Study Of The Binding Mechanism Between The Tumor Suppressor DLG1 And APC

The tumor suppressor DLG1 (the human discs large 1) is a member of the membrane associated guanylate kinase (MAGUK) family, consisting of three PDZ domains, each PDZ is made up of ~90 amino acids, they mediate protein-protein interactions by specifically recognizing the extreme carboxy terminal residues of target proteins. The APC (adenomatous polyposis coli) is found and named in the study of the family adenomatous polyposis in recent years, whose C-terminal motif VTSV constitutes a PDZ recognition site and can interact with two of three PDZ domains of DLG1, PDZ1 and PDZ2. The DLG1/APC complex inhibits the cell cycle progression from the G0/G1 to the S phase, regulates epithelial cell migration and morphogenesis, and is required for polarization of the microtubule cytoskeleton. However, the molecular details of how DLG1 recognizes APC is not clear.In this study, we first constructed, expressed and purified recombinant proteins of PDZ1 and PDZ2, mixed with the APC protein C-terminal 11 peptide and crystallized, respectively. Fortunately, We successfully determined the crystal structures of PDZ1/APC-C11 complex and PDZ2/APC-C11 complex. which reveal the amino acid residues and forces involved in PDZ interactions with the APC. The results show that in addition to those canonical associations, there are more interactions observed betweenβ2/β3 loops of PDZ1 and APC residues upstream of the PDZ-binding S/T-X-V motif. Although we did not observe these non-canonical interactions in the PDZ2/APC-C11 complex structure, it is possible that the crystallization condition we used for producing crystals of the PDZ2/APC-C11 complex was not favorable for these interactions. Most of the residues in PDZ1 mediating non-canonical binding with APC-C11 are conserved in PDZ2. Therefore, it is reasonable to surmise that these non-canonical associations might also exist between PDZ2 and APC-C11.We then constructed a series of wild-type or PDZ mutant recombinant proteins and various C-terminal peptides of APC, combined with the GST pull-down assay as well as isothermal titration calorimetry (ITC) assay to analyze the interactions between them. The results indicate that APC binds to the first and the second PDZ domains of DLG1 with higher affinity for PDZ2, no appreciable binding to PDZ3 could be measured, the mutant of PDZ2 deleting theβ2/β3 loop or point mutation of Q340P make the interaction with APC-C11 weak or disappeared, and the pure exsit of the binding motif VTSV of APC carboxy terminal significantly reduced in binding affinity of PDZ. These results further indicate that theβ2/β3 loops of PDZ1 and PDZ2 play important roles in contributing to the binding affinity for the APC through interacting with APC residues upstream of the canonical PDZ-binding S/T-X-V motif. Our study thus provides molecular level informations, as well as new evidence and interpretation on the interaction between PDZ domains of DLG1 and the extreme carboxy terminal residues of APC protein.