Dynamics of the RING domain from human TRAF6 by 15N NMR spectroscopy: implications for biological function.

Activation of transcription factor NF-kappaB requires Lys63-linked polyubiquitination of the E3 ubiquitin ligase TRAF6 via protein-protein interactions mediated by a RING domain. In this study, intra- and intermolecular chemical exchange processes of the TRAF6 RING domain were analyzed by (15)N NMR spectroscopy. Micro- to millisecond time scale motions were assessed through R 1, R 2, NOE, and cross-correlated relaxation measurements, and the kinetics of these motions were quantified with relaxation dispersion. The relaxation experiments indicate that the protein core is rigid, consistent with the functional requirement that RING domains form a binding scaffold for E2 ubiquitin conjugation enzymes. Chemical exchange is observed at the C-terminal end of the main alpha-helix of the RING domain. The C-terminal end of the main alpha-helix from the RING domain is involved in E2-E3 interactions, and modulation of slow motions for this region of the helix may be a general mechanism by which these interactions achieve ubiquitin transfer. Chemical shift mapping indicates that the TRAF6 RING domain does not self-associate in solution. Numerous RING domains are homo- or heterodimeric, and this is thought to be a functional necessity for recruitment of substrates for ubiquitination, or recruitment of multiple E2 enzymes for efficient substrate ubiquitination. However, lack of self-association for the RING domain from TRAF6, and the observation that the intact protein is a trimer, suggests that close association of RING domains within a homodimeric scaffold may not be a fundamental requirement for biological function.