NMR-based investigation of intrinsically disordered regions of modular proteins for tailored drug-design

Numerous RNA-binding proteins (RBPs) exhibit modular structures containing folded domains and intrinsically disordered regions (IDRs). Investigating the role of these domains and their potential interaction is essential for understanding protein function and developing intervention strategies. The Nucleocapsid protein (N) of SARS-CoV-2 is a pivotal example of RBP. Its complex structure encompasses two folded domains and three IDRs. In particular, the globular N-terminal domain (NTD) is responsible for the viral RNA interaction and the two flanking IDRs play an important synergic role (1). We herein report the design and synthesis of peptides able to interfere with the protein function, monitoring the interaction through solution NMR titrations. In particular, taking into account the structural propensity of the nucleocapsid protein, a first peptide was designed to simulate the main interactions driving the viral RNA binding. The peptide is indeed characterized by the presence of negatively charged residues and an aromatic one to mimic electrostatic and π-π stacking interactions. The first peptide was tested by NMR titrations with the NTD and a Nucleacapsid protein construct comprising also two flanking IDRs. Different NMR experiments were performed to enable the simultaneous observation of globular and disordered regions at atomic resolution. In particular, interaction studies were followed by 1H-15N HSQC and 13C experiments to investigate flexible regions. The interaction was improved with a designed peptide-PNA chimera in which some amino acid residues of the first-in-class peptide have been replaced with four PNA building blocks, selecting four G as nucleobases (2) with the aim to mimic the RNA nature. This promising synthetic peptide-PNA was tested with both protein constructs in the same experimental conditions. Up to now, the take-home message of our study is based on two unequivocal results: 1) the peptide-PNA chimera revealed a significantly greater affinity than the first designed peptide sequence; 2) IDRs in the protein NTR construct has shown visibly more pronounced effects, detected by HSQC, both in the presence of the first-in-class peptide and the PNA chimera, suggesting an important contribution of these flexible domains in the interaction