The all-atom model of Rac1 was constructed with CHARMM-GUI (34), using the crystal structure of the Rac2-RhoGDI complex (PDB ID: 1DS6), on the basis of our previous result that this structure well represents the ground-state structure of GDP-bound Rac1 in the uncomplexed state (7, 8). The Rac2 sequence was converted to that of Rac1 with or without the N92I mutation, and the structure including the bound GDP and Mg2+ was solvated in a periodic water box with a size of 74 Å by 74 Å by 74 Å, using TIP3P water molecules. The system was then neutralized with about 150 mM KCl. Molecular dynamics simulations were performed using the NAMD2.12 software (35) and the CHARMM36 parameter set (36). A cutoff distance of 12 Å was used for the van der Waals and short-range electrostatic interactions, and the long-range electrostatic interactions were computed with the particle mesh Ewald method (37). Bonds containing hydrogen atoms were restrained with the SHAKE algorithm (38). Bonded and short-range electrostatic interactions were computed every 2 fs, and long-range electrostatic interactions were computed every 4 fs. The system was first energy-minimized for 500 steps and then equilibrated under isothermal-isobaric (NPT) conditions for 5 ns at 1 atm and 310 K. The production simulations were performed for 50 ns at 1 atm and 310 K. The trajectories were analyzed using the Visual Molecular Dynamics 1.9.3 software (39).

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