Deciphering the structural stability and oligomeric state of the HERC5 HECT domain by saxs

ISG15, interferon-stimulated gene 15, is a ubiquitin-like protein induced by type I interferon (IFN). The functions and interactions of ISG15 within the cell are determined by two tandem ubiquitin-like domains. Similar to ubiquitin, ISG15 binds to cellular proteins via the action of the E1-E2-E3 enzyme complex, known as ISGylation. HERC5 (HECT And RLD Domain Containing E3 Ubiquitin Protein Ligase 5) is an E3 ubiquitin ligase that plays a critical role in the conjugation of ISG15 and enhances the antiviral response by inhibiting viral replication. The HECT (Homologous to the E6-AP Carboxyl Terminus) domain is the one of the domains of HERC5 that catalyzes the conjugation process via catalytic binding of cysteine at the 944th position. There is no consensus regarding the initiation site and the length of the HECT region. Some researchers have proposed the inclusion of an additional N-terminal alpha helix structure within this region. In Chapter 2, HERC5 HECT domain were produced and purified using different expression plasmids (pQE-60, pET42), fusion tags (SUMO, GST), expression hosts (E. coli Rosetta 2 strain for both pQE-60 and pET42, and Gami strain for pQE-60), a range of temperatures (30°C, 25°C, 21°C, and 16°C), and varying IPTG concentrations (0.2 μMand 0.04 μM) to investigate factors affecting the stability of the boundaries of the HECT domain. Proteins were purified by affinity chromatography, the gravity-flow column with Ni-NTA resin. All samples were visualized by sodium dodecyl-sulfate polyacrylamide gel electrophoresis with 15% gels.