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

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

Publication date: Nov 30, 2024

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 A 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.

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Concepts Keywords
Crystal atomic-resolution structure
Immunogenicity Coronavirus Nucleocapsid Proteins
Recombinant Coronavirus Nucleocapsid Proteins
Target COVID-19
Viral COVID-19
Crystallography, X-Ray
DNA, Single-Stranded
DNA, Single-Stranded
Humans
mass photometry
Models, Molecular
N-terminal domain
nucleocapsid phosphoprotein, SARS-CoV-2
nucleocapsid protein
Nucleocapsid Proteins
Nucleocapsid Proteins
Phosphoproteins
Phosphoproteins
Protein Binding
Protein Domains
protein expression
protein purification
protein–DNA interactions
Recombinant Proteins
Recombinant Proteins
SARS-CoV-2
SARS-CoV-2
X-ray crystallography

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