Active site mutants of human cyclophilin A separate peptidyl-prolyl isomerase activity from cyclosporin A binding and calcineurin inhibition

Protein Sci. 1992 Sep;1(9):1092-9. doi: 10.1002/pro.5560010903.


Based on recent X-ray structural information, six site-directed mutants of human cyclophilin A (hCyPA) involving residues in the putative active site--H54, R55, F60, Q111, F113, and H126--have been constructed, overexpressed, and purified from Escherichia coli to homogeneity. The proteins W121A (Liu, J., Chen, C.-M., & Walsh, C.T., 1991a, Biochemistry 30, 2306-2310), H54Q, R55A, F60A, Q111A, F113A, and H126Q were assayed for cis-trans peptidyl-prolyl isomerase (PPIase) activity, their ability to bind the immunosuppressive drug cyclosporin A (CsA), and protein phosphatase 2B (calcineurin) inhibition in the presence of CsA. Results indicate that H54Q, Q111A, F113A, and W121A retain 3-15% of the catalytic efficiency (kcat/Km) of wild-type recombinant hCyPA. The remaining three mutants (R55A, F60A, and H126Q) each retain less than 1% of the wild-type catalytic efficiency, indicating participation by these residues in PPIase catalysis. Each of the mutants bound to a CsA affinity matrix. The mutants R55A, F60A, F113A, and H126Q inhibited calcineurin in the presence of CsA, whereas W121A did not. Although CsA is a competitive inhibitor of PPIase activity, it can complex with enzymatically inactive cyclophilins and inhibit the phosphatase activity of calcineurin.

Publication types

  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Amino Acid Isomerases / chemistry
  • Amino Acid Isomerases / genetics*
  • Amino Acid Isomerases / metabolism*
  • Amino Acid Sequence
  • Base Sequence
  • Binding Sites
  • Calcineurin
  • Calmodulin-Binding Proteins / pharmacology*
  • Carrier Proteins / chemistry
  • Carrier Proteins / genetics*
  • Carrier Proteins / metabolism*
  • Circular Dichroism
  • Cyclosporine / metabolism*
  • Humans
  • Kinetics
  • Molecular Sequence Data
  • Mutagenesis, Site-Directed*
  • Oligodeoxyribonucleotides
  • Peptidylprolyl Isomerase
  • Phosphoprotein Phosphatases / pharmacology*
  • Protein Binding
  • Protein Conformation*
  • Protein Structure, Secondary*
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / metabolism
  • Spectrometry, Fluorescence


  • Calmodulin-Binding Proteins
  • Carrier Proteins
  • Oligodeoxyribonucleotides
  • Recombinant Proteins
  • Cyclosporine
  • Calcineurin
  • Phosphoprotein Phosphatases
  • Amino Acid Isomerases
  • Peptidylprolyl Isomerase