Fig. 1: MavL is a macro domain protein that converts ADPR-Ub into ADP-ribose and ubiquitin.

a Hydrolysis of ADPR-Ub into ADPR and Ub by MavL DupA or DupB. Recombinant proteins were incubated with ADPR-Ub, and the production of native Ub was detected by native polyacrylamide gel electrophoresis (upper panel). Native Ub, PR-Ub, and ADPR-Ub were loaded separately as controls. Identical samples separated by SDS-PAGE were detected by CBB staining, phosphoprotein stain, or immunoblotting with an ADPR-specific antibody (lower three panels). b Mutational analysis of residues important for the de-ADP ribosylation activity of MavL. Recombinant MavL or its mutants were incubated with ADPR-Ub, and the reduction of the reactant was detected by immunoblotting with an ADPR-specific antibody. c Binding of Ub, ADPR-Ub, and ADPR to MavL_40-404 or its mutants. Binding affinity was evaluated using a low-volume Nano ITC set at 20 °C. d Ribbon diagram representation of the MavL_{(40-404)D315A}-ADPR-Ub complex. Ub, ADPR, and MavL are colored in pink, yellow, and cyan, respectively. The recognition of ADPR-Ub by MavL, as well as the interactions between the two proteins, are shown in the middle panel. The 2Fo−Fc (blue) and Fo−Fc (green) electron-density maps of the key residues of MavL, Ub, and water surrounding the ribose moiety involved in forming the catalytic center are contoured at the 1.0σ and 3.0σ levels and shown in 1 and 2 of the right panel, respectively. It represents the transient state of the substrate catalyzed by MavL. The N-glycosidic bond between the side chain of R42 in Ub and the ADPR moiety was cleaved, as indicated by the red dashed lines and arrow. e The overall structure of MavL_{(40-404)D315A}-ADPR-Ub and its comparison to Apo MavL (gray) and MavL-ADPR (blue). The conformational changes that contribute to the opening of the catalytic pocket to facilitate the binding of ADPR-Ub and the subsequent reaction were shown. Residues that cause steric hindrance between Apo MavL and Ub were marked with red dashed circles. f MavL reduces the level of ADPR-Ub in infected cells. The indicated L. pneumophila strains were used to infect cells expressing 3xHA-Ub, and the accumulation of ADPR-Ub was detected after HA antibody immunoprecipitation. Expression of Flag-MavL and its mutant was detected with Flag antibody and isocitrate dehydrogenase (ICDH) was probed as a loading control.