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1 Supporting Information Arai et al /pnas SI Text Protein Expression and Purification. Myb3 (mouse, residues ) was expressed in Escherichia coli as a fusion with the B1 domain of protein G and purified using a Ni-NTA column. The fusion protein was cleaved on column using thrombin and Myb3 was separated using reverse-phase HPLC. The resulting protein (GYNDEDPEKEKRIKELELLLMSTENELKGQQAL) contains an additional Gly at the N terminus. Binding Simulations in the Absence of MLL. When the number of Myb3 binding sites on KIX is two, the binding can be described by an independent two-site binding model as shown below (4): CD Measurement. CD spectra of Myb3 and Myb5 (100 μm) were measured on an Aviv 6DS spectropolarimeter at 30 C in 0 mm Tris-acetate buffer (ph 7.0) containing 50 mm NaCl. ITC Measurement. Titrations of Myb3 (0.8 mm or 0.6 mm) into KIX (45 μm) in the presence or absence of MLL8 (90 μm) were performed at 30 C, using a MicroCal Omega VP-ITC instrument as previously described (1, 3). Samples were prepared in 0 mm Tris-acetate (ph 7.0) and 50 mm NaCl. Typically, two injections of 5 μl were followed by 8 injections of 10 μl until a molar ratio of was obtained. The dilution heats are typically small and were subtracted from the calorimetric data. Integration of the thermogram and subtraction of the blanks yielded a binding isotherm that was fitted to a one-site binding model using the MicroCal Origin software or to a two-site binding model using an in-house fitting program to determine the stoichiometric ratios, N 1 and N, the dissociation constants, K d1 and K d, and the changes in enthalpy of the interaction, ΔH 1 and ΔH, for the primary and secondary binding, respectively. To improve fits, correction for protein concentration was implemented in the fitting program (4). The change in entropy, ΔS, was then calculated according to Eq. S1, 1 ΔG = RT ln = ΔH TΔS, [S1] K d where R is the gas constant, T is the absolute temperature, and ΔG is the free energy change from the free to the bound form. All experiments were performed in duplicate. NMR Measurement. NMR spectra were recorded using Bruker 500-, 600-, and 800-MHz spectrometers and analyzed using NMRPipe (39) and NMRView (40). Backbone resonances of free Myb3 and the KIX domain in complex with Myb3 at a 1:1 ratio were assigned using standard 3D NMR experiments with 500 μm 13 C, 15 N-labeled protein in 0 mm Tris-d 11 -acetate-d 4 (ph 7.0), 50 mm NaCl, mm NaN 3, and 10% (vol/vol) D O at 30 C. Backbone resonances of Myb3 in complex with KIX at a 1:1 ratio in the presence of MLL8 were assigned using 1 H- 15 N HSQC, 15 N TOCSY-HSQC, and 15 N NOESY-HSQC spectra. 15 N R relaxation dispersion data were acquired on Bruker 500- and 800-MHz spectrometers, using relaxation-compensated constant-time Carr Purcell Meiboom Gill (CPMG) pulse sequences (10, 38) with a constant relaxation delay of 40 ms or 60 ms. Errors in R were estimated from duplicate measurements (10). The samples contained 0 mm Tris-d 11 -acetate-d 4 (ph 7.0), 50 mm NaCl, mm NaN 3, 10% (vol/vol) D O, 0.7 mm Myb3, mm KIX, and mm MLL8. The weighted q average ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi 15 N/ 1 H chemical shift change was calculated as Δδ av = ðδδ H Þ + ðδδ N =5Þ (4). If K d1 and K d are known, the fractions of the F, B1, B, and B1 forms, f F, f B1, f B, and f B1, respectively, are calculated, using K d1 of 0.13 μm and K d of 46 μm, as f F = K d1 K d K d1 K d + ðk d1 + K d Þ½LŠ + ½LŠ K d ½LŠ f B1 = K d1 K d + ðk d1 + K d Þ½LŠ + ½LŠ K d1 ½LŠ f B = K d1 K d + ðk d1 + K d Þ½LŠ + ½LŠ f B1 = ½LŠ K d1 K d + ðk d1 + K d Þ½LŠ + ½LŠ, where [L] is the concentration of the free form of Myb3, which is a solution of the cubic equation ½LŠ 3 + a½lš + b½lš + c = 0 a = ½PŠ tot ½LŠ tot + K d1 + K d b = ½PŠ tot ½LŠ tot ðkd1 + K d Þ + K d1 K d c = K d1 K d ½LŠ tot, [S] where [P] tot and [L] tot are the total concentrations of KIX and Myb3, respectively (4). The closed-form solution of Eq. S has been reported (41), where a ½LŠ = 3 + qffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi ða 3 3bÞcos θ 3, θ = arccos a3 + 9ab 7c q ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi. ða 3bÞ 3 There are three Myb3 species; the free form (C0), Myb3 bound to the primary site (C1), and Myb3 bound to the secondary site Arai et al. 1of1

