Siah ubiquitin ligase is structurally related to TRAF and modulates TNF-α signaling

Article metrics


Members of the Siah (seven in absentia homolog) family of RING domain proteins are components of E3 ubiquitin ligase complexes that catalyze ubiquitination of proteins. We have determined the crystal structure of the substrate-binding domain (SBD) of murine Siah1a to 2.6 Å resolution. The structure reveals that Siah is a dimeric protein and that the SBD adopts an eight-stranded β-sandwich fold that is highly similar to the TRAF-C region of TRAF (TNF-receptor associated factor) proteins. The TRAF-C region interacts with TNF-α receptors and TNF-receptor associated death-domain (TRADD) proteins; however, our findings indicate that these interactions are unlikely to be mimicked by Siah. The Siah structure also reveals two novel zinc fingers in a region with sequence similarity to TRAF. We find that the Siah1a SBD potentiates TNF-α-mediated NF-κB activation. Therefore, Siah proteins share important similarities with the TRAF family of proteins, including their overall domain architecture, three-dimensional structure and functional activity.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.

Figure 1: Primary structure of Siah.
Figure 2: Siah1a SBD forms a dimer in solution.
Figure 3: Structure of Siah1a SBD.
Figure 4: Similarities between Siah and TRAF.
Figure 5: Surface features of Siah SBD.

Accession codes


Protein Data Bank


  1. 1

    Ciechanover, A. The ubiquitin-proteasome pathway: on protein death and cell life. EMBO J. 17, 7151–7160 (1998).

  2. 2

    Lorick, K.L. et al. RING fingers mediate ubiquitin-conjugating enzyme (E2)-dependent ubiquitination. Proc. Natl. Acad. Sci. USA 96, 11364–11369 (1999).

  3. 3

    Joazeiro, C.A. et al. The tyrosine kinase negative regulator c-Cbl as a RING-type, E2-dependent ubiquitin-protein ligase. Science 286, 309–312 (1999).

  4. 4

    Zheng, N., Wang, P., Jeffrey, P.D. & Pavletich, N.P. Structure of a c-Cbl-UbcH7 complex: RING domain function in ubiquitin-protein ligases. Cell 102, 533–539 (2000).

  5. 5

    Carthew, R.W. & Rubin, G.M. Seven in absentia, a gene required for specification of R7 cell fate in the Drosophila eye. Cell 63, 561–577 (1990).

  6. 6

    Tang, A.H., Neufeld, T.P., Kwan, E. & Rubin, G.M. PHYL acts to down-regulate TTK88, a transcriptional repressor of neuronal cell fates, by a SINA-dependent mechanism. Cell 90, 459–467 (1997).

  7. 7

    Li, S., Li, Y., Carthew, R.W. & Lai, Z.C. Photoreceptor cell differentiation requires regulated proteolysis of the transcriptional repressor Tramtrack. Cell 90, 469–478 (1997).

  8. 8

    Dong, X. et al. ebi regulates epidermal growth factor receptor signaling pathways in Drosophila. Genes Dev. 13, 954–965 (1999).

  9. 9

    Boulton, S.J., Brook, A., Staehling-Hampton, K., Heitzler, P. & Dyson, N. A role for ebi in neuronal cell cycle control. EMBO J. 19, 5376–5386 (2000).

  10. 10

    Matsuzawa, S., Takayama, S., Froesch, B.A., Zapata, J.M. & Reed, J.C. p53-inducible human homologue of Drosophila seven in absentia (Siah) inhibits cell growth: suppression by BAG-1. EMBO J. 17, 2736–2747 (1998).

  11. 11

    Hu, G. et al. Mammalian homologs of seven in absentia regulate DCC via the ubiquitin- proteasome pathway. Genes Dev. 11, 2701–2714 (1997).

  12. 12

    Hu, G. & Fearon, E.R. Siah-1 N-terminal RING domain is required for proteolysis function, and C-terminal sequences regulate oligomerization and binding to target proteins. Mol. Cell. Biol. 19, 724–732 (1999).

