SMAD4 nanobodies as tools for investigating SMAD structures and modulating SMAD function

In the canonical TGF-β signaling pathway, SMADs are transducers of receptor-activated signals. These proteins contain two well-structured domains (MH1 and MH2 domains) connected by a long, functional, unstructured linker. Beyond the physiological role of TGF-β signaling, its dysregulation is implicated in several pathologies, with many disease-causing mutations clustering within the SMAD4 MH2 domain. Here, we have discovered and structurally characterized (via X-ray crystallography) two nanobodies (Nbs) against SMAD4, with applications to study the effects of SMAD4 in TGF-β signaling. We found that NbA1 binds a region of SMAD4 MH2 domain that overlaps the R-SMAD interaction surface, preventing the formation of SMAD4/R-SMAD complexes, whereas NbA7 binding allows the formation of functional complexes. Applications of these two Nbs comprise detection of SMAD4 complexes, enabling study of the impact of disease-associated variants on complex formation, facilitating high-resolution cryo-EM structure determination, and opening avenues for therapeutic and diagnostic development.