Fig. 5: An aspartate-rich patch located in the linker is important for AIP56 translocation but not for pore formation. | Nature Communications

Fig. 5: An aspartate-rich patch located in the linker is important for AIP56 translocation but not for pore formation.

From: Unconventional structure and mechanisms for membrane interaction and translocation of the NF-κB-targeting toxin AIP56

Fig. 5

a V5 plus His-tagged AIP56 modified in the aspartate-rich motif (AIP56D274S/D276-278S and AIP56D274N/D276-278N) is unable to cleave p65 in intact cells. Cleavage of p65 was assessed by western blotting and protein loading by Ponceau S staining. The result shown is representative of six (n = 6) independent experiments. b V5 plus His-tagged AIP56D274S/D276-278S and AIP56D274N/D276-278N were unable to translocate across the host cell membrane in response to acidification. In all experiments, mock-treated cells were used as controls. NF-kB p65 cleavage was analyzed by western blotting. The result shown is representative of five (n = 5) independent experiments. The plot shows the quantification of intact NF-kB p65 normalized for Ponceau S. Statistical significance was tested by one-way ANOVA and p values for the individual comparisons were calculated using Tukey’s HSD test. Data are presented as mean values ± SD. Actual p values from left-to-right and upwards for pH 7.0: p < 0.001, p = 0.91, p > 0.99, p = 0.005, p = 0.90, p = 0.81; for pH 4.5: p = 0.02, p > 0.99, p > 0.99, p > 0.99, p = 0.78, p = 0.36; ns non-significant. Open (without concanamycin A) or closed (with concanamycin A) symbols as well as color coding have been added to facilitate the reading of the experimental conditions, as specified below the graph. Samples were derived from the same experiment and the blots processed in parallel. NF-kB p65, nuclear factor kappa-light-chain-enhancer of activated B cells subunit p65; ConcA, concanamycin A (black); AIP56, purple, AIP56D274S/D276-278S, red; AIP56D274N/D276-278N, green. c AIP56D274S/D276-278S and AIP56D274N/D276-278N retained the ability to interact with black lipid bilayers. Proteins were used at a final concentration of 14 nM. Membrane activity was induced by acidification (pH 4.8; red arrows) of the aqueous phase at the cis-side of the chamber. Each result shown is representative of at least three (n = 3) independent measurements. Source data for (b) and (c) are provided in the Source data file.

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