Structural Insights into the Trimeric Assembly of the HERC5 HECT Domain through SAXS Analysis

HERC5 is an interferon-induced E3 ubiquitin ligase that mediates ISGylation, a critical post-translational modification involved in regulating antiviral immune responses. Its HECT (Homologous to the E6-AP Carboxyl Terminus) domain is essential for substrate recognition and the catalytic transfer of ubiquitin or ubiquitin-like proteins. Members of the HERC protein family are structurally defined by a C-terminal HECT domain and N-terminal RCC1-like (Regulator of Chromosome Condensation 1) domains. Despite its biological importance, the oligomerization behavior of the HERC5 HECT domain remains poorly understood. In this study, we examined the oligomeric state of the HERC5 HECT domain in solution using affinity and size-exclusion chromatography, followed by small-angle X-ray scattering (SAXS) at a protein concentration of 2.0 mg/ml and temperature of 10{degrees}C. SAXS data analysis revealed that the HERC5 HECT domain predominantly adopts a trimeric assembly. This conclusion is supported by Guinier and Kratky analyses, distance distribution functions, and ab initio shape reconstructions. These findings suggest that trimerization may play a regulatory role in the functional activity of HERC5, contributing to a deeper understanding of the structural mechanisms governing HECT-type E3 ligases.