Fig. 5

MANF inhibits nucleotide exchange and ATP-induced substrate release from BiP. a Representative plot of fluorescence against time of pre-formed complexes of MABA-ADP and BiP NBD (1.25 µM) challenged at t = 0 with buffer solution containing ATP (125 µM) and MANF or its SAP domain (200 µM). b Bar diagram of MABA-ADP release rates from BiP NBD in the presence of MANF, the isolated SAP or SAPLIP domain or the indicated mutant forms of MANF. Bars represent mean values ± SD from three to five independent experiments (****P<0.0001, unpaired Student’s t test). c As in (b) but measuring the MABA-ADP release from full-length BiP in the presence of either ADP or ATP as the competitor. d As in (b) but with additional presence of the NEF Grp170 (1.25 µM) and physiological concentrations of calcium (0.5 mM). e Bar diagram of rate of MABA-ATP loading onto BiP in the absence or presence of the indicated proteins (final: 1.25 µM BiP, 1.25 µM MABA-ATP, 100 µM MANF or mutants). Bars represent mean values ± SD from three independent experiments (***P<0.001 and ****P<0.0001, unpaired Student’s t test). f Plot of k_obs for binding of MABA-ATP to BiP (as in e) against final concentration of MANF or SAP. The mean values and SD bars of three independent experiments are plotted. Single exponential best fit lines are shown. g Bar diagram of k_off of fluorescently bound substrate peptide (peptide-LY) from BiP. BiP–substrate complexes were pre-formed in presence of ADP and dissociation of pre-formed BiP–peptide complexes was induced by the introduction of ATP in the absence or presence of MANF derivatives (200 µM). Bars represent mean values ± SD from three independent experiments (***P<0.001 and ****P<0.0001, unpaired Student’s t test). h Plot of k_off for ATP-induced dissociation of BiP–substrate complex (as in g) against final concentration of MANF. The mean values and SD bars of three independent experiments are plotted. Single exponential best fit lines are shown. Source data are provided as a Source Data file