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W3123265702 abstract "Article Figures and data Abstract Introduction Results Discussion Materials and methods Appendix 1 Data availability References Decision letter Author response Article and author information Metrics Abstract Shprintzen–Goldberg syndrome (SGS) is a multisystemic connective tissue disorder, with considerable clinical overlap with Marfan and Loeys–Dietz syndromes. These syndromes have commonly been associated with enhanced TGF-β signaling. In SGS patients, heterozygous point mutations have been mapped to the transcriptional co-repressor SKI, which is a negative regulator of TGF-β signaling that is rapidly degraded upon ligand stimulation. The molecular consequences of these mutations, however, are not understood. Here we use a combination of structural biology, genome editing, and biochemistry to show that SGS mutations in SKI abolish its binding to phosphorylated SMAD2 and SMAD3. This results in stabilization of SKI and consequently attenuation of TGF-β responses, both in knockin cells expressing an SGS mutation and in fibroblasts from SGS patients. Thus, we reveal that SGS is associated with an attenuation of TGF-β-induced transcriptional responses, and not enhancement, which has important implications for other Marfan-related syndromes. Introduction Shprintzen–Goldberg syndrome (SGS) is a multisystemic connective tissue disorder. Common features observed in SGS patients include craniofacial, skeletal, and cardiovascular anomalies, ranging from heart valve defects to thoracic aortic aneurysms, all of which are also characteristic of Marfan syndrome (MFS) and Loeys–Dietz syndrome (LDS) (Cook et al., 2015a; Verstraeten et al., 2016; Loeys et al., 2005; Williams et al., 2007). In addition, SGS patients present with craniosynostosis, intellectual disability, and skeletal muscle hypotonia (Shprintzen and Goldberg, 1982; Glesby and Pyeritz, 1989; Greally et al., 1998). All three syndromes have been linked to deregulation of the transforming growth factor β (TGF-β) signaling pathway (Cannaerts et al., 2015). The TGF-β family of ligands comprises the TGF-βs themselves, Activins, Nodal, bone morphogenetic proteins (BMPs), and growth differentiation factors (GDFs) and they play pleiotropic roles in embryonic development and tissue homeostasis. In addition, their signaling is deregulated in diverse pathologies (Miller and Hill, 2016). They exert their action by binding to type I and type II serine/threonine kinase receptors at the cell surface (TGFBR1 and TGFBR2, respectively, for the TGF-βs) (Massagué, 2012). In the resulting ligand-bound heterotetrameric receptor complex, the type II receptor phosphorylates and activates the type I receptor, which in turn phosphorylates the intracellular mediators, the receptor-regulated SMADs (R-SMADs). Once phosphorylated, the R-SMADs (SMAD2 and SMAD3 in the case of TGF-β, Activin, and Nodal) associate with the common mediator of the pathway, SMAD4. The resulting heterotrimeric complexes accumulate in the nucleus where they interact with other transcriptional regulators to activate or repress target gene expression (Massagué, 2012). Two highly related co-repressors, SKI and SKIL (formerly known as SnoN), act as negative regulators in the pathway (Deheuninck and Luo, 2009; see below). The role of deregulated TGF-β signaling in Marfan-related syndromes is controversial. MFS is caused by loss-of-function mutations in the extracellular matrix protein, Fibrillin 1 (FBN1) (Dietz et al., 1991). These mutations are thought to increase the bioavailability of TGF-β ligands, as FBN1 binds the latent form of the TGF-βs (Neptune et al., 2003; Kaartinen and Warburton, 2003). Supporting the idea that excessive TGF-β signaling contributes