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

Authors

  • Lynne D. Zydowsky,

    1. Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115
    Search for more papers by this author
  • Felicia A. Etzkorn,

    1. Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115
    Search for more papers by this author
  • Howard Y. Chang,

    1. Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115
    Search for more papers by this author
  • Stephen B. Ferguson,

    1. Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115
    Search for more papers by this author
  • Lesley A. Stolz,

    1. Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115
    Search for more papers by this author
  • Susanna I. Ho,

    1. Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115
    Search for more papers by this author
  • Christopher T. Walsh

    Corresponding author
    1. Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115
    • Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, 240 Longwood Avenue, Boston, Massachusetts 02115
    Search for more papers by this author

Abstract

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.

Ancillary