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

• Published in: Protein science 1992-09, Vol.1 (9), p.1092-1099
• Main Authors: Zydowsky, Lynne D., Etzkorn, Felicia A., Chang, Howard Y., Ferguson, Stephen B., Stolz, Lesley A., Ho, Susanna I., Walsh, Christopher T.
• Format: Article
• Language: English
• Subjects: active sites; Amino Acid Isomerases - chemistry; Amino Acid Isomerases - genetics; Amino Acid Isomerases - metabolism; Amino Acid Sequence; Base Sequence; binding; Binding Sites; Calcineurin; Calmodulin-Binding Proteins - pharmacology; Carrier Proteins - chemistry; Carrier Proteins - genetics; Carrier Proteins - metabolism; Circular Dichroism; cyclophilin; cyclophilin A; cyclosporin A; Cyclosporine - metabolism; enzymatic activity; Humans; immunosuppression; inhibition; Kinetics; man; Molecular Sequence Data; Mutagenesis, Site-Directed; mutants; Oligodeoxyribonucleotides; Peptidylprolyl Isomerase; peptidyl‐prolyl isomerase; Phosphoprotein Phosphatases - pharmacology; Protein Binding; Protein Conformation; Protein Structure, Secondary; Recombinant Proteins - chemistry; Recombinant Proteins - metabolism; specificity; Spectrometry, Fluorescence
• ISSN: 0961-8368; 1469-896X
• 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.