Exploring DIX-DIX homo- and hetero-oligomers in Wnt Signaling with AlphaFold2

Wnt<b> </b>signaling is involved in embryo development and cancer. The binding events between the DIX domains of three proteins (Axin1/2, Dishevelled1/2/3, and Coiled-coil-DIX1) are essential to the mechanism of action downstream of the Wnt/b-catenin signaling pathway. Structural and biological studies have revealed that DIX domains are polymerized through head-to-tail interface interaction, which is indispensable for activating b-catenin Wnt signaling. There is growing evidence that DVL or AXIN isoforms display both redundant and specific functions in the Wnt signaling pathway. Due to technical challenges, however, the binding and specificity of DIX-mediated interactions still need to be discovered. We employ AlphaFold2(AF2) to predict six homodimers and 22 heterodimers of DIX domains without templates and compare them with the reported X-ray complex structures. The results indicate that AF2 predicts the complex structures of homodimers and heterodimers accurately, compared to the reported X-ray structures. PRODIGY is used to calculate the binding affinities of these DIX complexes. The results show that the binding affinity (K_D) of the Axin2 DIX(DAX2) homodimer is stronger than that of the Axin1 DIX(DAX1) homodimer. For Dishevelled (Dvl) proteins, Dvl1 DIX homodimer (DIX1-DIX1) shows higher binding comparable to Dvl2 DIX(DIX2) and Dvl3 DIX(DIX3) homodimers. For the positive Wnt signaling regulator Coiled-coil-DIX1(Ccd1), the K_D value of the Ccd1 DIX(DC1) homodimer is lower than that of the Axin1 DIX(DAX1) homodimer. Depending on the isoforms of DIX proteins, the heterodimer interactions are estimated to be stronger than the homodimer ones. Based on the binding affinities of DIX-driven interactions, we discuss the molecular mechanism of the Wnt signaling pathway. This study suggests that AF2-powered protein complex prediction and PRODIGY-powered binding affinity prediction could provide crucial insights into protein-protein interactions in signaling pathways.