Structural Insights into TRIM21 Ubiquitination Mechanisms
The attachment of ubiquitin (Ub) to proteins is one of the most abundant and versatile of all posttranslational modifications and affects outcomes in essentially all physiological processes. During ubiquitination, RING E3 ligases direct E2 Ub-conjugating enzymes to substrates to catalyze their ubiquitination. The cytosolic antibody receptor TRIM21 possesses unique ubiquitination activity that drives broad-spectrum anti-pathogen targeting and underpins the protein depletion technology Trim-Away. Understanding how TRIM21 functions as an E3 ligase mechanistically is required to explain how it achieves such broad-specificity and targets unrelated viruses and proteins for degradation. Moreover, it is required to further evolve TRIM21-based technologies such as Trim-Away. The aim of my PhD was to understand how TRIM21 acts as an E3 ubiquitin ligase. In particular, I wanted to determine how TRIM21 selectively recruits only a subset of E2 enzymes and how it utilizes these for ubiquitin chain formation. Previous work suggested that TRIM21 requires the E2 enzymes Ube2W and Ube2N/Ube2V2 to build K63-linked ubiquitin chains and drive anti-viral function. My work reveals how TRIM21 facilitates ubiquitin transfer and differentiates Ube2N from other closely related enzymes. A tri-ionic motif allows TRIM21 to wrap an Ube2N~Ub around its RING domain, promoting ubiquitin discharge. The tri-ionic motif is exclusively required for Ube2N but not other E2 enzymes and provides a generic E2-specific recruitment mechanism for RING E3s. In addition, I have determined how TRIM21 forms a K63-linked ubiquitin chain on itself. By analyzing a catalytically trapped structure showing the initiation of TRIM21 RING-anchored ubiquitin chain elongation, I have uncovered the chemical mechanism of ubiquitin conjugation. Moreover, this mechanism enables TRIM21 to perform targeted protein degradation in cells. My Thesis explains how TRIM21 catalyses formation of the K63-ubiquitin chains required for its function. More broadly, these findings help to illuminate the mechanism of other K63-specific RING E3s ligases.