R-Smads are effectors of the transforming growth factor β (TGFβ) superfamily and along with Smad4 form trimers to interact with other transcription factors and with DNA. The 5GC-DNA complexes determined here by X-ray crystallography for Smad5 and Smad8 proteins corroborate that all MH1 domains bind SBE and 5GC sites similarly, although Smad2/3/4 MH1 domains bind DNA as monomers whereas Smad1/5/8 form helix-swapped dimers. To examine the relevance of the dimerization phenomenon and to exclude a possible crystallography-induced dimeric state, we studied these MH1 domains in solution. The results show that Smad5/8 domains populate dimers and open monomers in equilibrium, whereas their Smad2/3/4 counterparts adopt monomeric closed conformations. We also found that swapping the loop1 sequence between Smad5 and Smad3 results in the Smad5 chimera-DNA complex crystallizing as a monomer, revealing that the loop1 sequence determines the monomer/dimer propensity of Smad MH1-domains. We propose that distinct MH1-dimerization status of TGFβ and BMP activated Smads influences the interaction with specific loci genome-wide by distinct R-Smad and Smad4 complexes.Significance TGFβ- and BMP-activated R-Smads were believed to have different preferences with respect to the recognition of DNA motifs and to respond to specific activation inputs. However, recent results indicate that several types of R-Smads can be activated by similar receptors and that all Smads might recognize various DNA motifs. These results pose new questions as to why different types of R-Smads have been conserved for more than 500 million years if they could have a redundant function. They also raise questions as to how different Smad complexes recognize specific clusters of DNA motifs genome-wide.Here, using structural biology approaches, we elucidated some of the rules that help define the dimers of Smad-DNA complexes and propose how dimers and monomers could influence the composition of Smad complexes as well as the recognition of specific cis -regulatory elements genome-wide.Highlights R-Smads and Smad4 interact with 5GC and GTCT sites using a conserved binding mode.Functional differences of TGFβ- and BMP-activated R-Smads are not exclusively related to DNA specificity.Dimer/monomer propensities are detected in solution and in the absence of DNA.Footnotes Supplemental information is available for this article.

Unveiling the dimer/monomer propensities of Smad MH1-DNA complexes