2 (C). The concentrations of these species, [C0], [C1], and [C], respectively, are obtained as ½C0Š = f F ½LŠ tot ½C1Š = ðf B1 + f B1 Þ½LŠ tot ½CŠ = ðf B + f B1 Þ½LŠ tot, which are shown in Fig. S4 A C. Binding Simulations in the Presence of MLL. When MLL8 was added to the Myb3:KIX complex, the binding model is described as shown below: The concentrations of three Myb3 species were calculated as ½C0Š = ½Myb3Š tot ½C1Š ½CŠ ½C1Š = ½S1Š + ½D1Š + ½DŠ ½CŠ = ½SŠ + ½D1Š + ½D3Š. Similarly, there are three MLL8 species: the free form (M0), MLL8 bound to the primary site (M1), and MLL8 bound to the secondary site (M). The concentrations of these species, [M0], [M1], and [M], respectively, are obtained as ½M0Š = ½MLLŠ tot ½M1Š ½MŠ ½M1Š = ½S3Š + ½D3Š + ½D4Š ½MŠ = ½S4Š + ½DŠ + ½D4Š, which are shown in Fig. S4 D G. To study the direct binding of Myb3 to the primary, high-affinity site on KIX, it is desirable to observe only the S1 and D forms, in which Myb3 binds only to the primary site. Here, the following equations hold, F + S1 + S + S3 + S4 + D1 + D + D3 + D4 ½KIXŠ tot = 0 C + S1 + S + D1 + D + D3 ½Myb3Š tot = 0 M + S3 + S4 + D + D3 + D4 ½MLL8Š tot = 0 F C K d1 S1 = 0 F C K d S = 0 F M K d3 S3 = 0 F M K d4 S4 = 0 S C K d1 D1 = 0 S4 C K d1 D = 0 S1 C K d D = 0 S3 C K d D3 = 0 S M K d3 D3 = 0 S4 M K d3 D4 = 0 S1 M K d4 D = 0 S3 M K d4 D4 = 0, where [KIX] tot, [Myb3] tot, and [MLL8] tot are the total concentrations of KIX, Myb3, and MLL8, respectively; C is the free Myb3 concentration; M is the free MLL8 concentration; and F, S1 S4, and D1 D4 denote the concentrations of the corresponding forms. By solving these simultaneous equations using the Newton Raphson method (4), the concentrations (fractions) of various KIX, Myb3, and MLL8 forms are obtained. Here, we used the following numbers: K d1 and K d are 0.13 μm and46μm, respectively, and K d3 and K d4 are.1 μmand 90 μm, respectively, as determined previously (3). Analysis of R Relaxation Dispersion Data. The R relaxation dispersion data were analyzed using the fortran version of the inhouse fitting program GLOVE (43), named GLOVEf. The fitting equation for the two-state model (including both models 1 and 3b) is where R eff = R ½KIXŠk ON + k OFF 1 cosh 1 ½D + coshðη τ + Þ CP D cosðη ÞŠ, " # D ± = 1 ±1 + Ψ + Δω FB pffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi Ψ + ξ sffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi qffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi 1 η ± = τ CP ±Ψ + Ψ + ξ Ψ = ð½kixšk ON + k OFF Þ Δω FB ξ = Δω FB ð½kixšk ON k OFF Þ, where R 0 is an intrinsic relaxation rate, [KIX] is the concentration of free KIX, k ON and k OFF are the association and dissociation rates, respectively, τ CP is the interval between 180 pulses in CPMG pulse sequences, and Δω FB is the chemical shift difference between the free and bound forms (44). The fitting equation for the induced-fit model (model ) and the conformational selection model (model 3a) is R eff = R 0 1 τ CP lnðλ 1 Þ, where λ 1 is the largest eigenvalue of the matrix 0 h Re exp h Im exp A τ CP A τ CP exp exp A p τ i CP A p τ i CP h Im exp h Re exp A τ CP A τ CP exp exp A p τ i CP 1 C A p τ i A, CP Arai et al. of1