to the manifestations of MFS, a TGF-β neutralizing antibody significantly improved the lung phenotype in a mouse model of MFS (homozygous Fbn1mgΔ) (Neptune et al., 2003; Cannaerts et al., 2015) and reduced the occurrence of aortic aneurysms in the Fbn1C1039G/+ mouse model of MFS (Habashi et al., 2006). Contradicting these results, others have shown that the aortopathy in the Fbn1C1039G/+ mouse model is not mediated by excessive TGF-β signaling and in fact is exacerbated by loss of TGF-β signaling in smooth muscle cells (Wei et al., 2017). Furthermore, TGF-β signaling protects against abdominal aortic aneurysms in angiotensin II-infused mice (Angelov et al., 2017). This controversy emphasizes the importance of understanding exactly how TGF-β signaling is impacted in MFS. Furthermore, the related syndrome LDS is caused by pathogenic mutations in several different components of the TGF-β pathway, TGFBR1, TGFBR2, SMAD2, SMAD3, and the ligands, TGFB2 and TGFB3. These mutations all cause missense amino acid substitutions that have been either verified in vitro, or are predicted to be loss of function, implying that LDS is caused by attenuated TGF-β signaling (Horbelt et al., 2010; Cardoso et al., 2012; Schepers et al., 2018). However, paradoxically, histological and biochemical studies of aortic tissue derived from LDS patients reveal an apparent high TGF-β signaling signature (van de Laar et al., 2012; Gallo et al., 2014; Lindsay et al., 2012). SGS is caused by mutations in SKI, and both SMAD-mediated and non-SMAD-mediated TGF-β signaling has been reported to be increased in primary dermal fibroblasts from SGS patients (Doyle et al., 2012). The co-repressors SKI and SKIL play important roles in a number of different cellular processes including proliferation, differentiation, transformation, and tumor progression (Bonnon and Atanasoski, 2012). They are dimeric proteins that interact with both phosphorylated SMAD2 and SMAD3 (PSMAD2 or PSMAD3) via short motifs at their N-termini, and with SMAD4 via a SAND domain (named after Sp100, AIRE-1, NucP41/75, DEAF-1) in the middle of both proteins (Deheuninck and Luo, 2009). Between these two domains lies a Dachshund homology domain (DHD), which is thought to also be important for R-SMAD binding (Wilson et al., 2004; Ueki and Hayman, 2003). SKI and SKIL both contain a leucine zipper domain in their C-termini, through which they dimerize (Deheuninck and Luo, 2009). They are negative regulators of TGF-β/Activin signaling, with two distinct mechanisms of regulation having been proposed. In one model, SKI and SKIL bind with SMAD4 to SMAD binding elements (SBEs) of TGF-β/Activin target genes, and recruit co-repressors such as NCOR1 or SIN3A (Tokitou et al., 1999; Nomura et al., 1999; Stroschein et al., 1999; Deheuninck and Luo, 2009). They thus maintain the transcription of these target genes suppressed in the absence of signal. Upon TGF-β/Activin signaling, SKI and SKIL are rapidly degraded by the E3 ubiquitin ligase, RNF111 (formerly known as Arkadia), a process that requires SKI/SKIL binding to PSMAD2 or PSMAD3 (Le Scolan et al., 2008; Levy et al., 2007; Nagano et al., 2007). This then allows the activated SMAD3–SMAD4 complexes to bind the exposed SBEs and activate target gene transcription (Levy et al., 2007; Stroschein et al., 1999). In the competing model, SKI and SKIL act as repressors of active signaling simply by binding to PSMAD2 or PSMAD3 and SMAD4 in such a way as to disrupt the activated PSMAD2/PSMAD3–SMAD4 complexes (Luo, 2004; Ueki and Hayman, 2003; Wu et al., 2002). The heterozygous missense mutations that cause SGS have been mapped in SKI to the N-terminal R-SMAD-binding domain, with some small deletions and point mutations also found in the DHD, which is also necessary for R-SMAD