PDBsum entry 1g88

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protein Protein-protein interface(s) links
Transcription PDB id
Protein chains
221 a.a. *
242 a.a. *
* Residue conservation analysis
PDB id:
Name: Transcription
Title: S4afl3arg515 mutant
Structure: Smad4. Chain: a, b, c. Fragment: smad4 active fragment. Synonym: dpc4. Mutation: yes
Source: Homo sapiens. Human. Organism_taxid: 9606
Biol. unit: Trimer (from PQS)
3.00Å     R-factor:   0.203     R-free:   0.263
Authors: B.M.Chako,B.Qin,S.S.Lam,J.J.Correia,K.Lin
Key ref:
B.M.Chacko et al. (2001). The L3 loop and C-terminal phosphorylation jointly define Smad protein trimerization. Nat Struct Biol, 8, 248-253. PubMed id: 11224571 DOI: 10.1038/84995
16-Nov-00     Release date:   29-Nov-00    
Go to PROCHECK summary

Protein chain
Pfam   ArchSchema ?
Q13485  (SMAD4_HUMAN) -  Mothers against decapentaplegic homolog 4
552 a.a.
221 a.a.*
Protein chains
Pfam   ArchSchema ?
Q13485  (SMAD4_HUMAN) -  Mothers against decapentaplegic homolog 4
552 a.a.
242 a.a.*
Key:    PfamA domain  Secondary structure  CATH domain
* PDB and UniProt seqs differ at 2 residue positions (black crosses)

 Gene Ontology (GO) functional annotation 
  GO annot!
  Cellular component     intracellular   1 term 
  Biological process     regulation of transcription, DNA-dependent   1 term 


DOI no: 10.1038/84995 Nat Struct Biol 8:248-253 (2001)
PubMed id: 11224571  
The L3 loop and C-terminal phosphorylation jointly define Smad protein trimerization.
B.M.Chacko, B.Qin, J.J.Correia, S.S.Lam, Caestecker, K.Lin.
Smad proteins mediate the transforming growth factor beta responses. C-terminal phosphorylation of R-Smads leads to the recruitment of Smad4 and the formation of active signaling complexes. We investigated the mechanism of phosphorylation-induced Smad complex formation with an activating pseudo-phosphorylated Smad3. Pseudo-phosphorylated Smad3 has a greater propensity to homotrimerize, and recruits Smad4 to form a heterotrimer containing two Smad3 and one Smad4. The trimeric interaction is mediated through conserved interfaces to which tumorigenic mutations map. Furthermore, a conserved Arg residue within the L3 loop, located near the C-terminal phosphorylation sites of the neighboring subunit, is essential for trimerization. We propose that the phosphorylated C-terminal residues interact with the L3 loop of the neighboring subunit to stabilize the trimer interaction.
  Selected figure(s)  
Figure 3.
Figure 3. The sulfate binding site within the L3 loop is critical for Smad heteromeric interaction. a, The sulfate binding sites shown in detail. The location of the sulfate ions in the context of the trimeric S4AF structure is shown in Fig. 2b. b, Mutation of the sulfate binding site at the L3 loop specifically reduces heteromeric interaction between S3LC(3E) and S4AF. S3LC(3E) and the sulfate binding site mutants of S4AF were mixed at a 1:1 mole ratio and loaded on the size exclusion column. The eluted fractions were analyzed by SDS-PAGE and the gels were stained with Coomassie blue. c, Stereo view of the F[o] - F[c] omit map at the L3 loop of the S4AF(R515S) mutant. The mutant and wild type coordinates are shown in black and gray, respectively. d, The Smad4 Arg 515 mutant only weakly activates Smad3/4 dependent transcriptional responses. Smad4 null MDA-MB468 cells were transfected with SBE-Lux along with the indicated FLAG-Smad3(3E) and Smad4-Myc mutant constructs. e, Homotrimerization of S3LC(3E) is abolished by mutation of residues at the trimer interface or the conserved Arg residue within the L3 loop. Proteins (50 M) were loaded onto the size-exclusion column and the eluted fractions were analyzed by SDS-PAGE and the gels were stained by Coomassie-Blue.
Figure 4.
