![]() The curves were fitted with a single exponential model values are shown in Supplementary Table 1. i, 0.3 µM BiP-NBD and 2 µM MABA-ADP were chased with 0.5 mM ADP in the absence of Bap (gray), with 6 µM Bap (black) or 6 µM Bap-C (blue). h, 0.3 µM BiP and 2 µM MABA-ADP were chased with 0.5 mM ADP (black curve) and Bap-N (green curve). 0.3 µM BiP and 2 µM MABA-ADP were chased with 0.5 mM ADP (black curve) in the presence of various Bap concentrations (f, gray curves) or Bap-C concentrations (g, blue curves). Nucleotide-exchange kinetics of Bap were determined by stopped-flow experiments at 20 ☌. f–j, Nucleotide-exchange activity of Bap. The inset shows a representative association trace of 1 μM Bap with 0.9 μM BiP-NBD in the presence of 1 mM AMP-PNP at 20 ☌. The difference in the fluorescence anisotropy signal Δr between nonbound and fully bound Bap was plotted against the Bap concentration to derive a Kd = 1.0 ± 0.2 μM (red line association kinetic was fitted using a single exponential model). e, Fluorescence anisotropy measurements of IAEDANS-labeled BiP-NBD-167 with increasing Bap concentrations. AUC measurements of BiP and BiP and Bap or Bap-C in the presence of 1 mM ATP (c) and with 1 mM ADP (d). The raw data are shown in Supplementary Fig. Fitting of s20,w value versus the Bap and Bap-C concentrations yielded Kd values of 0.14 ± 0.03 μM for BiP–Bap (black), 0.16 ± 0.05 μM for BiP–Bap-C (red) and 0.9 ± 0.3 μM for Bip-NBD–Bap (blue). Analysis of AUC measurements of 0.4 µM BiP-167–638 (black and red) or 0.5 µM BiP-NBD-167 (blue) titrated with different concentrations of Bap (black and blue) or Bap-C (red) in the presence of 1 mM AMP-PNP are shown in b. Experiments in the absence of nucleotide (apo) are shown in a. ![]() Complex formation by sedimentation velocity runs of 0.5 µM Atto488-labeled BiP alone (black line), in presence of 5 µM Bap (red line) or 0.4 µM Atto488-labeled BiP with 4 µM Bap-C (dark yellow line) were performed. Interaction of BiP with Bap and its effect on nucleotide exchangeĪ–d, AUC assays and dissociation constants of BiP-167–638 with Bap and Bap-C. The preferential interaction with BiP in its ADP state places Bap at a late stage of the chaperone cycle, in which it coordinates release of substrate and ADP, thereby resetting BiP for ATP and substrate binding. Thus, Bap is a conformational regulator affecting both nucleotide and substrate interactions. The largely unstructured Bap N-terminal domain promotes the substrate release from BiP. ![]() Here, single-molecule FRET experiments with mammalian proteins reveal that Bap affects the conformation of both BiP domains, including the lid subdomain, which is important for substrate binding. However, stimulation of the BiP ATPase activity requires full-length Bap, suggesting a complex interplay of these two factors. The interaction of the Bap C-terminal domain with the BiP ATPase domain is sufficient for its weak NEF activity. BiP is regulated by several co-chaperones including the nucleotide-exchange factor (NEF) Bap (Sil1 in yeast). BiP is the endoplasmic member of the Hsp70 family. ![]()
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