Abstract
SMAD transcription factors, the main effectors of the TGFβ (transforming growth factor β) network, have a mixed architecture of globular domains and flexible linkers. Such a complicated architecture precluded the description of their full-length (FL) structure for many years. In this study, we unravel the structures of SMAD4 and SMAD2 proteins through an integrative approach combining Small-angle X-ray scattering, Nuclear Magnetic Resonance spectroscopy, X-ray, and computational modeling. We show that both proteins populate ensembles of conformations, with the globular domains tethered by disordered and flexible linkers, which defines a new dimension of regulation. The flexibility of the linkers facilitates DNA and protein binding and modulates the protein structure. Yet, SMAD4FL is monomeric, whereas SMAD2FL is in different monomer-dimer-trimer states, driven by interactions of the MH2 domains. Dimers are present regardless of the SMAD2FL activation state and concentration. Finally, we propose that SMAD2FL dimers are key building blocks for the quaternary structures of SMAD complexes.