Structural and functional basis for TRIM25 E3 ligase catalytic activity and NS1-mediated suppression

Abstract Ubiquitination is a post-translational modification of proteins with broad regulatory roles across cellular biology. This process involves the addition of ubiquitin molecules on target proteins, altering their cellular role and properties. Ubiquitination is performed by an enzymatic cascade consisting of three enzymes: ubiquitin activating enzymes (E1), ubiquitin conjugating enzymes (E2) and ubiquitin ligases (E3). The tripartite motif (TRIM) family of proteins constitutes one of the largest subfamilies of RING E3 ligases and the majority of them are contributing to the regulation of innate immune responses. They are characterized by a conserved tripartite motif in their N-terminal region which comprises a RING domain, one or two B-box domains and a coiled-coil region. Self-association is believed to be crucial for catalytic activity of TRIM proteins, however, the precise molecular mechanism underlying this observation remains elusive. The work presented in this thesis provides insights into the E3 ligase function of TRIM25 and shows how its oligomeric state is linked to its catalytic activity. The crystal structure of a complex between the TRIM25 RING domain and a ubiquitin-loaded E2 identifies the structural and mechanistic features that promote activation of E2~Ub allowing us to propose a model for the regulation of activity in the full-length protein. In the second part of this thesis, the molecular details of Influenza A NS1-mediated TRIM25 inhibition are presented. The crystal structures of NS1 bound to TRIM25 along with biochemical analysis allowed us to identify the interacting domains and propose a model for the inhibition of substrate ubiquitination during viral infection. The results of this project extend our understanding of the mechanism, structure and regulation of TRIM E3 ligases and their substrates, leading to increased chances of targeting specific steps of the ubiquitination pathway during disease