Energetic and structural features of SARS-CoV-2 N-protein co-assemblies with nucleic acids

Author:

Zhao, Huaying

Nguyen, Ai

Li, Yan

Adao, Regina C.

Valkov,Eugene

Patterson, George H.

Piszczek, Grzegorz

Schuck, Peter
Author Address

Natl Inst Biomed Imaging & Bioengn, Dynam Macromol Assembly Sect, Lab Cellular Imaging & Macromol Biophys, 13 South Dr, Bethesda, MD 20892 USA.NHLBI, Biophys Core Facil, 50 South Dr, Bethesda, MD 20892 USA.NINCDS, Prot Peptide Sequencing Facil, NIH, Bethesda, MD 20892 USA.NCI, Messenger RNA Regulat & Decay Sect, RNA Biol Lab, Ctr Canc Res, Bldg 560,Room 21-105A, Frederick, MD 21702 USA.Natl Inst Biomed Imaging & Bioengn, Sect Biophoton, NIH, Bethesda, MD 20892 USA.

Abstract:

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.

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