3 where Re[ ] and Im[ ] are functions to extract the real or imaginary elements, respectively, of the complex matrix (10). A is a 3 3 evolution matrix, and A * is its complex conjugate. For the induced-fit model, 0 A ½KIXŠk 1 ON k OFF 0 ½KIXŠk ON k OFF + k IB iδω FI k BI A, 0 k IB k BI iδω FB where k IB and k BI are the folding and unfolding rate constants between the intermediate and bound forms, respectively, and Δω FI is the chemical shift difference between the free and intermediate forms. For the conformational selection model, 0 A k 1 UH k HU 0 k UH k HU + ½KIXŠk ON iδω UH k OFF A, 0 ½KIXŠk ON k OFF iδω UB where k UH and k HU are the folding and unfolding rate constants between the unfolded and helical forms, respectively, and Δω UH and Δω UB are the chemical shift difference between the unfolded and helical forms and between the unfolded and bound forms, respectively. The free KIX concentration is obtained by ½KIXŠ = 1 K d + c K ½KIXŠ tot c M ½Myb3Š tot qffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi + Kd c K ½KIXŠ tot + c M ½Myb3Š tot + 4cK ½KIXŠ tot K d, where c K and c M are correction factors for [KIX] tot and [Myb3] tot, respectively. For the two-state model, for the induced-fit model, K d = k OFF k ON ; K d = k OFF k ON k BI k BI + k IB ; and for the conformational selection model, K d = k OFF k ON k UH + k HU k UH. The fitting parameters are R 0, k ON, k OFF, Δω FB, c K, and c M for all three models and, in addition, k IB, k BI, and Δω FI for the inducedfit model and k UH, k HU, and Δω UH for the conformational selection model. Fitting Procedures. In the present study, 8 dispersion curves (four concentration ratios at two magnetic fields) were obtained for each residue, and each curve contained 17 data points. Because 1 residues show smooth dispersion curves, a total of 96 dispersion curves containing 1,63 data points were used for data analysis. Fits to the above fitting equations were done by the nonlinear least-squares method. There are three types of fitting as follows: Local fit: Dispersion curves for only one residue (8 curves) are globally fitted with common rate constants and common chemical shift differences. R 0 is a local parameter for each curve. Correction for protein concentrations was not done (c K and c M were fixed to 1). Full global fit: Dispersion curves for all residues (96 curves) are globally fitted with common rate constants and common correction factors for protein concentrations. Chemical shift differences are common for the curves of the same residue. R 0 is a local parameter for each curve. Subglobal fit: Dispersion curves for all residues (96 curves) are globally fitted with a common correction factor(s) for protein concentration(s). All rate constants and chemical shift differences are common for the curves of the same residue. R 0 is a local parameter for each curve. The nonlinear least-squares fitting requires good estimates of initial values. Therefore, many fitting runs were carried out from various initial parameter values (grid search). Then, the best fit was selected, that is, the fit with a small reduced χ, which indicates the goodness of fit, and with reasonable parameter sets. We initially performed local fits for each residue and searched for the best fit by a grid search. Then, the median of the rate constants obtained for all residues was calculated and was used as an initial value for the subsequent full global fit. In the full global fit, a grid search around the medians of local fits was performed. If the full global fit gives a reasonable fit, a subglobal fit was performed, in which only a correction factor(s) for protein concentration(s) is a global parameter(s). Here, the results of the full global fit were used as initial parameter values, and the random and Monte Carlo minimizations were performed as described previously (43). Fitting errors were obtained by a covariance matrix. Finally, we checked whether the fitted results fulfill the following: The K d value obtained by fitted rate constants is close to the K d obtained by ITC, the population of the bound form is larger than that of the free and intermediate forms, and the bound form is more structured than other forms. After the full global fits, we found that only the two-state model gives reasonable fits (main text and Fig. S7). Therefore, the subglobal fits were performed only for the two-state model. To obtain accurate estimates of correction factors for protein concentrations, c M and c K, in the subglobal fits to the two-state model, we performed a grid search by slightly changing the fixed c M and c K values. Here, c M and c K were fixed in each fit of the grid search, because fitting was unstable if both parameters were set as variables. The parameters obtained in the best fit among the grid searches are shown in the main text and in Fig. 5 (c M = 1.030; c K = 0.959). The apparent variation in k ON for different residues (Fig. 5A) is not statistically significant. On the other hand, residues L30, T305, N307, E308, and L309 in the C-terminal part of the c-myb helix have lower than average values of k OFF (Fig. 5B); the average k OFF is 1.7 ± 0.4 (SE) for residues and 16.6 ± 0.6 for residues Several variants of the two-state fits were performed in which clusters of residues were constrained to have the same values of k ON and k OFF. The results of these fits are given in Table S1. The best fits were obtained when k ON and k OFF were allowed to vary independently for each residue or when k ON was constrained globally (for residues ) or constrained separately for clusters of residues in the N- and C-terminal regions of c-myb. In all such fits, lower than average dissociation rates were observed consistently for the C-terminal residues. However, fits in which k OFF was constrained to a global value or constrained for separate clusters of residues in the N-terminal and C-terminal regions were noticeably poorer, as judged by χ (Table S1). Data Treatment for Fig. 5D. The correlation between the association rates of Giri et al. (7) and the predicted percentage of helix according to the program AGADIR () is valid only for some of the mutation sites. The association rates of the Lys/Ala, Lys/Gly, and Arg/Ala mutant peptides are systematically lower than wild type by an average 1.85 (± 0.31) μm 1 s 1, presumably because of the reduction in charge. In the complex, these residues, especially K93 and R94, are close to a negatively charged patch on the surface of KIX. The rates for the K93A, K93G, R94A, and K96A peptides were therefore corrected, k ON = k ON(obs) μm 1 1 s (Fig. 5D, green triangles). All other rates are as in table 1 of Giri et al. (7) (Fig. 5D, red circles). The line (slope = 0., R = 0.85) Arai et al. 3of1

