UBCH7 reactivity profile reveals parkin and HHARI to be RING/HECT hybrids

摘要

Although the functional interaction between ubiquitin-conjugating enzymes (E2s) and ubiquitin ligases (E3s) is essential in ubiquitin (Ub) signalling, the criteria that define an active E2-E3 pair are not well established. The human E2 UBCH7 (also known as UBE2L3) shows broad specificity for HECT-type E3S~1, but often fails to function with RING E3s in vitro despite forming specific complexes~(2-4). Structural comparisons of inactive UBCH7-RING complexes with active UBCH5-RING complexes reveal no defining differences~(3,4), highlighting a gap in our understanding of Ub transfer. Here we show that, unlike many E2s that transfer Ub with RINGs, UBCH7 lacks intrinsic, E3-independent reactivity with lysine, explaining its preference for HECTs. Despite lacking lysine reactivity, UBCH7 exhibits activity with the RING-in-between-RING (RBR) family of E3s that includes parkin (also known as PARK2) and human homologue of ariadne (HHARI; also known as ARIH1)~(5,6). Found in all eukaryotes~7, RBRs regulate processes such as translation~8 and immune signalling~9. RBRs contain a canonical C3HC4-type RING, followed by two conserved Cys/His-rich Zn~(2+)-binding domains, in-between-RING (IBR) and RING2 domains, which together define this E3 family~7. We show that RBRs function like RING/HECT hybrids: they bind E2s via a RING domain, but transfer Ub through an obligate thioester-linked Ub (denoted ~Ub), requiring a conserved cysteine residue in RING2. Our results define the functional cadre of E3s for UBCH7, an E2 involved in cell proliferation~(10) and immune function~(11), and indicate a novel mechanism for an entire class of E3s.