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Comparative Study
. 2005 Oct 24;579(25):5663-8.
doi: 10.1016/j.febslet.2005.09.038. Epub 2005 Sep 30.

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

Chung-ke Chang  1 , Shih-Che Sue, Tsan-hung Yu, Chiu-Min Hsieh, Cheng-Kun Tsai, Yen-Chieh Chiang, Shin-jye Lee, Hsin-Hao Hsiao, Wen-Jin Wu, Chi-Fon Chang, Tai-huang Huang
Affiliations
Comparative Study

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

Chung-ke Chang et al. FEBS Lett. .

Abstract

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

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Figures

Figure 1
Figure 1
(A) 15N‐HSQC spectrum of u‐15N‐NP248–365. (B) Summary of the NMR parameters employed for secondary structure prediction. Dots at the top indicate residues’ NH protons are protected from deuterium exchange after 24 h. (C) Secondary structure profile of the SARS‐CoV N protein. 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].
Figure 2
Figure 2
(A) Stripe plots showing the intermolecular NOE connectivities in the β‐sheet (left panel) and between the side chain resonances of residues in the β2 strand of one monomer (labeled on top of the stripes) and side chain resonances of residues in E helix in the other monomer (indicated by arrows) (right panel). The stripes on the left panel were selected from the F1[13C, 15N]‐filtered, F3‐15N‐edited 3D NOESY‐HSQC spectrum and the stripes on the the right panel were selected from the F1[13C, 15N]‐filtered, F3‐13C‐edited 3D NOESY‐HSQC. (B) NOE connectivities of the β‐sheet forming the dimer interface. The shaded arrows and the connecting loops represent the two β hairpins of the two monomers. The two‐headed arrows show the observed NOE pairs and the dotted lines are the proposed hydrogen bonds stabilizing the β hairpins, as well as the dimer interface between the two β hairpins. The dotted rectangular boxes represent the positions of the two helices which interact with the four‐stranded β‐sheet. The boxed residues are those involved in hydrophobic interaction with the helices. The NOEs between β1 and β2 (also β1′ and β2′ in the other monomer) were obtained from 3D 15N‐NOESY‐HSQC spectrum of u‐15N‐NP248–365 sample and the interfacial NOEs between β2 and β2′ were obtained from 15N‐filtered 3D NOESY‐HSQC spectrum of sample containing u‐(2H, 13C, 15N)‐NP248–365(65% Deuteration)/unlabeled NP248–365 hetero‐dimer.
Figure 3
Figure 3
(A) Schematic representation of the structure of the dimer interface of SARS‐CoV N protein. The relative orientation between the anti‐parallel β‐sheet and the E helix is defined by the six NOEs identified as shown on Fig. 2A. Residues involved in these NOEs are shown in stick and ball representations. (B) Helical wheel plot of helix E, showing the amphipathic nature of the helix. The hydrophobic face is defined by the four hydrophobic residues (colored green). (C) Ribbon representation of the structure of the dimer interface of the C‐terminal domain of the nucleocapsid protein of porcine reproductive and respiratory syndrome virus (PRRSV) (PDB ID: 1P65). (D) Ribbon representation of the structure of the dimer interface of the capsid protein of bacteriophage MS2 (PDB ID: 1AQ3). The ribbon representations are prepared with the MOLMOL program.
Figure 4
Figure 4
(A) Light scattering results of NP248–365 and NP281–365. Estimated particle radii and molecular weights are listed. (B) Chemical cross‐linking of NP248–365 (lanes 1 and 2) and NP281–365 (lanes 3 and 4). Lanes 1 and 3: without cross‐linker. Lanes 2 and 4: with cross‐linker. (C) 15N‐edited HSQC spectra of NP248–365 (left) and NP281–365 (right).
Figure 5
Figure 5
Alignment of the amino acid sequences of various coronavirus N proteins. The alignment shows only the regions corresponding to the dimer interface region of SARS‐CoV. From top to bottom: SARS‐CoV, porcine transmissible gastroenteritis virus (TGEV), feline coronavirus (FCoV), human coronavirus strain 229E (HCoV 229E), bovine coronavirus (BCoV), human coronavirus strain OC43 (HCoV OC43), porcine hemagglutinating encephalomyelitis virus (PHEV), murine hepatitis virus 1 (MHV‐1) and avian infectious bronchitis virus (IBV). JPred secondary structure predictions of the sequences are shown below the sequences. E and H represent the predicted secondary structure of a particular amino acid as β‐strand or α helix, respectively.
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