Abstract
Tumor necrosis factor (TNF) receptor–associated factor (TRAF)-6 mediates Lys63-linked polyubiquitination for NF-κB activation via its N-terminal RING and zinc finger domains. Here we report the crystal structures of TRAF6 and its complex with the ubiquitin-conjugating enzyme (E2) Ubc13. The RING and zinc fingers of TRAF6 assume a rigid, elongated structure. Interaction of TRAF6 with Ubc13 involves direct contacts of the RING and the preceding residues, and the first zinc finger has a structural role. Unexpectedly, this region of TRAF6 is dimeric both in the crystal and in solution, different from the trimeric C-terminal TRAF domain. Structure-based mutagenesis reveals that TRAF6 dimerization is crucial for polyubiquitin synthesis and autoubiquitination. Fluorescence resonance energy transfer analysis shows that TRAF6 dimerization induces higher-order oligomerization of full-length TRAF6. The mismatch of dimeric and trimeric symmetry may provide a mode of infinite oligomerization that facilitates ligand-dependent signal transduction of many immune receptors.
This is a preview of subscription content, access via your institution
Access options
Subscribe to this journal
Receive 12 print issues and online access
$189.00 per year
only $15.75 per issue
Buy this article
- Purchase on Springer Link
- Instant access to full article PDF
Prices may be subject to local taxes which are calculated during checkout
Similar content being viewed by others
References
Wu, H. Assembly of post-receptor signaling complexes for the tumor necrosis factor receptor superfamily. Adv. Protein Chem. 68, 225–279 (2004).
Pineda, G., Ea, C.K. & Chen, Z.J. Ubiquitination and TRAF signaling. Adv. Exp. Med. Biol. 597, 80–92 (2007).
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).
McWhirter, S.M. et al. Crystallographic analysis of CD40 recognition and signaling by human TRAF2. Proc. Natl. Acad. Sci. USA 96, 8408–8413 (1999).
Ye, H. et al. Distinct molecular mechanism for initiating TRAF6 signalling. Nature 418, 443–447 (2002).
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).
Wang, C. et al. TAK1 is a ubiquitin-dependent kinase of MKK and IKK. Nature 412, 346–351 (2001).
Lamothe, B. et al. Site-specific Lys-63-linked tumor necrosis factor receptor-associated factor 6 auto-ubiquitination is a critical determinant of IκB kinase activation. J. Biol. Chem. 282, 4102–4112 (2007).
Hershko, A. & Ciechanover, A. The ubiquitin system. Annu. Rev. Biochem. 67, 425–479 (1998).
Pickart, C.M. & Eddins, M.J. Ubiquitin: structures, functions, mechanisms. Biochim. Biophys. Acta 1695, 55–72 (2004).
Hochstrasser, M. Lingering mysteries of ubiquitin-chain assembly. Cell 124, 27–34 (2006).
Dye, B.T. & Schulman, B.A. Structural mechanisms underlying posttranslational modification by ubiquitin-like proteins. Annu. Rev. Biophys. Biomol. Struct. 36, 131–150 (2007).
Pickart, C.M. & Fushman, D. Polyubiquitin chains: polymeric protein signals. Curr. Opin. Chem. Biol. 8, 610–616 (2004).
Eddins, M.J., Carlile, C.M., Gomez, K.M., Pickart, C.M. & Wolberger, C. Mms2-Ubc13 covalently bound to ubiquitin reveals the structural basis of linkage-specific polyubiquitin chain formation. Nat. Struct. Mol. Biol. 13, 915–920 (2006).
Chen, Z.J. Ubiquitin signalling in the NF-κB pathway. Nat. Cell Biol. 7, 758–765 (2005).
VanDemark, A.P., Hofmann, R.M., Tsui, C., Pickart, C.M. & Wolberger, C. Molecular insights into polyubiquitin chain assembly: crystal structure of the Mms2/Ubc13 heterodimer. Cell 105, 711–720 (2001).
Moraes, T.F. et al. Crystal structure of the human ubiquitin conjugating enzyme complex, hMms2-hUbc13. Nat. Struct. Biol. 8, 669–673 (2001).
McKenna, S. et al. Noncovalent interaction between ubiquitin and the human DNA repair protein Mms2 is required for Ubc13-mediated polyubiquitination. J. Biol. Chem. 276, 40120–40126 (2001).
Ardley, H.C. & Robinson, P.A. E3 ubiquitin ligases. Essays Biochem. 41, 15–30 (2005).
Eletr, Z.M., Huang, D.T., Duda, D.M., Schulman, B.A. & Kuhlman, B. E2 conjugating enzymes must disengage from their E1 enzymes before E3-dependent ubiquitin and ubiquitin-like transfer. Nat. Struct. Mol. Biol. 12, 933–934 (2005).
