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

HERC5 is an interferon-induced E3 ubiquitin ligase involved in ISGylation, a crucial post-translational modification regulating antiviral responses. The HECT domain of HERC5 plays a key role in substrate recognition and ubiquitin transfer. In this study, we employed small-angle X-ray scattering (SAXS) to investigate the molecular organization and oligomeric state of the HERC5 HECT domain in solution. The protein was expressed and purified using affinity and size-exclusion chromatography, followed by SAXS measurements at 2.0 mg/ml and 10°C. Data analysis revealed a trimeric assembly, supported by Guinier and Kratky analyses, distance distribution functions, and ab initio modeling. Structural overlays with the AlphaFold model confirmed the SAXS-derived envelope, providing insights into the conformational stability and trimeric self-assembly properties of HERC5 HECT. These findings suggest that trimerization may play a regulatory role in HERC5 function, contributing to a broader understanding of HECT-type E3 ligase mechanisms.