Extended Data Fig. 5: Mutational assessment of native MYCBP2 E2-E3 transthiolation activity and demonstration of attenuated E2~Ub reactivity.
From: Structural basis for RING-Cys-Relay E3 ligase activity and its role in axon integrity
a, Proposed intramolecular role for Ub I36 in maintaining a closed-like E2~Ub conformation. The interaction between Ub I36 and L71 is maintained in the “closed-like” E2~Ub conformation. Superposition of the RCR E2~Ub and RNF4 E2~Ub (PDB 4AP4) complexes. The gap between Ub I36 and L71 has decreased by 0.7 Å in the RCR complex. The RCR, E2 and Ub are in cartoon representation with select residues in ball and stick representation: Ub I36, L71, R72 and the engineered linker (gray), E2 C85 (mauve), RCR K4441 (blue) and C4520 (orange). The RNF4 E2~Ub complex is in cartoon representation with select residues in stick representation: Ub I36, L71, R72 (pink), R181 (purple). b, For the selected mutants, MYCBP2 activity was assessed using single turnover E2~Ub discharge assays mediated by the presence of wild type MYCBP2 and threonine (50 mM). c, Single turnover E2~Ub discharge assay mediated by the presence of wild type MYCBP2 and threonine (50 mM) for E2 Asn114Ala and Asn114Gln mutants. Experiment repeated twice with similar results. d, Quantification for selected mutants, (n = 3-4 independent experiments performed with identical purified proteins). e-i native single-turnover WT MYCBP2 and threonine dependent E2~Ub discharge assay. Observed rate constants tabulated in Extended Data Fig. 4 were obtained from the one-phase exponential association equation using the routine within Graphpad Prism. Blue squares and black circles correspond to experiments where E3 was added or withheld, respectively. (n = 3 independent experiments performed with identical purified proteins) j, Quantification of lysine discharge assay in the presence of a transthiolation-defective RCR A4520 mutant or the canonical RING E3 RNF4 (n = 3 independent experiments performed with identical purified proteins). Although efficient lysine discharge was observed with the RCR C4520K mutant, the structural context of this acceptor lysine templates the reaction which can increase the reaction rate by multiple orders of magnitude, thereby reconciling the lack of activity towards free lysine which would be diffusion-limited39,60.
