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

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 monomers in equilibrium, whereas their Smad2/3/4 counterparts adopt monomeric 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 the 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 elucidate 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. Loop1 sequence determines the propensity of R-Smads to form monomers or dimers in complexes with DNA. Author Contributions L.R., Z.K., and T.G. designed and performed most experiments and coordinated collaborations with other authors. L.R. and E.A. cloned, expressed and purified all proteins, L.R., R.F., C.T., N.M., and J.C performed EMSA experiments. L.R., E.A., T.G., T.C., P.M.M., and M.J.M. performed the SAXS and NMR measurements and analyzed the data. P.M.M. analyzed the clustering of DNA motifs in ChIP-Seq data. Z.K. B.B. and R.P. screened crystallization conditions, collected X-ray data and determined the structures. Z.K., R.F. R.P., T.G., J.A.M., and M.J.M. analyzed the structures. All authors contributed ideas to the project. M.J.M. and R.P. supervised the project. M.J.M. wrote the manuscript with contributions from all other authors. The authors declare no conflict of interest. Data Deposition NMR assignments and chemical shifts have been deposited in the Biological Magnetic Resonance Data Bank, BMRB entry 27548, and the Small-angle scattering data and models have been deposited in SASBDB, entries SASDE32 (Smad5) and SASDE42 (Smad8). Densities and coordinates have been deposited in the Protein Data Bank, entries 6FZS (Smad5), 6FZT (Smad8), 6TBZ (Smad5\_3 chimera), 6TCE (Smad5\_gly mutant). This article contains supporting information. ### Competing Interest Statement The authors have declared no competing interest.