Fig. 7: The HOIL-1 RING2 domain contains a Zn2/Cys6 binuclear cluster.

a Sequence alignment of RING2 domains from HOIL-1, RNF216 and Parkin. Zinc binding residues are highlighted blue and green. Position of the catalytic triad residues are indicated by black arrowheads. The RING2 domains of HOIL-1 and RNF216 contain distinctive zinc binding extensions or insertions (underlined in orange). Conversely, Parkin has a canonical RING2 domain, with two zinc-binding motifs and a conserved catalytic triad of Cys, His, Glu. b Structure of the HOIL-1 RING2 domain showing the Zn2/Cys6 binuclear cluster and active site residues (with active site Cys460 mutated to Ala). c Structure of the Parkin RING2 domain (PDB: 4BM9)⁴³ with canonical zinc binding arrangement and active site residues. d Schematics showing arrangement of HOIL-1 and Parkin RING2 zinc-coordinating residues. e Time-course of HOIL-1 catalysed E2-Ub discharge assay with HOIL-1 RING2 mutants. The red arrowhead indicates the accumulation of the E3-Ub thioester intermediate that is reduced by addition of DTT shown in the lower panels, as well as a small amount of HOIL-1 autoubiquitination product that is stable under reducing conditions. The asterisk indicates E2 disulfide dimer that is also present in the WT reaction and reduced by addition of DTT shown in the lower panels. The right panel shows quantification of three independent experiments. f Time-course of maltose ubiquitination by HOIL-1 WT and mutants. The red arrowhead indicates autoubiquitination of HOIL-1 H510A. The right panel shows quantification of three independent experiments. Source data are provided as a Source data file.