Structural insights into the specific recognition and transfer of ISG15

Expression of the ubiquitin-line protein ISG15 is induced during the host response to viral and bacterial infections. Covalent conjugation of ISG15 to lysine residues in substrate proteins — a process known as ISGylation — inhibits viral replication and modulates host damage and repair response pathways. Similar to ubiquitin (Ub) and other Ub-like modifiers, the attachment of ISG15 to target proteins requires the consecutive action of a highly specific cascade comprising E1, E2 and E3 enzymes. ISG15 is activated by the E1-activating enzyme UBA7 (also known as UBE1L) by adenylation and the formation of thioester bonds, followed by transfer to the catalytic cysteine of the E2-conjugating enzyme UBCH8 (also known as UBE2L6), and ultimately ISG15 is ligated to substrate proteins by one of the E3 ligases (HERC5 (HERC6 in mice), HHARI (ARIH1 in mice) or TRIM25).

ISG15 is composed of two Ub-like domains separated by a flexible linker. The N- and C-terminal domains of ISG15 share some sequence similarity, and much more structural similarity, with Ub. Despite these commonalities between Ub and ISG15, ISG15 is exclusively activated by UBA7. This raises the question, what are the molecular mechanisms of this specific ISG15 activation and UBA7–UBE2L6–ISG15 interaction? Two recent studies in Nature Communications by Afsar et al.¹ and Wallace et al.² have tackled outstanding questions in the ISGylation field, providing the molecular basis of the activity, specificity and mechanism of action of UBA7–UBE2L6 recognition of ISG15. Both groups follow a similar approach, and provide cryo-electron microscopy (cryo-EM) structures of chemically trapped UBA7–UBE2L6 complex bound to adenylated ISG15², or of a ‘doubly-loaded’ UBA7–UBE2L6 in complex with ISG15 adenylate and ISG15 thioester intermediates¹.

In both structures, the ISG15 N-terminal domain is weakly ordered. Wallace et al.² show that the N-terminal domain can extend outward from the E1 enzyme and is dispensable for UBA7 activity in biochemical assays, whereas Afsar et al.¹ use 3D variability analysis to implicate the N-terminal domain in promoting the proximal conformation of the ubiquitin-fold domain (UFD), required for E1–E2–ISG15 thioester transfer. The Thr125 patch of ISG15 — a region that corresponds to the Ile44 patch of Ub — is significantly more polar and determines specificity towards UBA7. By swapping these and adjacent hydrophobic residues with the equivalent residues of Ub, Wallace et al.² use biochemical and cellular studies to show that ISG15 negatively selects against mis-activation through UBA1. The results of both studies also highlight several determinants of E2 selectivity, narrowing down the specificity determinants of the UBA7–UBE2L6 pair to the hydrophobic interface between the N-terminal α-helix A of UBE2L6 and the UFD of UBA7, involving a specific set of residues compared with the UBA1 UFD and E2 enzymes in the Ub pathway. The structures presented by Afsar et al.¹ reveal unique conformations of the ISG15-adenylate and ISG15-thioester intermediates that promote the active conformational state of the UBA7–UBE2L6–ISG15 complex and provide an additional mechanism as to why the double-loaded complex is more efficient during thioester transfer. Structure-based mutagenesis and biochemical experiments reinforce the importance of the catalytically relevant residues and confirm the molecular basis of the UBA7 specificity for ISG15 activation and thioester transfer to UBE2L6.

These studies provide important mechanistic insights into the initial catalytic steps of the ISG15 conjugation pathway, which distinguish it from the canonical Ub pathway, and future investigations will hopefully unravel downstream steps, including ISG15 transfer to the E3 enzymes, ligation to substrates, how the targeted modification of proteins is read and how it is functionally interpreted.