Fig. 3: Residues E218, H222, H231 and E234 in the D209-K247 hairpin control the low pH-triggered conformational changes required for AIP56 membrane interaction and translocation. | Nature Communications

Fig. 3: Residues E218, H222, H231 and E234 in the D209-K247 hairpin control the low pH-triggered conformational changes required for AIP56 membrane interaction and translocation.

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

Fig. 3: Residues E218, H222, H231 and E234 in the D209-K247 hairpin control the low pH-triggered conformational changes required for AIP56 membrane interaction and translocation.The alternative text for this image may have been generated using AI.

a Localization of the putative pH-sensing residues selected for replacement on the three-dimensional structure of the AIP56 catalytic domain. Cartoon (left) and surface representation of AIP56 catalytic domain with (middle) or without (right) the middle domain. The catalytic residues (H165, E166 and H169) are shown in red, the putative pH-sensing residues in orange and the D209-K247 hairpin in marine blue. The cysteine residues (pink sticks) forming the disulfide bridge (yellow) are also shown. b Analysis of NF-kB p65 cleavage in mouse bone marrow-derived macrophages (mBMDM) by V5 plus His-tagged AIP56 variants. Cleavage of p65 was assessed by western blotting (upper panel; chromogenic detection) and protein loading by staining the membranes with Ponceau S (lower panel). The result shown is representative of six (n = 6) independent experiments. c Peak ANS (8-Anilino-1-naphthalenesulfonic acid) fluorescence measured at 475 nm for the indicated pH, normalized by subtracting the corresponding values at pH 7 (fluorescence due to conformational changes caused by the mutation and not due to acidification). The measurement curves for each pH at different wavelengths are shown in Supplementary Fig. 7a. The results shown are representative of at least three (n = 3) independent experiments. AIP56, black; AIP56E214K, blue; AIP56E218K, purple; AIP56H222K, green; AIP56H231K, brown; AIP56E234K, pink; AIP56H231K/E234K, gray; AIP56E214K/E218K/H222K, orange; d Coomassie Blue-stained SDS-PAGE gels from limited proteolysis of AIP56 and AIP56H231K/E234K by Proteinase K. A and B mark the bands corresponding to the catalytic and receptor-binding domains, respectively. The results shown are representative of two (n = 2) independent experiments. e Interaction of AIP56 or AIP56 variants with black lipid bilayers. Single-channel recordings of DiPhPC/n-decane membranes after addition of the indicated proteins to one side of the black lipid bilayer 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), (c) and (d) are provided in the Source data file.

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