Thermodynamic characterization between encounter complex and saturated bound state of the cdM2-1/P90-110 interaction of hRSV

The human Respiratory Syncytial Virus (hRSV) infection is the most common cause of severe lower track respiratory diseases in young children, older adults, and immunocompromised patients worldwide. The United Nations has produced a guide entitled "Preventing the Next Pandemic", which highlights hRSV as one of the viruses that require vigilance. In the viral replication cycle, 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 viral polymerase replication complex 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 (P_90-110). In the present study, fluorescence anisotropy and NMR (15N-HSQC and CMPG) experiments were performed at three different temperatures to characterize and compare the thermodynamics of the cdM2-1/ P_90-110 interaction in the saturated bound state and in the encounter complex. The encounter complex was investigated using subsaturation conditions, that is, the P_90-110 peptide concentration employed in the experiments was only 10% equivalent to the molar concentration of cdM2-1. In the saturated-bound state condition, the anisotropy results indicate a cooperative binding of P_90-110 to cdM2-1 with a dissociation constant of hundreds of nanomolar. The thermodynamic analysis revealed an enthalpic term close to zero and a dominant entropic contribution to the negative Gibbs free energy of the cdM2-1/P_90-110 complex, suggesting that the electrostatic interactions play a key role in the formation of this complex. The 15N-HSQC and CMPG results showed that cdM2-1 residues participating in the interaction in the encounter complex and saturated-bound states present differences and partially overlap. At subsaturation conditions, the CMPG experiments displayed a thermodynamic behavior of cdM2-1/P_90-110 encounter complex different from that observed for the saturated-bound state. The findings of the present study provide molecular details of the cdM2-1/P_90-110 interaction not only in the context of saturated binding, but also in a subsaturation condition typical of an encounter complex, which together provide the deepest detail of an essential viral protein-protein interaction with potential as a target for antiviral development.