Figure 4. Proposed model of phosphorylation induced Smad protein activation. Left, phosphorylation induced heterotrimer between Smad4 and R-Smad subunits. Right, phosphorylation induced homotrimer of R-Smad. The Smad4 subunit is shown in pink. The R-Smad subunits are shown in blue. The conserved L3 loops are shown in black. The phosphorylated C-terminal tails of the R-Smad subunits are represented by red arrows.
  The above figures are reprinted by permission from Macmillan Publishers Ltd: Nat Struct Biol (2001, 8, 248-253) copyright 2001.  
  Figures were selected by an automated process.  

Literature references that cite this PDB file's key reference

  PubMed id Reference
19594322 S.Kokabu, J.Nojima, K.Kanomata, S.Ohte, T.Yoda, T.Fukuda, and T.Katagiri (2010).
Protein phosphatase magnesium-dependent 1A-mediated inhibition of BMP signaling is independent of Smad dephosphorylation.
  J Bone Miner Res, 25, 653-660.  
19557331 C.Wang, L.Chen, L.Wang, and J.Wu (2009).
Crystal structure of the MH2 domain of Drosophila Mad.
  Sci China C Life Sci, 52, 539-544.
PDB code: 3gmj
19441058 I.L.Blitz, and K.W.Cho (2009).
Finding partners: How BMPs select their targets.
  Dev Dyn, 238, 1321-1331.  
19224984 K.N.Retting, B.Song, B.S.Yoon, and K.M.Lyons (2009).
BMP canonical Smad signaling through Smad1 and Smad5 is required for endochondral bone formation.
  Development, 136, 1093-1104.  
19489734 M.K.Tarrant, and P.A.Cole (2009).
The chemical biology of protein phosphorylation.
  Annu Rev Biochem, 78, 797-825.  
19135894 S.Dupont, A.Mamidi, M.Cordenonsi, M.Montagner, L.Zacchigna, M.Adorno, G.Martello, M.J.Stinchfield, S.Soligo, L.Morsut, M.Inui, S.Moro, N.Modena, F.Argenton, S.J.Newfeld, and S.Piccolo (2009).
FAM/USP9x, a deubiquitinating enzyme essential for TGFbeta signaling, controls Smad4 monoubiquitination.
  Cell, 136, 123-135.  
19032343 T.Tabata, K.Kokura, P.Ten Dijke, and S.Ishii (2009).
Ski co-repressor complexes maintain the basal repressed state of the TGF-beta target gene, SMAD7, via HDAC3 and PRMT5.
  Genes Cells, 14, 17-28.  
18024957 E.Cocolakis, M.Dai, L.Drevet, J.Ho, E.Haines, S.Ali, and J.J.Lebrun (2008).
Smad signaling antagonizes STAT5-mediated gene transcription and mammary epithelial cell differentiation.
  J Biol Chem, 283, 1293-1307.  
18506886 R.Zheng, Q.Xiong, B.Zuo, S.Jiang, F.Li, M.Lei, C.Deng, and Y.Xiong (2008).
Using RNA interference to identify the different roles of SMAD2 and SMAD3 in NIH/3T3 fibroblast cells.
  Cell Biochem Funct, 26, 548-556.  
18061509 S.Ross, and C.S.Hill (2008).
How the Smads regulate transcription.
  Int J Biochem Cell Biol, 40, 383-408.  
17340614 K.A.Brown, J.A.Pietenpol, and H.L.Moses (2007).
A tale of two proteins: differential roles and regulation of Smad2 and Smad3 in TGF-beta signaling.
  J Cell Biochem, 101, 9.  
17668048 K.Singh, D.Mogare, R.O.Giridharagopalan, R.Gogiraju, G.Pande, and S.Chattopadhyay (2007).
p53 target gene SMAR1 is dysregulated in breast cancer: its role in cancer cell migration and invasion.
  PLoS ONE, 2, e660.  
17563385 M.R.Pratt, E.C.Schwartz, and T.W.Muir (2007).
Small-molecule-mediated rescue of protein function by an inducible proteolytic shunt.
  Proc Natl Acad Sci U S A, 104, 11209-11214.  
18056415 T.Morita, T.Mayanagi, and K.Sobue (2007).
Dual roles of myocardin-related transcription factors in epithelial mesenchymal transition via slug induction and actin remodeling.
  J Cell Biol, 179, 1027-1042.  
16449645 A.Kurisaki, K.Kurisaki, M.Kowanetz, H.Sugino, Y.Yoneda, C.H.Heldin, and A.Moustakas (2006).
The mechanism of nuclear export of Smad3 involves exportin 4 and Ran.
