Study of conformational dynamics of the encounter complex formed between the core domain of hRSV M2-1 protein and the intrinsically disordered region of viral phosphoprotein P using NMR spectroscopy

In the replication cycle of the human Respiratory Syncytial Virus (hRSV), the interaction between the M2-1 anti-termination factor and phosphoprotein P is important for the success of viral transcription. The M2-1 protein plays a crucial role in preventing the RNA-dependent RNA polymerase replication complex, viral polymerase plus the cofactor phosphoprotein, from disassociating upon reaching the codon stop, increasing transcriptional efficiency. The M2-1/P interaction occurs between the core domain of M2-1 (cdM2-1) and the intrinsically disordered region (IDR) of residues 90–110 of P (P90-110). The present work aimed to characterize the conformation dynamics of the encounter complex formed between the cdM2-1 and the IDR P90-110 using NMR spectroscopy. For this purpose, it was analyzed the changes in the chemical shift perturbation (CSP) of the 15N cdM2-1 at different concentrations of the peptide P90-110 until reached saturation, as well as analyzed 15N CPMG relaxation dispersion (RD) at undersaturation concentration of the peptide. The CSP and CPMG-RD results revealed concomitantly that the residues Val127, Asn138, Gln144, Lys150, and Val156 in helix α3, α4, and α6 of the cdM2-1 are directly involved in the M2-1/P interaction interface, presenting the highest values of CSP and significant relaxation dispersion profiles, while 15N CPMG-RD data identified exclusively the residues Ser133, Thr145, Leu149, Arg151, and Thr160 involved in the encounter complex formed between the protein and the IDR. These results indicate that the formation of the M2-1/P complex is dynamic with both cdM2-1 and IDR P90-110 undergoing structure, population, and interconversion rate changes in order to interact. The study of such complexes helps in the structural understanding of proteins and intrinsically disordered regions (IDPs and IDRs) enabling new strategies for drug discovery. In further experiments, such dynamics will be investigated by targeting the P90-110 instead of the cdM2-1.