Structures of Natural and Strategic Mutants reveal a Core Network crucial for Integrity of the SARS-CoV-2 Nucleocapsid NTD

The SARS-CoV-2 nucleocapsid protein is indispensable for processing of the viral RNA genome. The N-terminal domain (NTD) was suggested to mediate specific RNA-interactions, but the lack of high-resolution structures with viral RNA prohibits insights into the precise mechanism of complex formation. Similarly, mutations in the nucleocapsid NTD since 2019 have not been addressed molecularly. Systematically combining crystallography and solution NMR, we here investigate the influence of six naturally occurring plus strategic NTD mutations on structural integrity and RNA-binding. We find that both features rely on a core network of residues conserved in Betacoronaviruses. This core network proves important for protein stability and communication between flexible loop-regions, key for RNA-recognition. Our comprehensive structural analysis demonstrates that crucial contacts within the expanded network steer selective interaction with target RNAs. We propose the core network renders the NTD evolutionarily robust in stability and plasticity for its versatile RNA genome-processing functions.