle governing the self-association of coronavirus N protein dimer is also unknown. We have employed nuclear magnetic resonance (NMR) techniques to investigate the structure of the dimerization domain of SARS-CoV N protein. We report our results in this communication.

Plasmid construction
SARS-CoV TW1 strain cDNA clones were kindly provided to us by Dr. P.-J. Chen of National Taiwan University Hospital [10]. The a SARS-CoV nucleocapsid protein fragment consisting of residues 281-365 (NP 281-365 ) and a SARS-CoV nucleocapsid protein fragment consisting of residues 248-365 (NP 248-365 ) clones were obtained by polymerase chain reaction (PCR) on a RoboCycler Gradient 96 (Stratagene, CA, USA) using appropriate primers. The resulting PCR fragment contained an NcoI site at one end and a BamHI site at the other. After restriction enzyme digestion, the resulting fragment was cloned into the pET6H plasmid, which contains a His-tag coding region. The resultant protein fragment included an extra MHHHHHHAMG sequence at the N-terminus.

Protein expression and purification
The fragments corresponding to residues 248-365 (NP 248-365 ) and 281-365 (NP 281-365 ) of SARS-CoV N proteins were expressed in Escherichia coli BL21(DE3) strain. Isotopically labeled protein samples were prepared by culturing the cells in standard M9 media, supplemented with 15 NH 4 Cl (1 g/L) (For 15 N-labeling) and/or u-13 C-glucose (2 g/L) (For 13 C-labeling) and appropriately labeled Isogro (0.5 g/L) (Isotec, OH, USA). Perdeuterated isotopically labeled protein samples were prepared by culturing the cells in the same media in D 2 O (80% D 2 O for samples used in filtered experiments) and supplemented with deuterated Isogro and glucose. Deuteration rates for all clones were on the order of 85% (65% for samples used in filtered experiments) as measured by mass spectrometry. The cells were broken with a microfluidizer and the protein purified through a Ni-NTA affinity column (Qiagen, CA, USA) in buffer (50 mM sodium phosphate, 150 mM NaCl, and pH 7.4) containing 7 M urea. The protein was then allowed to refold by dialysis in liquid chromatography buffer (50 mM sodium phosphate, 150 mM NaCl, 1 mM EDTA, 0.01% NaN 3 , and pH 7.4).
Abbreviations: NP 248-365 , a SARS-CoV nucleocapsid protein fragment consisting of residues 248-365; NP 281-365 , a SARS-CoV nucleocapsid protein fragment consisting of residues 281-365; PRRSV, porcine reproductive and respiratory syndrome virus The two shaded areas represent the N-terminal and C-terminal structural domains. Secondary structure of the N-terminal domain was adapted from Huang et al. [27].
Renatured protein was loaded onto an AKTA-EXPLORER fast performance liquid chromatography (FPLC) system equipped with a Hi-Load 16/60 Superdex 75 column (Amersham Pharmacia Biotech, Sweden). Complete Protease Inhibitor cocktail (Roche, Germany) was added to the purified protein. Protein concentration was determined with the Bio-Rad Protein Assay kit as per instructions from the manufacturer (Bio-Rad, CA, USA). The correct molecular weight of the expressed protein was then confirmed by mass spectroscopy.