4 is a linear least-squares fit of all data except L300A and L301A. These latter mutants, which bind faster than wild type and have negative Φ values, are clear outliers (Fig. 5D, black circles) and were excluded from the least-squares fit. It should be noted that the slope is unchanged and the correlation coefficient is improved (R = 0.94) if the Lys and Arg mutants are also excluded. The linear correlation between association rate and predicted helicity is robust and is also observed when the population of helix is predicted for mutant Myb5 peptides with the N terminus acetylated (R = 0.8) or preceded by Gly residues (R = 0.86) to mimic the fusion protein used by Giri et al. (7) for stopped-flow kinetics. Arai et al. 4of1

5 Fig. S1. (A) 1 H- 15 N HSQC spectra of 15 N-labeled Myb3 showing chemical shift changes upon titration with KIX. Assignments are shown for residues showing fast-exchange shifts. The cross-peak color changes gradually from blue (free) to red (bound) according to the concentration ratios shown. (B) 1 H- 15 N HSQC spectra of 15 N-labeled KIX showing chemical shift changes upon titration with Myb3. The cross-peak color changes gradually from black (free) to magenta (bound) according to the concentration ratios shown. (C and D) Histograms of the averaged chemical shift differences Δδ av for the primary (C) and secondary (D) binding of Myb3 to KIX. The black horizontal line shows the mean of all Δδ av (0.144 ppm and ppm for primary and secondary binding, respectively). The residues are categorized into the following groups: red, greater than or equal to mean + SD; orange, mean + 1 SD to mean + SD; yellow, mean to Legend continued on following page Arai et al. 5of1

