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

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 (k cat/K m) 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.