Amino acid substitutions in the Runx1 Runt domain that specifically disrupt DNA and CBFβ binding. (A) Structures of the Runt domain and CBFβ are shown in gray and blue, respectively, and the DNA is purple.³⁶  The R174 side chain in the Runt domain is green, and the T161 and Y113 side chains are orange. (B) Urea denaturation monitored by tryptophan fluorescence for the wild-type Runt domain and point mutants thereof. Plotted is the fraction of unfolded Runt domain in the presence of increasing concentrations of urea. Data from the wild-type Runt domain, R174Q, and T161A are from Matheny et al.³⁰  (C) Diagram of the potential interactions between the Runt Domain, CBFβ, and DNA. (D) EMSA measuring the affinity of the Y113A/T161A Runt domain for DNA (K₂). Shown is a representative example of 3 experiments. Triangles indicate decreasing concentrations of the Runt domain (2 × 10⁻⁶ to 4 × 10⁻¹⁵ M). Arrow indicates the lane in which the Runt domain concentration approximates K₂. (E) Isothermal titration calorimetric measurements of CBFβ binding to the wild-type, T161A, and Y113A/T161A mutant Runt domains. Wild-type Runt domain (45 μM) was titrated with 440 μM CBFβ at 26°C, and 42 μM T161A or 38 μM Y113A/T161A were titrated with 396 μM CBFβ at 22°C. In each panel, the top portion is the raw data, and the bottom panel is a plot of the binding corrected for dilution enthalpy. Experimental data (squares) are fit to a one-site binding model (line). The equilibrium dissociation constants (K₁) are indicated in the plots. The average values from 2 independent measurements (± standard deviation [SD]) are given.