Extended Data Fig. 6: HBP binding is insufficient to render ALPK1-KD competent for substrate recognition and phosphate transfer.
From: Alpha-kinase 1 is a cytosolic innate immune receptor for bacterial ADP-heptose
a, BLI assay of HBP binding to in vitro-biotinylated apo-ALPK1-(N+K). Sensorgrams of the binding in different concentrations of HBP (colour lines) are shown. Grey lines are from model fits. b, Excess HBP competitively inhibits activation of ALPK1 by ADP-Hep. Flag–ALPK1 purified from 293T cells was incubated with purified TIFA–His6 and ADP-Hep in the presence of a titrating amount of HBP. c, d, Effects of HBP binding on ALPK1 phosphorylation and recognition of a 15-residue peptide substrate derived from TIFA. The N-terminal 15-residue sequences of TIFA were fused to GST (GST–TIFA.N15). d, GST-pulldown assay of ALPK1-(N+K) binding to GST–TIFA.N15. e, Effect of HBP binding on ATP hydrolysis activity of ALPK1. Apo-ALPK1-(N+K) (wild type or K/M) was incubated with HBP or ADP-LD-Hep, and further reacted with ATP. Percentages of ATP consumption at indicated reaction conditions are shown (mean ± s.d. from three technical replicates). Two-tailed unpaired Student’s t-test was performed (*P < 0.05; **P < 0.01; ***P < 0.001). f, Graphical representation of DSS-crosslinked residues between ALPK1-NTD and ALPK1-KD identified by chemical cross-linking coupled with mass spectrometry. Apo-ALPK1-(N+K) was left untreated, or incubated with HBP or ADP-LD-Hep. Crosslinking connections are depicted by straight lines, and the corresponding raw mass spectrometry data are in Supplementary Table 3. g, Crystal structure of TRPM7 kinase domain in complex with AMPPNP (PDB code, 1IA9). TRPM7 is shown in cartoons and AMPPNP is in sticks. The loop containing K1727 and I1736 (shown in sticks, corresponding to K1140 and K1149 in human ALPK1, respectively) is in yellow. Phosphorylation of TIFA and GST-TIFA.N15 was assayed by anti-pT9-TIFA immunoblotting (b, c). Apo-ALPK1-(N+K), TIFA–His6, and GST–TIFA.N15 were purified from E. coli BL21 (DE3) ΔhldE (a–f). Data shown are representative of three (a–e) and two (f) independent experiments.
