Fig. 3: FICD’s TPR domain is essential for BiP-AMP binding and deAMPylation.
a Representative BLI association–dissociation curves of FICD analytes from immobilised BiP (either AMPylated or unmodified) bound to ATP (n = 3 independent experiments). See Supplementary Fig. 4d. b Representative BLI analysis of TPR domain mutants of monomeric (i) and dimeric (ii) FICD binding to immobilised AMPylated BiP (n = 3 independent experiments). In both a and b the buffer used in the second dissociation step (see vertical dashed lines) was supplemented with 2 mM ATP. In b(ii) the second dissociation step traces (where applicable) are overlayed with the best-fit curves derived from a two-phase exponential decay model. See Supplementary Fig. 4e, f. c Analysis of the ability of different FICD variants to deAMPylate BiP. Left, Fluorescent polarisation-derived time courses of BiP-AMP(FAM) deAMPylation. Fits of the initial linear reaction phase are overlaid. Right, quantification of the approximate catalytic efficiencies of the different FICD variants. Mean values of approximate kcat/KM values for each FICD variant ± standard deviation (SD), from n = 4 independent experiments, are shown. See Supplementary Fig. 5. For reasons of experimental expedience, the Glu105Arg FICD mutant was not incorporated into an enzymatically competent FICD background and was, therefore, neither tested in this in vitro deAMPylation assay nor in the vitro AMPylation assays shown later (Fig. 5a). Source data are provided as a Source Data file.