binding (Carmignac et al., 2012; Doyle et al., 2012; Schepers et al., 2015). Thus, depending on the mechanism whereby SKI inhibits TGF-β/Activin signaling, loss of the interaction with PSMAD2/PSMAD3 would be predicted to have opposite effects on signaling output. If the PSMAD2/PSMAD3 interaction is required for SKI degradation, its loss would inhibit TGF-β signaling. However, if SKI binding to PSMAD2/PSMAD3 disrupts active SMAD complexes, then its loss would promote TGF-β signaling. Here we use a combination of genome editing, structural biology, biochemistry, and analysis of patient samples to elucidate the molecular mechanism underlying SGS and to resolve the paradox surrounding the role of TGF-β signaling in Marfan-related syndromes. We first determine at the molecular level how SKI/SKIL function in the TGF-β/Activin signaling pathways and show that an intact ternary phosphorylated R-SMAD–SMAD4 complex is required for ligand-induced SKI/SKIL degradation. We demonstrate that the SGS mutations in SKI abolish interaction with PSMAD2 and PSMAD3 and this results in an inability of SKI to be degraded in response to TGF-β/Activin signaling. We go on to show that SKI stabilization results in an attenuation of the TGF-β transcriptional response in both knockin HEK293T cells and fibroblasts from SGS patients. Our work unequivocally establishes that SGS mutations lead to an attenuated TGF-β response, which has major implications for all the Marfan-related syndromes. Results A PSMAD2/3–SMAD4 ternary complex is essential for TGF-β/Activin-induced degradation of SKI/SKIL To understand the consequences of SKI mutations in SGS and to resolve the paradox surrounding the function of TGF-β signaling in Marfan-related syndromes, we first set out to determine exactly how SKI and SKIL act as negative regulators of TGF-β and Activin signaling. We and others have previously demonstrated that SKI and SKIL are rapidly degraded upon TGF-β/Activin stimulation by the E3 ubiquitin ligase RNF111, and this requires PSMAD2 or PSMAD3 (Le Scolan et al., 2008; Levy et al., 2007; Nagano et al., 2007). Knockdown experiments suggested that SMAD4 was not necessary (Levy et al., 2007), but we subsequently showed that tumor cells deleted for SMAD4 or containing mutations in SMAD4 that abolish interactions with activated R-SMADs, abrogated TGF-β-induced degradation of SKI/SKIL (Briones-Orta et al., 2013). Whether the requirement for SMAD4 was direct or indirect was not clear. To define the role of SMAD4 in SKI/SKIL degradation, we used CRISPR/Cas9 technology to delete SMAD4 in transformed embryonic kidney cells HEK293T, which express both SKI and SKIL, and in the human keratinocyte cell line, HaCaT, which predominantly express SKIL (Levy et al., 2007; Figure 1—source data 1). In wild-type (WT) cells, TGF-β/Activin induced rapid SKI and SKIL degradation, compared to cells treated with the TGFBR1 inhibitor, SB-431542 (Inman et al., 2002; Figure 1A,B). Deletion of SMAD4 in multiple clones of both cell types abolished ligand-induced SKI/SKIL degradation (Figure 1A,B). We validated these SMAD4-null cell lines by demonstrating that transient expression of SMAD4 could rescue TGF-β/Activin-induction of the SMAD3–SMAD4 reporter, CAGA12-Luciferase (Figure 1—figure supplement 1A,B). Furthermore, we could show that loss of SMAD4 inhibited the ligand-induced expression of a number of endogenous TGF-β and BMP target genes (Figure 1—figure supplement 1C). By knocking out SMAD2 or SMAD3 individually or together, we also confirmed that these R-SMADs are absolutely required for TGF-β/Activin-induced degradation of SKI and SKIL and act redundantly (Figure 1C; Figure 1—source data 1). Thus, R-SMADs and SMAD4 are all essential for TGF-β/Activin-dependent SKI/SKIL degradation. Figure 