Peptide ligands to explore interactions with intrinsically disordered multidomain proteins: the case of SARS-CoV-2...

The SARS-CoV-2 nucleocapsid protein (N), essential for viral RNA packaging, comprises a structured RNA-binding domain, NTD(44-180), flanked by intrinsically disordered regions, IDR1 (1-43) and IDR2 (181-248). This extended, positively charged RNA-binding surface makes it an attractive target for synthetic ligands able to interfere with its function. In this study, peptide ligands were explored as synthetically accessible alternatives for modulating N interactions. After confirming the ability of a reference peptide (P0) in engaging IDRs in the interaction, we shifted our attention to key properties that modulate the interaction with the central domain NTD(44-180). Two new peptides (P1 and P2) were designed with identical amino acid compositions but distinct sequence arrangements. In each one a para-fluorinated phenylalanine residue was incorporated to introduce an aromatic component useful to mimic RNA bases and enable ¹⁹F NMR detection. Molecular dynamics simulations, circular dichroism, and NMR spectroscopy were combined to characterize the structural, dynamic and binding properties of P1 and P2. Both peptides involve the RNA-binding groove of the N protein (NTD(44-180)). While P1 demonstrated higher α-helical propensity and favorable electrostatic interactions, P2 displayed stronger interactions through enhanced conformational adaptability. ¹⁹F NMR experiments confirmed complex formation also from the ligand perspective. These findings support a modular peptide design strategy for targeting multidomain proteins.