Crystal structure of TRAF1 TRAF domain and its implications in the TRAF1-mediated intracellular signaling pathway

Abstract

TNF-receptor associated factor (TRAF) proteins are key adaptor molecules containing E3 ubiquitin ligase activity that play a critical role in immune cell signaling. TRAF1 is a unique family of TRAF lacking the N-terminal RING finger domain. TRAF1 is an important scaffold protein that participates in TNFR2 signaling in T cells as a negative or positive regulator via direct interaction with TRAF2, which has recently been identified as a pro-apoptotic regulator in neuronal cell death. Here, we report the first crystal structure of the TRAF1 TRAF domain containing both the TRAF-N coiled-coil domain and the TRAF-C domain. Our structure reveals both similarities and differences with other TRAF family members, which may be functionally relevant to TRAFs. We also found that the TRAF-N coiled-coil domain of TRAF1 is critical for the trimer formation and stability of the protein. Finally, we found that conserved surface residues on the TRAF1 TRAF domain that might be binding hot spots that are critical for interaction with signaling molecules.

Molecular basis for TANK recognition by TRAF1 revealed by the crystal structure of TRAF1/TANK complex

“…The seven known TRAF proteins, TRAF1-TRAF7, play distinct roles in regulating immune responses, as well as in critical cellular signaling events, such as NF-kappaB, JNK, and apoptosis signaling pathways [2,9,10]. This TRAF domain mediates protein-protein interactions [11][12][13]. This TRAF domain mediates protein-protein interactions [11][12][13].…”

“…Expression and purification details for TRAF1 TRAF domain have been described in our previous study [12]. Expression and purification details for TRAF1 TRAF domain have been described in our previous study [12].…”

“…The structures of TRAF domains of the proteins TRAF1-TRAF6 have Abbreviations ITC, isothermal titration calorimetry; LB, Luria-Bertani; RMSD, root-mean-square deviation; SEC, size-exclusion chromatography; SPR, surface plasmon resonance; TANK, TRAF family member-associated NF-kappaB activator; TRAF, tumor necrosis factor receptor-associated factor. been elucidated [11,12,[14][15][16]. TRAF domains are composed of seven to eight antiparallel b-strands folds (TRAF-C) and a coiled-coil region (TRAF-N).…”

“…Escherichia coli was used for the expression of TRAF1 TRAF domain (amino acid residues: 220-416) and TRAF2 TRAF domain (amino acid residues: 264-501). Expression and purification details for TRAF1 TRAF domain have been described in our previous study [12]. To obtain expression constructs of TRAF2, TRAF domain was cloned into pET24a vector containing the C-terminal hexahistidine (6xHIS) tag.…”

“…Although it has been suggested that TANK can interact with TRAF1, 2, and 3 using qualitative analysis, quantitative analysis of their interaction was reported from TRAF3 and TANK interaction studies [25]. Recently, when we reported the structure of TRAF domain of TRAF1, we introduced the tentative in vitro interaction between TRAF1 and TANK [12]. Based on these preliminary binding data, we thoroughly analyzed the interaction between TRAF domains of TRAF1 or TRAF2 and TANK peptide (SVPIQCTD KT) using ITC and SPR.…”