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

Attachment of the small protein ubiquitin (Ub) to substrates is a reversible covalent modification that regulates the stability, activity and localization of target proteins. Assembly of ubiquitin chains requires three enzyme activities: a Ub-activating (E1) enzyme, a Ub-conjugating (E2) enzyme and a Ub ligase (E3). E2 enzymes are responsible both for E3 selection and substrate modification and for much of the diversity of Ub cellular signalling. The E2 UbcH7 shows broad specificity for HECT-type E3, but often fails to function with RING E3s despite its ability to form specific complexes. Rachel Klevit and colleagues demonstrate that UbcH7 exhibits robust activity with a family of E3 ligases that are RING/HECT hybrids: they bind to E2s through a RING domain but transfer Ub through a HECT-like mechanism. They propose that the intrinsic chemical reactivity of an E2 is a powerful predictor in determining with which class of E3 it will function. 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 E3s1, but often fails to function with RING E3s in vitro despite forming specific complexes2,3,4. Structural comparisons of inactive UBCH7–RING complexes with active UBCH5–RING complexes reveal no defining differences3,4, 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 eukaryotes7, RBRs regulate processes such as translation8 and immune signalling9. RBRs contain a canonical C3HC4-type RING, followed by two conserved Cys/His-rich Zn2+-binding domains, in-between-RING (IBR) and RING2 domains, which together define this E3 family7. 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 proliferation10 and immune function11, and indicate a novel mechanism for an entire class of E3s.