Abstract
The ISGylation pathway, a key post-translational modification, plays a pivotal role in the innate immune response by covalently attaching ISG15 to target proteins. This cascade involves a series of enzymatic steps, including activation by the E1 enzyme Uba7, conjugation by the E2 enzyme UbcH8, and ligation by an E3 ligase. Central to this process is the ubiquitin-fold domain (UFD) of Uba7, which facilitates the transfer of ISG15 to UbcH8. However, the structural and mechanistic details of this interaction remain poorly understood. In this work, we present the solution NMR structure and functional analysis of the ubiquitin-fold domain of human Uba7, the E1 enzyme in the ISGylation cascade. Through detailed NMR titration experiments and mutational studies, we mapped the interaction surface of Uba7-UFD with UbcH8, identifying key residues and their contributions to the E1-E2 interaction. Our results reveal that Uba7-UFD exhibits flexibility in its free form, adopting a more ordered conformation upon binding UbcH8. Chemical shift perturbation and mutational analysis further demonstrate the importance of specific residues, particularly Cys996, in maintaining UFD's structural integrity and binding capacity. Additionally, mutations designed to alter the flexibility and length of the loop region between UFD and UbcH8 show significant effects on binding, indicating that these regions are crucial for efficient E2 recruitment. These findings provide new insights into the mechanistic basis of E2 enzyme selection in ISGylation and underscore the functional relevance of dynamic structural transitions in E1-E2 complex formation.