Fig. 2

NRX-1933 binds at the pSer33/Ser37 β-catenin:β-TrCP interface and potentiates the binding affinity of the interaction. a Overall structure of NRX-1933 (yellow spheres) bound to the β-catenin:β-TrCP complex (PDB: 6M93) (Supplementary Table 1). b Unbiased Fo-Fc electron density for NRX-1933 at 3.0 standard deviations above the mean is shown (green). The compound is shown as sticks (yellow), bound between the surface of the β-TrCP β-propeller (gray) and the monophosphorylated β-catenin degron peptide, shown as sticks (magenta). c Cutaway view of the hydrophobic pocket formed by residues Leu472 and Ala434 from β-TrCP and Ile35 and Leu31 from β-catenin. d Side view of the same interaction with the β-TrCP surface removed, highlighting the β-catenin and β-TrCP residues that interact with NRX-1933. e The binding affinities of the pSer33/Ser37 and pSer33/pSer37 β-catenin peptides for β-TrCP were measured in the TR-FRET assay by titrating BODIPY-FL-labeled β-catenin peptides (residues 17-48) against 300 pM β-TrCP at varying concentrations of NRX-1532. The binding curves for the pSer33/pSer37 β-catenin peptide titrations were fitted to one-site binding model. For the pSer33/Ser37 β-catenin peptide titrations, which do not reach saturation owing to limitations in the maximum amount of fluorophore compatible with this assay. Curves were fitted to the same one-site binding model, with the upper plateau constrained to that of the pSer33/pSer37 β-catenin peptide binding curve. The K_Ds are reported for both pSer33/Ser37 and pSer33/pSer37 β-catenin peptides