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Biochemistry 2010, 49, 8020–8032 DOI: 10.1021/bi101153w
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Structural Analysis of the Smad2-MAN1 Interaction That Regulates Transforming Growth Factor-β Signaling at the Inner Nuclear Membrane† Emilie Konde,‡,^ Benjamin Bourgeois,‡,^ Carine Tellier-Lebegue,‡ Wei Wu,§ Javier Perez, Sandrine Caputo,‡ Wika Attanda,‡ Sylvaine Gasparini,‡ Jean-Baptiste Charbonnier,‡ Bernard Gilquin,‡ Howard J. Worman,§ and Sophie Zinn-Justin*,‡ Laboratoire de Biologie Structurale et Radiobiologie, URA CNRS 2096, CEA Saclay, 91190 Gif-sur-Yvette, France, § Department of Medicine and Department of Pathology and Cell Biology, College of Physicians and Surgeons, Columbia University, New York, New York 10032, and Synchrotron SOLEIL, BP 48, 91192 Gif-sur-Yvette Cedex, France. ^These authors contributed equally to this work. )
‡
Received July 20, 2010; Revised Manuscript Received August 16, 2010 ABSTRACT: MAN1, an integral protein of the inner nuclear membrane, influences transforming growth factor-
β (TGF-β) signaling by directly interacting with R-Smads. Heterozygous loss of function mutations in the gene encoding MAN1 cause sclerosing bone dysplasias and an increased level of TGF-β signaling in cells. As a first step in elucidating the mechanism of TGF-β pathway regulation by MAN1, we characterized the structure of the MAN1 C-terminal region that binds Smad2. Using nuclear magnetic resonance spectroscopy, we observed that this region is comprised of a winged helix domain, a structurally heterogeneous linker, a U2AF homology motif (UHM) domain, and a disordered C-terminus. From nuclear magnetic resonance and small-angle X-ray scattering data, we calculated a family of models for this MAN1 region. Our data indicate that the linker plays the role of an intramolecular UHM ligand motif (ULM) interacting with the UHM domain. We mapped the Smad2 binding site onto the MAN1 structure by combining GST pull-down, fluorescence, and yeast two-hybrid approaches. The linker region, the UHM domain, and the C-terminus are necessary for Smad2 binding with a micromolar affinity. Moreover, the intramolecular interaction between the linker and the UHM domain is critical for Smad2 binding. On the basis of the structural heterogeneity and binding properties of the linker, we suggest that it can interact with other UHM domains, thus regulating the MAN1-Smad2 interaction. The transforming growth factor-β (TGF-β)1 family of cytokines comprises key regulators of metazoan embryonic development and adult tissue homeostasis. In the canonical pathway, ligands of both the TGF-β and bone morphogenetic protein members of this family bind to heteromeric serine/threonine kinase receptor complexes, which in turn phosphorylate R-Smad transcription factors at their C-terminal tails. This phosphorylation induces Smad1, -5, and -8 in the bone morphogenic protein pathway and Smad2 and -3 in the TGF-β pathway to accumulate in the nucleus and assemble transcriptional complexes that regulate hundreds of target genes (1, 2). Controlling the duration of TGF-β signaling is critical in development. This can be achieved by restricting the ability of Smad2 † H.J.W. and W.W. were supported in part on this work by a gift from Ms. Lyn Pickel. E.K. and W.A. were supported by Association Franc-aise contre les Myopathies (Ph.D. Grant AFM13401 and ANR06-MRAR-006-02). S.Z.-J., B.B., C.T.-L., S.G., J.-B.C., and B.G. were supported by Agence Nationale de la Recherche (ANR-06-MRAR-00602), Commissariat a l’Energie Atomique, and Centre National de la Recherche Scientifique. *To whom correspondence should be addressed: LBSR B^at. 144, CEA Saclay, 91190 Gif-sur-Yvette, France. Phone: þ33 169083026. Fax: þ33 169084712. E-mail:
[email protected]. 1 Abbreviations: TGF-β, transforming growth factor-β; MAPK, mitogenactivated protein kinase; UHM, U2AF homology motif; ULM, UHM ligand motif; NMR, nuclear magnetic resonance; SAXS, small angle X-ray scattering; ITC, isothermal titration calorimetry; CD, circular dichroı¨ sm; HSQC, heteronuclear single-quantum correlation; NOESY, nuclear Overhauser effect spectroscopy; PDB, Protein Data Bank; SIM, Smad interacting motif; TEV, tobacco etch virus.
