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.

Figure 1. UbcH7 does not react with free lysine

A) Coomassie-stained SDS-PAGE of UbcH7~Ub (left) and UbcH5c~Ub (right) incubated with amino acids lysine, serine, threonine, arginine, or buffer (-). Reactions were quenched in non-reducing loading buffer. ‘0’ indicates starting amounts of E2~Ub prior to amino acid addition. Reactivity with amino acids is indicated by loss of E2~Ub and concurrent increase in free Ub (denoted by asterisks) and free E2. B) Time course assays of UbcH5c~Ub and UbcH7~Ub incubated with lysine. Reactions were quenched in non-reducing loading buffer at the indicated times.

Figure 2. Lysine reactivity is multifactorial

A) Alignment of E2 active site residues (right). Structure of UbcH5c (green; PDB ID 2FUH) with active site residues represented as sticks and active site cysteine as spheres (left). B) Wildtype (WT) or mutant UbcH5c~Ub incubated with lysine as in Fig. 1B. C) Western blot of Flag BRCA1(1–304)/BARD1(26–327) (green) auto-ubiquitination with indicated E2 and T7-Ub (red). Time is measured as minutes post ATP addition. D) Same as in Fig 1B, except a 1:1 (E3:E2) equivalent of Flag-BRCA1(1–112)/BARD(26–140) was added to either UbcH5c-WT (left) or D87E(right) charged with HA-Ub-K0. Reactions were visualized by Western for HA (K0-Ub) and Flag (BRCA1) epitopes.

Figure 3. RBR E3s function via a HECT-like mechanism

A) HHARI_R1-IBR-R2 auto-ubiquitination assays with the indicated E2 were visualized on a Coomassie-stained, reducing, SDS-gel. Time is measured as minutes post ATP addition. B) Auto-ubiquitination assay of Parkin_R1-IBR-R2 with the E2s indicated. Products were visualized by Western blotting for the GST-tag on Parkin. C) UbcH7 was pre-charged with HA-Ub and mixed with HHARI_R1-IBR-R2. Reactions were quenched in SDS-buffer under reducing (+βME) and non-reducing conditions (−βME) and visualized by Western blotting for the HA-epitope on Ub. A βME-sensitive HA-Ub band corresponding to the molecular weight of HHARI_R1-IBR-R2-Ub appears at 10, 20, and 30 seconds post addition of UbcH7~Ub. Asterisk denotes a cross-reactive band.

Figure 4. Cysteine C357 is the active site of HHARI

A) Sequence alignment of RING2 domains from human RBR ligases. Conserved cysteine residues are colored green. Asterisks indicate Zn²⁺-liganding cysteines in the HHARI_R2 structure. B) Auto-ubiquitination assays of T7-tagged UbcH7 and the indicated construct of HHARI. Ubiquitination is measured as time post ATP-addition. Reaction products were visualized by Western blotting simultaneously for the GST-tag on HHARI (top panel) and the T7-tag on UbcH7 (bottom panel). C) E3 auto-ubiquitination assay with UbcH7 and the indicated construct of Parkin. D) GST-pulldowns of purified constructs of GST-tagged HHARI and T7-tagged UbcH7. Bound protein was eluted and visualized by Western blotting for T7 and GST epitopes simultaneously.