Cryo-EM structures of Uba7 reveal the molecular basis for ISG15 activation and E1-E2 thioester transfer

Abstract

ISG15 plays a crucial role in the innate immune response and has been well-studied due to its antiviral activity and regulation of signal transduction, apoptosis, and autophagy. ISG15 is a ubiquitin-like protein that is activated by an E1 enzyme (Uba7) and transferred to a cognate E2 enzyme (UBE2L6) to form a UBE2L6-ISG15 intermediate that functions with E3 ligases that catalyze conjugation of ISG15 to target proteins. Despite its biological importance, the molecular basis by which Uba7 catalyzes ISG15 activation and transfer to UBE2L6 is unknown as there is no available structure of Uba7. Here, we present cryo-EM structures of human Uba7 in complex with UBE2L6, ISG15 adenylate, and ISG15 thioester intermediate that are poised for catalysis of Uba7-UBE2L6-ISG15 thioester transfer. Our structures reveal a unique overall architecture of the complex compared to structures from the ubiquitin conjugation pathway, particularly with respect to the location of ISG15 thioester intermediate. Our structures also illuminate the molecular basis for Uba7 activities and for its exquisite specificity for ISG15 and UBE2L6. Altogether, our structural, biochemical, and human cell-based data provide significant insights into the functions of Uba7, UBE2L6, and ISG15 in cells.

Insights into the ISG15 transfer cascade by the UBE1L activating enzyme

“…3 , Supplementary Table 1 ). Similar to other E1 enzymes 30 , and as seen in a related study published while our paper was in revision 20 , the UBE1L structure consists of four distinct domains: the adenylation domain (AD; which contains both the inactive adenylation domain & active adenylation domain), first catalytic cysteine half domain (FCCH), second catalytic cysteine half domain (SCCH) and ubiquitin-fold domain (UFD) (Fig. 2a , Supplementary Fig.…”

“…2d ). In contrast, a recent study suggests the ISG15 N-lobe contacts the UFD 20 . Together these results highlight the flexibility of the N-lobe during the adenylation reaction, which is further supported by the low resolution of this domain in both structures (Supplementary Fig.…”

“…To prevent non-specific crosslinking, the non-catalytic UBE2L6 cysteine residues were mutated to serine (UBE2L6 C86only ; Supplementary Fig. 2a ), which was also utilized for UBE2L6 in a recent publication by Olsen and colleagues 20 . Importantly, UBE2L6 C86only retained its ability to form a UBE2L6 ~ ISG15 thioester bond with fluorescent ISG15 (Supplementary Fig.…”

“…This approach captures the ISG15 adenylate intermediate and reveals the structural features required for ISG15 and UBE2L6 recognition. A similar strategy was recently reported to solve another UBE1L-ISG15-UBE2L6 structure while this paper was under review 20 . In addition to the structures, biochemical analysis using viral effector proteins and site-directed mutagenesis probe the mechanistic basis of this specificity.…”

Enhanced ISGylation reduces respiratory distress followingFrancisella novicidainfection

“…Since there is not a discernable consensus sequence for ISG15 it is tempting to speculate that there could be additional ISG15 E3-ligases, distinct from HERC5, induced following bacterial infection. Recent structural studies may elucidate whether there is an E2 specific-binding surface that could confer specificity [85, 86].…”