Huang, D.T. et al. Structural basis for recruitment of Ubc12 by an E2 binding domain in NEDD8's E1. Mol. Cell 17, 341–350 (2005).
Mercier, P. et al. Structure, interactions, and dynamics of the RING domain from human TRAF6. Protein Sci. 16, 602–614 (2007).
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).
Zhang, M. et al. Chaperoned ubiquitylation–crystal structures of the CHIP U box E3 ubiquitin ligase and a CHIP-Ubc13-Uev1a complex. Mol. Cell 20, 525–538 (2005).
Xu, Z. et al. Structure and interactions of the helical and U-box domains of CHIP, the C terminus of HSP70 interacting protein. Biochemistry 45, 4749–4759 (2006).
Ohi, M.D., Vander Kooi, C.W., Rosenberg, J.A., Chazin, W.J. & Gould, K.L. Structural insights into the U-box, a domain associated with multi-ubiquitination. Nat. Struct. Biol. 10, 250–255 (2003).
Rothe, M., Wong, S.C., Henzel, W.J. & Goeddel, D.V. A novel family of putative signal transducers associated with the cytoplasmic domain of the 75 kDa tumor necrosis factor receptor. Cell 78, 681–692 (1994).
Nakano, H. et al. TRAF5, an activator of NF-κB and putative signal transducer for the lymphotoxin-β receptor. J. Biol. Chem. 271, 14661–14664 (1996).
Ishida, T.K. et al. TRAF5, a novel tumor necrosis factor receptor-associated factor family protein, mediates CD40 signaling. Proc. Natl. Acad. Sci. USA 93, 9437–9442 (1996).
Rothe, M., Pan, M.G., Henzel, W.J., Ayres, T.M. & Goeddel, D.V. The TNFR2-TRAF signaling complex contains two novel proteins related to baculoviral inhibitor of apoptosis proteins. Cell 83, 1243–1252 (1995).
Santoro, M.M., Samuel, T., Mitchell, T., Reed, J.C. & Stainier, D.Y. Birc2 (cIap1) regulates endothelial cell integrity and blood vessel homeostasis. Nat. Genet. 39, 1397–1402 (2007).
Mahoney, D.J. et al. Both cIAP1 and cIAP2 regulate TNFα-mediated NF-κB activation. Proc. Natl. Acad. Sci. USA 105, 11778–11783 (2008).
Svergun, D., Baraberato, C. & Koch, M.H. CRYSOL—a program to evaluate X-ray solution scattering of biological macromolecules from atomic coordinates. J. Appl. Cryst. 28, 768–773 (1995).
Kostic, M., Matt, T., Martinez-Yamout, M.A., Dyson, H.J. & Wright, P.E. Solution structure of the Hdm2 C2H2C4 RING, a domain critical for ubiquitination of p53. J. Mol. Biol. 363, 433–450 (2006).
Knipscheer, P. & Sixma, T.K. Protein-protein interactions regulate Ubl conjugation. Curr. Opin. Struct. Biol. 17, 665–673 (2007).
Gazdoiu, S. et al. Proximity-induced activation of human Cdc34 through heterologous dimerization. Proc. Natl. Acad. Sci. USA 102, 15053–15058 (2005).
Brzovic, P.S., Lissounov, A., Christensen, D.E., Hoyt, D.W. & Klevit, R.E.A. UbcH5/ubiquitin noncovalent complex is required for processive BRCA1-directed ubiquitination. Mol. Cell 21, 873–880 (2006).
Hao, B., Oehlmann, S., Sowa, M.E., Harper, J.W. & Pavletich, N.P. Structure of a Fbw7-Skp1-cyclin E complex: multisite-phosphorylated substrate recognition by SCF ubiquitin ligases. Mol. Cell 26, 131–143 (2007).
Tang, X. et al. Suprafacial orientation of the SCFCdc4 dimer accommodates multiple geometries for substrate ubiquitination. Cell 129, 1165–1176 (2007).
Peschard, P. et al. Structural basis for ubiquitin-mediated dimerization and activation of the ubiquitin protein ligase Cbl-b. Mol. Cell 27, 474–485 (2007).
Yang, J.K. et al. Crystal structure of MC159 reveals molecular mechanism of DISC assembly and FLIP inhibition. Mol. Cell 20, 939–949 (2005).
Carrington, P.E. et al. The structure of FADD and its mode of interaction with procaspase-8. Mol. Cell 22, 599–610 (2006).
Siegel, R.M. et al. SPOTS: signaling protein oligomeric transduction structures are early mediators of death receptor-induced apoptosis at the plasma membrane. J. Cell Biol. 167, 735–744 (2004).