NMR spectroscopy
Protein samples for NMR experiments contain between 0.5 and 3 mM protein in NMR buffer (10 mM sodium phosphate buffer, pH 6.0, containing 50 mM NaCl, 1 mM EDTA, 1 mM 2,2-dimethyl-2-silapentane-5-sulfonate (DSS), 0.01% NaN 3 , 10% D 2 O and complete protease inhibitor cocktail). All NMR data were acquired at 30°C on 500, 600 or 800 MHz Bruker AVANCE spectrometers equipped with a triple resonance TXI cryoprobe with an actively-shielded Z-gradient. Experimental parameters were set as described previously [11,12]. Sequential backbone resonance assignments for 1 H N , 15 N, 13 C a and 13 C b were derived from standard 3D HNCA, HN(CO)CA, HNCO, HN(CA)CO, CBCANH, and CBCA(CO)NH experiments [13]. (H m )C m CH-TOCSY experiments were also obtained to correct for iso-tope effect on 13 C chemical shifts [14]. The assignments of H a and H b resonances were achieved from analysis of the HBHA(CO)NH spectrum. H(CC)(CO)NH, CC(CO)NH and HCCH-TOCSY spectra were analyzed to obtain side chain assignments. To identify the interface region involved in dimer interactions, F 1 [ 13 C, 15 N]-filtered, F 3 -15 N-edited and F 1 [ 13 C, 15 N]-filtered, F 3 -13 C-edited 3D NOESY-HSQC spectra were obtained using a (u-2 H, 13 C, 15 N)NP 248-365 (65% deuteration)/ NP 248-365 hetero-dimer sample prepared by mixing labeled NP 248-365 sample with equal amount of unlabeled NP 248-365 sample [15,16]. The protein was denatured in 8 M urea and renatured by extensive dialysis in desired NMR buffer conditions. Data were processed with the XWINNMR suite and AURELIA software (Bruker, Germany) on SGI workstations or NMRPipe on Linux workstations [17]. The 1 H chemical shift was referenced to DSS at 0 ppm as suggested [18].

Static light scattering
Protein samples in NMR buffer were diluted to a concentration between 0.5 and 2 mg/ml. Prior to loading into the cuvette, samples were filtered through a 0.22 lm-cutoff filter. Data were acquired on a Dyn-aPro MS/X light scattering system equipped with a fixed-angle detector (Protein Solutions, NJ, USA) at 4°C. Analysis was carried out on the Dynamics V6 program suite included in the system on an IBM PCcompatible computer. Concentration effects were corrected within the program.

Chemical cross-linking
The homobifunctional amine cross-linker disuccinimidyl suberate was purchased from Sigma-Aldrich (MO, USA) and prepared in N,N-dimethylformamide (DMF) to a concentration of 25 mg/ml. Reactions were carried out with a final protein concentration of 0.35 mM and final cross-linker concentration of 5 mM. Mock reactions were set up as control which contained only the protein solution and DMF without cross-linker. The reaction mixtures in NMR buffer were allowed to react for 1 h at 4°C prior to quenching with 100 mM glycine. The results were visualized on SDS-PhastGel minigels (Pharmacia Biotech, Sweden).

Secondary structure of the dimerization domain
We have previously shown that SARS-CoV N protein consists of two structured domains, the RNA binding domain (a.a. 45-181) and the dimerization domain (a.a. 248-365), with the remainders of the sequence existing in disordered state [8]. NP 248-365 is the most stable domain which retains the dimer structure. Shortening the fragment causes structural changes and lengthening the fragment has no effect on the structure of NP 248-365 . Backbone assignment for most amino acids of NP 248-365 was achieved except for residues located at the Nterminus and H301 (Fig. 1A). Perdeuteration of NP 248-365 was necessary to obtain triple-resonance spectra due to short T 2 (transverse relaxation time) of the dimer. The secondary structure of NP 248-365 was determined from standard NMR parameters, such as the characteristic NOE patterns, the consensus chemical shift indices (CSI), the magnitude of the 3 J HNa value, and the H N exchange rates (Fig. 1B). The result shows that NP 248-365 consists of five a-helices (A, Val271-Phe274; B, Gln290-Gln295; C, Trp302-Phe308; D, Ala312-Gly317 and E, Phe347-Ala360) and two b strands (b1: Arg320-Thr326; b2: Gly329-Leu340) (see Fig. 1C).

