Comparison of Target Recognition by TRAF1 and TRAF2

Abstract

Although TRAF1 and TRAF2 share common receptors and have extremely conserved amino acid residues, recent studies have shown that key differences in receptor binding preferences with different affinities exist, which might be important for their different functions in TRAF-mediated signal transduction. To better understand TRAF1 and TRAF2 signaling, we analyzed and compared their receptor binding-affinities. Our study revealed that TRADD, TANK, and caspase-2 bind to both TRAF1 and TRAF2 with different affinities in vitro. Sequence and structural analyses revealed that S454 on TRAF2 (corresponding to A369 of TRAF1) is critical for the binding of TRADD, and F347 on TRAF1 (corresponding to L432 of TRAF2) is a critical determinant for high affinity binding of TANK and caspase-2.

Keywords: TRADD; TRAF; apoptosis; inflammation; protein interaction.

Figure 1

Domain organization and sequence comparison between TRAF1 and TRAF2. (a) General domains of the TRAF family. (b) Structural comparison of the TRAF family by superposition. All known structures of the TRAF domain from each TRAF member were superimposed using Pymol. (c) Domains of TRAF1 and TRAF2. (d) Sequence comparison of the TRAF domain between TRAF1 and TRAF2. Green highlighted and un-highlighted parts indicate TRAF-N and TRAF-C, respectively. Secondary structures are shown above the corresponding residues.

Figure 2

In vitro TRADD interactions between TRAF1 and TRAF2. (a) Schematic diagrams of the domain compositions of TRAF1 and TRAF2 with their tentative binding partner, TRADD. Binding region is indicated by red dotted box. (b) Native-PAGE. Lane1: TRAF1, Lane2: TRAF2, Lane3: TRADD-N, Lane4: TRAF1+TRADD-N, Lane5: TRAF2+TRADD-N. The white arrow indicates the position of the disappearance of TRADD-N due to complex formation. The black arrow indicates a newly produced band due to complex formation. (c,d) Size-exclusion chromatography profiles. Elution profiles of protein-complex peaks generated by mixing TRAF1 and TRADD-N (c) and TRAF2 and TRADD-N (d) and fractions loaded on SDS-PAGE (shown at the right part from the peaks). Loaded factions are indicated by black bar.

Figure 3

Quantitative affinity and structure analyses of the interaction between TRAFs and TRADD. (a,b) SPR characterization of the TRADD-N interaction with TRAF1 (a) and TRAF2 (b). TRADD-N was applied to TRAF1- and TRAF2-coupled sensor chips. (c,d) Structural models of TRAF1:TRADD-N complexes generated by the structural information of the previously solved TRAF2:TRADD-N complex. Light blue and pink colors indicate TRAF2 and TRAF1, respectively. Yellow color indicates TRADD-N that binds to TRAF2. Side view (c) and top view (d) are shown. (e) Two TRADD interacting regions on TRAF2. (f) Sequence alignment of the TRADD binding regions of TRAF1 and TRAF2. Red and blue colors indicate the TRADD interacting residues of TRAF1 and TRAF2 at Regions I and II, respectively. The red star indicates the amino acid residue that differs among the residues involved in the TRADD interaction. (g) Close-up view of the Region I interaction interface. Light blue and pink colors indicate TRAF2 and TRAF1, respectively. Yellow color indicates TRADD-N. The star marks the residues that differ among residues that are involved in the TRADD interaction. The interactions and distances are indicated by dashed line and number, respectively. The units of distance are in Å.

Figure 4

Quantitative interaction analyses of TANK and caspase-2 on TRAF1 and TRAF2. (a) TRAF1- and TRAF2-binding motif. Major motif Px(Q/E)E and minor motifs Px(Q/E)xxD or PxQxT are shown in the upper and lower panels, respectively. (b) Peptide sequences of TANK and caspase-2 that are used in the interaction study and contain TRAF1- and TRAF2-binding motifs, indicated as red color. (c–f) Isothermal titration calorimetry (ITC) used to assess peptide-protein interactions. The TANK peptide was titrated into a TRAF1 solution (c) and TRAF2 solution (d). Additionally, the caspase-2 peptide was titrated into a TRAF1 (e) and TRAF2 solution (f). Experimental fitting of the data to a single site interaction model is shown. Calorimetric titration and fitting data are shown in the upper and lower panels, respectively. The data presented are the average from two independent experiments.

Figure 5

Structure analyses of the interaction between TRAFs and receptors, including TANK and caspase-2. (a) Prototype trimeric structure of the TRAF-C domain of TRAFs. Three domains are shown with different colors. Receptor-interaction regions are marked with black stars. Receptor binding hot spots are indicated. Top and side views are shown in the left and right panels, respectively. (b) Receptor-binding hot spots and conserved amino acid residues in TRAF1 and TRAF2 which are involved in the interaction with various receptors. The amino acid residues that are not conserved are colored in red. (c) Comparison of the receptor-interacting region between TRAF1 and TRAF2. The structure of TRAF1 (yellow):TANK (gray peptide) complex was compared with the structure of TRAF2 (green):LMP1 (magenta peptide) complex by structural superposition. All the amino acid residues in TRAF2 involved in the interaction with the LMP1 peptide are labelled in green. Non-conserved amino acid residues are labelled in red. Amino acid positions of the TRAF-binding motif in the receptor peptides are labeled as P-2 and P0. Close-up views of binding hot spot1 and hot spot2 are shown in the left and right panels, respectively.