Structural Insight Into The Trim Family Of Ubiquitin E3Ligases

So far, there have been more than80members of TRIM family protein found inhuman genome. TRIM proteins contain three conserved motif in the N terminus, whichare RING domain, B-box domain and coiled-coil domain while the C-terminal domainis variable. For the involvement in innate immunity, transcription, apoptosis and manyother life events, TRIM protein shows more and more significance in its biological rolesrecently. Based on the existence of the conserved RING domain, many TRIM familymembers have been reported to function as an E3ligase in the ubiquitin modification.However, the mechanism of TRIM protein exert the E3ligase activity is still unclear.The B-Box domain which locates next to the RING domain may evolve from the RINGdomain according to their sequence similarity, while the functional study of B-Box inTRIM protein is only on very few members and not in depth. Coiled-coil domainmediate the homo-or hetero-oligomerization among TRIM family and other proteins,but the structural basis of coiled-coil domain oligomerization has not been well studiedBased on answering the unsolved questions above on TRIM protein, we usecrystallography to study the CCD (Coiled coil domain) of TRIM69. Quite differentfrom the former model proposed by the other group, TRIM69CCD has beendemonstrated to be an anti-parallel dimer but not a parallel dimer or trimer. Thesequence alignment by bioinformatics study illustrates the molecular mechanism ofhomo-or hetero-oligomerization of TRIM protein through their CCD domain. Based onthe structure, a model of full-length TRIM69protein has been proposed which may beapplicable for most members. Due to the conservation of TRIM protein, TRIM25withthe determined substrate is used to verify the model. After expression and purificationof the mutation, truncation and full-length TRIM25protein, a series of biochemical andcellular molecular studies have been used together with some in vivo and in vitroubiquitination assays to verify the model. During the study, two conserved Glutamateare found to be very essential for the E3ligase activities.In all, by using structural biology, biochemistry, molecular biology and cellularbiology, our studies on TRIM protein have proposed a new model that demonstratingthe mechanism of TRIM protein function as E3ligase and provide a broad view in the role that TRIM protein are playing in innate immunity, cancer, cell proliferation anddifferentiation.