Extended Data Fig. 5: Direct binding of ADP-Hep to ALPK1-NTD.
From: Alpha-kinase 1 is a cytosolic innate immune receptor for bacterial ADP-heptose
a, E. coli-purified ALPK1-(N+K) complex on a Coomassie blue-stained SDS–PAGE gel. b–e, HPLC–MS fractionation, NF-κB-inducing activity and mass spectrometry of small-molecule extracts from His6–ALPK1-NTD purified from wild-type E. coli BL21 (DE3). The small-molecule extracts, obtained by protein denaturing and precipitation (95 °C for 5 min), were analysed by HPLC–MS with 17 fractions obtained (b). The 17 fractions were used to treat 293T cells (c) or cells expressing Flag–TIFA (d); NF-κB luciferase activity (mean ± s.d. from three technical replicates) and anti-pT9-TIFA immunoblotting are in c and d, respectively. e, Mass spectrometry of fraction 6 identified three major ions corresponding to AMP, ADP and ADP-Hep. f, LC–MS/MS of ADP-Hep in fraction 6 (b, e) or synthetic ADP-LD-Hep standard. g, MS/MS spectra of the [M+H]+ product ions of ADP-Hep in fraction 6 in comparison with those of synthetic ADP-LD-Hep. The heptose of ADP-Hep was not shown owing to neutral loss. h, BLI assay of ADP-LD-Hep or S7P binding to ALPK1-(N+K). ALPK1-(N+K) complexes purified from E. coli BL21 (DE3) ΔhldE were biotinylated in vitro. Sensorgrams of the binding to ALPK1-(N+K) by different concentrations of the indicated sugar (colour lines) are shown. Grey lines are from model fits. Data shown are representative of three (a, h) or two (b–g) independent experiments.
