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.

ISG15 在先天免疫应答中起着至关重要的作用，由于其抗病毒活性和对信号转导、细胞凋亡和自噬的调节，已得到充分研究。ISG15 是一种泛素样蛋白，可被 E1 酶 （Uba7） 激活并转移到同源 E2 酶 （UBE2L6） 上，形成 UBE2L6-ISG15 中间体，该中间体与 E3 连接酶一起发挥作用，催化 ISG15 与靶蛋白偶联。尽管 Uba7 具有生物学重要性，但由于没有 Uba7 的可用结构，因此 Uba7 催化 ISG15 激活并转移到 UBE2L6 的分子基础尚不清楚。在这里，我们展示了人 Uba7 与 UBE2L6、ISG15 腺苷酸和 ISG15 硫酯中间体复合物的冷冻电镜结构，这些结构已准备好催化 Uba7-UBE2L6-ISG15 硫酯转移。与泛素偶联途径的结构相比，我们的结构揭示了复合物的独特整体结构，特别是在 ISG15 硫酯中间体的位置方面。我们的结构还阐明了 Uba7 活性的分子基础及其对 ISG15 和 UBE2L6 的精细特异性。总而言之，我们的结构、生化和基于人类细胞的数据为细胞中 Uba7、UBE2L6 和 ISG15 的功能提供了重要的见解。