Structure, interactions, and dynamics of the RING domain from human TRAF6

Abstract

A key step in the signaling cascade responsible for activation of the transcription factor NF-kB involves Lys63-linked polyubiquitination of TRAF6. Covalent attachment of ubiquitin (Ub) to TRAF6, and subsequent poly(Ub) chain synthesis, is catalyzed by the hUev1a-hUbc13 heterodimer. hUbc13 is a catalytically competent E2 enzyme, and hUev1a is an E2-like protein that binds substrate Ub. The hUev1a-hUbc13 heterodimer is targeted to TRAF6 through interactions between hUbc13 and the N-terminal RING domain from TRAF6. Nuclear magnetic resonance (NMR) spectroscopy was used to determine the solution state structure of the RING domain from human TRAF6, and the interaction between hUbc13 and TRAF6 was characterized using NMR chemical shift mapping. The main-chain dynamics of the RING domain from TRAF6 were studied using 15 N NMR relaxation. Analysis of the main-chain dynamics data indicates that residues within the a-helix and b-sheet of the RING domain are as rigid as regions of canonical secondary structure in larger proteins, consistent with the biological role of RING-domain E3 proteins, which requires that the E3 contain a recognition site for recruitment of E2 ubiquitin conjugation enzymes. The C-terminal MATH domain from TRAF2 and TRAF6 is involved in interactions with the cytoplasmic domains of TNF receptors (Wu and Arron 2003). The structure of the MATH or TRAF domain from TRAF6 has been determined using X-ray crystallography and forms an eight-stranded antiparallel b-sandwich (PDB: 1LB6) . The structure of the C-terminal region of TRAF2 is a trimer with a ''mushroom'' shape, whose self-association is due in part to the coiled-coil region, which forms the stalk of the mushroom (PDB: 1CA4) Reprint requests to: Leo Spyracopoulos, Department of Biochemistry, Room 416, Medical Sciences Building, University of Alberta, Edmonton, Alberta T6G 2H7, Canada; e-mail: leo.spyracopoulos@ ualberta.ca; fax: (780) 492-0886.Abbreviations: E1, ubiquitin-activating enzyme; E2, ubiquitin-conjugating enzyme; E3, ubiquitin protein ligase; HSQC, heteronuclear single quantum coherence; MATH domain, merpin and TRAF homology domain; NMR, nuclear magnetic resonance; NOE, nuclear Overhauser effect; RING, really interesting new gene; RMSD, root-mean-square deviation; R 1 , longitudinal relaxation rate; R 2 , transverse relaxation rate; TNF, tumor necrosis factor; TRAF, tumor necrosis receptor associated factor; TRAF6-RD, TRAF6-RING domain; Ub, ubiquitin.Article published online ahead of print. Article and publication date are at

The RING Domain and First Zinc Finger of TRAF6 Coordinate Signaling by Interleukin-1, Lipopolysaccharide, and RANKL

“…Previous structural studies using NMR revealed that the RING domain (residues 67-124) of TRAF6 presents a topology similar to those of other RING domains, indicating that the RING adopts a correctly folded structure in the absence of ZF1 (30). Interestingly, the NMR structure of TRAF6 RING revealed a very weak affinity for Ubc13 in the low millimolar range (30), which is too weak to be biologically relevant. This result suggests that other regions contiguous to the RING and possibly the first ZF domain may serve to increase the affinity of TRAF6 for Ubc13.…”

Structural basis for the recognition and degradation of host TRIM proteins by Salmonella effector SopA

“…( b , c , d , e ) TRIM56 RING domain was aligned with various closely related RINGs showing the clashes (circled) of SopA with the central α-helix of various RING domains ( b , TRIM32; c , TRAF6; d , RNF4; e , TRIM39). PDB codes of the structures used in the alignment are indicated in brackets: (2JMD)47, (4AP4)30. ( f ) SopA does not bind TRIM32, TRIM39 and RNF4.…”

Molecular Mechanisms of TNFR-associated Factor 6 (TRAF6) Utilization by the Oncogenic Viral Mimic of CD40, Latent Membrane Protein 1 (LMP1)

“…The TRAF-C domain mediates receptor binding to TNFR superfamily members (50,51), and the absence of this domain abolishes TRAF6 binding to members of this superfamily (29,54,55). Interestingly, the TRAF6 TRAF-C domain is not required for several important CD40-mediated TRAF6-dependent events, indicating that CD40 may use TRAF6 as a strictly CY adaptor protein in certain situations (29), similar to TRAF6 use by the Toll-like receptors (44).…”