1 with 1 supplement see all Download asset Open asset Requirement of SMAD2 or SMAD3 and SMAD4 for SKI and SKIL degradation. (A and C) The parental HEK293T cell line and two individual SMAD4 knockout clones (A) or two individual SMAD2, SMAD3 knockout clones, or two SMAD2 and SMAD3 double knockout clones (C) were incubated overnight with 10 μM SB-431542, washed out, then incubated with full media containing either SB-431542 or 20 ng/ml Activin A for 1 hr, as indicated. Whole-cell extracts were immunoblotted with the antibodies indicated. (B) Parental HaCaT and four individual SMAD4 knockout clones were treated as above, except that they were treated with 2 ng/ml TGF-β for 1 hr instead of Activin A. Nuclear lysates were immunoblotted using the antibodies indicated. SB, SB-431542; A, Activin A; T, TGF-β; S2, SMAD2; S3, SMAD3; S2/3, SMAD2 and SMAD3; S4, SMAD4; KO, knockout; dKO, double knockout. Figure 1—source data 1 Sequences of knockout alleles made in HEK293T cells. https://cdn.elifesciences.org/articles/63545/elife-63545-fig1-data1-v2.docx Download elife-63545-fig1-data1-v2.docx In addition to forming a ternary complex with PSMAD2 or PSMAD3, SMAD4 has also been shown to interact directly with SKI and SKIL through their SAND domains (Walldén et al., 2017; Wu et al., 2002). To determine which of these SMAD4 interactions were important for TGF-β/Activin-induced SKI/SKIL degradation, we stably reintroduced enhanced GFP (EGFP) fusions of WT or mutated SMAD4 into HaCaT SMAD4-null cells. We selected two missense mutations on opposite faces of the C-terminal Mad homology 2 (MH2) domain of SMAD4: Asp351->His (D351H) and Asp537-->Tyr (D537Y) (Shi et al., 1997). These have been shown to occur naturally in the human colorectal cancer cell lines CACO-2 and SW948, and have lost the ability to bind phosphorylated R-SMADs (De Bosscher et al., 2004). In addition, we used the crystal structure of the MH2 domain of SMAD4 and the SAND domain of SKI, to design two mutations Ala433-->Glu (A433E) and Ile435-->Tyr (I435Y), that would be expected to abolish SMAD4 binding to SKI and SKIL (Wu et al., 2002). We confirmed that these SMAD4 mutants behaved as expected in the rescue cell lines by testing their interaction with SKIL and R-SMADs by immunoprecipitation. As endogenous RNF111 triggers SKIL degradation in TGF-β/Activin-dependent manner, the stable SMAD4-expressing HaCaT rescue cell lines were incubated with the proteasome inhibitor, MG-132 for 3 hr prior to TGF-β stimulation, to block SKIL degradation. As predicted, the D351H and D537Y SMAD4 mutants had lost their ability to bind SMAD2 upon TGF-β induction, but retained the interaction with SKIL. By contrast, A433E and I435Y SMAD4 mutants were unable to bind SKIL, but could interact with SMAD2 upon TGF-β stimulation (Figure 2A). Furthermore, as expected, D351H and D537Y SMAD4 mutants failed to rescue the ability of TGF-β to induce expression of CAGA12-Luciferase in HaCaT SMAD4-null cells or rescue TGF-β-induced transcription of target genes, but the A433E and I435Y SMAD4 mutants rescued these responses almost as well as WT SMAD4 (Figure 2—figure supplement 1A,B). Figure 2 with 1 supplement see all Download asset Open asset Characterization of the role of SMAD4 in TGF-β-induced SKIL degradation. (A–C) HaCaT SMAD4 knockout (S4 KO) cells were stably transfected with EGFP alone, or EGFP SMAD4 (WT) or with four different EGFP-SMAD4 mutants (D351H, D537Y, which abolish interaction with the R-SMADs, and A433E and I435Y, which do not interact with SKIL). (A) Cells were incubated overnight with 10 µM SB-431542, washed out and pre-incubated with 25 μM MG-132 for 3 hr, and then treated either with 10 μM SB-431542 or 2 ng/ml TGF-β for 1 hr. Whole-cell extracts were immunoprecipitated (IP) with GFP-trap