Intramolecular loops control SARS-CoV-2 nucleocapsid protein self-association and nucleic acid binding dependent on phosphorylation

Abstract

The nucleocapsid protein of SARS-CoV-2 scaffolds genomic RNA into ribonucleoprotein complexes (RNP) for assembly in the virion, but also fulfills critical intracellular functions in replication and the suppression of host defense. It is comprised of a folded nucleic acid binding domain (NTD) and a dimerization domain, connected by a disordered linker containing a serine/arginine-rich (SR) region and a leucine-rich sequence (LRS). The switch between intracellular and assembly functions of N-protein is controlled by phosphorylation of the SR region, but the molecular details are unclear. Here we describe a model in which two mutually exclusive intramolecular loops bind the NTD and dynamically control self-association and nucleic acid binding properties dependent on the SR linker phosphorylation state. The model is supported by biophysical properties and interactions of full-length protein, point mutants, and peptide fragments. We find SR linker phosphorylation compacts the protein and inhibits nucleic acid binding and RNP formation, while enhancing self-association through promotion of transient coiled-coils in the LRS of the linker. These changes shift the nucleocapsid protein to a configuration poised for multi-valent interactions that support intracellular functions.