High-affinity interactions of tumor necrosis factor receptor-associated factors (TRAFs) and CD40 require TRAF trimerization and CD40 multimerization

Signaling by some TNF receptor family members, including CD40, is mediated by TNF receptor-associated factors (TDAFs) that interact with receptor cyto plasmic domains following ligand- induced receptor oligomerization. Here we have defined the oligomeric structure of recombinant TRAF domains that dir ectly interact with CD40 and quantitated the affinities of TRAF2 and TRAF3 for CD40. Biochemical and biophysical analyses demonstrated that TRAF domai ns of TRAF1, TRAF2, TRAF3, and TRAF6 formed homo-trimers in solution. N-ter minal deletions of TRAF2 and TRAF3 defined minimal amino acid sequences nec essary for trimer formation and indicated that the coiled coil TRAF-N regio n is required for trimerization. Consistent with the idea that TRAF trimeri zation is required for high-affinity interactions with CD40, monomeric TRAF -C domains bound to CD40 significantly weaker than trimeric TRAFs. In surfa ce plasmon resonance studies, a hierarchy of affinity of trimeric TRAFs for trimeric CD40 was found to be TRAF2 > TRAF3 much greater than TRAF1 and TR AF6. CD40 trimerization was demonstrated to be sufficient for optimal NF-ka ppa B and p38 mitogen activated protein kinase activation through wild-type CD40. In contrast, a higher degree of CD40 multimerization was necessary f or maximal signaling in a cell line expressing a mutated CD40 (T254A) that signaled only through TRAF6. The affinities of TRAF proteins for oligomeriz ed receptors as well as different requirements for degree of receptor multi merization appear to contribute to the selectivity of TRAF recruitment to r eceptor cytoplasmic domains.