  13. 13

    Zhang, J., Guenther, M.G., Carthew, R.W. & Lazar, M.A. Proteasomal regulation of nuclear receptor corepressor-mediated repression. Genes Dev. 12, 1775–1780 (1998).

  14. 14

    Germani, A. et al. SIAH-1 interacts with α-tubulin and degrades the kinesin Kid by the proteasome pathway during mitosis. Oncogene 19, 5997–6006 (2000).

  15. 15

    Matsuzawa, S. & Reed, J.C. Siah-1, SIP, and Ebi collaborate in a novel pathway for β-catenin degradation linked to p53 responses. Mol. Cell 7, 915–926 (2001).

  16. 16

    Liu, J. et al. Siah-1 mediates a novel β-catenin degradation pathway linking p53 to the adenomatous polyposis coli protein. Mol. Cell 7, 927–936 (2001).

  17. 17

    Germani, A. et al. hSiah2 is a new Vav binding protein which inhibits Vav-mediated signaling pathways. Mol. Cell. Biol. 19, 3798–3807 (1999).

  18. 18

    Relaix, F. et al. Pw1/Peg3 is a potential cell death mediator and cooperates with Siah1a in p53-mediated apoptosis. Proc. Natl. Acad. Sci. USA 97, 2105–2110 (2000).

  19. 19

    Pickart, C.M. Ubiquitin enters the new millenium; meeting review. Mol. Cell 8, 499–504 (2001).

  20. 20

    Ulrich, H.D. & Jentsch, S. Two RING finger proteins mediate cooperation between ubiquitin-conjugating enzymes in DNA repair. EMBO J. 19, 3388–3397 (2000).

  21. 21

    Deng, L. et al. Activation of the IκB kinase complex by TRAF6 requires a dimeric ubiquitin-conjugating enzyme complex and a unique polyubiquitin chain. Cell 103, 351–361 (2000).

  22. 22

    Wang, C. et al. TAK1 is a ubiquitin-dependent kinase of MKK and IKK. Nature 412, 346–351 (2001).

  23. 23

    Narayan, V.A., Kriwacki, R.W. & Caradonna, J.P. Structures of zinc finger domains from transcription factor Sp1. Insights into sequence-specific protein–DNA recognition. J. Biol. Chem. 272, 7801–7809 (1997).

  24. 24

    Liew, C.K. et al. Solution structures of two CCHC zinc fingers from the FOG family protein U-shaped that mediate protein–protein interactions. Structure Fold Des. 8, 1157–1166 (2000).

  25. 25

    Holm, L. & Sander, C. Protein structure comparison by alignment of distance matrices. J. Mol. Biol. 233, 123–138 (1993).

  26. 26

    Ni, C.Z. et al. Molecular basis for CD40 signaling mediated by TRAF3. Proc. Natl. Acad. Sci. USA 97, 10395–10399 (2000).

  27. 27

    Inoue, J. et al. Tumor necrosis factor receptor-associated factor (TRAF) family: adapter proteins that mediate cytokine signaling. Exp. Cell Res. 254, 14–24 (2000).

  28. 28

    Zapata, J.M. et al. A diverse family of proteins containing tumor necrosis factor receptor-associated factor domains. J. Biol. Chem. 276, 24242–24252 (2001).

  29. 29

    Park, Y.C., Burkitt, V., Villa, A.R., Tong, L. & Wu, H. Structural basis for self-association and receptor recognition of human TRAF2. Nature 398, 533–538 (1999).

  30. 30

    McWhirter, S.M. et al. Crystallographic analysis of CD40 recognition and signaling by human TRAF2. Proc. Natl. Acad. Sci. USA 96, 8408–8413 (1999).

  31. 31

    Park, Y.C. et al. A novel mechanism of TRAF signaling revealed by structural and functional analyses of the TRADD-TRAF2 interaction. Cell 101, 777–787 (2000).

