The structure of the B state was modeled with the Xplor-NIH software, version 2.48 (19, 31). The initial structure was constructed on the basis of the crystal structure of the Rac2-Rho GDI complex (PDB ID: 1DS6) (8). First, the Rac1 model structure was constructed with SWISS-MODEL (32) using the atomic coordinates of Rac2 in the complex structure, and then Glu31, Asp63, Gln74, and Leu160 were substituted with Cys residues conjugated with MTSL using Xplor-NIH. A three-conformer ensemble representation for the Cys-MTSL groups was used to account for their flexibility. Because the exchange process is in the fast exchange limit and the observed PREs represent the population-weighted averages of the PREs from two exchanging states, we conducted ensemble simulated annealing calculations by assuming a two-state ensemble, in which each ensemble member represents the structure of state A or B. In the ensemble member corresponding to the A state, only the side chains of the MTSL-conjugated Cys were allowed to move, and positions of the rest of the residues were fixed. In the ensemble member corresponding to the B state, the residues around the switch1 region (residues 24 to 47) and the side chains of the MTSL-conjugated Cys were allowed to move, and the rest of the residues were held fixed. The ensemble weights were set to 0.6 for the A state and 0.4 for the B state. The total energy function minimized in the structure calculations contained the PRE pseudo-potential, the term for nonbonded interatomic contacts, the covalent geometry terms for bonds, bond angles, and improper dihedral angles, and a statistical potential to restrain dihedral angles. Hydrogen-bonding distance restraints to preserve the integrity of the β2-β3 sheet portion were also included. The side chains of MTSL-conjugated Cys were prevented from clashing with backbone and Cβ, Hβ atoms, but were allowed to overlap with other side chains to account for the mobility of the tags. During the simulated annealing calculations, PRE back calculations were performed using the Solomon-Bloembergen (SB) equation during the high-temperature stage, which was switched to the SB model-free equation during the cooling phase (19). The PRE correlation time was set to 14 ns, which was estimated from the rotational correlation time of Rac1 calculated using the dipolar and chemical shift anisotropy cross-correlated relaxation rates. In total, 200 ensembles were calculated, and the structures were ranked according to the total energy.

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