title: UBCH7 reactivity profile reveals parkin and HHARI to be RING/HECT hybrids.
abstract: 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, but often fails to function with RING E3s in vitro despite forming specific complexes. Structural comparisons of inactive UBCH7-RING complexes with active UBCH5-RING complexes reveal no defining differences, 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). Found in all eukaryotes, RBRs regulate processes such as translation and immune signalling. 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. 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 and immune function, and indicate a novel mechanism for an entire class of E3s.
authors.affiliation: Department of Biochemistry, University of Washington, Seattle, Washington 98195, USA.
authors.forename: Dawn M
authors.initials: DM
authors.lastname: Wenzel
authors.forename: Alexei
authors.initials: A
authors.lastname: Lissounov
authors.forename: Peter S
authors.initials: PS
authors.lastname: Brzovic
authors.forename: Rachel E
authors.initials: RE
authors.lastname: Klevit
mesh_terms: D000595:Amino Acid Sequence; D002352:Carrier Proteins / Q000737:chemistry / Q000378:metabolism*; D020134:Catalytic Domain; D003545:Cysteine / Q000737:chemistry; D006801:Humans; D008239:Lysine / Q000378:metabolism; D008969:Molecular Sequence Data; D050506:Mutant Chimeric Proteins / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D016415:Sequence Alignment; D025801:Ubiquitin / Q000378:metabolism; D044763:Ubiquitin-Conjugating Enzymes / Q000737:chemistry / Q000378:metabolism*; D044767:Ubiquitin-Protein Ligases / Q000737:chemistry / Q000378:metabolism*
chemical_list: D002352:Carrier Proteins; D050506:Mutant Chimeric Proteins; D025801:Ubiquitin; C079437:UBE2L3 protein, human; D044763:Ubiquitin-Conjugating Enzymes; C419193:ARIH1 protein, human; D044767:Ubiquitin-Protein Ligases; C111567:parkin protein; D008239:Lysine; D003545:Cysteine