Cyclophilin residues that affect noncompetitive inhibition of the protein serine phosphatase activity of calcineurin by the cyclophilin.cyclosporin A complex

Mutation of three cationic surface residues of human cyclophilin A (hCyPA), R69, K125, and R148, to both anionic and neutral residues left its intrinsic peptidyl-prolyl isomerase (PPIase) activity and cyclosporin A (CsA) binding unaffected, but altered its ability to inhibit the serine phosphatase a...

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Bibliographic Details
Published in:Biochemistry (Easton) 1994-03, Vol.33 (9), p.2380
Main Authors: Etzkorn, F A, Chang, Z Y, Stolz, L A, Walsh, C T
Format: Article
Language:English
Subjects:
Amino Acid Isomerases - chemistry
Amino Acid Isomerases - pharmacology
Base Sequence
Calcineurin
Calmodulin-Binding Proteins - antagonists & inhibitors
Carrier Proteins - chemistry
Carrier Proteins - pharmacology
Cyclosporine - chemistry
Cyclosporine - pharmacology
Humans
In Vitro Techniques
Kinetics
Macromolecular Substances
Magnetic Resonance Spectroscopy
Models, Molecular
Molecular Sequence Data
Mutagenesis, Site-Directed
Peptidylprolyl Isomerase
Phosphoprotein Phosphatases - antagonists & inhibitors
Structure-Activity Relationship
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Summary:Mutation of three cationic surface residues of human cyclophilin A (hCyPA), R69, K125, and R148, to both anionic and neutral residues left its intrinsic peptidyl-prolyl isomerase (PPIase) activity and cyclosporin A (CsA) binding unaffected, but altered its ability to inhibit the serine phosphatase activity of calcineurin (CN). R69E was 13-fold less effective (Ki = 3400 nM) than wild-type hCyPA (Ki = 270 nM) in presenting CsA for calcineurin phosphatase inhibition, while R148E was 17-fold more effective (Ki < or = 16 nM), and human CyPB was 13-fold better (Ki < or = 21 nM), establishing that a composite drug/protein surface is being recognized. The phosphoserine phosphatase reaction catalyzed by CN using unlabeled phosphoserine RII19 peptide was coupled to a continuous spectrophotometric assay to measure inorganic phosphate production using the enzyme purine ribonucleoside phosphorylase and the substrate N7-methyl-2-thioguanosine [Webb, M. R. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 4884-4887]. With this assay, we have determined that human cyclophilin A complexed with the immunosuppressive drug cyclosporin A is a noncompetitive inhibitor of calcineurin phosphatase activity. This mutational analysis identified hCyPA residues that interact with CN, and comparison to similar data on FKBP allowed us to begin to map out the CN recognition surface. The p-nitrophenylphosphatase activity of CN was stimulated ca. 3-fold by CyP.CsA, presumably reflecting altered active site geometry and selective access of this small substrate.
ISSN:0006-2960