Protein-protein interactions in TRAF3.

TNF-receptor-associated factors (TRAFs) are intracellular proteins that bind to the cytoplasmic portion of TNF receptors and mediate downstream signaling. The six known TRAF proteins play overlapping yet distinct roles in controlling immune responses as well as cellular processes such as activation of NF-kappaB and JNK signaling pathways. For example, CD40 binds to TRAF2, TRAF3 and TRAF6 to control B cell differentiation, proliferation and growth. In contrast, binding of lymphotoxin-beta receptor (LTbetaR) to TRAF2 and TRAF5 propagates signals leading to activation of NF-kappaB, while binding to TRAF3 induces negative regulation of this pathway and leads to apoptosis in tumor cells. Binding recognition is mediated by specific contacts of a consensus recognition sequence in the partner with residues in a hydrophobic crevice on the TRAF molecule. Since each of these protein-protein interactions occurs within this same binding crevice, it appears that TRAF-mediated cellular mechanisms may be regulated, in part, by the level of expression or recruitment of the adaptor proteins or receptors that are competing for the crevice. The specific contacts of CD40, LTbetaR and BAFF-R have been defined in crystal structures of the complex with TRAF3. In addition, the downstream regulator TANK and the viral oncogenic protein LMP1 from the Epstein Barr virus also bind to the same TRAF crevice and these contacts have also been described crystallographically. Comparison of these five crystal structures has revealed that the recognition motifs in each of these proteins are accommodated in one TRAF3 binding crevice and that the binding interface is structurally and functionally adaptive. In this chapter, the molecular details of the interactions will be described and correlated with the functional implications for multiple TRAF3 roles in cellular regulation.

Molecular basis for regulation of Src by the docking protein p130Cas.

The docking protein p130Cas (Cas) becomes tyrosine-phosphorylated in its central substrate domain in response to extracellular stimuli such as integrin-mediated cell adhesion, and transmits signals through interactions with various intracellular signaling molecules such as the adaptor protein Crk. Src-family kinases (SFKs) bind a specific site in the carboxyl-terminal region of Cas and subsequently SFKs phosphorylate progressively the substrate domain in Cas. In this study crystallography, mutagenesis and binding assays were used to understand the molecular basis for Cas interactions with SFKs. Tyrosine phosphorylation regulates binding of Cas to SFKs, and the primary site for this phosphorylation, Y762, has been proposed. A phosphorylated peptide corresponding to Cas residues 759MEDpYDYVHL767 containing the key phosphotyrosine was crystallized in complex with the SH3-SH2 domain of the SFK Lck. The results provide the first structural data for this protein-protein interaction. The motif in Cas 762pYDYV binds to the SH2 domain in a mode that mimics high-affinity ligands, involving dual contacts of Y762 and V765 with conserved residues in SFK SH2 domains. In addition, Y764 is in position to make an electrostatic contact after phosphorylation with a conserved SFK arginine that mediates interactions with other high-affinity SH2 binders. These new molecular data suggest that Cas may regulate activity of Src as a competing ligand to displace intramolecular interactions that occur in SFKs (between the C-terminal tail and the SH2 domain) and restrain and down-regulate the kinase in an inactive form.