Fig. 6: Analysis of the allosteric Ub binding sites in HOIL-1 and RNF216.
From: The unifying catalytic mechanism of the RING-between-RING E3 ubiquitin ligase family
a HOIL-1 allosteric Ub binding site. The Ub I44 hydrophobic patch binds to a conserved allosteric site formed by the HOIL-1 RING1-IBR helix and IBR. Key interacting residues and M1 and K63 that would form an (iso-)peptide bond with the distal Ub in di-Ub are shown in ball and stick depiction. b RNF216 allosteric Ub binding site. The Ub I44 hydrophobic patch binds to a conserved allosteric site formed by the RNF216 RING1-IBR helix and IBR. Key interacting residues and K63 are shown in ball and stick depiction. c WT and I358R HOIL-1 catalysed UbcH7-Ub discharge assay in the presence of increasing concentrations of M1-linked di-Ub. The bottom panel shows quantification of three independent experiments for EC50 determination. Nonlinear regression curves were fitted using the [agonist] vs. normalised responses—variable slope model. d Time-course of HOIL-1 catalysed E2-Ub discharge assay using constructs with (helix-RBR) or without (RBR) the N-terminal helix in the presence or absence of M1-linked di-Ub. The graph shows quantification of three independent experiments. e RNF216 catalysed UbcH7-Ub discharge assay using constructs with (helix-RBR-helix) or without (RBR-helix) the N-terminal helix, in the presence of increasing concentrations of K63-linked di-Ub. The bottom panel shows quantification of three independent experiments for EC50 determination. Nonlinear regression curves were fitted using the [agonist] vs. normalised responses—variable slope model. Source data are provided as a Source data file.
