Fig. 5: Acidic residues in the β3-RING loop make extensive contacts with Ub^D.

a UbcH5b and Ark2C are represented as grey cartoons with the basic β3-RING region shown as a black dashed line. Ub^R and Ub^D are shown as a surfaces coloured to highlight their electrostatic potential with contours at ±5.0 kT/e. The position of relevant residues is highlighted. b Multiturnover assays showing that mutation of Lys11 to Ala (K11A) and Asp (K11D) impedes activity. For these assays, UbArk2C was utilised to ensure the Ub^R binding site was occupied by WT ubiquitin. c A 1:1 UbArk2C–UbcH5b~Ub complex (50 µM) was incubated with EDC crosslinker for 45 min and then resolved by SDS-PAGE. d Analysis of the excised band revealed a cross-link between Lys11 in ubiquitin and Glu287/Asp289 in the β3-RING loop. MS/MS spectrum of the EDC cross-linked peptide with relevant b and y fragment ions highlighted on the spectrum and the peptide sequence. The colours of the fragment marker indicate its relative intensity. e Multiturnover assays comparing the activity of WT Ark2C with WT ubiquitin and ubiquitin in which Lys29 and Lys33 were mutated to Ala (K29A/K33A) and Glu (K29E/K33E) as indicated. f Multiturnover assays comparing the activity of WT Ark2C and a mutant in which the acidic residues (Asp283, Glu284, Glu286 and Glu287) were mutated to lysine (referred to as 4 K). Both WT ubiquitin and a mutant in which Lys29 and Lys33 were mutated to Glu (KK/EE) were utilised. Gels were stained with Coomassie Blue and molecular weight standards are indicated. g Close-up view of the contacts between the TRIM21 RING and Lys11 in Ub^D of the TRIM21–E2~Ub complex (PDB: 6S53), and the equivalent region of the UbArk2C–UbcH5b~Ub structure showing interactions between the C-terminal residues in the β3-RING loop and Lys11 in Ub^D. h Structure of MDM2 in complex with an E2~Ub conjugate (PDB: 6SQS) showing contacts between a phosphorylated residue (Ser429) and Lys33 in Ub^D (left) and the equivalent region from the UbArk2C–UbcH5b~Ub structure showing the position of Lys33 in Ub^D. The β3-RING loop is shown as a dotted line and the acidic residues in the centre of the loop are indicated. i Ub^D is shown as a ribbon with a surface and the key contacts that stabilised the activated conformation in MDM2, TRIM21 and Ark2C are shown. The figure was prepared by superimposing Ub^D from the activated complexes with MDM2 (PDB: 6SQS), TRIM21 (PDB: 6S53) and Ark2C. Key contact residues from the E3s are shown as blue sticks. Contacts mediated by Ark2C bound Ub^R from this study are shown as grey and yellow sticks, respectively.