Characterizing two nanobodies for SMAD4

In metazoans, TGF-β signaling regulates essential cellular processes via the SMAD proteins. Many biophysical and cellular assays of SMAD-driven signaling require molecular tools that can recognize them in their native folded state. In this context, we report two nanobodies (Nbs), NbS4A1 and NbS4A7, which target the 3D structure of the human SMAD4-MH2 domain with high affinity and specificity. X-ray structures of the Nb-SMAD4 complexes reveal that each nanobody recognizes the SMAD4-MH2 domain through distinct but overlapping sites, precluding their simultaneous binding. Furthermore, NbS4A1 impedes SMAD4 binding with R-SMADs while NbS4A7 recognizes SMAD4 even when bound to R-SMADs. These distinct interaction modes will enable the study of folded SMAD complexes with cofactors and/or DNA in different experimental contexts.