(A) Representation of the KIX structure in the ternary KIX·MLL·c-Myb complex (PDB 2AGH []). The six mutation sites are mapped onto the surface of KIX, color coded to indicate the effect of Ala substitution on K_d for MLL binding: yellow, 2-15× increase in K_d; orange, 25-40× increase; red, 100× increase. Backbones of the bound MLL (2842-2860 only) and c-Myb are shown in blue and green, respectively. The figure was prepared using MOLMOL []. (B) Far-UV CD spectra, (C) urea-induced equilibrium unfolding transition, and (D) MLL binding isotherms of the wild-type and mutant KIX. Color codes are shown in (D). In (C) and (D), continuous lines are the curves fitted to a two-state transition and a one-site binding model, respectively.

(A) Regions of the ¹H-¹⁵N HSQC spectrum of the KIX-L628A mutant showing chemical shift changes upon titration with MLL. The cross-peak color changes gradually from black (free) to magenta according to the concentration ratio as shown in the bar above the figure. (B) ¹H and ¹⁵N chemical shift titration curves for a subset of KIX-L628A resonances (colored points corresponding to residues according to the legend) upon titration with increasing amounts of MLL. The lines represent a global fit to the titration data, using a two-site binding model with K_d1 = 220 μM and K_d2 = 1.63 mM. (C,D) MLL binding sites on KIX-L628A. The weighted average chemical shift differences [ ] for KIX-L628A amide resonances between the free form and the bound form, in which the primary (C) or secondary binding site (D) is occupied, are mapped onto the surface of KIX in the ternary complex, using colors to indicate changes in chemical shift greater than 2 × standard deviation (SD) from the mean (red and blue), between 1 and 2 × SD from the mean (orange and cyan), and between mean and 1 × SD from the mean (yellow and green).

(A, upper) Histogram showing Δδ(N,H)_av between the wild-type and the L628A mutant of KIX in the free form. Color codes are the same as in . (A, lower) Histogram showing the secondary ¹³Cα chemical shifts calculated by subtraction of random coil values [] from the experimental data for the wild-type KIX (open bars) and the L628A mutant (gray bars) in the free form. Regions showing positive secondary Cα chemical shifts indicate a helical conformation. Secondary structure elements in the ternary KIX·MLL·c-Myb complex [] are depicted at the top. (B) The same as in (A), but in the presence of MLL (1:1 and 1:2 KIX:MLL ratios for the wild-type KIX and the L628A mutant, where 94% and 74% of KIX are in the MLL-bound form, respectively). (C,D) The KIX structures in the binary KIX·c-Myb complex (PDB 1SB0 []) and in the ternary KIX·MLL·c-Myb complex, respectively. Residues having large Δδ(N,H)_av are shown by spheres of radius corresponding to the van der Waals radius of each atom. In (D), the G₂ helix is colored cyan.

(A) Regions of the ¹H-¹⁵N HSQC spectrum of wild-type KIX showing chemical shift changes upon titration with MLL. b and f indicate the peaks for the bound and free forms, respectively. (B) ¹H and ¹⁵N chemical shift titration curves for a subset of KIX resonances upon titration with increasing amounts of MLL. The lines represent a global fit to the titration data, using a two-site binding model with K_d1 = 2.14 μM and K_d2 = 920 μM. (C,D) MLL binding sites on the wild-type KIX, as shown in . Δδ(N,H)_av for primary binding was obtained from the HSQC spectra at 1:0 and 1:1 KIX:MLL ratios. Δδ(N,H)_av for secondary binding was obtained from a global fit of titration curves.