How do TRIM21 and TRIM5¿ execute dual antiviral effector and signalling functions? (2016–2018)
Abstract:
TRIM21 and TRIM5¿ are intracellular proteins that provide important antiviral defence. Both TRIM21 and TRIM5¿ have intrinsic E3 ubiquitin ligases activity, and associate with multiple E2 ubiquitin-conjugating enzymes to catalyse the formation of poly-ubiquitin chains with different ubiquitin linkages. Distinct ubiquitin polymers trigger innate immune signalling and recruit the proteasome to directly block the replication of susceptible viruses. Currently the molecular mechanisms behind E2 recruitment, interactions and enzymatic regulation are unknown. TRIM21 and TRIM5¿ are characterized by a conserved tripartite motif that comprises an E3 RING, B-Box and coiled-coil domain. We hypothesize that distinct interfaces within this motif are used to interact with different E2 ubiquitin-conjugating enzymes in order to catalyse formation of distinct ubiquitin polymers, however, no structures of the intact region exist, hampering mutagenesis studies to probe TRIM function. This project will structurally characterize constructs covering the tripartite motif of TRIM21 and TRIM5¿ by X-ray crystallography, SAXS, and other biophysical techniques to determine the supra-domain structure and oligomeric state. The molecular determinants for interactions between TRIM21/TRIM5¿ and a range of diverse E2 enzymes will be identified by co-crystallization and NMR experiments providing insight on the role of the tripartite motif in E2 recruitment and enzymatic regulation. The gained structural information will inform mutagenesis to selectively disrupt interactions with E2s. The effect of these mutations on immune signalling and blocking viral replication will be determined in cell culture based infection assays to validate the involvement of specific interactions in each function. This work will provide mechanistic insight into how TRIM21 and TRIM5¿, an emerging class of dual effector and signalling molecules, use distinct ubiquitination pathways to perform the two antiviral activit...