agarose beads. The IPs were immunoblotted using the antibodies shown. Inputs are shown below. (B) Nuclear lysates were prepared from the HaCaT S4 KO cells stably transfected with EGFP alone or with EGFP-SMAD4 constructs as indicated, treated as in (A), but without the MG-132 step and immunoblotted using the antibodies shown. On the right the quantifications are the normalized average ± SEM of five independent experiments. The quantifications are expressed as fold changes relative to SB-431542-treated S4 KO cells. (C) Levels of SKIL in the EGFP-positive S4 KO rescue cell lines treated as in (B), assayed by flow cytometry. Each panel shows an overlay of the indicated treatment conditions. The red line indicates the SB-431542-treated sample, whereas the cyan line indicates the TGF-β-treated sample. Quantifications are shown bottom right. For each group, the percentage of the median fluorescence intensity normalized to the SB-431542-treated sample is quantified. Data are the mean ± SEM of five independent experiments. The p-values are from one-way ANOVA with Sidak’s post hoc correction *p<0.05; ****p<0.0001. SB, SB-431542; T, TGF-β. Figure 2—source data 1 Quantification of Western blot for HaCaT S4 KO rescue cell lines, as presented in Figure 2B. https://cdn.elifesciences.org/articles/63545/elife-63545-fig2-data1-v2.xlsx Download elife-63545-fig2-data1-v2.xlsx Figure 2—source data 2 Flow cytometry data for HaCaT S4 KO rescue cell lines, as presented in Figure 2C. https://cdn.elifesciences.org/articles/63545/elife-63545-fig2-data2-v2.xlsx Download elife-63545-fig2-data2-v2.xlsx Having demonstrated that these mutants behaved as designed, we asked which were able to mediate TGF-β-induced SKIL degradation, using three different assays. In a Western blot assay using nuclear extract, we found that reintroduction of WT SMAD4 in SMAD4-null cells caused a 50% reduction in SKIL levels in TGF-β-induced cells compared to those treated with SB-431542 (Figure 2B). However, none of the four SMAD4 mutants could rescue TGF-β-induced SKIL degradation (Figure 2B). We then established a flow cytometry assay to quantify SKIL protein stability in EGFP/EGFP-SMAD4-expressing cells (Figure 2C; Figure 2—figure supplement 1C). Treatment with TGF-β for 1 hr caused a 52% reduction in the relative median fluorescence intensity in the EGFP-SMAD4 WT-expressing cells, reflecting SKIL levels, compared to cells treated with SB-431542 (Figure 2C). However, for all four SMAD4 mutants tested, the median fluorescence was not decreased by TGF-β treatment (Figure 2C). Finally, we used an immunofluorescence analysis to monitor SKIL protein stability following TGF-β exposure. SMAD4-null cells showed strong nuclear staining of SKIL in the non-signaling condition (SB-431542), which remained unchanged by TGF-β treatment (Figure 3). Reintroduction of WT EGFP-SMAD4 conferred the ability to degrade SKIL upon TGF-β treatment, whereas none of the mutant SMAD4s were able to rescue SKIL degradation (Figure 3, arrows). Thus, all three assays demonstrate that a ternary R-SMAD–SMAD4 complex is absolutely necessary for TGF-β-induced SKIL degradation, as is the ability of SMAD4 to interact with SKIL itself. This suggests that within a canonical activated ternary SMAD complex, the R-SMAD component binds to the N-terminal region of SKIL/SKI, whilst SMAD4 binds the SAND domain, and both interactions are absolutely required for SKIL/SKI degradation. Figure 3 Download asset Open asset Visualization of TGF-β-induced SKIL degradation. HaCaT SMAD4 knockout (S4 KO) cells or those stably expressing EGFP SMAD4 WT or EGFP SMAD4 mutants were incubated overnight with 10 μM SB-431542, washed out, and incubated for 1 hr with 10 µM SB-431542 or with 2 ng/ml TGF-β. Cells were fixed and stained for EGFP (for SMAD4), SKIL, and with DAPI (blue) to mark nuclei and imaged by confocal microscopy. The merge combines SKIL, SMAD4, and DAPI staining. Arrows indicate examples of EGFP-expressing cells and corresponding levels of nuclear SKIL. Scale bar corresponds to 50 µm. SGS mutations inhibit the interaction of SKI with phosphorylated R-SMADs We next investigated the consequences of the SGS mutations on SKI and SKIL’s ability to interact with the R-SMADs. SKI and SKIL share a highly conserved region at their N-terminus comprising the domain known to be important for R-SMAD binding (Deheuninck and Luo, 2009; Figure 4—figure supplement 1A). We first determined the minimal region of SKI required for R-SMAD binding using peptide pulldown assays with biotinylated SKI peptides and whole-cell extract from uninduced and TGF-β-treated HaCaT cells. This revealed that amino acids 11–45 of SKI are sufficient for binding to PSMAD2 and PSMAD3 upon TGF-β stimulation, whilst the unphosphorylated SMADs did not bind to any of the SKI peptides (Figure 4—figure supplement 1B). SMAD4 is also pulled down in these assays in a ligand-induced manner, by virtue of its interaction with the phosphorylated R-SMADs. The SGS mutations discovered so far mostly cluster within this 11–45 region of SKI and a few deletions and point mutations have additionally been mapped in the DHD domain (Carmignac et al., 2012; Doyle et al., 2012; Schepers et al., 2015). The residues mutated are completely conserved, both between species, and also in the related protein, SKIL (Figure 4—figure supplement 1A; Carmignac et al., 2012). To determine the effect of these mutations on R-SMAD interaction, we introduced six different SGS mutations into the SKI peptide 11–45 and showed that they all prevented binding of PSMAD2 and PSMAD3, and as a result, also SMAD4 (Figure 4A). These results were also confirmed with the equivalent mutations in SKIL (Figure 4B). We proved that the interaction with SMAD2 was mediated via its MH2 domain using a mouse embryonic fibroblast cell line that expresses a truncated SMAD2 protein comprising just the MH2 domain (Piek et al., 2001; Das et al., 2009; Figure 4C). We confirmed this using recombinant human phosphorylated SMAD2 MH2 domain produced in insect cells by co-expressing the SMAD2 MH2 domain with the kinase domain of TGFBR1 (Figure 4D). In both cases, the SGS mutations prevented interaction of the SKI peptide with the SMAD2 MH2 domain. Figure 4 with 1 supplement see all Download asset Open asset SGS mutations inhibit binding of SKI to SMAD2/3. (A and B) HaCaT cells were treated or not with 2 ng/ml TGF-β. A peptide pulldown assay was performed on whole cells extracts and pulldowns were immunoblotted with the antibodies indicated. Inputs are shown on the right. (A) Wild-type (WT) SKI peptides corresponding to amino acids 11–45 or containing SGS point mutations as shown in red were used. (B) WT SKIL peptides corresponding amino acids 80–120 or containing mutations (in red) corresponding to SGS mutations in SKI were used. (C) WT SKI peptides or those containing SGS point mutations were used in pulldown assays with whole-cell extracts of SMAD2-null mouse embryonic fibroblasts that express just the MH2 domain of SMAD2 (MEF SMAD2Δex2) (Das et al., 2009), treated with 2 ng/ml TGF-β. The untreated sample is only shown for the WT SKI peptide. A PSMAD2 immunoblot is shown. (D) A recombinant trimer of phosphorylated SMAD2 MH2 domain was used in a peptide pulldown assay with WT and G34D SKI peptides. A PSMAD2 immunoblot is shown, with inputs on the right. (E) Mutational peptide array of SKI peptides (amino acids 11–45), mutated at all residues between amino acids 19 and 35, was probed with a recombinant PSMAD3–SMAD4 complex, which was visualized using a SMAD2/3 