Structure of Smad1 MH1/DNA complex reveals distinctive rearrangements of BMP and TGF-β effectors

Abstract

Smad1 is a downstream effector of the BMP signaling pathway that binds regulatory DNA to execute gene expression programs leading to, for example, the maintenance of pluripotency in mice. On the contrary, the TGF-β-activated Smad3 triggers strikingly different programs such as mesodermal differentiation in early development. Because Smad1 and Smad3 contain identical amino acids at the DNA contact interface it is unclear how they elicit distinctive bioactivities. Here, we report the crystal structure of the MH1 domain of Smad1 bound to a palindromic Smad binding element. Surprisingly, the DNA contact interface of Smad1 is drastically rearranged when compared to Smad3. The N-terminal helix 1 of Smad1 is dislodged from its intramolecular binding site and adopts a domain swapped arrangement with a symmetry-related molecule. As a consequence, helix 2 kinks away from the double helix disabling several key phosphate backbone interactions. Thermal melting analysis corroborates a decompacted conformation of Smad1 and DNA binding assays indicate a lower overall affinity of Smad1 to DNA but increased cooperativity when binding to palindromic DNA motifs. These findings suggest that Smad1 and Smad3 evolved differential qualities to assemble on composite DNA elements and to engage in co-factor interactions by remodeling their N-termini.

Changing POU dimerization preferences converts Oct6 into a pluripotency inducer

“…Next, we extended the analysis and included a total of six TFs belonging to POU classes I, II, III, V, and VI (Figs 1E and F, and EV1B). We quantified the cooperativity of the homodimeric Oct–Oct and heterodimeric Sox–Oct binding of the POU proteins to the MORE ( OctOct ) and SoxOct elements using previously derived equations 27, 40. Cooperativity represents the ratio of the equilibrium binding constant for the binding of a factor to free DNA to the binding of DNA in the presence of another protein (Sox2 for heterodimers and another POU factor for homodimers).…”

“…Cooperativity factors (ω) were calculated after quantifying the fraction of bound DNA in EMSAs using the below equations. Equations were derived using Boltzmann weights and principles of statistical mechanisms as detailed in 27, 40.…”

Transcriptional Control by the SMADs

“…In all cases, an 11-residue b-hairpin was shown to be responsible for DNA sequence recognition, and is embedded in the major groove of DNA ( Fig. 1C -E) (Shi et al 1998;Baburajendran et al 2010Baburajendran et al , 2011. The amino acids responsible for specific contacts with the bases are conserved in all three MH1 domains ( Fig.…”

“…The crystal structure of the SMAD1 MH1 domain revealed that the DNA contact interface of SMAD1 is rearranged compared with that of SMAD3, and, as a consequence, several key DNA backbone interactions are inhibited (Baburajendran et al 2010). In the SMAD1 MH1 domain, the amino-terminal a1 helix is dislodged from the intramolecular binding site seen in the SMAD3 MH1 domain (Fig.…”

“…This results in a decreased affinity for DNA, but additional cooperativity when binding to palindromic DNA motifs (Baburajendran et al 2010). Interestingly, isolated MH1 domains of SMAD1 can bind to GC-rich compressed palindromes, such as GGCGCC, as efficiently as they do to GTCT palindromes, suggesting that the preferential binding of SMAD1/5-SMAD4 complexes to the GC-rich motifs in vivo is not determined by DNA selectivity of the MH1 domains per se (Baburajendran et al 2010). What exactly determines the DNA-binding specificity of different types of SMAD complexes (SMAD1/5-SMAD4 versus SMAD3 -SMAD4 complexes) in vivo is still not fully understood, but the cooperative nature of the SMAD1 MH1 DNA binding may be crucial.…”