Extended Data Fig. 3: ATP is the ligand and actin is the host activator of LnaB.
From: Legionella effector LnaB is a phosphoryl-AMPylase that impairs phosphosignalling
a, Representative screening results of the effectors responsible for producing ADPRR42-Ub from PRR42-Ub. PRR42-Ub was treated with the cell lysates of 293T cells transfected with the plasmids encoding the indicated effectors and analysed by SDS–PAGE. b, Other representative screening results of the effectors responsible for producing ADPRR42-Ub from PRR42-Ub. c, Discrimination of PRR42-Ub and ADPRR42-Ub by 13% phosphate-affinity Phos-tag SDS–PAGE. d, GFP-LnaB immunoprecipitated from 293T cells was incubated with PRR42-Ub with the ligand mixture of AMP, ADP, ATP and NAD. Effects of depletion of each ligand in the reactions were examined by immunoblotting. e, Immunoblotting analysis of the treatment of PRR42-Ub by recombinant GST–LnaB protein that was purified from E. coli. f, GST and GST-tagged LnaB purified from E. coli were incubated with 293T cell lysates. After pull-down, GST or GST-tagged LnaB reacted with PRR42-Ub in the presence of ATP. ADPRR42-Ub was examined by immunoblotting with the anti-ADPR antibody. g, PRR42-Ub was incubated with recombinant LnaB protein purified from E. coli and the proteinase K-treated or untreated 293T cell lysates. The samples were analysed by SDS–PAGE. h, Recombinant GST–LnaB protein purified from E. coli was incubated with the 293T cell lysates. After GST pull-down, the samples were analysed by SDS–PAGE and silver staining. The band of actin on the gel is highlighted with an arrow. i, List of the proteins identified by mass spectrometry analysis of the protein band bound by LnaB on SDS–PAGE in (h). j, Interactions of the LnaB fragments with endogenous actin in 293T cells. k, The activities of the LnaB fragments. PRR42-Ub was treated by the cell lysates of 293T cells expressing GFP-tagged LnaB fragments. l, LnaB and LHLS inhibit yeast growth. LnaB HE/AA, the double mutant of H305A and E309A of LnaB; LHLS HE/AA, the double mutant of H287A and E291A of LHLS.
