Heme-oxidised IRP2 ubiquitin ligase 1 (HOIL-1) is a central player in inflammatory and immune signalling. HOIL-1 deficient patients exhibit autoinflammation and immunodeficiency but also, unexpectedly, amylopectinosis characterised by pathological deposition of unbranched glycogen in the patients’ muscles. A recent study shows HOIL-1 can ubiquitinate glycogen and its subunit, oligosaccharides via oxyester linkage in vitro, opening the possibility HOIL-1 may directly ubiquitinate non-proteinaceous substrates to prevent amylopectinosis. Other studies suggest HOIL-1 catalyses oxyester-linked (O-linked) ubiquitination of protein Ser/Thr residues. It is therefore interesting to understand HOIL-1’s substrate preference for proteinaceous and non-proteinaceous substrates as well as its catalytic mechanism enabling its noncanonical activity.
HOIL-1 is a RING-between-RING (RBR) E3 ligase with functionally conserved catalytic RBR domains. RBRs catalyse substrate ubiquitination by transferring Ub from E2 enzymes to their own RING2 domain catalytic Cys before transferring the Ub to substrates. The molecular details of how HOIL-1 performs both Ub transfer steps will elucidate on HOIL-1’s O-linked ubiquitination mechanism.
We show E2/HOIL-1 Ub transfer is allosterically activated by specific Ub species. A structure of HOIL-1 RBR captured in action with E2-Ub and allosteric Ub reveals a unique fold in HOIL-1 RING2 domain not found in any other mammalian protein. The unique fold structurally completes the missing piece of HOIL-1’s non-conserved catalytic triad which has distinct roles in the two-step Ub transfer. Structural differences of lone and Ub bound HOIL-1 RING2 provide a glimpse on how HOIL-1’s catalytic sites rearrange for substrate ubiquitination. Our in vitro studies demonstrate HOIL-1 ubiquitinates Ser preferentially over Thr but does not distinguish glucosaccharide isomers outstandingly. HOIL-1 also targets various physiological monosaccharides for ubiquitination with low specificity, suggesting to us additional cellular cofactors may be involved in substrate recognition and ubiquitination. Our research addresses HOIL-1’s catalytic mechanisms and regulation, laying the foundations for studying HOIL-1’s functions in physiological contexts.