Cryo-EM structures of UBA6 reveal mechanisms of E1–E2 specificity and dual FAT10/ubiquitin thioester transfer

UBA1 and UBA6 define parallel ubiquitin (Ub) activation systems that perform non-overlapping roles in Ub and ubiquitin-like protein (Ubl) signaling. Whereas UBA1 supports the canonical Ub pathway, UBA6 also activates the Ubl FAT10, linking Ub signaling to immune-regulated proteostasis. In addition to selective Ub/Ubl activation, UBA1 and UBA6 engage distinct sets of E2s, yet how these enzymes achieve selective E2 engagement has remained unclear. Using chemical trapping and high-resolution cryo-EM, we determine four structures of UBA6–E2 complexes representing the thioester-transfer step with either FAT10 or Ub, revealing how this E1 distinguishes its cognate partners. UBA6 achieves E2 specificity through coordinated contributions of the UFD and SCCH domains, a dual-domain mechanism that contrasts with the UFD-dominated selectivity of UBA1. The structures further show that an existing inositol hexakisphosphate (InsP₆)–binding site, unique to UBA6, stabilizes an expanded SCCH cleft that pre-organizes the enzyme for selective engagement of UBA6-specific E2s. These findings define principles for E1–E2 recognition and identify InsP₆ as a cofactor shaping specificity within the Ub-like conjugation network. Specificity in ubiquitin (Ub) and Ub-like protein signaling is essential. Here, the authors use cryo-EM to show how UBA6 selectively engages its cognate E2 for dual Ub and FAT10 transfer, revealing a role for an InsP₆-binding site and illuminating molecular rules governing pathway specificity.