SnoN Stabilizes the SMAD3/SMAD4 Protein Complex

Abstract

TGF-β signaling regulates cellular processes such as proliferation, differentiation and apoptosis through activation of SMAD transcription factors that are in turn modulated by members of the Ski-SnoN family. In this process, Ski has been shown to negatively modulate TGF-β signaling by disrupting active R-SMAD/Co-SMAD heteromers. Here, we show that the related regulator SnoN forms a stable complex with the R-SMAD (SMAD3) and the Co-SMAD (SMAD4). To rationalize this stabilization at the molecular level, we determined the crystal structure of a complex between the SAND domain of SnoN and the MH2-domain of SMAD4. This structure shows a binding mode that is compatible with simultaneous coordination of R-SMADs. Our results show that SnoN, and SMAD heteromers can form a joint structural core for the binding of other transcription modulators. The results are of fundamental importance for our understanding of the molecular mechanisms behind the modulation of TGF-β signaling.

Figure 1. Analysis of SnoN/SMAD Interactions.

Affinity purification of a complex including truncated forms of untagged SnoN (M1-S356, 38.6 kDa), Strep II tagged SMAD3 (MH2 domain (S423E, S425D), 33.6 kDa) and His tagged SMAD4 (MH2 domain, 28.3 kDa) (A–C). From left, SnoN-^StrepSMAD3-^HisSMAD4 input; sample not binding to the Strep-Tactin column (Strep flow-through); Strep-Tactin eluate (Strep eluate); Strep-Tactin elute was loaded directly onto the Ni-NTA column; any proteins not binding to the Ni-NTA column (Ni-NTA flow-through/wash); and Ni-NTA eluate containing the isolated complex of SMAD3, SMAD4 and SnoN. Integrity and identity of SMAD4 was confirmed by MALDI-MS/MS mass spectrometry analysis of trypsinated protein extracted from the gel (Supplemental information). (A) Coomassie stained gel. (B) Western blot analysis using anti-hexahistidine tag antibody recognizing ^HisSMAD4. The lowest band corresponds to SMAD4, as confirmed by mass spectrometry. (C) Western-blot analysis using anti-Strep II tag antibody recognizing ^StrepSMAD3. (D) Affinity purification as in (A–C) but excluding SnoN. (E) Stoichiometry of SnoN-^StrepSMAD3-^HisSMAD4 complex assessed by fluorescence labeling of cysteines in the NiNTA eluate seen in A by a maleimide derivative of IRDye 800CW (LI-COR Biotechnology GmbH). Error bars are for the standard deviation of three individual measurements, see Supplementary information. SeeBlue^® Plus2 Pre-stained Protein Standard (Invitrogen) and Mark12 unstained standard (Thermo Fisher Scientific) were used for (A–C and D), respectively, with relevant molecular weights indicated to the left of each gel. IgG antibody conjugated to alkaline phosphatase was used for blots in (B and C). Please see the experimental procedures section for further details.

Figure 2. Overall Structure of SnoN-SMAD4.

(A) Content of asymmetric unit, with SnoN-SMAD4 as trimers of heterodimers, with SMAD4 (grey) mediating most of the trimer contacts, one open (beige) and two SnoN molecules in closed conformation (cyan). Bound Zn ions are indicated in brown and blue for open and closed conformation SnoN, respectively. (B) Open and closed conformations of SnoN with the interacting SMAD4s superimposed. (C) Close-up of open-conformation SnoN. (D) Close-up of closed-conformation SnoN. Secondary structure elements are indicated as α-helix (α) and β-strand (β) with numbers increasing from the N-terminal to the C-terminal. Color codes as in (A).

Figure 3. Interactions Between SnoN and SMAD4.

I(A) Open conformation interactions between SnoN (beige) and SMAD4 (grey). (B) Closed conformation interactions between SnoN (cyan) and SMAD4 (grey). (C) Additional interactions between SnoN (cyan) and SMAD4 (grey), which are part of SnoN-SMAD4 interactions in the closed conformations only. (D) Open conformation of SnoN. Residues involved in SMAD4 interactions in SnoN, according to the buried surface model as implemented in PISA, are colored according to level of evolutionary conservation, going from fully conserved (violet-red) to no conservation (blue-green), calculated using the Consurf server (http://consurf.tau.ac.il/). (E) ‘Closed’ conformation of SnoN. Same coloring as in (D).

Figure 4. Surface plasmon resonance-based analysis of SMAD4-binding to SnoN.

Corrected response of various concentrations of SnoN on SMAD4. Sensorgrams with curve fittings (black continuous line) for each titration experiment are shown. SnoN concentrations used for each titration/sensorgram are indicated.

Figure 5. SnoN-SMAD4 Superimposed on the SMAD3-SMAD4 Heterotrimer Structure.

(A) Open conformation of SnoN (beige) superimposed based on the interacting SMAD4 with the structure of SMAD3-SMAD4 (green-grey) (PDB code 1U7F34) (B) Close-up of (A). Glu267 is shown in both the conformation found in our complex structure and in its most preferred rotamer (50% transparent), which is compatible with the binding of an activated SMAD3-SMAD4 heteromer. (C) Closed conformation of SnoN (cyan) superimposed based on the interacting SMAD4 in the structure of SMAD3-SMAD4 (green-grey). (D) Close-up of (C). Phosphoserine residues of SMAD3 indicated as pSer423 and pSer425, respectively.