Svergun, D.I. Restoring low resolution structure of biological macromolecules from solution scattering using simulated annealing. Biophys. J. 76, 2879–2886 (1999).
Otwinowski, Z. & Minor, W. Processing of X-ray diffraction data collected in oscillation mode. Methods Enzymol. 276, 307–326 (1997).
Hendrickson, W.A. Analysis of protein structures from diffraction measurements at multiple wavelengths. Trans. Am. Crystallogr. Assoc. 21, 11 (1985).
Terwilliger, T. SOLVE and RESOLVE: automated structure solution, density modification and model building. J. Synchrotron Radiat. 11, 49–52 (2004).
Emsley, P. & Cowtan, K. Coot: model-building tools for molecular graphics. Acta Crystallogr. D Biol. Crystallogr. 60, 2126–2132 (2004).
Brunger, A.T. et al. Crystallography & NMR system: a new software suite for macromolecular structure determination. Acta Crystallogr. D. Biol. Crystallogr. 54, 905–921 (1998).
Collaborative Computational Project N. The CCP4 suite: programs for protein crystallography. Acta Crystallogr. D Biol. Crystallogr. 50, 760–763 (1994).
Evans, S.V. SETOR: hardware-lighted three-dimensional solid model representations of macromolecules. J. Mol. Graph. 11, 134–138 (1993).
Myszka, D.G. Improving biosensor analysis. J. Mol. Recognit. 12, 279–284 (1999).
Iyer, R.R. et al. The MutSα-proliferating cell nuclear antigen interaction in human DNA mismatch repair. J. Biol. Chem. 283, 13310–13319 (2008).
Konarev, P.V., Volkov, V.V., Sokolova, A.V., Koch, M.H.J. & Svergun, D.I. PRIMUS: a Windows PC-based system for small-angle scattering data analysis. J. Appl. Crystallogr. 36, 1277–1282 (2003).
Svergun, D.I. Determination of the regularization parameter in indirect-transform methods using perceptual criteria. J. Appl. Crystallogr. 25, 495–503 (1992).
Nagar, B. et al. Organization of the SH3–SH2 unit in active and inactive forms of the c-Abl tyrosine kinase. Mol. Cell 21, 787–798 (2006).
Svergun, D.I., Petoukhov, M.V. & Koch, M.H. Determination of domain structure of proteins from X-ray solution scattering. Biophys. J. 80, 2946–2953 (2001).
Kozin, M.B. & Svergun, D.I. Automated matching of high- and low-resolution structural models. J. Appl. Crystallogr. 34, 33–41 (2001).
Glater, O. & Kratky, O. Small Angle X-ray Scattering (Academic, London, 1982).
Acknowledgements
We thank T. Min and J.Y. Chung for their earlier work on the project, X. Jiang and X. Wang of the Sloan-Kettering Institute for purified E1, Z. Chen of the University of Texas Southwestern Medical School for the expression constructs of Ubc13 and Uev1A, R. Abramowitz and J. Schwanof of X4A of the National Synchrotron Light Source for data collection and J. Wu for maintaining our X-ray and computer equipment. This work was supported by the US National Institutes of Health (RO1 AI045937 to H.W. and RO1 AR053540 to B.G.D.), the US Department of Defense (DOE Contract DE-AC02-05CH11231 for G.H.), the Intramural Research Program of the US National Institute of Allergy and Infectious Diseases (to L.Z. and M.J.L.) and institutional start-up funds to B.G.D., S.-C.L. and Y.-C.L. from the Cancer Research Institute and to M.L. from the American Heart Association.
Author information
Authors and Affiliations
Corresponding author
Supplementary information
Supplementary Text and Figures
Supplementary Figures 1–7 and Supplementary Methods (PDF 1798 kb)
Rights and permissions
About this article
Cite this article
Yin, Q., Lin, SC., Lamothe, B. et al. E2 interaction and dimerization in the crystal structure of TRAF6. Nat Struct Mol Biol 16, 658–666 (2009). https://doi.org/10.1038/nsmb.1605
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1038/nsmb.1605
This article is cited by
-
Epstein-Barr virus-driven B cell lymphoma mediated by a direct LMP1-TRAF6 complex
Nature Communications (2024)
-
TRAF6 regulates the abundance of RIPK1 and inhibits the RIPK1/RIPK3/MLKL necroptosis signaling pathway and affects the progression of colorectal cancer
Cell Death & Disease (2023)
-
The protease calpain2a limits innate immunity by targeting TRAF6 in teleost fish
Communications Biology (2023)
-
Intrinsic disorder in proteins associated with oxidative stress-induced JNK signaling
Cellular and Molecular Life Sciences (2022)
-
Kruppel-like factor 13 inhibits cell proliferation of gastric cancer by inducing autophagic degradation of β-catenin
Discover Oncology (2022)