The dimer interface is composed of a b-sheet stabilized by
helix E The short T 2 of the NP 248-365 dimer (MW = 28 kD) precluded full assignment of side chain resonances due to weak signals even on cryoprobe-equipped spectrometers, thus hampered complete 3D structure determination of the dimer to high resolution. However, most H a , H b and aliphatic sidechain nuclei could be assigned from (H m )C m CH-TOCSY, HCCH-TOCSY and 13 C-edited NOESY spectra. Further analysis of the intramolecular dimer-interface NOEs identified a number of contacts between the two b strands, which allowed us to define the b hairpin structure (Fig. 2A). This information allowed us to manually assign the intermolecular NOEs at the dimer interface from analysis of the F 1 [ 13 C, 15 N]-filtered, F 3 -15 N-edited and F 1 [ 13 C, 15 N]-filtered, F 3 -13 C-edited 3D NOESY-HSQC spectra using u-( 2 H, 13 C, 15 N)NP 248-365 (65% deuteration)/unlabeled-NP 248-365 sample. Our results indicate that the dimer interface is composed of a continuous fourstranded b-sheet, formed by extensive hydrogen bond interactions between the two long b strands of the two b-hairpins, contributed one from each of the two monomers (Figs. 2B and 3A). The dimer is further stabilized by hydrophobic interactions between residues on one side of the amphipathic long helix E and the b-sheet (Fig. 3B). The presence of the extensive interactions between the two monomers in a dimer provides strong stabilization force and explains why the two monomers cannot be separated without denaturing the protein, even though there is no cysteine in the dimerization domain to form a covalent disulfide bond. This arrangement is reminiscent of the dimer-interface of the porcine reproductive and respiratory syndrome virus (PRRSV) nucleocapsid protein (Fig. 3C) [19], the coat protein of bacteriophage MS2 (Fig. 3D) [19] and the peptide recognition domain of the human histocompatibility antigen (HLA) [20,21].

The stable dimer interface is an ideal common building block
for dimer interfaces It has been postulated that although coronaviruses are evolutionary related to arteriviruses, the large size discrepancy of their nucleocapsid proteins most likely implied that they had different folds [22]. However, we show that there are common principles that underlie the architecture of a nucleocapsid protein in both SARS-CoV and PRRSV. They both contain two regions, one for RNA-binding and the other for dimerization or oligomerization [8,23], albeit the two domains in SARS-CoV N protein is linked by a much longer flexible linker of 120 a.a. Most importantly, the structures of the dimer interface of the two viruses are very similar. The presence of extensive interactions in the dimer interface may render this  dimer interface to become self-contained, i.e., it is less likely to be dependent on the global structure of the protein. In fact, we have found that a.a. NP 281-365 still retained dimerization potential, even though structural differences were evident from NMR spectra (Fig. 4). Similar observations have been reported in the literature [6]. It is evident that the tight dimer interface structure permits certain extended perturbation in the global structure without affecting the dimer structure. Such characteristics make this particular fold ideal as a common building block for dimer interfaces in a variety of proteins.
3.4. The dimerization mechanism may be common among coronavirus nucleocapsid proteins To investigate whether all coronavirus N proteins share this dimerization mechanism we have used ClustalX to align the sequences of other coronaviruses [24] and the resulting subsequences were then submitted to the JPred server for secondary structure prediction [25]. Sequence alignment coupled with secondary structure prediction show that many share the bba topology observed in SARS-CoV (Fig. 5). In particular, the long b-strand and the long C-terminal helix are predicted to be present in all cases. Most of them also contain the short b-strand, with the exceptions of BCoV, HCoV and PHEV. These results raise the possibility that all coronavirus employ the same interface mechanism for dimerization and they belong to the same structural class, however this cannot be verified by the class-dependent prediction algorithm because of the lack of known tertiary structure [26].
In conclusion, we have determined the secondary structure of the dimerization domain of SARS-CoV N protein and have mapped out the residues involved in the interface. We show that the interface of SARS-CoV N protein dimer is a four-stranded b-sheet, superposed by two long helices. The topology closely resembles that of the PRRSV nucleocapsid protein and the coat protein of bacteriophage MS2. This type of dimer interfaces is highly stable and could serve as one of the common building blocks for dimer interfaces in nature. Sequence alignment and secondary structure prediction suggest that other coronavirus N proteins also adopt a similar dimerization mechanism.