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E2 interaction and dimerization in the crystal structure of TRAF6


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.

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Figure 1: Biochemical characterization of the TRAF6–Ubc13 interaction and structure of the N-terminal region of TRAF6.
Figure 2: Structural analysis of the TRAF6–Ubc13 interaction.
Figure 3: Cellular effects of TRAF6 mutants that fail to interact with Ubc13.
Figure 4: TRAF6 dimerization is crucial for its ability to promote polyubiquitin chain synthesis.
Figure 5: Cellular effects of TRAF6 mutants that fail to dimerize.
Figure 6: TRAF6 oligomerization.

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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.

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Correspondence to Hao Wu.

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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).

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