Crystal structure of a transcriptionally active Smad4 fragment.
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.
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Department of Pharmacology and Molecular Toxicology, University of Massachusetts Medical School, Worcester 01655, USA.
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D000595:Amino Acid Sequence; D000818:Animals; D018360:Crystallography, X-Ray; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000378:metabolism*; D006801:Humans; D046911:Macromolecular Substances; D051379:Mice; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry* / Q000378:metabolism*; D020033:Protein Isoforms / Q000737:chemistry / Q000378:metabolism; D020836:Protein Structure, Quaternary; D017433:Protein Structure, Secondary; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D015398:Signal Transduction; D051901:Smad4 Protein; D012984:Software; D015534:Trans-Activators / Q000737:chemistry* / Q000378:metabolism*
D004268:DNA-Binding Proteins; D046911:Macromolecular Substances; D010446:Peptide Fragments; D020033:Protein Isoforms; D011994:Recombinant Proteins; C497796:SMAD4 protein, human; D051901:Smad4 Protein; C490810:Smad4 protein, mouse; C490811:Smad4 protein, rat; D015534:Trans-Activators