Structural insights into the binding of human TGIF1 with SMAD2 MH2 domain

Homeobox protein TGIF1 plays crucial roles in human development and body functions, partly by functioning as a corepressor in TGFβ signaling pathway. TGIF1 interacts with the MH2 domain of SMAD2 and is subsequently recruited to SMAD‐binding elements to repress TGFβ‐responsive gene expression. Here, through NMR titration, HDX‐MS, and AlphaFold3 modeling, we reveal that a vertebrate‐conserved short motif (I302‐L310) of TGIF1 binds to a groove on the surface of SMAD2‐MH2. The TGIF1‐binding sites of SMAD2 overlap with those for its coactivators. BiFC assays verified that α2‐β8 loop of SMAD2‐MH2 plays a key role in binding to TGIF1. This study provides structural insight into the mechanism by which TGIF1 acts as a corepressor of SMAD2, probably through competing with coactivators for binding.