Fig. 2: The interaction between MavC and UBE2N.

a Overview of the interface between MavC and UBE2N in the MavC/UBE2N/Ub complex. b–d Detailed interfaces of MavC–UBE2N interaction as marked in a, including A2 b, B2 c, and C2 d. Hydrogen bonds are represented as red dashed lines. e UBE2N and its mutants were incubated with MavC and Ub for 10 min at 37 °C. Then the samples were treated as in Fig. 1i. f Disrupting MavC–UBE2N interactions decreased the activity of MavC. MavC and the mutants were incubated with UBE2N and Ub for 10 min at 37 °C. Then the samples were treated as in Fig. 1i. g Kinetic analysis of MavC and the mutants targeting the UBE2N-binding surface. The percentage of ubiquitinated UBE2N was calculated by the amount of produced UBE2N–Ub divided by the sum of produced UBE2N–Ub and free UBE2N, at different reaction time points. Data shown are mean values ± SEM (n = 3 independent experiments). The representative gels used for the quantification are shown in Supplementary Fig. 8. h Ni pull-down experiment, combined with mutagenesis experiments to evaluate the roles of UBE2N residues in MavC–UBE2N interaction. i MavC mutants disrupting MavC–UBE2N interactions did not show dampened deamidase activity. MavC and its mutants were incubated with Ub for 2 h at 37 °C. Then the samples were subjected to native gel, followed by Coomassie blue staining. Source data are provided as a Source Data file. Experiments in e, f, h, and i were repeated independently three times with similar results.