Energetic and structural features of SARS-CoV-2 N-protein co-assemblies with nucleic acids
Huaying Zhao,
Di Wu,
Ai Nguyen,
Yan Li,
Regina C. Adão,
Eugene Valkov,
George H. Patterson,
Grzegorz Piszczek,
Peter Schuck
Affiliations
Huaying Zhao
Dynamics of Macromolecular Assembly Section, Laboratory of Cellular Imaging and Macromolecular Biophysics, National Institute of Biomedical Imaging and Bioengineering, 13 South Drive, Bethesda, MD 20892, USA
Di Wu
Biophysics Core Facility, National Heart, Lung, and Blood Institute, 50 South Drive, Bethesda, MD 20892, USA
Ai Nguyen
Dynamics of Macromolecular Assembly Section, Laboratory of Cellular Imaging and Macromolecular Biophysics, National Institute of Biomedical Imaging and Bioengineering, 13 South Drive, Bethesda, MD 20892, USA
Yan Li
Protein/Peptide Sequencing Facility, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD 20892, USA
Regina C. Adão
Dynamics of Macromolecular Assembly Section, Laboratory of Cellular Imaging and Macromolecular Biophysics, National Institute of Biomedical Imaging and Bioengineering, 13 South Drive, Bethesda, MD 20892, USA
Eugene Valkov
Messenger RNA Regulation and Decay Section, RNA Biology Laboratory, Center for Cancer Research, National Cancer Institute, Building 560, Room 21-105A, Frederick, MD 21702, USA
George H. Patterson
Section on Biophotonics, National Institute of Biomedical Imaging and Bioengineering, National Institutes of Health, Bethesda, MD 20892, USA
Grzegorz Piszczek
Biophysics Core Facility, National Heart, Lung, and Blood Institute, 50 South Drive, Bethesda, MD 20892, USA
Peter Schuck
Dynamics of Macromolecular Assembly Section, Laboratory of Cellular Imaging and Macromolecular Biophysics, National Institute of Biomedical Imaging and Bioengineering, 13 South Drive, Bethesda, MD 20892, USA; Corresponding author
Summary: Nucleocapsid (N) protein of the SARS-CoV-2 virus packages the viral genome into well-defined ribonucleoprotein particles, but the molecular pathway is still unclear. N-protein is dimeric and consists of two folded domains with nucleic acid (NA) binding sites, surrounded by intrinsically disordered regions that promote liquid-liquid phase separation. Here, we use biophysical tools to study N-protein interactions with oligonucleotides of different lengths, examining the size, composition, secondary structure, and energetics of the resulting states. We observe the formation of supramolecular clusters or nuclei preceding growth into phase-separated droplets. Short hexanucleotide NA forms compact 2:2 N-protein/NA complexes with reduced disorder. Longer oligonucleotides expose additional N-protein interactions and multi-valent protein-NA interactions, which generate higher-order mixed oligomers and simultaneously promote growth of droplets. Phase separation is accompanied by a significant change in protein secondary structure, different from that caused by initial NA binding, which may contribute to the assembly of ribonucleoprotein particles within macromolecular condensates.