6 mean + 1 SD; and gray, less than mean. (C) 1 H and 15 N chemical shifts of KIX for the c-myb bound form at a 1:0.8 KIX:Myb3 ratio were subtracted from those of free KIX to get Δδ H and Δδ N. The data at a 1:0.8 ratio (rather than at 1:1) were used for primary binding to eliminate the effect of secondary c-myb binding to KIX. (D) Δδ H and Δδ N obtained from the titration analysis (Fig. S3A) were used to calculate Δδ av. The data at a 1:0.8 ratio were used as a reference. (E) 1 H- 15 N HSQC spectra of 15 N-labeled Myb3 showing chemical shift changes upon titration with the KIX-L68A mutant. The cross-peak color changes gradually from blue (free) to red (bound) according to the concentration ratios shown. Fast-exchange shifts are observed for some peaks in the titrations of small amounts of KIX, suggesting the presence of secondary c-myb binding on KIX. Fig. S. (A and B) ITC titration profiles (Upper) and binding isotherms (Lower) for the Myb3:KIX interactions in the absence (A) and the presence (B) of MLL8 [0 mm Tris-acetate (ph 7.0), 50 mm NaCl, 30 C]. (A) 0.8 mm Myb3 was titrated into 45 μm KIX. Two titration curves from duplicate measurements were globally fitted assuming a two-site binding model. Thermodynamic parameters are K d1 = 0.3 ± 0.06 μm, N 1 = 1.1 ± 0., ΔG 1 = 9. ± 0. kcal/mol, ΔH 1 = 5 ± 1 kcal/mol, TΔS 1 = 4 ± 1 kcal/mol, K d = 43 ± 14 μm, N = 0.8 ± 0., ΔG = 6.1 ± 0. kcal/mol, ΔH = 7 ± 3 kcal/mol, TΔS = 1 ± 3 kcal/mol. A correction factor for the Myb3 concentration was 1.1 ± 0.. (B) 0.6 mm Myb3 was titrated into 45 μm KIXinthepresenceof90μM MLL8. The titration curve was fitted to a one-site binding model. Thermodynamic parameters are K d = 0.13 ± μm, N = 1.04 ± 0.01, ΔG = 9.3 ± 0. kcal/mol, ΔH = 9.5 ± 0.1 kcal/mol, and TΔS = 0.3 ± 0.1 kcal/mol. Arai et al. 6of1

7 Fig. S3. (A) Global fitting of the titration curves for the 15 N-KIX titration with unlabeled Myb3, referenced to the shifts at a 1:0.8 KIX:Myb3 ratio (4). 1 H(A, Left) and 15 N(A, Right) chemical shift changes of HSQC cross-peaks of 15 N-KIX are plotted as a function of the Myb3/KIX concentration ratio. Only the peaks showing clear fast-exchange shifts were used for fitting (154 curves for 77 assigned peaks). Color codes are shown (A, Right) along with the residue number. In the fitting, we assumed that K d1 is 0.13 μm, which was obtained by the ITC experiment for the Myb3 binding to the primary site on KIX in the presence of excess MLL8 and that Δδ H and Δδ N in the primary binding site are zero (3). The global fitting gave a K d of 46 ± 1 μm. A correction factor for the Myb3 concentration was ± 0.00 (4). (B) 15 N R relaxation dispersion curves for 15 N-Myb3 in the free form (B, Left) and for 15 N-Myb3 in the presence of twofold excess of MLL8 (B, Right) measured with an 800-MHz spectrometer at 0.7 mm Myb3. Color codes are shown in each panel along with the residue number. Arai et al. 7of1

8 Fig. S4. (A) Concentrations of various Myb3 species dependent on a KIX:Myb3 concentration ratio from 0 to 1.5 at 0.7 mm Myb3. Red, Myb3 bound to the primary c-myb binding site on KIX; blue, Myb3 bound to the secondary c-myb binding site on KIX; and green, free Myb3. (B) Fractions of various Myb3 species under the conditions for R relaxation dispersion experiments (from 1:0.95 to 1:1.10 Myb3:KIX concentration ratios at 0.7 mm Myb3), plotted in a logarithmic scale. (C) The results show that 3 9% of Myb3 binds to the secondary, low-affinity site. See SI Text for details of the calculations. (D F) Fractions of various KIX (D), MLL8 (E), and Myb3 (F) species under the conditions for R relaxation dispersion experiments plotted on a logarithmic scale (the KIX:Myb3: MLL8 concentration ratio = 1:0.95:1.90, 1:1.00:.00, 1:1.05:.10, and 1:1.10:.0 at 0.7 mm Myb3). (D) The D form, in which Myb3 and MLL bind at the primary (c-myb/pkid) and secondary (MLL) sites, respectively, is the most dominant form under these conditions. See SI Text for the definitions of various KIX forms. (E) Almost half of MLL8 binds to the MLL site, and the rest is mostly in the free form. Less than % of MLL8 binds to the primary site. Such a small amount of secondary MLL8 binding to KIX will not affect the Myb3 binding to the primary site on KIX. (F and G) More than 94% of Myb3 is bound to the primary site, % is in the free form, and only less than 0.5% is bound to the secondary site. Such a small fraction of secondary Myb3 binding to KIX will not be detected in the R relaxation dispersion experiments. Arai et al. 8of1

