Insights into the ISG15 transfer cascade by the UBE1L activating enzyme

The attachment of the ubiquitin-like protein ISG15 to substrates is a well-established antiviral signalling mechanism of the innate immune response. However, despite the identification of thousands of substrates and clear roles in antiviral immunity, a molecular understanding of ISG15 selection and transfer through its cognate E1-E2- E3 enzyme cascade is largely unknown. Here, we present a 3.45 [A] cryo-EM structure of a chemically trapped UBE1L-UBE2L6 complex bound to activated ISG15. This structure reveals the details of the first steps of ISG15 recognition and UBE2L6 recruitment by UBE1L. Taking advantage of viral effector proteins from severe acute respiratory coronavirus 2 (SARS-CoV-2) and influenza B virus (IBV), we validated the structure and confirmed the importance of the ISG15 C-terminal ubiquitin-like domain in the adenylation reaction. Moreover, biochemical characterization of the UBE1L-ISG15 and UBE1L-UBE2L6 interactions enabled the design of ISG15 and UBE2L6 mutants with altered selectively for the ISG15 and ubiquitin conjugation pathways. Together, our study provides much needed insight into the specificity determinants that ensure the fidelity of ISG15 signalling during the antiviral response.