Extended Data Fig. 3: Interactions between SdeA mART and SdeA PDE are essential for the activity of SdeA mART.

a, Overview of the interactions between SdeA mART and SdeA PDE. SdeA is coloured as in Fig. 1b. The major interaction region between the two domains is outlined. b, An expanded view of the region outlined in a. Interaction residues are shown in stick representation and the red dashed lines represent polar interactions. The plug loop in SdeA mART is indicated. c, The interaction between SdeA mART and SdeA PDE. SdeA mART and SdeA PDE are shown in cartoon and surface electrostatic models, respectively. d, A view of the interaction from c rotated by 180 degrees. In this view, SdeA mART and SdeA PDE are shown as surface electrostatic and cartoon models, respectively. e, Testing the ability of SdeA PDE to process ADPR-Ub into PR-Ub. SdeA(231–588), wild-type ∆NC SdeA or the H277A mutant were incubated with ADPR-Ub and RAB33B for 30 min. The samples were stained with Coomassie and Pro-Q diamond phosphoprotein stain. f, Testing the importance of domain interaction for the activity of SdeA mART. Various SdeA segments, and mixtures of SdeA(231–588) and SdeA(597–935) or SdeA(193–935)^H277A, were incubated with RAB33B, NAD⁺ and Ub for 30 min. The samples were analysed using Coomassie staining, immunoblotting with anti-Ub antibodies and Pro-Q diamond phosphoprotein staining. e, f, Similar results were obtained in three independent experiments. Uncropped blots and gel images are shown in Supplementary Fig. 1.