Structural basis for suramin binding to the C-terminal domain of the SARS-CoV-2 nucleocapsid protein

Abstract

The global threat posed by COVID-19 persists, largely due to the high mutability of SARS-CoV-2 and the limited availability of effective antiviral therapeutics. The nucleocapsid (N) protein of SARS-CoV-2 is an attractive drug target because of its high degree of sequence conservation and essential role in viral replication. In this study, we show that suramin, a polysulfonated antiviral compound, binds to the C-terminal domain (N-CTD) of the N protein and interferes with its interaction with RNA. Biolayer interferometry (BLI) shows that suramin has a higher binding affinity for N-CTD (Kd, 3.30 μM) than for RNA (Kd, 10.12 μM). Electrophoretic mobility shift assays (EMSAs) further confirms that suramin effectively displaces RNA from N-CTD. NMR titration experiments and site-directed mutagenesis identify the α1-η1 helix (residues 248–262) as the primary suramin binding region, with residues K256, R259 and R262 playing critical roles in ligand recognition. In addition, NMR relaxation and model-free analyses reveal that the α1-η1 helix is highly flexible on the picosecond to nanosecond timescale, a dynamic feature that likely facilitates ligand binding. Furthermore, ITC and EMSA experiments demonstrate that suramin can bind to the full-length N protein at multiple sites and dissociate RNA from the N protein. Taken together, these findings provide structural and biophysical insights into the mechanism of action of suramin and establish a rational basis for the development of targeted antiviral therapies against SARS-CoV-2.

1. Introduction

Remarkable progress has been made in understanding COVID-19 in terms of virology, treatment and prevention strategies. However, the high mutability of its causative agent, SARS-CoV-2, continues to present formidable challenges. SARS-CoV-2 harbors a large positive-sense RNA genome encoding four structural proteins—a spike (S), an envelope (E), a membrane (M), a nucleocapsid (N) and sixteen non-structural proteins (nsp1–16) [1–3]. Among these proteins, the N protein is highly conserved and plays a central role in the packaging and assembly of viral particles [4]. It binds to the viral RNA genome to form ribonucleoprotein (RNP) complexes, which are essential for efficient viral replication [5]. In addition, the N protein modulates host immune responses by interacting with innate immune signaling pathways [6,7], making it a promising therapeutic target.

The full-length N protein consists of 419 amino acids and has two structured domains: the N-terminal domain (N-NTD, residues 44–180) and the C-terminal domain (N-CTD, residues 247–364), which are separated and flanked by intrinsically disordered regions (IDRs: residues 1–43, 181–246, and 365–419) [8]. Both N-NTD and N-CTD exhibit RNA-binding capabilities with enriched positively charged residues [9–11]. In our previous investigation, we demonstrated that suramin, a polysulfonated naphthylurea compound, binds to the N-NTD of SARS-CoV-2 and disrupts its interaction with RNA [12]. Other studies have shown that suramin inhibits SARS-CoV-2 replication and reduces the viral load in human lung epithelial cells [13]. Although suramin has also been shown to inhibit key viral enzymes, such as RNA-dependent RNA polymerase (RdRp) and the 3CL protease [14,15], its precise mechanism of action remains poorly defined.

Drug repurposing strategies, particularly those involving suramin, offer a rapid and efficient route to antiviral development. In this study, we show that the C-terminal domain of the SARS-CoV-2 N protein also binds to suramin and competitively displaces RNA. Our findings elucidate the structural basis of this interaction and provide a rational framework for the design of targeted antivirals against SARS-CoV-2.