Figure 3.

Fragment 28 is the most potent stabilizer of the Pin1_72/14-3-3 complex. (A) Schematic representation of the difference between dissociation constant of the binary complex (K_D, red line), concentration specific stabilization of the ternary complex (SF_i, purple line), and α-factor of the ternary complex (dark green line). Gray lines directly relate to panel E, where the apparent K_D at a specific concentration of fragment is represented (light to dark gray indicates fragment concentration as per the legend). The stabilization factor shows the shift in the apparent K_D relative to the binary complex at a set concentration of compound. The α-factor is a measure of the maximum stabilizing response at saturation of the compound and measures the maximal cooperativity of the ternary complex. This schematic illustrates the limitation of only using a stabilization factor as different compounds have differing cooperativity with the complex. (B) Chemical structures of 13, 23, 27, and 28. (C) Crystal structure of the 13/Pin1_72/14-3-3σΔC complex (PDB code 7BDT). Hydrogen bonds are shown as yellow dashes, and the electron density map is shown as a blue mesh (contoured at 1σ). (D) FA protein titrations in the presence of 100 μM fragment as indicated or DMSO control (n = 3). (E) 2D protein titrations in the presence of increasing concentrations of 28 (n = 1). (F) Ratio of K_D/apparent K_D plotted against the fragment concentrations. The data are derived from 2D titrations (see panel E and Figure S5). The saturation of the ratio represents the α-factor. (G) Comparison of stabilization factors in the presence of 100 μM compound (SF₁₀₀) and the α-factor. (H) Crystal structure of the 28/Pin1_72/14-3-3σΔC complex (PDB code 7BFW). Hydrogen bonds are shown as yellow dashes, and the electron density map is shown as a blue mesh (contoured at 1σ). (I) Overlay of FCA (PDB code 4JDD; ERα-peptide and 14-3-3σ hidden for clarity) and the Pin1_72/14-3-3 complex shows a steric clash of Trp+1 of Pin1_72 and FCA.