  Mol Cell Biol, 26, 1318-1332.  
17074756 M.Simonsson, M.Kanduri, E.Grönroos, C.H.Heldin, and J.Ericsson (2006).
The DNA binding activities of Smad2 and Smad3 are regulated by coactivator-mediated acetylation.
  J Biol Chem, 281, 39870-39880.  
16611366 N.Kaivo-oja, L.A.Jeffery, O.Ritvos, and D.G.Mottershead (2006).
Smad signalling in the ovary.
  Reprod Biol Endocrinol, 4, 21.  
17074053 U.Ueberham, E.Ueberham, H.Gruschka, and T.Arendt (2006).
Altered subcellular location of phosphorylated Smads in Alzheimer's disease.
  Eur J Neurosci, 24, 2327-2334.  
16766264 Y.A.Yang, G.M.Zhang, L.Feigenbaum, and Y.E.Zhang (2006).
Smad3 reduces susceptibility to hepatocarcinoma by sensitizing hepatocytes to apoptosis through downregulation of Bcl-2.
  Cancer Cell, 9, 445-457.  
17051103 Z.Gao, Z.Wang, Y.Shi, Z.Lin, H.Jiang, T.Hou, Q.Wang, X.Yuan, Y.Zhao, H.Wu, and Y.Jin (2006).
Modulation of collagen synthesis in keloid fibroblasts by silencing Smad2 with siRNA.
  Plast Reconstr Surg, 118, 1328-1337.  
15817471 A.Morén, T.Imamura, K.Miyazono, C.H.Heldin, and A.Moustakas (2005).
Degradation of the tumor suppressor Smad4 by WW and HECT domain ubiquitin ligases.
  J Biol Chem, 280, 22115-22123.  
15799969 H.B.Chen, J.G.Rud, K.Lin, and L.Xu (2005).
Nuclear targeting of transforming growth factor-beta-activated Smad complexes.
  J Biol Chem, 280, 21329-21336.  
16109720 S.Gao, J.Steffen, and A.Laughon (2005).
Dpp-responsive silencers are bound by a trimeric Mad-Medea complex.
  J Biol Chem, 280, 36158-36164.  
16212511 X.H.Feng, and R.Derynck (2005).
Specificity and versatility in tgf-beta signaling through Smads.
  Annu Rev Cell Dev Biol, 21, 659-693.  
15210694 I.Yakymovych, C.H.Heldin, and S.Souchelnytskyi (2004).
Smad2 phosphorylation by type I receptor: contribution of arginine 462 and cysteine 463 In the C terminus of Smad2 for specificity.
  J Biol Chem, 279, 35781-35787.  
14729983 J.Qing, C.Liu, L.Choy, R.Y.Wu, J.S.Pagano, and R.Derynck (2004).
Transforming growth factor beta/Smad3 signaling regulates IRF-7 function and transcriptional activation of the beta interferon promoter.
  Mol Cell Biol, 24, 1411-1425.  
15009204 M.Lutz, K.Krieglstein, S.Schmitt, P.ten Dijke, W.Sebald, A.Wizenmann, and P.Knaus (2004).
Nerve growth factor mediates activation of the Smad pathway in PC12 cells.
  Eur J Biochem, 271, 920-931.  
14729957 R.A.Randall, M.Howell, C.S.Page, A.Daly, P.A.Bates, and C.S.Hill (2004).
Recognition of phosphorylated-Smad2-containing complexes by a novel Smad interaction motif.
  Mol Cell Biol, 24, 1106-1121.  
12794086 A.Morén, U.Hellman, Y.Inada, T.Imamura, C.H.Heldin, and A.Moustakas (2003).
Differential ubiquitination defines the functional status of the tumor suppressor Smad4.
  J Biol Chem, 278, 33571-33582.  
14555996 B.Y.Qin, C.Liu, S.S.Lam, H.Srinath, R.Delston, J.J.Correia, R.Derynck, and K.Lin (2003).
Crystal structure of IRF-3 reveals mechanism of autoinhibition and virus-induced phosphoactivation.
  Nat Struct Biol, 10, 913-921.
PDB code: 1qwt
12764135 J.He, S.B.Tegen, A.R.Krawitz, G.S.Martin, and K.Luo (2003).
The transforming activity of Ski and SnoN is dependent on their ability to repress the activity of Smad proteins.
