ATP and nucleic acids competitively modulate LLPS of the SARS-CoV2 nucleocapsid protein

Abstract

SARS-CoV-2 nucleocapsid (N) protein with very low mutation rates is the only structural protein which not only functions to package viral genomic RNA, but also manipulates host-cell machineries, thus representing a key target for drug development. Recent discovery of its liquid-liquid phase separation (LLPS) opens up a new direction for developing anti-SARS-CoV-2 strategies/drugs. However, so far the high-resolution mechanism of its LLPS still remains unknown. Here by DIC and NMR characterization, we have demonstrated: 1) nucleic acids modulate LLPS by dynamic and multivalent interactions over both folded NTD/CTD and Arg/Lys residues within IDRs; 2) ATP with concentrations > mM in all living cells but absent in viruses not only binds NTD/CTD, but also Arg residues within IDRs with a Kd of 2.8 mM; and 3) ATP dissolves nucleic-acid-induced LLPS by competitively displacing nucleic acid from binding the protein. Our study deciphers that the essential binding of N protein with nucleic acid and its LLPS are targetable by small molecules including ATP, which is emerging as a cellular factor controlling the host-SARS-CoV-2 interaction. Fundamentally, our results imply that the mechanisms of LLPS of IDR-containing proteins mediated by ATP and nucleic acids appear to be highly conserved from human to virus.

Adenosine Triphosphate: The Primordial Molecule That Controls Protein Homeostasis and Shapes the Genome–Proteome Interface

“…Detailed NMR investigations, including ATP/S2m competition experiments, elucidated this mechanism [85]. Notably, due to S2m binding to N (1-249) with a Kd of ~µM, considerably higher than ATP (with a Kd of ~mM), ATP at 20 mM remained insufficient to completely displace S2m from binding to N (1-249) [103].…”

“…Furthermore, the C-terminal folded domain (CTD) not only facilitates dimerization, but also enhances oligomerization, potentially reinforcing LLPS but also exacerbating aggregation, akin to observations in the hydrophobic region of the TDP-43 PLD. While turbidity measurement and DIC imaging revealed the biphasic effects of ATP and the nucleic acids of both specific and non-specific sequences on the LLPS of the entire N protein, the aggregation-prone complexity hinders detailed residue-specific investigations using NMR techniques [103]. However, a recent NMR characterization of a dissected fragment, N (1-249) composed of the folded NTD and two IDRs, elucidated the modulating mechanism of LLPS by ATP and a 32-mer S2m ssDNA derived from the stem-loop II motif of SARS-CoV-2 [103].…”

How does severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) achieve immune evasion?: A narrative review

“…Therefore, small molecules disrupting the interaction between N protein and stress granule-related proteins may serve as a direction for developing new antiviral drugs. [7] Dang et al [42] believe that small molecules regulating the interaction between the N protein and nucleic acids are crucial for intervening in the virus lifecycle, and thus, these molecules hold promise for the development of novel antiviral drugs. Furthermore, targeting the impact of the N protein on the immune system may provide insights into antiviral therapy.…”

Molecular Mechanisms and Potential Antiviral Strategies of Liquid–Liquid Phase Separation during Coronavirus Infection

“…ATP induces LLPS at low concentrations and dissociates LLPS at high concentrations by specifically binding to arginine/lysine residues in the RNA-binding domains of the N proteins. S2m, a 32-oligomer stem-loop II nucleic acid motif derived from SARS-CoV-1, shares highly overlapping binding sites in the folded NTD and IDR of N proteins and interacts with ATP in the same way to regulate the LLPS of N proteins [49].…”

“…ATP binds not only to nucleic acids but also to arginine residues in the proteins, competitively dislodging the nucleic acids from the proteins and solubilizing the LLPS. S2m (derived from the 32-polymer stem-loop II of the SARS-CoV-2) and A24 (a 24-oligomer non-specific nucleic acid) can modulate LLPS through dynamic and multivalent interactions with arginine/lysine residues in the structural domains and disordered regions of proteins [49]. Moreover, a single-stranded DNA aptamer (N-Apt17) and its circular bivalent form (cb-N-Apt17) can effectively disrupt the LLPS of the SARS-CoV-2 N protein, which presents a potential inhibitory ability on viral replication [58].…”