Fig 2: Unique inhibiting strategies of Lpg2149 against MavC and MvcA.a The dimer-to-monomer transition of Lpg2149 upon binding to MavC/MvcA. b Conformational changes of MavC (left) and MvcA (right) in their apo and Lpg2149-bound states. The Catalytic domain is the reference point of structural superimpositions for both proteins. c Isothermal titration calorimetry (ITC) assays to test binding of Lpg2149 to MavC (left) and MvcA (right). KDs and Ns are mean ± SEM (n = 2). d, e Inhibition activities of various Lpg2149 mutants for d MavC- and e MvcA-mediated Ub deamidation. f, g Inhibition activities of various Lpg2149 mutants for f MavC- and g MvcA-mediated UBE2N~Ub deubiquitination. h Lpg2149 antagonized MavC’s inhibitory effect on NF-?B activation in cells, and the mutations of Lpg2149 (m1: F11D/F12D/Y15D; m2: V20D/L23D/L24D) decreased the antagonism. Plasmids encoding Lpg2149 (or its mutants) and MavC were co-transfected with NF-?B luciferase reporter system plasmids. Cells were mock-treated or treated with TNF-a. The luciferase activities were measured for the activation of NF-?B reporter (see details in Methods). Data presented are means ± SEM of three independent experiments. **p < 0.01, unpaired and two-tailed t test. Source data are provided as a Source Data file. Experiments in c–h were repeated independently three times with similar results.

Fig 3: Structure of MavC in complex with conjugated UBE2N~Ub.a Domain organization of MavC and conjugated UBE2N~Ub. b, c Two views of cartoon (upper) and surface (lower) representations of the MavC+UBE2N~Ub complex, with the same color code as in a. The isopeptide bond between UBE2N-K92 and Ub-Q40 was highlighted in red circles. d Structural superimposition of MavC in apo (PDB: 5TSC) and UBE2N-bound states and conformational changes of the Insertion domain upon binding to UBE2N. e Detailed hydrogen bonding interactions between MavC catalytic residues and the K92–Q40 isopeptide.

Fig 4: Interactions between MavC and Ub.a Overall view of Ub (cartoon; yellow) binding within the cleft of Catalytic (surface; green) and HBD (surface; blue) domains of MavC. b–d Three major regions of hydrogen bonding interactions between MavC and Ub, same color code as in a. The hydrogen bonds were shown in black dash lines. e, f Two major regions of hydrophobic interactions between MavC and Ub, same color code as in a. g–i In vitro ubiquitination reactions for various MavC mutants on different MavC–Ub inter-molecular interacting surfaces. The reaction mixtures were subjected to SDS-PAGE and stained with Coomassie dye. j Indicated Ub proteins were incubated with MavC and UBE2N. The reaction mixtures were subjected to SDS-PAGE and stained with Coomassie dye. k In vitro Ub deamidation reactions for various MavC mutants. The reaction mixtures were subjected to native-PAGE and stained with Coomassie dye. l Mutations on MavC decreased its inhibition ability to NF-?B activation in cells. Plasmids encoding MavC or its mutants were co-transfected with NF-?B luciferase reporter system plasmids. Cells were mock-treated or treated with TNF-a. The luciferase activities were measured for the activation of NF-?B reporter (see details in Methods). Data presented are means ± SEM of three independent experiments. **p < 0.01, unpaired and two-tailed t test. Source data are provided as a Source Data file. Experiments in g–l were repeated independently three times with similar results.

Fig 5: Interactions between MavC and UBE2N.a Overall view of UBE2N–Catalytic and UBE2N–Insertion interacting surfaces, with the same color code as in Fig. 1a. b The aa 87–100 loop region of UBE2N (cartoon; purple) binds within a critical groove of MavC Catalytic domain (surface; green). c Detailed hydrophobic interactions between MavC-M317 (green) and the residues from UBE2N (purple). d Conformational changes of UBE2N upon binding to MavC. The K92 residues were highlighted as spheres. e Detailed hydrophobic interactions between MavC Insertion domain (cyan) and UBE2N (purple). f, g In vitro ubiquitination reactions for various MavC mutants in both Catalytic (f) and Insertion (g) domains. The reaction mixtures were subjected to SDS-PAGE and stained with Coomassie dye. h, i Elution profiles of SEC runs on Superdex 200 10/300 column to test binding of UBE2N with MavC/MvcA (h) or their isolated Insertion domains (i). Note that only MavC or MavC–Insertion, but not MvcA or MvcA–Insertion, has a considerable shift in elution volume upon binding to UBE2N. Black asterisks indicate the fractions analyzed by SDS-PAGE. Source data are provided as a Source Data file. Experiments in f–i were repeated independently three times with similar results.