ISGylation Mechanism Uncovers Conformational Specificity for HECT-family E3 ligase

Interferon-stimulated gene 15 (ISG15), composed of N-terminal and C-terminal ubiquitin-like domains (NTD/CTD), plays a critical role in antiviral immunity. Although the ubiquitination mechanism is well established, the mechanisms governing ISG15 transfer, particularly from E2 to E3 and subsequent lysine conjugation, remain unknown. Here, we reveal that UbcH8(E2)∼ISG15 exhibits striking specificity for HECT-family E3 ligases (particularly HERC5) but is inactive with RING or RBR E3. In contrast, UbcH8∼Ub preferentially engages RBR E3, indicating a switched E2–E3 specificity depending on the conjugated ubiquitin-like modifier. Structural and biochemical studies uncover how a unique closed conformation of UbcH8∼ISG15 enables trans-thiolation mediated by selective HECT-family E3 ligases. We further demonstrate that HERC5’s C-lobe specifically recognizes donor ISG15 for lysine conjugation, explaining its exclusive ISGylation activity and lack of ubiquitination function. These findings delineate the molecular basis of ISG15 conjugation and reveal how its pathway has evolved distinct mechanisms from ubiquitination, offering new avenues for therapeutic intervention in infection and immunity.