Structural determinants for FAT10 activation and transfer from UBA6 to E2 enzymes
Cryo-EM captures FAT10 activation and transfer from UBA6 to E2 UBE2Z, revealing FAT10 monopolises UBA6. Efficient transfer requires E2 engagement of both FAT10 UBL domains, particularly UBL2, and co-ordination of InsP₆ i
Attachment of the ubiquitin-like protein FAT10 onto substrates targets them for proteasomal degradation. FAT10 is activated by the E1 enzyme UBA6 and then transferred to E2 enzymes, but the mechanisms controlling ubiquitin versus FAT10 activation by UBA6 and FAT10 transfer onto E2s remain unclear. Using cryo-EM, we visualise all stages of FAT10 E1-E2 handover: adenylation, thiolation and transthiolation. We find that FAT10 monopolises UBA6 by out-competing ubiquitin for thiolation and blocking the adenylation domain, preventing further UBL recruitment and promoting FAT10 signalling. We profiled UBA6-compatible E2 enzymes and found FAT10 transfer is restricted to a select subset associated with specific cellular pathways. UBE2Z (USE1) showed highest activity followed by UBE2D2, UBE2J2 and UBE2S. Capturing FAT10 or ubiquitin transfer from UBA6 to UBE2Z reveals UBE2Z is highly specialised for FAT10 transfer. It simultaneously engages both FAT10 domains (UBL1 and UBL2) and co-ordinates the metabolite inositol hexakisphosphate (InsP6) bound within the UBA6 catalytic domain. This InsP6 co-ordination extends to other FAT10 compatible E2s. Our structural and biochemical analyses reveal regulatory mechanisms underpinning FAT10 activation and transfer. We define principles governing selective FAT10 transfer, highlighting favourable interactions with FAT10 C-terminal domain (UBL2) and stable UBA6 binding, ensuring controlled conjugation onto substrates.