Extended Data Fig. 8: Modelling of E2-MYCBP2(cat) complex.

a, Ubiquitin adduct formation for catalytic mutants of GST-tagged MYCBP2(cat). The H4583N mutant undergoes near-quantitative ubiquitin-adduct formation. The adduct is largely removed after thiol treatment, indicating that ubiquitin is linked to the E3 via a thioester. The diffuse nature of the upper band might be due to the presence of a trapped thioester-linked ubiquitin on C4520, and C4572, as the H4583N mutation prevents substrate deprotonation. The C4572S/H4583N double mutant forms only a single ubiquitin adduct that is thioester-linked, presumably to the C4520 residue. This indicates that formation of the engineered ester-linked adduct on a mutated S4572 residue is dependent on the presence of a general base. C4520 does not appear to have a base in its proximity, hence its activity could be due to its intrinsic pK_a which results in it being nucleophilic at physiological pH in the absence of a general base. This could explain why we failed to produce an engineered ester adduct on a mutant S4520 residue, as serine is fully protonated at physiological pH. The experiment was repeated twice with similar results. b, Superposition of the RING domain from the RBR E3 ligase HOIP in complex with E2 (PDB ID: 5EDV; ubiquitin linked to E2 has been omitted owing to a direct clash with the tandem cysteine domain¹⁸) allows modelling of the E2 into our structure (grey cartoon representation). The catalytic C85 residue in E2 (mutated in silico from Lys to Cys¹⁸) is proximal to C4520, which undergoes transthiolation with E2–Ub. Right, a top-down close up of the mediator loop region. The eight missing residues that form the mediator loop are shown schematically in brown text. c, Model of the proposed ubiquitin relay intermediate as shown in Fig. 5e but from an alternative perspective. In the experimental structure, tandem-cysteine-domain residues are shown in orange and mediator-loop residues are in dark brown. In the model, tandem-cysteine-domain residues are in light orange and mediator-loop residues are in mauve. The modelled E2, based on the superposition in d, is in grey cartoon. Essential cysteines C4520 and C4572 are in yellow and coloured by atom type. Ubiquitin residues G75–G76 are in blue ball-and-stick representation and are coloured by atom type. Gly residues in the mediator loop that are likely to be important for loop mobility are displayed in mauve ball and stick and coloured by atom type. N4570 and H4583 side chains have been rotated by the specified angles to relieve steric clash. d, As in c, but amino acid side chains that have been flipped to relieve steric clash with the modelled mediator loop are labelled in blue. e, All phi and psi angles in the modelled structure fall within accepted values as determined by Ramachandran analysis with the RAMPAGE server⁴¹.