Figure 4. Unfolded protein CH1 binds to canonical BiP substrate binding domain without binding to UPR luminal domains.
(A) MST binding curves for CH1 binding to full-length BiP (Kd = 8.7 μM), BiP's ATPase domain (no binding), BiP's substrate binding domain (Kd = 5.1 μM), Ire1 luminal domain (no binding) and Perk luminal domain (no binding). (B) Pull down experiment showing CH1 binding to both full-length and substrate binding domain of BiP only, with no interaction observed to luminal domains, reaffirming the data for CH1 interactions using MST in part A.
Figure 4—figure supplement 1. Assessing the role of unfolded protein peptide mimic (ΔEspP) in UPR stress sensing.
(A–C) ITC binding curves for ΔEspP titration into (A) Ire1 luminal domain (Kd = 6.4 μM; N = 1.97), (B) Perk luminal domain (Kd = 9.3 μM; N = 1.98), (C) full-length BiP (no binding). (D) SEC MALS analyses showing Ire1 luminal domain is dimeric in solution (MW = 104.5 kDa) and the addition of ΔEspP has no effect on Ire1 luminal domain oligomeric state (MW = 103.2 kDa). (E) SEC MALS analyses showing Perk luminal domain is dimeric in solution (MW = 67.8 kDa) and the addition of ΔEspP has no effect on Perk luminal domain oligomeric state (MW = 67.6 kDa). (F) Pull down assay to test the effect of ΔEspP on His6-tagged full-length BiP-luminal domain complexes. ΔEspP has no visible effect upon BiP-luminal domain complexes.