Characterization of MLL-IBD interaction and the solution structure of MLL-IBD complex. (A) HSQC spectrum for 70 μM IBD (green) titrated with 35 μM (cyan), 120 μM (blue), and 280 μM MLL₁₆₀ (black). (B) Determination of IBD-MLL₁₆₀ binding affinity from NMR titrations. Representative titration curves for 3 residues are shown, and the average K_d is reported. (C) Schematics of MLL constructs used for mapping the IBM1 and IBM2 binding motifs. (D) The 20 lowest energy structures of MLL-IBD. Well-ordered MLL fragments corresponding to residues 124 to 133 (IBM1) and 147 to 152 (IBM2) are shown in green, and IBD (residues 349-428) is blue. (E) Representative structure of MLL-IBD; coloring is the same as in panel D. (F) Details of IBM2-IBD interaction. Key residues involved in the contacts are green (IBM2) and gray (IBD). (G) Characterization of binding affinity using ITC. Equimolar complexes of menin with MLL₁₆₀, MLL₁₆₀ F129A, and MLL₁₆₀ FLAA were titrated with IBD. Experiments were performed twice, and K_d and stoichiometry (N) are shown for representative experiments. (H) Model of menin-MLL-LEDGF complex based on solution MLL-IBD structure and previously published crystal structure (PDB code 3U88).