pubs.acs.org/Biochemistry
Published on Web 08/17/2010
and Smad3 to accumulate in the nucleus. Smad2 constitutively shuttles between the cytoplasm and the nucleus, in particular through binding to nucleoporins (3). However, these nucleoporins compete with the cytoplasmic retention factor SARA and the nuclear Smad2 partner FAST-1 for binding to a hydrophobic corridor on the MH2 surface of Smad2 (3, 4). SARA overexpression forces Smad2 into clustered locations in the cytoplasm, and FAST-1 overexpression forces Smad2 into the nucleus. On the basis of these insights, it has been proposed that localization of Smad2 is regulated by its binding to different nuclear and cytoplasmic proteins. TGF-β signaling does not influence Smad2 shuttling by changing its affinity for nucleoporins but by controlling its association with retention factors (3). Smad proteins exhibit multiple phosphorylation sites that could participate in the regulation of their binding properties. In particular, phosphorylation of Xenopus Smad2 in its linker region restricts the time during which it remains in the nucleus (5). Multiple kinases have been identified that mediate the phosphorylation of Smad linkers (6-10). For example, in response to mitogens, the mitogen-activated protein kinase (MAPK) extracellular signal-regulated kinase catalyzes the phosphorylation of Smad linkers in vivo (6, 7). This phosphorylation results in inhibition of Smad1 activity (6, 8) and a decreased level of nuclear accumulation (6). Similarly, Smad2 linker phosphorylation causes MAPK-mediated attenuation of Smad2 nuclear accumulation and activity (7). Thus, the MAPK pathway influences the ability of Smads to transduce TGF-β signals. r 2010 American Chemical Society
Article
Biochemistry, Vol. 49, No. 37, 2010
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FIGURE 1: Architecture of the main protein fragments cited in this study: MAN1C, the C-terminal nucleoplasmic region of MAN1; MAN1whL consisting of the WH domain and the linker; MAN1Luhm containing the linker, the UHM domain, and the C-terminus; two mutants of MAN1Luhm, namely, MAN1LuhmWQ and MAN1LuhmΔ21; S2MH2 comprising the MH2 domain of Smad2; S2LMH2 consisting of the linker and the MH2 domain, and a mutant of S2LMH2, namely S2LMH2EEE, mutated at the three C-terminal serines of the SSXS motif.
