TRAF6 coiled-coil domain mediates its trimerization

Tumor necrosis factor receptor-associated factor 6 (TRAF6) is an adaptor protein that plays a critical role in innate immune signaling. TRAF6 consists of an N-terminal RING domain, five zinc fingers, a coiled-coil domain, and a C-terminal TRAF domain. Although structural and oligomeric information for coiled-coiled domains in TRAF2-5 is known, information on TRAF6s coiled-coiled domain is relatively unknown. Here, we characterized the oligomeric state of the human TRAF6 domain (residues 288-348) using size-exclusion chromatography (SEC) and size-exclusion chromatography coupled multi-angle light scattering (SEC-MALS). Both His-tagged and tagless proteins displayed molecular masses consistent with a trimer. Coiled-coil computational tools identified a heptad repeat pattern that supported the trimeric findings. AlphaFold-Multimer modelling further showed a symmetric, trimeric coiled-coiled domain with a conserved hydrophobic core. These results provide experimental support for TRAF6 coiled-coiled domain trimerization and help establish a structural framework for understanding how TRAF6 assembles into higher-order structures.