Extended Data Fig. 5: AMPylase activities of LnaB family members.
From: Legionella effector LnaB is a phosphoryl-AMPylase that impairs phosphosignalling
a, Recombinant GST-tagged LnaB and its truncated mutant (residues 1–327), which were purified from E. coli, were incubated with the Acanthamoeba castellanii cell lysate. The GST pull-down assays were analysed by SDS–PAGE and immunoblotting with an anti-α-actin antibody recognizing the amoebic actin. b, The actin-contained cell lysates of A. castellanii amoebae activated the AMPylase activity of LnaB. c, The intracellular growth of the L. pneumophila strains in amoebae and macrophage cells. Acanthamoeba castellanii and U937 cells were infected with the indicated L. pneumophila strains. Experiments were performed in triplicate and repeated three times with similar results. error bars represent s.d. (n = 3). d, Multiple-sequence alignment of the residues around the two conserved catalytic consensuses of LnaB family members. 40 homologous sequences were selected for analysis. e,f, Interactions of the fragments of LnaB (e,f), LHLS (e), LHEI (e) and LHBA proteins (f), respectively, with endogenous actin in transfected 293T cells. g,h, The actin-activated AMPylase activities of the LnaB homologues. PRR42-Ub was incubated by recombinant LnaB, LHLS (g), LHEI (g) and LHBA proteins (h), respectively, with actin and ATP. *, the degraded fragments of GST-LHEI. i, The AlphaFold2-predicted structure of LnaB. The prediction quality is indicated by the pLDDT (predicted Local Distance Difference Test) scores ranging from blue for high confidence to red for low confidence. The catalytic domain of LnaB is highlighted by a black dashed box. j, Sequence coverage scores of LnaB in the structure prediction. k, The pLDDT scores of the five AlphaFold2-predicted LnaB models. l, Comparison of the AlphaFold2-predicted structures of the catalytic domains of LHLS, LHBA and LHEI with that of LnaB.
