Fig. S7.
Correlation of 15N chemical shift differences of 15N-Myb32 determined from the R2 relaxation dispersion experiments (ΔωN) with equilibrium chemical shift differences of 15N-Myb32 between the free and KIX-bound forms in the presence of MLL28 determined from HSQC spectra (ΔδN). (A) The results obtained by the fits to the induced-fit mechanism (model 2). Fitted parameters are kON = (1.8 ± 0.1) × 107 M−1⋅s−1, kOFF = 15.5 ± 0.3 s−1, Kd = 0.81 ± 0.06 μM, kIB = 0.74 ± 0.09 s−1, kBI = 15.4 ± 0.4 s−1, and a correction factor for the Myb32 concentration = 1.072 ± 0.003. kIB < kBI, indicating that the population of c-Myb in the intermediate state (88%) is much higher than that in the fully bound state (4%), which is unreasonable. The ΔωN values between the free and bound forms obtained by R2 dispersion experiments (red) were significantly larger than ΔδN obtained from equilibrium HSQC. However, the ΔωN values between the free and intermediate forms (blue) were well correlated with ΔδN between the free and bound forms, which is inconsistent. Therefore, the fits to the induced-fit model are unreasonable. (B) The results obtained by the fits to the slow conformational selection mechanism (model 3a). Fitted parameters are kON = (2.1 ± 0.2) × 108 M−1⋅s−1, kOFF = 21.0 ± 0.6 s−1, Kd = 0.7 ± 0.1 μM, kHU = 12,000 ± 1,000 s−1, kUH = 1,900 ± 100 s−1, and a correction factor for the Myb32 concentration = 1.095 ± 0.004. The ΔωN values between the unfolded and helical forms obtained by fitting the R2 dispersion experiments (blue) were significantly larger than ΔδN between the free and bound forms obtained from equilibrium HSQC spectra, suggesting that the conformational ensemble of free c-Myb contains a state that is more highly structured than KIX-bound c-Myb. Therefore, the fits to the conformational selection model 3a are physically unrealistic.