Fig. 5: Binding interface of MavC with Ube2N.

a–c Detailed view of interactions in MavC regions 1, 2, and 3 with Ube2N. Key residues are labeled and represented as stick models. MavC is depicted in burgundy and Ube2N in green. Hydrogen-bonding interactions are given in black and hydrophobic interactions in red. d Comparison of the ubiquitinating activity of wild-type MavC versus mutant proteins using Ube2N-SS-Ub as the substrate. Reactions were subjected to SDS-PAGE and visualized with Coomassie Blue. A control reaction without MavC is included. e MavC-mediated ubiquitination of Ube2N during L.p infection. Cells infected with the indicated L. pneumophila strains were lysed with 0.2% saponin and lysate separated by SDS-PAGE, probed by immunoblotting with antibodies specific for Ube2N (upper panel) and MavC (lower panel). respectively. f The effects of MavC and its mutants on NFκB activation. HEK293T cells were transfected with plasmids expressing a luciferase reporter responsive to NF-κB and Flag-MavC or its mutant. At the same time, a plasmid expressing Renilla luciferase used as an internal control and stimulator TRAF6 were co-transfected. NF-κB activity was determined by dual luciferase assay. The expression of MavC and its mutants was probed in lysates of transfected cell while tubulin was detected as a loading control. Three independent experiments were done with similar results. Error bars indicate standard error of the mean (SEM).