PH Titration of the histidine residues of cyclophilin and FK506 binding protein in the absence and presence of immunosuppressant ligands

Abstract

Histidine residues in immunophilins, particularly His-126 of cyclophilin (CyP) and His-87 of the FK506 binding protein (FKBP), have been suggested to play important roles in ligand binding and peptidyl prolyl cis-trans isomerase (PP iase) catalysis. The charged states of the histidine residues in FKBP and CyP, which were characterized by their p K a values, have been determined in the absence and presence of the immunosuppressant ligands, ascomycin and cyclosporin A (CsA), respectively, by using a heteronuclear two-dimensional NMR method. Overall, the histidine residues in FKBP and CyP are very acidic with p K a values ranging from ≤ 2.8 to 6.5, indicating that they are predominantly uncharged at physiological pH. To our knowledge, the p K a value of ≤ 2.8 determined from this study is the lowest p K a reported for the free imidazole ring of the histidine residues in proteins. The abnormally acidic p K a 's of His-25 in FKBP and His-54 in CyP could be explained by their highly positively charged environments. His-87 of FKBP, which is located in the FK506 binding pocket, was found to exist in two forms in free FKBP with p K a values of 5.9 and 6.5 for the major and minor forms, respectively. His-126, which is part of the CsA and substrate binding site, has a p K a of 6.3 in free CyP. The p K a values of these two histidine residues in the free proteins are higher than the p K a 's obtained for the peptidyl prolyl cis-trans isomerase (PP iase) activity of these enzymes, indicating that the acid/base characters of His-87 of FKBP and His-126 of CyP are not essential in the PPiase catalysis. The hydrogen bonding of the histidine imidazole rings and the effect of hydrophobicity upon changes in p K a values are discussed.