Structural Basis of Heteromeric Smad Protein Assembly in TGF-β Signaling

The formation of protein complexes between phosphorylated R-Smads and Smad4 is a central event in the TGF-β signaling pathway. We have determined the crystal structure of two R-Smad/Smad4 complexes, Smad3/Smad4 to 2.5 Å, and Smad2/Smad4 to 2.7 Å. Both complexes are heterotrimers, comprising two phosphorylated R-Smad subunits and one Smad4 subunit, a finding that was corroborated by isothermal titration calorimetry and mutational studies. Preferential formation of the R-Smad/Smad4 heterotrimer over the R-Smad homotrimer is largely enthalpy driven, contributed by the unique presence of strong electrostatic interactions within the heterotrimeric interfaces. The study supports a common mechanism of Smad protein assembly in TGF-β superfamily signaling.