MAN1 is an integral protein of the inner nuclear membrane with an N-terminal nucleoplasmic region, two transmembrane segments, and a C-terminal nucleoplasmic region (11). The C-terminal region of MAN1 directly interacts with Smad2 and Smad3 and represses TGF-β signaling (12-16). Heterozygous loss of function mutations in the gene encoding MAN1 cause the sclerosing bone dysplasias osteopoikilosis, Buschke-Ollendorff syndrome, and nonsporadic melorheostosis, which likely result from enhanced TGF-β signaling in cells (12). The MAN1-Smad interaction may lead to the cytoplasmic accumulation of Smad2 and Smad3, thus causing inhibition of TGF-β signaling (14). However, the mechanism by which the nucleoplasmic MAN1 C-terminal region would stimulate Smad2 and Smad3 nuclear export remains unclear. One hypothesis, as proposed for the MAN1 paralog NET25 (17), is that MAN1 functions as a scaffold protein recruiting Smad2, Smad3, and kinases to the inner nuclear membrane. Phosphorylation of Smad linker residues by kinases such as MAPKs would then lead to enhanced nuclear export. Alternatively, MAN1 may more simply act as a “nuclear envelope sink” for activated nuclear Smad proteins, sequestering them at the inner nuclear membrane and competitively inhibiting interactions with other nuclear factors involved in gene regulation. To improve our understanding of the function of MAN1, we initiated structural studies of its C-terminal region that binds to Smad2 and Smad3. We previously showed that this region contains a winged helix (WH) DNA binding domain (18). In this study, we characterized the three-dimensional (3D) structure of the whole C-terminal region of MAN1 also comprising a structurally heterogeneous linker, a U2AF homology motif (UHM) domain, and a disordered C-terminus. Our data revealed that the linker plays the role of an intramolecular UHM ligand motif (ULM), and that its positioning onto the MAN1 UHM domain is critical for Smad2 binding. EXPERIMENTAL PROCEDURES Protein Preparation. We generated various protein fragments of human MAN1 that are shown in Figure 1: MAN1C corresponding to the C-terminal nucleoplasmic region of MAN1, MAN1whL consisting of the WH domain and the linker, MAN1Luhm containing the linker, the UHM domain, and the C-terminus, and several mutants of MAN1Luhm, such as MAN1LuhmWQ and MAN1LuhmΔ21. Similarly, several regions of Smad2 were produced: S2MH2 comprising the MH2 domain of Smad2, S2LMH2 consisting of the linker and the MH2 domain, and a
mutant of S2LMH2, namely S2LMH2EEE, mutated at the three C-terminal serines of the SSXS motif. MAN1C was overexpressed in Escherichia coli strain BL21 DE3 Star transformed with a plasmid that encodes a ZZ domain, a cleavage site for tobacco etch virus protease, and MAN1C. MAN1whL was overexpressed in E. coli strain DE3 pLys S transformed with a construct generated in pGEX-4T-1 (GE Healthcare) that encodes glutathione S-transferase (GST) and a thrombin site fused to MAN1whL. Both proteins were produced and purified as described previously (18). MAN1 constructs MAN1Luhm (755-911) and MAN1LuhmΔ21 (755-890) and the mutants of MAN1Luhm (W765A/ Q766A, P778A/P779A, and K864A/R870A) were overexpressed in E. coli strain BL21 DE3 Star transformed with a plasmid that encodes a six-histidine tag and the protein of interest. Fusion proteins were purified using a His trap column (GE Healthcare) and a gel filtration column (Superdex 200, Hi-Load, 120 mL, GE Healthcare). Their circular dichroism (CD) spectra were recorded on a Jobin Yvon CD6 instrument between 190 and 250 nm at 5 C on 15 μM protein samples in a buffer containing 50 mM Tris-H2SO4 (pH 8), 75 mM NaF, and 5 mM β-mercaptoethanol. CD spectra obtained for wild-type MAN1Luhm and its variants analyzed in this study are similar, suggesting no overall structural changes due to the deletions or mutations (Figure S0 of the Supporting Information). S2MH2, S2LMH2, and S2LMH2EEE were produced in E. coli strain BL21 DE3 Star transformed with a plasmid that encodes a six-histidine tag and the protein of interest. The fusion proteins were purified using a His trap column (GE Healthcare) and a gel filtration column (Superdex 200, Hi-Load, 120 mL, GE Healthcare). GST-S2MH2 and GST-S2LMH2 were overexpressed in E. coli strain BL21 DE3 Star transformed with a plasmid that encodes GST and the protein of interest. They were purified on a glutathioneSepharose 4B column and by gel filtration (Superdex 200, Hi-Load, 120 mL, GE Healthcare). Nuclear Magnetic Resonance (NMR) Spectroscopy. For NMR experiments, proteins were produced in M9 minimal medium containing 15NH4Cl (Cortecnet and Eurisotop) as the only nitrogen source or 15NH4Cl and [13C]glucose (Cortecnet and Eurisotop) to yield 15N-labeled or 15N- and 13C-labeled proteins, respectively. NMR samples were prepared in 50 mM Tris buffer (pH 6.7) containing 150 mM NaCl (90% H2O/ 10% D2O), 1 mM EDTA, 1 mM PMSF, and 1 mM NaN3. 3-(Trimethylsilyl)[2,2,3,3-2H4]propionate was added as a chemical
