Structure–function relationship of inhibitory Smads: Structural flexibility contributes to functional divergence

Abstract

Smads are a small family of eukaryotic transcription regulators that play key roles in the transforming growth factor-beta signaling cascade. Smad6 and Smad7, the inhibitory or I-Smads, inhibit signaling downstream of TGF-beta type I receptors, thereby acting as negative regulators of signaling mediated by TGF-beta superfamily of ligands. Smad6 is known to specifically inhibit BMP type I receptor mediated signaling, while Smad7 is a more general inhibitor, able to block signaling mediated by a set of related TGF-beta type I receptors, including type I receptors for BMP and TGF-beta/Activin. In this study we have sought to understand the structural basis for this functional divergence of I-Smads. We have created homology-based models for the MH1 and MH2 domains of the two I-Smads and have carried out detailed molecular dynamics (MD) simulations of these proteins in explicit solvent to investigate the flexibility of the domains. The molecular models show that the I-Smads have lost many of the secondary structural elements found in the R-Smads, giving rise to longer loops in the tertiary structure of Smad6 and Smad7. Detailed analysis of the structural models and the MD trajectories clearly reveal that compared to Smad6, Smad7 has a more flexible overall folding, marked by the presence of highly flexible amino acid residues in functionally important regions of the protein. Interestingly, three of these residues-Phe411, Lys401, and Cys406, map to L3 loop of Smad7 MH2 domain, which is a critical structural determinant in Smad-type I receptor interactions. The increased structural flexibility of Smad7, arising out of longer, more flexible loops in its MH2 domain, might enable Smad7 to interact with a set of related yet structurally diverse type I receptors. Taken together with experimental evidence published in recent literature that hint at structural factors underlying the generic nature of inhibition by Smad7, our results strongly suggest that structural flexibility could be a prime contributor to the functional differences between Smad6 and Smad7. Additionally, we have been able to use the Smad7 structural model to successfully rationalize the results of in vitro site-specific mutagenesis experiments in published literature. This also provides biological validation for our model. Apart from this, analysis of the MH1 molcular model of Smad6 delineates a basic patch on the surface of the domain that might take part in nonspecific DNA binding by Smad6. This finding is consistent with earlier experimental data and is relevant since the characteristic beta-hairpin DNA binding element of R-Smads is completely absent in the I-Smads. Finally, the molecular models described here can serve to guide future biochemical and genetic studies on I-Smads.

Molecular modeling‐based evaluation of dual function of IκBζ ankyrin repeat domain in toll‐like receptor signaling

“…Among these, 13 residues (Met477, His481, Met482, Lys486, Cys521, Arg548, Asn550, Gln610, Glu644, Pro646, Ser647, Cys648, and Leu649) are thermodynamically flexible. Such thermodynamically flexible residues identified in smad7 and CYBP61 have been shown to interact with their receptor and substrate, respectively, (Li et al, 2004;Hariharan and Pillai, 2008), which further validates our docking model.…”

“…MD simulations were conducted to assess the structural flexibility as previously described (Hariharan and Pillai, 2008) using the AMBER 99 force field distributed in MOE starting from the energy minimized IkBz structure in each case. The MD simulation was conducted in explicit solvent and gas phase.…”

Emodin protects rat liver from CCl4-induced fibrogenesis via inhibition of hepatic stellate cells activation

“…Smads 6, 7, 8 are inhibitory factors of these Smads. When TGF-β1 binds to its receptor, Smad 2/3 is phosphorylated and binds with Smad4 and together they move into the nucleus for translation and expression of the target gene [43,44] . Smad signal transduction pathways are thought to play a crucial role in the process of liver damage and recovery, as well as liver fibrosis.…”

Mechanisms driving neural crest induction and migration in the zebrafish andXenopus laevis

“…In addition to BMP secretion, BMP signaling is controlled by secreted antagonists 7 as well as intracellular regulators. 8 The simple intermediate-gradient model of neural crest induction implies that neural crest induction occurs after neuroectodermal induction, but there is clear evidence that neural crest induction begins during gastrulation, concomitantly with neural induction. 9 …”