9 Fig. S5. 15 N R ex of Myb3 in the presence of KIX and MLL8 measured by the R relaxation dispersion experiments with 500-MHz (Left) and 800-MHz (Right) spectrometers. 15 N-Myb3:KIX:MLL8 concentration ratios are shown in each panel. R ex was estimated from the difference in R eff at the lowest and highest 1/τ cp values. Arai et al. 9of1

10 Fig. S6. 15 N R relaxation dispersion curves for 1 residues of 15 N-Myb3 in complex with KIX and MLL8 used for global analysis. In each panel, the Myb3:KIX concentration ratios and the color codes are shown. The MLL8 concentration was twofold excess of the KIX concentration. Solid lines are the fits to the twostate binding model. Arai et al of 1

11 Fig. S7. Correlation of 15 N chemical shift differences of 15 N-Myb3 determined from the R relaxation dispersion experiments (Δω N ) with equilibrium chemical shift differences of 15 N-Myb3 between the free and KIX-bound forms in the presence of MLL8 determined from HSQC spectra (Δδ N ). (A) The results obtained by the fits to the induced-fit mechanism (model ). Fitted parameters are k ON = (1.8 ± 0.1) 10 7 M 1 s 1, k OFF = 15.5 ± 0.3 s 1, K d = 0.81 ± 0.06 μm, k IB = 0.74 ± 0.09 s 1, k BI = 15.4 ± 0.4 s 1, and a correction factor for the Myb3 concentration = 1.07 ± k IB < k BI, 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 Δω N values between the free and bound forms obtained by R dispersion experiments (red) were significantly larger than Δδ N obtained from equilibrium HSQC. However, the Δω N values between the free and intermediate forms (blue) were well correlated with Δδ N 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 k ON = (.1 ± 0.) 10 8 M 1 s 1, k OFF = 1.0 ± 0.6 s 1, K d = 0.7 ± 0.1 μm, k HU = 1,000 ± 1,000 s 1, k UH = 1,900 ± 100 s 1, and a correction factor for the Myb3 concentration = ± The Δω N values between the unfolded and helical forms obtained by fitting the R dispersion experiments (blue) were significantly larger than Δδ N 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. Fig. S8. (A and B) Helicities of the TADs of c-myb (A) and pkid (B) predicted by AGADIR () at 303 K and 88 K, respectively. (C and D) Deviations of 13 C α (red) and 13 C (blue) chemical shifts from sequence-corrected (19) random coil shifts for intrinsically disordered proteins (0), applied to Myb3 (C, chemical shifts at 303 K) and pkid (D, chemical shifts at 88 K) (9). The predicted population of helix between residues 90 and 301 of Myb3 (66%) is in close agreement with experiment (70%, estimated from 13 C α and 13 C shifts). For pkid, the population of helix between residues 117 and 19 (spanning helix αa of pkid bound to KIX) (6) estimated from the secondary chemical shifts is 46%. Residues (helix αb of bound pkid) have no measurable propensity to adopt regular helical structure in the free pkid. Arai et al of 1

12 Table S1. Variants of the subglobal fits to the two-state model in which clusters of residues are constrained to have the same values of k ON and k OFF Fitted parameter values Correlation between Δω FB and Δδ FB Fit type Global parameters, in addition to c M and c K Local parameters, in addition to 0 Δω FB and R No. Average k ON parameters Reduced χ ( 10 7 M 1 s 1 ) N-terminal average k OFF,s 1 * C-terminal average k OFF,s 1 c M c K Slope r 1 k ON, k OFF ± ± ± ± a k ON1 (90 303), k ON ( ) k OFF ± ± ± ± b k ON1 (90 301), k ON (30 309) k OFF ± ± ± ± k ON (90 309) k OFF ± ± ± ± a k OFF1 (90 303), k OFF ( ) k ON ± ± ± ± b k OFF1 (90 301), k OFF (30 309) k ON ± ± ± ± a k ON (90 309), k OFF1 (90 303), k OFF ( ) ± ± ± ± b k ON (90 309), k OFF1 (90 301), k OFF (30 309) ± ± ± ± a k ON1 (90 303), k ON ( ), k OFF1 (90 303), k OFF ( ) ± ± ± ± b kon1(90 301), kon(30 309), koff1(90 301), koff(30 309) ± ± ± ± k OFF (90 309) k ON ± ± ± k ON (90 309), k OFF (90 309) ± ± ± *k OFF1 or the average of k OFF for residues k OFF or the average of koff for residues Correlation coefficient. The results of this type of fit are shown in Fig.. Arai et al. 1 of 1