Crystal structure of a transcriptionally active Smad4 fragment

Structure. 1999 Dec 15;7(12):1493-503. doi: 10.1016/s0969-2126(00)88340-9.


Background: Smad4 functions as a common mediator of transforming growth factor beta (TGF-beta) signaling by forming complexes with the phosphorylated state of pathway-restricted SMAD proteins that act in specific signaling pathways to activate transcription. SMAD proteins comprise two domains, the MH1 and MH2 domain, separated by a linker region. The transcriptional activity and synergistic effect of Smad4 require a stretch of proline-rich sequence, the SMAD-activation domain (SAD), located N-terminal of the MH2 domain. To understand how the SAD contributes to Smad4 function, the crystal structure of a fragment including the SAD and MH2 domain (S4AF) was determined.

Results: The structure of the S4AF trimer reveals novel features important for Smad4 function. A Smad4-specific sequence insertion within the MH2 domain interacts with the C-terminal tail to form a structural extension from the core. This extension (the TOWER) contains a solvent-accessible glutamine-rich helix. The SAD reinforces the TOWER and the structural core through interactions; two residues involved in these interactions are targets of tumorigenic mutation. The solvent-accessible proline residues of the SAD are located on the same face as the glutamine-rich helix of the TOWER, forming a potential transcription activation surface. A tandem sulfate-ion-binding site was identified within the subunit interface, which may interact with the phosphorylated C-terminal sequence of pathway-restricted SMAD proteins.

Conclusions: The structure suggests that the SAD provides transcriptional capability by reinforcing the structural core and coordinating with the TOWER to present the proline-rich and glutamine-rich surfaces for interaction with transcription partners. The sulfate-ion-binding sites are potential 'receptors' for the phosphorylated sequence of pathway-restricted SMAD proteins in forming a heteromeric complex. The structure thus provides a new model that can be tested using biochemical and cellular approaches.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Crystallography, X-Ray
  • DNA-Binding Proteins / chemistry*
  • DNA-Binding Proteins / metabolism*
  • Humans
  • Macromolecular Substances
  • Mice
  • Models, Molecular
  • Molecular Sequence Data
  • Peptide Fragments / chemistry*
  • Peptide Fragments / metabolism*
  • Protein Isoforms / chemistry
  • Protein Isoforms / metabolism
  • Protein Structure, Quaternary
  • Protein Structure, Secondary
  • Rats
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / metabolism
  • Sequence Alignment
  • Sequence Homology, Amino Acid
  • Signal Transduction
  • Smad4 Protein
  • Software
  • Trans-Activators / chemistry*
  • Trans-Activators / metabolism*


  • DNA-Binding Proteins
  • Macromolecular Substances
  • Peptide Fragments
  • Protein Isoforms
  • Recombinant Proteins
  • SMAD4 protein, human
  • Smad4 Protein
  • Smad4 protein, mouse
  • Smad4 protein, rat
  • Trans-Activators

Associated data

  • PDB/1DD1