The starting configurations for the US windows were obtained as snapshots from the individual pulling trajectories. For systems 1 and 2, 26 umbrella windows were used with a 0.05 nm spacing along the reaction coordinate for the first 1 nm of COM separation between the proteins and a 0.1 nm spacing for the next 0.5 nm. For system 3, 21 umbrella windows with a 0.05 nm spacing were used for 1 nm COM separation between the peptide and protein.