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

• Suramin binds to the C-terminal domain (N-CTD) of SARS-CoV-2 nucleocapsid protein with higher affinity (Kd = 3.30 μM) than to RNA (Kd = 10.12 μM), and effectively displaces RNA from N-CTD. • NMR and mutagenesis identify the α1-η1 helix (residues 248–262) as the primary suramin binding site, with residues K256, R259, and R262 critical for recognition. • The α1-η1 helix exhibits high flexibility on the picosecond-to-nanosecond timescale, likely facilitating ligand binding. • Suramin binds to full-length N protein at multiple sites and dissociates RNA, providing a rational basis for developing targeted antiviral therapies against SARS-CoV-2.