  J Biol Chem, 278, 30540-30547.  
14527958 M.Funaba, T.Ikeda, M.Murakami, K.Ogawa, K.Tsuchida, H.Sugino, and M.Abe (2003).
Transcriptional activation of mouse mast cell Protease-7 by activin and transforming growth factor-beta is inhibited by microphthalmia-associated transcription factor.
  J Biol Chem, 278, 52032-52041.  
12426322 M.Mizuide, T.Hara, T.Furuya, M.Takeda, K.Kusanagi, Y.Inada, M.Mori, T.Imamura, K.Miyazawa, and K.Miyazono (2003).
Two short segments of Smad3 are important for specific interaction of Smad3 with c-Ski and SnoN.
  J Biol Chem, 278, 531-536.  
14534577 R.Derynck, and Y.E.Zhang (2003).
Smad-dependent and Smad-independent pathways in TGF-beta family signalling.
  Nature, 425, 577-584.  
12809600 Y.Shi, and J.Massagué (2003).
Mechanisms of TGF-beta signaling from cell membrane to the nucleus.
  Cell, 113, 685-700.  
12440701 A.Mehra, and J.L.Wrana (2002).
TGF-beta and the Smad signal transduction pathway.
  Biochem Cell Biol, 80, 605-622.  
12154125 B.Y.Qin, S.S.Lam, J.J.Correia, and K.Lin (2002).
Smad3 allostery links TGF-beta receptor kinase activation to transcriptional control.
  Genes Dev, 16, 1950-1963.
PDB codes: 1mjs 1mk2
12374795 G.J.Inman, and C.S.Hill (2002).
Stoichiometry of active smad-transcription factor complexes on DNA.
  J Biol Chem, 277, 51008-51016.  
12191474 G.J.Inman, F.J.Nicolás, and C.S.Hill (2002).
Nucleocytoplasmic shuttling of Smads 2, 3, and 4 permits sensing of TGF-beta receptor activity.
  Mol Cell, 10, 283-294.  
11920677 P.Ten Dijke, M.J.Goumans, F.Itoh, and S.Itoh (2002).
Regulation of cell proliferation by Smad proteins.
  J Cell Physiol, 191, 1.  
11782434 R.A.Randall, S.Germain, G.J.Inman, P.A.Bates, and C.S.Hill (2002).
Different Smad2 partners bind a common hydrophobic pocket in Smad2 via a defined proline-rich motif.
  EMBO J, 21, 145-156.  
12185845 S.Souchelnytskyi, A.Moustakas, and C.H.Heldin (2002).
TGF-beta signaling from a three-dimensional perspective: insight into selection of partners.
  Trends Cell Biol, 12, 304-307.  
11779505 B.Y.Qin, B.M.Chacko, S.S.Lam, Caestecker, J.J.Correia, and K.Lin (2001).
Structural basis of Smad1 activation by receptor kinase phosphorylation.
  Mol Cell, 8, 1303-1312.
PDB code: 1khu
11553622 D.Maurice, C.E.Pierreux, M.Howell, R.E.Wilentz, M.J.Owen, and C.S.Hill (2001).
Loss of Smad4 function in pancreatic tumors: C-terminal truncation leads to decreased stability.
  J Biol Chem, 276, 43175-43181.  
11779503 J.W.Wu, M.Hu, J.Chai, J.Seoane, M.Huse, C.Li, D.J.Rigotti, S.Kyin, T.W.Muir, R.Fairman, J.Massagué, and Y.Shi (2001).
Crystal structure of a phosphorylated Smad2. Recognition of phosphoserine by the MH2 domain and insights on Smad function in TGF-beta signaling.
  Mol Cell, 8, 1277-1289.
PDB code: 1khx
  11691834 S.L.Stroschein, S.Bonni, J.L.Wrana, and K.Luo (2001).
Smad3 recruits the anaphase-promoting complex for ubiquitination and degradation of SnoN.
  Genes Dev, 15, 2822-2836.  
The most recent references are shown first. Citation data come partly from CiteXplore and partly from an automated harvesting procedure. Note that this is likely to be only a partial list as not all journals are covered by either method. However, we are continually building up the citation data so more and more references will be included with time. Where a reference describes a PDB structure, the PDB code is shown on the right.