The pulling simulation was performed with a pull rate of 0.000033 nm/ps and a spring constant of 1000 kJ/mol/nm 2 . Frames were saved every picosecond. For umbrella sampling simulations the distance between protein chains was restrained with the force constant of 10000 kJ/mol/nm 2 . In all the simulations C α atoms of the static protein chain were restrained to their initial positions with the force constant of 1000 kJ/mol/nm 2 . C α atoms of the moving protein chain were restrained to their initial positions with the force constant of 1000 kJ/mol/nm 2 in x-and y-directions, as proposed by Doudou et al. To estimate the PMF along the reaction coordinate from the US simulations, the WHAM implementation from Grossfield lab version 2.0.11 39 was used.