antibody conjugated to Alexa 488. On each row, the indicated amino acid is substituted for every other amino acid. A representative example is shown. See Figure 4—figure supplement 1C and Figure 4—source data 2 for quantification of the peptide arrays. Figure 4—source data 1 Peptide sequences for peptide array. https://cdn.elifesciences.org/articles/63545/elife-63545-fig4-data1-v2.pdf Download elife-63545-fig4-data1-v2.pdf Figure 4—source data 2 Quantification of peptide arrays. https://cdn.elifesciences.org/articles/63545/elife-63545-fig4-data2-v2.xlsx Download elife-63545-fig4-data2-v2.xlsx We next used a peptide array to gain a better understanding of which amino acids can be tolerated at the positions found to be mutated in SGS and to determine which other amino acids in this region of SKI are essential for the R-SMAD interaction. The SKI peptide corresponding to amino acids 11–45 was synthesized as an array on a cellulose sheet such that each residue in the sequence between residues 19 and 35 was substituted with all 19 alternative amino acids (Figure 4E; Figure 4—source data 1). The array was probed with a recombinant PSMAD3–SMAD4 trimer, generated by co-expressing SMAD3 and SMAD4 with the TGFBR1 kinase domain in insect cells. The PSMAD3–SMAD4 complex was then detected using a fluorescently-labeled SMAD2/3 antibody. Eight residues are intolerant to almost any amino acid substitution (Thr20, Leu21, Phe24, Ser28, Ser31, Leu32, Gly34, and Pro35). Strikingly, six of these residues are the amino acids known to be mutated in SGS patients, and the array results readily explain why these residues are mutated to a number of different amino acids in SGS (Figure 4E; for quantification, see Figure 4—figure supplement 1C and Figure 4—source data 2). In addition, Thr20 and Phe24 are also crucial residues for binding the PSMAD3–SMAD4 complex, but have not yet been reported as disease mutations. Mutations in the other nine amino acids do not impair the binding, and almost any other amino acid apart from proline can be tolerated at these positions. Crystal structure of the SKI peptide with the phosphorylated SMAD2 MH2 domain To discover why these eight amino acids were so crucial for R-SMAD binding, and also to understand why SKI and SKIL only recognize phosphorylated R-SMADs, we solved the crystal structure of the SKI peptide (amino acids 11–45) with a phosphorylated homotrimer of the SMAD2 MH2 domain, produced in insect cells as described above. We confirmed using SEC-MALLS that the phosphorylated SMAD2 MH2 domain was indeed trimeric in solution (Figure 5—figure supplement 1A). Analysis of the binding affinity of the SKI peptide to the SMAD2 MH2 domain trimer indicated that the dissociation constant (Kd) was in the low nanomolar range (Figure 5—figure supplement 1B). The structure was determined by molecular replacement and refined at 2 Å resolution and readily explained why the crucial amino acids identified in the peptide were required for SMAD2 binding (Figure 5; Figure 5—figure supplement 1C). Figure 5 with 1 supplement see all Download asset Open asset Crystal structure of PSMAD2 MH2 domain and N-terminal SKI peptide. (A) Crystal structure of the phosphorylated SMAD2 MH2 domain trimer (the three monomers are shown in bright green, cyan, and olive) with the N-terminal SKI peptide amino acids 11–45 (magenta). A ribbon representation is shown. The C-terminal phosphates are indicated with a ball and stick representation (red and magenta). (B–F) Close ups on key residues for SKI binding. SKI residues are shown in magenta, and SMAD2 residues are in green. In (B–D), a ribbon representation is shown. In (E and F), SMAD2 is shown as a surface representation and SKI as a ribbon. (G) A detail from the structure of monomeric SMAD2 MH2 