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shift reference. The protein concentration was between 0.1 and 0.3 mM. NMR experiments were performed at 20 and 30 C on Bruker (Saclay, France) 600 and 700 MHz spectrometers equipped with a triple-resonance cryoprobe. Spectra allowing chemical shift assignment and structural characterization were acquired as described previously (19). Two-dimensional (2D) 1H-15N heteronuclear single-quantum correlation (HSQC) and 1H-15N nuclear Overhauser effect spectroscopy (NOESY) were conducted with MAN1C, MAN1whL, and wild-type and mutated MAN1Luhm fragments. 3D HNCO, HNCA, CBCA(CO)NH, HNCACB, HBHA(CO)NH, HNHA, 1H-15N NOESY-HSQC, and 1H-13C NOESY-HSQC spectra of MAN1Luhm were recorded. All NMR spectra were analyzed and exploited with Sparky (T. D. Goddard and D. G. Kneller, University of California, San Francisco, CA). Chemical shift assignment was accelerated by the use of MARS (20). Small Angle X-ray Scattering (SAXS) Experiments. SAXS samples were prepared in 50 mM Tris buffer (pH 8.0) containing 150 mM NaCl and 10 mM DTT. Their initial protein concentrations yielded between 12 and 14 mg/mL. Before data collection, they were injected on a KW402-5-4F gel filtration column (Shodex) to avoid the presence of aggregates. The SAXS measurements were taken just after protein elution at a concentration of ∼1 mg/mL. Synchrotron radiation X-ray scattering data were collected at 17 C following standard procedures (21) on the SWING line at Synchrotron SOLEIL (Gif-sur-Yvette, France). The scattering due to buffer was subtracted by averaging the buffer measurements enclosing the actual protein measurement. All data manipulations were performed with PRIMUS (22). GNOM (23) provided access to the radius of gyration and the maximal distance of the molecule. It also provided a value of the SAXS intensity at the origin I0, from which the mass was calculated using water as a reference, following M ¼ I0 =K N=ðcΔFm 2 Þ where M is expressed in kilodaltons, K is the ratio between theoretical (0.0164 cm-1) and measured water scattering, N is Avogadro’s number, c is the mass concentration in grams per liter, and ΔFm is the protein electron density (taken to be 2 1010 cm/g, which corresponds to a protein partial specific volume of 0.74). CRYSOL (24) was used to generate SAXS curves from calculated models. Molecular Modeling. MetaPrDos was ran on the server http://prdos.hgc.jp/cgi-bin/meta/top.cgi. The solution structure of the MAN1 WH domain (663-753) was previously determined by NMR in our laboratory [PDB entry 2CH0 (18)]. The MAN1 UHM domain (785-889) was modeled on the basis of the structure of the U2AF65 UHM domain (PDB entry 10OP; 28% identical to MAN1) and secondary structure constraints given by TALOS (25) using Modeler 9v7 (26). To position the linker onto the UHM domain, the globular domain was centered at the origin and the linker (755-784) in a completely extended conformation was located 20 A˚ from the center of mass of the globular domain parallel to the x-axis. To generate 5000 initial structures of the fragment comprising the linker and the UHM domain, the globular domain was randomly rotated around its center of mass and the linker was rotated 10 by 10 around the x-axis. The peptide bond between residues 784 and 785 was deleted and replaced by a distance restraint.
Konde et al. The C-terminus (890-910) was generated in a completely extended conformation. Ambiguous distance restraints were derived from chemical shift perturbation mapping analysis of mutant MAN1LuhmWQ. The 30 models of MAN1Luhm with the lowest distance restraint energies were selected, and the SAXS curves corresponding to these models were calculated with CRYSOL (24). Comparison with the experimental SAXS curves showed that 10 models are characterized by χ values of
