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A structural basis for mutational inactivation of the tumour suppressor Smad4

Abstract

The Smad4/DPC4 tumour suppressor1 is inactivated in nearly half of pancreatic carcinomas2 and to a lesser extent in a variety of other cancers2,3,4. Smad4/DPC4, and the related tumour suppressor Smad2, belong to the SMAD family of proteins that mediate signalling by the TGF-β/activin/BMP-2/4 cytokine superfamily from receptor Ser/Thr protein kinases at the cell surface to the nucleus5,6,7. SMAD proteins, which are phosphorylated by the activated receptor, propagate the signal, in part, through homo- and hetero-oligomeric interactions8,9,10,11,12,13. Smad4/DPC4 plays a central role as it is the shared hetero-oligomerization partner of the other SMADs. The conserved carboxy-terminal domains of SMADs are sufficient for inducing most of the ligand-specific effects, and are the primary targets of tumorigenic inactivation. We now describe the crystal structure of the C-terminal domain (CTD) of the Smad4/DPC4 tumour suppressor, determined at 2.5 Å resolution. The structure reveals that the Smad4/DPC4 CTD forms a crystallographic trimer through a conserved protein–protein interface, to which the majority of the tumour-derived missense mutations map. These mutations disrupt homo-oligomerization in vitro and in vivo, indicating that the trimeric assembly of the Smad4/DPC4 CTD is critical for signalling and is disrupted by tumorigenic mutations.

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Figure 1: The structure of the Smad4/DPC4 CTD consists of a β-sandwich with a three-helix bundle on one end and a collection of three large loops and an α-helix on the other.
Figure 2: SMAD C-terminal domains are highly conserved and are targeted by tumorigenic and developmental mutations.
Figure 3: In the crystals, the Smad4/DPC4 CTD forms a trimer that is disrupted by tumorigenic mutations and is probably important for SMAD function.
Figure 4: Size-exclusion chromatography showing that wild-type full-length Smad4/DPC4, but not tumour-derived mutants, has an apparent Mrconsistent with that of a trimer.
Figure 5: One face of the disk-like trimer structure may mediate hetero-oligomerization.

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Acknowledgements

This work was supported by the NIH, the Pew Charitable Trusts, the Arnold and Mabel Beckman Foundation, the Dewitt Wallace Foundation, the Samuel and May Rudin Foundation, and the Howard Hughes Medical Institute.

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Correspondence to Nikola P. Pavletich.

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Shi, Y., Hata, A., Lo, R. et al. A structural basis for mutational inactivation of the tumour suppressor Smad4. Nature 388, 87–93 (1997). https://doi.org/10.1038/40431

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