domain with a peptide from ZFYVE9 (formerly called SARA) (Wu et al., 2000). Note that the β1’ strand that contains Tyr268 is locked in a hydrophobic pocket, forcing Trp448 into flattened orientation, incompatible with SKI binding. (H) A detail from the structure in (A) indicating how SMAD2 complex formation shifts the position of the β1’ strand and more particularly, Tyr268, allowing Trp448 to flip 90°, enabling it to stack with SKI residues Phe24 and Pro35. Figure 5—source data 1 Structure validation report for crystal structure (ID: 6ZVQ). https://cdn.elifesciences.org/articles/63545/elife-63545-fig5-data1-v2.pdf Download elife-63545-fig5-data1-v2.pdf The SKI peptide binds on the outside face of the MH2 domain at the so-called three helix bundle, comprising helices 3, 4, and 5 (Wu et al., 2001; Figure 5A). The N-terminal helix of SKI packs against helix 3 of SMAD2, and the C-terminal portion of the SKI peptide, which contains the critical Gly34 and Pro35, forms a sharp turn that is stabilized by pi-stacking coordination between Phe24 of SKI, Trp448 of SMAD2, and Pro35 of SKI (Figure 5B). Moreover, the NE1 of the Trp448 side chain forms a H-bond to the main chain carbonyl group of Gly33, which in turn positions Pro35 for the interaction with Trp448 (Figure 5B). Furthermore, Glu270 in SMAD2 provides a pocket, which has a negatively charged base that ties down SKI Gly34 through hydrogen-bonding to its main chain amides. Other key interactions involving amino acids identified above as crucial for binding include the main chain carbonyl of Ser31, which forms a hydrogen bond to the ND1 of Asn387 in helix 3 (Figure 5C), and the hydroxyl group of SKI Thr20, which forms a hydrogen bond with the Gln455 at the end of helix 5 of SMAD2, and is nearly completely buried in the interface (Figure 5D). The two leucine residues (Leu21 and Leu32) that are mutated in SGS are both buried in the structure (Figure 5E,F). The structure we obtained is consistent with a SKI–SMAD2 MH2 domain structure that was published by others, while this work was in progress (Miyazono et al., 2018). In that case, a pseudo-phosphorylated SMAD2 MH2 domain produced in Escherichia coli was used, complexed with a SKI peptide containing a C-terminal acidic tag (Ser-Asp-Glu-Asp). Since our structure was generated with phosphorylated SMAD2, we were able to explore why SKI only binds phosphorylated R-SMADs and not monomeric unphosphorylated R-SMADs. To do this we compared the structure of the unphosphorylated SMAD2 MH2 domain bound to a region of ZFYVE9 (formerly called SARA) (Wu et al., 2000) with our current structure of phosphorylated SMAD2 MH2 domain complexed with SKI. It was clear that in the unphosphorylated SMAD2 structure, Tyr268 in the so-called β1’ strand (amino acids 261–274) is locked in a stable conformation in a hydrophobic pocket, and also forms a number of hydrogen bonds (Figure 5G). Crucially, this conformation forces Trp448 into flattened orientation, which is incompatible with SKI binding through the pi-stacking involving SKI Phe24, SMAD2 Trp448, and SKI Pro35 (Figure 5G). MH2 domain trimerization generates a new binding site for the β1’ strand on the adjacent MH2 domain subunit (Figure 5H; Video 1). The central residue driving this is Tyr268. In the trimer, the hydroxy group of" @default.
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W3123265702 date "2020-12-24" @default.
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W3123265702 title "Author response: Mutations in SKI in Shprintzen–Goldberg syndrome lead to attenuated TGF-β responses through SKI stabilization" @default.
W3123265702 doi "https://doi.org/10.7554/elife.63545.sa2" @default.
W3123265702 hasPublicationYear "2020" @default.
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