Figure 5. AMPylation does not alter non-substrate interactions between BiP and a model J-domain.

Bio-layer interferometry (BLI) experiment based on the experiment outlined in Figure 4C to measure the functionally relevant non-substrate interactions between an ATPase and substrate binding BiP mutant protein (BiP^T229A-V461F) and a model J-domain fused to GST (GST-J). The cartoons on the left depict the anticipated interactions between BiP in solution and the J-domain on the sensor surface during the association step and plots of BLI binding signals against time are shown on the right. After an initial equilibration step the sensors were saturated with biotinylated GST-J followed by another wash step to achieve a stable baseline signal (not shown). The sensors were then exposed to solutions containing different concentrations of unmodified or AMPylated BiP^T229A-V461Fproteins to measure their association with the sensors. Dissociation was detected in protein-free solutions containing ATP. Representative plots of recorded binding signals of the association and dissociation steps are shown. The insets present plots of the plateau binding amplitudes during the association step against BiP concentrations of three independent experiments as well as the obtained dissociation constant (K_D) values with the corresponding standard deviations and the R² values of the fits.

Figure 5—figure supplement 1. The non-substrate interaction between BiP^T229A-V461F and the BLI sensor requires a functional J-domain.

Bio-layer interferometry (BLI) experiment as in Figure 5 to measure the functionally relevant non-substrate interactions between an ATPase activity- and substrate binding-deficient mutant BiP protein (BiP^T229A-V461F) and a wildtype model J-domain fused to GST (GST-J) or a mutant thereof carrying an inactivating amino acid substitution in the conserved HPD motif (GST-J^QPD). After an initial equilibration step the sensors were saturated with biotinylated GST-J followed by a baseline step (not shown). The sensors were then exposed to solutions containing unmodified or AMPylated BiP^T229A-V461F to measure their association with the sensors. Dissociation was detected in protein-free solutions containing either ATP or ADP. A plot of the recorded binding signals against time of the association and dissociation steps is shown. The insets present the calculated dissociation rate constants (k_off) during the dissociation steps of the indicated samples from three independent repeats of the experiment. Bars indicate the standard deviations. Note that the dissociation rate constants are the same in presence of ATP or ADP.

Figure 5—figure supplement 2. The non-substrate interaction between BiP^T229A-V461F and the J-domain is sensitive to elevated salt concentrations.

(A) Bio-layer interferometry (BLI) experiment as in Figure 5 to measure the functionally relevant non-substrate interactions between an ATPase activity- and substrate binding-deficient mutant BiP protein (BiP^T229A-V461F) and a wildtype model J-domain fused to GST (GST-J) in presence of increasing concentrations of salt (potassium chloride; KCl). The sensors were saturated with biotinylated GST-J and washed to a stable baseline (not shown). The sensors were then exposed to solutions containing unmodified BiP^T229A-V461F in presence of the indicated salt concentrations to measure its association with the sensors. Dissociation was detected in protein-free solutions containing ATP. A plot of the binding signals against time of the association and dissociation steps is shown. The inset presents a plot of the obtained binding amplitudes against the salt concentration of three independent repeats of the experiment. The line represents a best-fit curve. Note the non-linear correlation of binding and salt concentration. (B) BLI experiment as in Figure 4C. A plot of the binding signals against time of the association and dissociation steps is shown. The sensors were loaded with biotinylated GST-J corresponding to a binding signal difference of ~0.4 nm followed by saturation with biotinylated P15 BiP substrate peptide and a baseline step. The sensors were then introduced into solution containing wildtype BiP protein until steady state binding was established. Dissociation of BiP from the sensor was measured in protein-free solutions containing ADP (or ATP as a control) and different salt concentrations as indicated. The dotted lines in the dissociation step represent single-exponential fits of the curves used to calculate the corresponding dissociation rate constants (k_off) shown on the graph below. The k_off values of four independent repeats are shown and bars represent standard deviations. Note that the dissociation of BiP in presence of ADP is unaffected by increasing salt concentrations.