All 1H-detected experiments were performed on Bruker AVANCE 500, 600, or 800 spectrometers equipped with cryogenic probes. 31P-detected experiments were performed on a Bruker AVANCE 400 spectrometer equipped with a BBFO probe. All spectra were processed by the Bruker TopSpin 2.1 or 3.1 software, and the data were analyzed using Sparky (T. D. Goddard and D. G. Kneller, Sparky 3, University of California, San Francisco, CA). Backbone resonance and side-chain methyl resonance assignments were obtained by combining out-and-back type triple-resonance experiments (30), NOE analyses based on the crystal structure, and mutagenesis approaches.

[1H-1H] NOESY-[15N-1H]-TROSY spectra were obtained using u-[2H, 15N] Rac1 samples at 25°C with a Bruker AVANCE 600 spectrometer. The mixing time was set to 200 ms.

The 1H-15N RDCs were obtained from the difference in the 15N-1H J-coupling constants in aligned (pentaethylene glycol monododecyl ether/hexanol) and isotropic media. The J-coupling constants were measured from the differences between the TROSY and heteronuclear single-quantum coherence peak positions. The measurements were performed at 20°C with a Bruker AVANCE 600 spectrometer, using u-[2H, 15N] Rac1 samples. The alignment tensors and the calculated RDCs were obtained by singular value decomposition with the dipolar coupling computation server developed by the Ad Bax group (National Institutes of Health) (https://spin.niddk.nih.gov/bax/nmrserver/dc/svd.html) by using the coordinates of the Rac1 structure modeled from the crystal structure of the Rac2-RhoGDI complex (PDB ID: 1DS6). In these analyses, the region with structural changes upon the binding of RhoGDI (residues 34 to 42) was not used.

To obtain the backbone 1H (amide NH), 13C (Cα, Cβ, and CO), and 15N (amide N) chemical shifts for δ2D analyses (13), we prepared u-[13C, 15N] Rac1 samples and conducted triple-resonance experiments at 35°C (for the N92I mutant) or 40°C (for the wild type) with a Bruker AVANCE 500 spectrometer.

The 13C single-quantum (SQ) and 1H triple-quantum (3Q) Carr-Purcell-Meiboom-Gill (CPMG) RD analyses were recorded at 25°C with Bruker AVANCE 600 and 800 spectrometers, using {u-[2H, 15N]; Alaβ, Ileδ1, Metε-[13CH3], Leu, and Val-[13CH3, 12C2H3]} Rac1 samples (31, 32). The constant-time CPMG relaxation period T was set to 20 ms for the 13C SQ experiments and 5 ms for the 1H 3Q experiments. The νCPMG values were varied between 50 and 1500 Hz for 13C SQ and between 200 and 3000 Hz for 1H 3Q RD experiments. The values of the effective relaxation rates measured in the presence of a νCPMG Hz CPMG pulse train, R2,effCPMG), were calculated using Eq. 1, where ICPMG) and I (0) represent the peak intensities with and without the relaxation period T, respectively.R2,eff(νCPMG)=1Tln{I(νCPMG)I(0)}(1)

For residue-specific fitting, the 13C SQ and 1H 3Q RD curves obtained with two static magnetic fields were simultaneously fitted to the Luz-Meiboom equation (Eq. 2) (33), where ΦX (X = 13C or 1H) denotes the dispersion amplitude parameter, R2,0 denotes the intrinsic transverse relaxation rate, kex denotes the exchange rate, ΔωX (X = 13C or 1H) denotes the chemical shift difference, and pB denotes the B state population (7)R2,eff=R2,0+ΦXkex(14νCPMGkextanh(kex4νCPMG))ΦC=pB(1pB)ΔωC2ΦH=pB(1pB)(3ΔωH)2(2)

Backbone amide NH and Trp ε-NH 1H temperature coefficients were calculated from the 1H-15N TROSY spectra obtained at 293, 298, 303, and 308 K with a Bruker AVANCE 500 spectrometer, using u-[2H, 15N] Rac1 samples.

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