Comparison of the peptide binding preferences of three closely related TRAF paralogs: TRAF2, TRAF3, and TRAF5

Abstract

Tumor necrosis factor receptor-associated factors (TRAFs) constitute a family of adapter proteins that act in numerous signaling pathways important in human biology and disease. The MATH domain of TRAF proteins binds peptides found in the cytoplasmic domains of signaling receptors, thereby connecting extracellular signals to downstream effectors. Beyond several very general motifs, the peptide binding preferences of TRAFs have not been extensively characterized, and differences between the binding preferences of TRAF paralogs are poorly understood. Here we report a screening system that we established to explore TRAF peptidebinding specificity using deep mutational scanning of TRAF-peptide ligands. We displayed single-and double-mutant peptide libraries based on the TRAF-binding sites of CD40 or TANK on the surface of Escherichia coli and screened them for binding to TRAF2, TRAF3, and TRAF5. Enrichment analysis of the library sequencing results showed differences in the permitted substitution patterns in the TANK versus CD40 backgrounds. The three TRAF proteins also demonstrated different preferences for binding to members of the CD40 library, and three peptides from that library that were analyzed individually showed striking differences in affinity for the three TRAFs. These results illustrate a previously unappreciated level of binding specificity between these close paralogs and demonstrate that established motifs are overly simplistic. The results from this work begin to outline differences between TRAF family members, and the experimental approach established herein will enable future efforts to investigate and redesign TRAF peptide-binding specificity.

TRAF Molecules in Inflammation and Inflammatory Diseases

“…TRAF3 is closely related to TRAF2 in terms of both structure and function [5,9]. TRAF3 is ubiquitously expressed in various cell types, and is pivotal in regulating inflammation in macrophages, dendritic cells (DCs), neutrophils, B cells, T cells, hepatocytes, cardiocytes, osteoclasts, microglia, and neurons (Fig.…”

“…The uniqueness and specificity of the binding of each TRAF molecule to various receptors is mainly mediated by minor structural differences in the TRAF-C domain that recognizes major and minor consensus sequences of the cytoplasmic tails of receptors or their associated adaptor proteins [7,8]. However, a recent study points out that the binding preferences of TRAF proteins may be more complicated than previously appreciated as TRAF molecules also exhibit binding preferences beyond the established core motifs [9], thus warranting further investigation on this aspect. In addition to their role as adaptor proteins, TRAFs (including TRAF2, 3, 5 and 6) also act as E3 ubiquitin ligases [3–5].…”

Comparison of Target Recognition by TRAF1 and TRAF2

“…As the sequences of the binding hot spots are conserved in TRAF1 and TRAF2, they also share the same binding consensus motifs, namely, a major motif, Px(Q/E)E, and two minor motifs, Px(Q/E)xxD and PxQxT (Figure 4a) [3]. Although possessing nearly identical receptor-binding sites, a previous deep mutational scanning study revealed key differences in the binding preferences and affinities between TRAF1 and TRAF2 [34]. In this regard, we wondered how TRAF1 and TRAF2 bind differently even though they share almost identical receptor-binding motifs.…”

“…Seven TRAF family members have been identified in mammals and each member performs distinct functions, especially in immune cell signaling. Although containing nearly identical receptor-binding environments composed of extremely conserved amino acid residues, especially TRAF1, TRAF2, TRAF3, and TRAF5, a recent study showed that key differences in binding preferences with different affinities exist, which might be important for TRAF-mediated signal transduction [34]. To understand how TRAF1 and TRAF2 have adaptor and receptor preferences with different affinities-even though they share almost identical binding motifs-we analyzed and compared adaptor (TRADD) and receptor (TANK and caspase-2) affinities for TRAF1 and TRAF2.…”

Roles of TRAFs in Ischemia-Reperfusion Injury

“…TRAF5 is much less studied than TRAF3, although TRAF5 shares the highest sequence with TRAF3 in the TRAF family (Foight and Keating, 2016;Kim et al, 2020). Similarly, TRAF5 contains an N-terminal RING domain, five zinc fingers at the middle of its sequence, a coiled-coil domain, and a MATH domain included in the C-terminal TRAF domain (Xu et al, 2020).…”