  32. 32

    Rothe, M. et al. I-TRAF is a novel TRAF-interacting protein that regulates TRAF-mediated signal transduction. Proc. Natl. Acad. Sci. USA 93, 8241–8246 (1996).

  33. 33

    Takeuchi, M., Rothe, M. & Goeddel, D.V. Anatomy of TRAF2. Distinct domains for nuclear factor-κB activation and association with tumor necrosis factor signaling proteins. J. Biol. Chem. 271, 19935–19942 (1996).

  34. 34

    Song, H.Y., Rothe, M. & Goeddel, D.V. The tumor necrosis factor-inducible zinc finger protein A20 interacts with TRAF1/TRAF2 and inhibits NF-κB activation. Proc. Natl. Acad. Sci. USA 93, 6721–6725 (1996).

  35. 35

    Lee, S.Y. & Choi, Y. TRAF-interacting protein (TRIP): a novel component of the tumor necrosis factor receptor (TNFR)- and CD30-TRAF signaling complexes that inhibits TRAF2-mediated NF-κB activation. J. Exp. Med. 185, 1275–1285 (1997).

  36. 36

    Song, H.Y., Regnier, C.H., Kirschning, C.J., Goeddel, D.V. & Rothe, M. Tumor necrosis factor (TNF)-mediated kinase cascades: bifurcation of nuclear factor-κB and c-jun N-terminal kinase (JNK/SAPK) pathways at TNF receptor-associated factor 2. Proc. Natl. Acad. Sci. USA 94, 9792–9796 (1997).

  37. 37

    Relaix, F., Wei, X.J., Wu, X. & Sassoon, D.A. Peg3/Pw1 is an imprinted gene involved in the TNF-NFκB signal transduction pathway. Nature Genet. 18, 287–291 (1998).

  38. 38

    Shi, C.S., Leonardi, A., Kyriakis, J., Siebenlist, U. & Kehrl, J.H. TNF-mediated activation of the stress-activated protein kinase pathway: TNF receptor-associated factor 2 recruits and activates germinal center kinase related. J. Immunol. 163, 3279–3285 (1999).

  39. 39

    Arch, R.H., Gedrich, R.W. & Thompson, C.B. Tumor necrosis factor receptor-associated factors (TRAFs) — a family of adapter proteins that regulates life and death. Genes Dev. 12, 2821–2830 (1998).

  40. 40

    Kim, S.J., Jeong, D.G., Chi, S.W., Lee, J.S. & Ryu, S.E. Crystal structure of proteolytic fragments of the redox-sensitive Hsp33 with constitutive chaperone activity. Nature Struct. Biol. 8, 459–466 (2001).

  41. 41

    Schulman, B.A. et al. Insights into SCF ubiquitin ligases from the structure of the Skp1-Skp2 complex. Nature 408, 381–386 (2000).

  42. 42

    Stebbins, C.E., Kaelin, W.G. Jr. & Pavletich, N.P. Structure of the VHL-ElonginC-ElonginB complex: implications for VHL tumor suppressor function. Science 284, 455–461 (1999).

  43. 43

    Johnson, M.L., Correia, J.J., Yphantis, D.A. & Halvorson, H.R. Analysis of data from the analytical ultracentrifuge by nonlinear least-squares techniques. Biophys. J. 36, 575–588 (1981).

  44. 44

    Ralston, G.B. OMMENU (University of Sydney, Australia; 1994).

  45. 45

    Perkins, S.J. Protein volumes and hydration effects. The calculations of partial specific volumes, neutron scattering matchpoints and 280-nm absorption coefficients for proteins and glycoproteins from amino acid sequences. Eur. J. Biochem. 157, 169–180 (1986).

  46. 46

    Hayes, D.B., Laue, T. & Philo, J. SEDNTERP. (University of New Hampshire, USA; 1995).

  47. 47

    Otwinowski, K. & Minor, W. Processing of X-ray diffraction data collected in oscillation mode. Methods Enzymol. 276, 307–326 (1997).

