UbcH7 reactivity profile reveals Parkin and HHARI to be RING/HECT hybrids

Although the functional interaction between ubiquitin conjugating enzymes (E2s) and ubiquitin ligases (E3s) is essential in ubiquitin (Ub) signaling, the criteria that define an active E2–E3 pair are not well-established. The human E2 UbcH7 (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. 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 and human homologue of ariadne (HHARI)(5,6). Found in all eukaryotes(7), RBRs regulate processes such as translation(8) and immune signaling(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). Here 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 suggest a novel mechanism for an entire class of E3s.