Study Of The Folding Characteristics Of Human Smad7 N-terminal Domain (NTD)

Smad7, a member of the class of inhibitory Smads, negatively regulates the TGF-βsignaling pathway by competing with receptor-regulated Smads for interaction with type I TGF-βreceptors, and by mediating the ubiquitination and degradation of TGF-βreceptors. The N-terminal domain (NTD) of Smad7 is critical for its function by bridging the E3 ubiquitin ligase Smurf2 and the E2 ubiquitin-conjugating enzyme UbcH7 together. However, we know little about the biochemical and structural properties of Smad7-NTD.In this study, we analyzed the folding of human Smad7-NTD by folding propensity prediction, secondary structure prediction, and sequence alignment. In addition, we made different constructs of Smad7-NTD with various deletions of residues from the N-terminus or from an internal loop of Smad7, and analyzed the biochemical properties of these Smad7-NTD deletion constructs. Our results revealed that the N-terminal ~20 residues of Smad7-NTD was critical for its solubility, suggesting that it played an important role in the folding of Smad7-NTD domain. Furthermore, our bioinformatic and biochemical results indicated that there was a large intrinsically unfolded loop in Smad7-NTD, which caused it to be unstable and susceptible for proteolytic degradation. Removal of this loop remarkably improved the stability and monomericity of Smad7-NTD domain protein, which would be suitable for future crystallographic studies.We also analyzed the predicted folding properties of Smad6 which is highly homologous to Smad7, and found that they might share the same folding properties. In addition, we purified and made initial crystallization trials on the Smad2-MH1 domain, which is highly homologous to other R-Smad-MH1 but has different functions.