  48. 48

    Brünger, A.T. et al. Crystallography & NMR system: A new software suite for macromolecular structure determination. Acta Crystallogr. D 54, 905–921 (1998).

  49. 49

    Collaborative Computational Project, Number 4. The CCP4 suite: Programs for protein crystallography. Acta Crystallogr. D 50, 760–763 (1994).

  50. 50

    Laskowski, R.A., MacArthur, M.W., Moss, D.S. and Thornton, J.M. PROCHECK: a program to check the stereochemical quality of protein structures. J. Appl. Crystallogr. 26, 283–291 (1993).

  51. 51

    Luthy, R., Bowie, J.U. & Eisenberg, D. Assessment of protein models with three-dimensional profiles. Nature 356, 83–85 (1992).

  52. 52

    Duyao, M.P., Buckler, A.J. & Sonenshein, G.E. Interaction of an NF-κB-like factor with a site upstream of the c-myc promoter. Proc. Natl. Acad. Sci. USA 87, 4727–4731 (1990).

  53. 53

    Seed, B. & Sheen, J.Y. A simple phase-extraction assay for chloramphenicol acyltransferase activity. Gene 67, 271–277 (1988).

  54. 54

    Lee, F.S., Hagler, J., Chen, Z.J. & Maniatis, T. Activation of the IκB α kinase complex by MEKK1, a kinase of the JNK pathway. Cell 88, 213–222 (1997).

  55. 55

    Ishikawa, K. et al. Competitive interaction of seven in absentia homolog-1A and Ca2+/calmodulin with the cytoplasmic tail of group 1 metabotropic glutamate receptors. Genes Cells 4, 381–390 (1999).

  56. 56

    Sourisseau, T. et al. Alteration of the stability of Bag-1 protein in the control of olfactory neuronal apoptosis. J. Cell Sci. 114, 1409–1416 (2001).

  57. 57

    Tanikawa, J. et al. p53 suppresses the c-Myb-induced activation of heat shock transcription factor 3. J. Biol. Chem. 275, 15578–15585 (2000).

  58. 58

    Barton, G.J. ALSCRIPT: a tool to format multiple sequence alignments. Protein Eng. 6, 37–40 (1993).

  59. 59

    Esnouf, R.M. Further additions to MolScript version 1.4, including reading and contouring of electron-density maps. Acta Crystallogr. D. 55, 938–940. (1999).

  60. 60

    Nicholls, A., Sharp, K.A. & Honig, B. Protein folding and association: insights from the interfacial and thermodynamic properties of hydrocarbons. Proteins 11, 281–296 (1991)

Download references


We thank K. Wilson, F. Katsis and B. Cromer (St. Vincent's Institute of Medical Research) for assistance with mass spectrometry and protein expression, and M. Pardee for N-terminal sequencing. We thank H. Tong and other staff at BioCARS for their help with data collection during our visit to the Advanced Photon Source. Access to BioCARS Sector 14 at the Advanced Photon Source at Argonne, Illinois, was provided by the Australian Synchrotron Research Program, which is funded by the Commonwealth of Australia as a Major National Research Facility. BioCARS Sector 14 is supported by the U.S. National Institutes of Health, National Center for Research Resources. The Advanced Photon Source is supported by the U.S. Department of Energy, Basic Energy Sciences, Office of Energy Research. M.W.P. is an Australian Research Council Senior Research Fellow, D.D.L.B. is a National Health and Medical Research Council of Australia Senior Research Fellow and D.S. is an American Heart Association Kenner Fellow. This work was additionally supported by grants from the NIH (NCI) (D.S.).

Author information

Correspondence to David D.L. Bowtell.

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Polekhina, G., House, C., Traficante, N. et al. Siah ubiquitin ligase is structurally related to TRAF and modulates TNF-α signaling. Nat Struct Mol Biol 9, 68–75 (2002) doi:10.1038/nsb743

Download citation

Further reading