Structural basis for the lack of E2 interaction in the RING domain of TRAF2

Abstract

TRAF proteins are intracellular signal transducers for a number of immune receptor superfamilies. Specifically, TRAF2 interacts with members of the TNF receptor superfamily and connects the receptors to downstream signaling proteins. It has been assumed that TRAF2 is a ubiquitin ligase like TRAF6 and mediates K63-linked polyubiquitination of RIP1, a kinase pivotal in TNFalpha-induced NF-kappaB activation. Here we report the crystal structure of the RING and the first zinc finger domains of TRAF2. We show that the TRAF2 RING structure is very different from the known TRAF6 RING structure. The differences are multifaceted, including amino acid differences at the critical Ubc13-interacting site, local conformational differences, and a unique nine-residue insertion between the RING domain and the first zinc finger in TRAF2. These structural differences prevent TRAF2 from interacting with Ubc13 and other related E2s via steric clash and unfavorable interfaces. Our structural observation should prompt a re-evaluation of the role of TRAF2 in TNFalpha signaling and may indicate that TRAF2-associated proteins such as cIAPs may be the ubiquitin ligases for NF-kappaB signaling.

FIGURE 1

Structure of the human TRAF2 RING and first zinc finger domains (RZ₁). (A) Domain organization of TRAF2. Z₁–Z₄: zinc finger domains 1–4; CC: coiled coil domain. (B) A ribbon diagram of TRAF2 RZ₁ colored in a rainbow mode from N- to C-termini. Secondary structure elements are labeled. (C) A region of SAD-phased electron density at 1.5 σ superimposed with the final model. (D) Cα trace of TRAF2 RZ₁ showing the zinc-coordinating residues. (E) Sequence alignment of TRAF2 from different species and with TRAF3, TRAF5 and TRAF6. Gray: zinc-coordinating residues; cyan: TRAF2-unique insertion; red: TRAF2 residues that would have been in clash with Ubc13; green: TRAF2 dimerization interface residues; yellow: residues in TRAF6 that contact Ubc13 and their equivalents in other TRAFs.

FIGURE 2

TRAF2 dimerization in the crystal and in solution. (A) TRAF2 RZ₁ dimer shown in a ribbon diagram colored in a rainbow mode. (B) superposition of the TRAF2 RZ₁ dimer (red and cyan) with the TRAF6 RZ₁₂₃ dimer (PDB code 3HCS, magenta and green). (C) TRAF2 RZ₁ dimer shown with residues at the dimerization interface. The region that is shown in detail in (D) is blocked by a rectangle. (D) Details of the TRAF2 dimerization interaction. Critical residues are labeled on one side of the symmetrical interface. (E) Superposition of gel filtration profiles of WT TRAF2 RZ₁ (High: at higher concentration; Low: at lower concentration), and Q46A, F91A, N94A and R98A mutants of TRAF2 RZ₁. MALS measurement results for WT TRAF2 RZ₁, the F91A mutant and the R98A mutant, as well as elution positions of molecular weight standards, are shown. (F) TRAF2 RING dimerization mutants rescued TNFα-induced IκBα phosphorylation in TRAF2/TRAF5 double knockout MEFs.

FIGURE 3

Comparison between TRAF2 RZ₁ (Cyan) and TRAF6 RZ₁₂₃ (magenta). (A) and (B) Superposition of TRAF2 and TRAF6 RING domains, showing the location of the unique insertion of TRAF2 in two roughly orthogonal orientations. (C) Superposition of the first zinc finger domains of TRAF2 and TRAF6. (D) and (E) Superposition of TRAF2 RZ₁ with TRAF6 RZ₁₂₃, showing the difference in relative orientation of the zinc finger domain relative to the RING domain. In (E), the Z₁–Z₃ domains of TRAF6 were superimposed onto the Z₁ of TRAF2 to show more clearly the relative rotation of ~20°.

FIGURE 4

Lack of interaction between TRAF2 and E2s. (A) Native PAGE of TRAF2 RZ₁ with Ubc13 using 8–25% gradient PhastGel on a PhastSystem (GE Healthcare). A similar native PAGE of TRAF6 RZ₁₂₃ with Ubc13 is shown as a positive control. The gels were stained with coomassie blue. (B) TRAF2 residues (in red) that would have been in steric clash with Ubc13 if the interaction follows the mode of the TRAF6: Ubc13 interaction (PDB code 3HCU). (C) A close up view of the superposition of the RING domains of TRAF2 (cyan) and TRAF6 (magenta). TRAF6 residues in direct contact with Ubc13 and their equivalents for TRAF2 are shown in yellow for TRAF2 and in gray for TRAF6. (D) Conformational difference at the region immediately preceding the RING between TRAF2 and TRAF6. (E) Superposition of UbcH5b (PDB code 2CLW) onto the hypothetical TRAF2: Ubc13 complex, showing that the TRAF2 residues in clash with Ubc13 would have been in clash with Ubc5Hb as well.

FIGURE 5

Ubiquitination assays. (A) Auto-ubiquitination and free chain polyubiquitin synthesis in vitro by TRAF2, TRAF3, TRAF5 and TRAF6 with a series of E2s. The indicated GST-fusion proteins bound to glutathione-agarose beads were subjected to an in vitro ubiquitination assay in the presence of the indicated E2s. Following the ubiquitination assay, a portion of the supernatant was subjected to SDS-PAGE and immunoblotted with anti-ubiquitin (middle panels). The beads were washed, subjected to SDS-PAGE, and immunoblotted with anti-ubiquitin (top panels). The membranes were stained with Ponceau S and one representative membrane is shown (bottom panel). G: GST; 6: GST-TRAF6; 2: GST-TRAF2; 3: GST-TRAF3; 5: GST-TRAF5. (B) cIAP1 and cIAP2 undergo RING-dependent auto-ubiquitination in the presence of the Ubc13/Uev1A E2 complex. C571R: RING mutant of cIAP1. C557R: RING mutant of cIAP2.