Fig. 5: Crystal structure analysis of the interaction between FBW7 and FIR/PUF60-UHM.

a The amino sequence LNGRYFGGRVVKA in SPF45 is similarto sequences in the C-terminal domains of FIR and U2AF65. A comparison of two crystal structures indicates that the positions and configurations of W425 and D399 in FBW7 are considerably similar to those of SAP155. FBW7 has many WD-motifs, and most of the motifs are involved in the conformational stabilization of the WD-repeated domain (molecules indicated in yellow). The binding structure between SF3B1 and one of the splicing factors containing UHM, SPF45 (human splicing factor 45), has previously been clarified by X-ray crystal analysis (PDB code: #2PEH)²⁹. In the 2PEH structure, the crystal unit cell contains two SPF45 recombinant proteins (amino acids: 301–401) and two SF3B1 partialpe ptides (aa: 333–342). b Amino acids sequence of U2AF65 (475aa). Similarity among SPF45, FIR, and U2AF65. c There is an extra pair of W425 and D399 at the center of the WD-repeated domain (molecules indicated in magenta). Since the binding pocket of FBW7 contains the WD-motif that is expected to interact with FIRΔexon2, the chemical skeleton of the two synthesized compounds is regarded as a WD-mimicking form. A low molecular weight artificial chemical, BK697, that inhibits FIRΔexon2, as shown by in silico analysis, was synthesized. d The isothermal titration calorimetry (ITC) measurement of FBW7 with FIRΔexon2 suggested the molecular interaction between two proteins. The exothermic peaks were observed in the initial 17 injections.The peak level was decreased in the later injections. Due to the quick upward change in the ITC thermogram, the association constant is >108 M⁻¹. Since the binding reaction is exothermic, the binding of FBW7 and FIRΔexon2 is enthalpically driven.This result is compatible with the importance of Asp of FBW7 in the molecular binding because Asp usually contributes to hydrophilic interactions, such as hydrogen bond formation, between protein molecules.