Affinity Tag-Free Purification of SARS-CoV-2 N Protein and Its Crystal Structure in Complex with ssDNA

Abstract

The nucleocapsid (N) protein is one of the four structural proteins in SARS-CoV-2, playing key roles in viral assembly, immune evasion, and stability. One of its primary functions is to protect viral RNA by forming the nucleocapsid. However, the precise mechanisms by which the N protein interacts with viral RNA and assembles into a nucleocapsid remain unclear. Compared to other SARS-CoV-2 components, targeting the N protein has several advantages: it exhibits higher sequence conservation, lower mutation rates, and stronger immunogenicity, making it an attractive target for antiviral drug development and diagnostics. Therefore, a detailed understanding of the N protein's structure is essential for deciphering its role in viral assembly and developing effective therapeutics. In this study, we report the expression and purification of a soluble recombinant N protein, along with a 1.55 Å resolution crystal structure of its nucleic acid-binding domain (N-NTD) in complex with ssDNA. Our structure revealed new insights into the conformation and interaction of the flexible N-arm, which could aid in understanding nucleocapsid assembly. Additionally, we identified residues that are critical for ssDNA interaction.

Keywords: COVID-19; N-terminal domain; SARS-CoV-2; X-ray crystallography; atomic-resolution structure; mass photometry; nucleocapsid protein; protein expression; protein purification; protein–DNA interactions.

Figure 1

(a) Schematic representation of SARS-CoV-2 virus and its structural proteins (S, N, M, and E) with genomic RNA [created with BioRender.com]. (b) The N protein is composed of several distinct regions: the N-terminal domain (N-NTD) and the C-terminal domain (N-CTD) connected by a flexible linker, and intrinsically disordered regions like the N-arm and the C-tail.

Figure 2

SARS-CoV-2 N protein purified by polyethyleneimine precipitation followed by SP Sepharose ion-exchange and finally size-exclusion chromatography. (a) FPLC size-exclusion chromatography and SDS-PAGE analysis of the purified N protein. The blue line represents the UV absorption spectrum of the eluted protein. The dashed green box highlights the absorption of the collected pure N protein. ‘N’ indicates the purified full-length N protein, while ‘M’ represents the protein marker. (b) Mass photometry analysis revealed that the N protein appeared as a monodisperse dimer (~93 kD) in-solution.

Figure 3

Comparisons of coronavirus N protein structures. (a) Our N protein structure (digested full-length N) showing N-NTD with part of the flexible N-arm (PDB ID:9CJ6; teal color, cartoon) in complex with ssDNA (blue, stick). A 2Fo–Fc electron density map contoured at 1σ is shown in light blue around the ssDNA. (b) Superimposition of our structure with other published SARS-CoV-2 N-NTD structures (PDB ID:7XWZ; light pink color, PDB ID:7CDZ; orange color, PDB ID:7N0R; limon color). (c) Superimposition of our structure with closely related SARS-CoV N-NTD (PDB ID: 2OFZ; navy blue color) and MERS-CoV- N-NTD (PDB ID: 4UD1; purple color) structures. N and C indicate the N- and C-terminal ends of the protein, respectively. The 5′ and 3′ represent the 5′ end and 3′ end of ssDNA. ß and η represent the ß-strand and short 3₁₀ helix, respectively.

Figure 4

Crystal packaging of digested N protein. (a) In the crystal, four neighboring protein molecules are colored as teal, hot pink, split pea, and yellow-orange, showing crystal packing at different 90° angles. (b) The N-arm extends to neighboring molecules and is anchored between two molecules through hydrogen bonding. Gray dashed lines represent the hydrogen bonds between amino acids.

Figure 5

Interactions of N protein with nucleic acids. (a) Superimposition of our structure (teal, PDB ID: 9CJ6) with the crystal structure of the N-NTD complex with dsRNA (light pink, PDB ID: 7XWZ). (b) Superimposition of our structure (teal, PDB ID: 9CJ6) with the NMR structure of the N-NTD complex with ssRNA (yellow, PDB ID: 7ACT). RNA is shown as a cartoon and ssDNA as a stick. ssDNA binds to N-NTD at the RNA binding pocket. (c) Stereo-view of detailed interaction of ssDNA with N-NTD (teal, our structure). (d) Stereo view of the detailed interaction of dsRNA with N-NTD (light pink, PDB ID: 7XWZ). ssDNA and RNA are represented as sticks, and water is represented as the red sphere. Gray dashed lines represent the hydrogen bonds. The nucleic acid bases of ssDNA interact with protein through direct and water-mediated hydrogen bonds. The RNA interacts with protein through hydrogen bonds in the phosphate backbone.