Fig. 5.

ER chaperones interact with a conserved positive electrostatic surface of cyclophilin B. A, chemical shift changes of ¹⁵N-labeled cyclophilin B upon binding to N-terminal 36-amino acid peptide of ERp72 (left panel) and C-terminal 21-amino acid peptide of GRp94 (right panel), plotted as a function of cyclophilin B sequence. B, the shifted residues of cyclophilin B Lys⁶, Lys⁹, and Lys³⁵ (bold type) are identical from worm to human (left panel) and localize to a positively charged surface of cyclophilin B (right panel). Red indicates negative electrostatic potential, and blue indicates positive electrostatic potential. C, affinity purification of chaperones from rat ER with recombinant GST-cyclophilin B and its mutants K6A/K35A and PentaK-A (K4A/K5A/K6A/K9A/K35A) demonstrate that the positive electrostatic surface represented by conserved lysine residues of cyclophilin B seen in B is indispensable for cyclophilin B-chaperone interactions. Glutathione eluates were analyzed by SDS-PAGE and colloidal Coomassie staining (CB) in the left panel and in the right panel by SDS-PAGE and Western blotting (WB) with α-GRp94, α-ERp72, and α-calreticulin (CALR) antibodies. The fold of each cyclophilin B mutant was verified by one-dimensional NMR. (See also supplemental Fig. S9.)