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. 1992 Sep;1(9):1092–1099. doi: 10.1002/pro.5560010903

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

L D Zydowsky 1, F A Etzkorn 1, H Y Chang 1, S B Ferguson 1, L A Stolz 1, S I Ho 1, C T Walsh 1
PMCID: PMC2142182  PMID: 1338979

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.

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Selected References

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  1. Blumenthal D. K., Takio K., Hansen R. S., Krebs E. G. Dephosphorylation of cAMP-dependent protein kinase regulatory subunit (type II) by calmodulin-dependent protein phosphatase. Determinants of substrate specificity. J Biol Chem. 1986 Jun 25;261(18):8140–8145. [PubMed] [Google Scholar]
  2. Brenner B., Francis C. W., Totterman S., Kessler C. M., Rao A. K., Rubin R., Kwaan H. C., Gabriel K. R., Marder V. J. Quantitation of venous clot lysis with the D-dimer immunoassay during fibrinolytic therapy requires correction for soluble fibrin degradation. Circulation. 1990 Jun;81(6):1818–1825. doi: 10.1161/01.cir.81.6.1818. [DOI] [PubMed] [Google Scholar]
  3. Emmel E. A., Verweij C. L., Durand D. B., Higgins K. M., Lacy E., Crabtree G. R. Cyclosporin A specifically inhibits function of nuclear proteins involved in T cell activation. Science. 1989 Dec 22;246(4937):1617–1620. doi: 10.1126/science.2595372. [DOI] [PubMed] [Google Scholar]
  4. Fesik S. W., Gampe R. T., Jr, Eaton H. L., Gemmecker G., Olejniczak E. T., Neri P., Holzman T. F., Egan D. A., Edalji R., Simmer R. NMR studies of [U-13C]cyclosporin A bound to cyclophilin: bound conformation and portions of cyclosporin involved in binding. Biochemistry. 1991 Jul 2;30(26):6574–6583. doi: 10.1021/bi00240a030. [DOI] [PubMed] [Google Scholar]
  5. Fischer G., Wittmann-Liebold B., Lang K., Kiefhaber T., Schmid F. X. Cyclophilin and peptidyl-prolyl cis-trans isomerase are probably identical proteins. Nature. 1989 Feb 2;337(6206):476–478. doi: 10.1038/337476a0. [DOI] [PubMed] [Google Scholar]
  6. Flanagan W. M., Corthésy B., Bram R. J., Crabtree G. R. Nuclear association of a T-cell transcription factor blocked by FK-506 and cyclosporin A. Nature. 1991 Aug 29;352(6338):803–807. doi: 10.1038/352803a0. [DOI] [PubMed] [Google Scholar]
  7. Friedman J., Weissman I. Two cytoplasmic candidates for immunophilin action are revealed by affinity for a new cyclophilin: one in the presence and one in the absence of CsA. Cell. 1991 Aug 23;66(4):799–806. doi: 10.1016/0092-8674(91)90123-g. [DOI] [PubMed] [Google Scholar]
  8. Handschumacher R. E., Harding M. W., Rice J., Drugge R. J., Speicher D. W. Cyclophilin: a specific cytosolic binding protein for cyclosporin A. Science. 1984 Nov 2;226(4674):544–547. doi: 10.1126/science.6238408. [DOI] [PubMed] [Google Scholar]
  9. Harding M. W., Galat A., Uehling D. E., Schreiber S. L. A receptor for the immunosuppressant FK506 is a cis-trans peptidyl-prolyl isomerase. Nature. 1989 Oct 26;341(6244):758–760. doi: 10.1038/341758a0. [DOI] [PubMed] [Google Scholar]
  10. Harrison R. K., Stein R. L. Mechanistic studies of peptidyl prolyl cis-trans isomerase: evidence for catalysis by distortion. Biochemistry. 1990 Feb 20;29(7):1684–1689. doi: 10.1021/bi00459a003. [DOI] [PubMed] [Google Scholar]
  11. Hayano T., Takahashi N., Kato S., Maki N., Suzuki M. Two distinct forms of peptidylprolyl-cis-trans-isomerase are expressed separately in periplasmic and cytoplasmic compartments of Escherichia coli cells. Biochemistry. 1991 Mar 26;30(12):3041–3048. doi: 10.1021/bi00226a009. [DOI] [PubMed] [Google Scholar]
  12. Kallen J., Spitzfaden C., Zurini M. G., Wider G., Widmer H., Wüthrich K., Walkinshaw M. D. Structure of human cyclophilin and its binding site for cyclosporin A determined by X-ray crystallography and NMR spectroscopy. Nature. 1991 Sep 19;353(6341):276–279. doi: 10.1038/353276a0. [DOI] [PubMed] [Google Scholar]
  13. Ke H. M., Zydowsky L. D., Liu J., Walsh C. T. Crystal structure of recombinant human T-cell cyclophilin A at 2.5 A resolution. Proc Natl Acad Sci U S A. 1991 Nov 1;88(21):9483–9487. doi: 10.1073/pnas.88.21.9483. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Kofron J. L., Kuzmic P., Kishore V., Colón-Bonilla E., Rich D. H. Determination of kinetic constants for peptidyl prolyl cis-trans isomerases by an improved spectrophotometric assay. Biochemistry. 1991 Jun 25;30(25):6127–6134. doi: 10.1021/bi00239a007. [DOI] [PubMed] [Google Scholar]
  15. Kunkel T. A. Rapid and efficient site-specific mutagenesis without phenotypic selection. Proc Natl Acad Sci U S A. 1985 Jan;82(2):488–492. doi: 10.1073/pnas.82.2.488. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Liu J., Albers M. W., Chen C. M., Schreiber S. L., Walsh C. T. Cloning, expression, and purification of human cyclophilin in Escherichia coli and assessment of the catalytic role of cysteines by site-directed mutagenesis. Proc Natl Acad Sci U S A. 1990 Mar;87(6):2304–2308. doi: 10.1073/pnas.87.6.2304. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Liu J., Chen C. M., Walsh C. T. Human and Escherichia coli cyclophilins: sensitivity to inhibition by the immunosuppressant cyclosporin A correlates with a specific tryptophan residue. Biochemistry. 1991 Mar 5;30(9):2306–2310. doi: 10.1021/bi00223a003. [DOI] [PubMed] [Google Scholar]
  18. Liu J., Farmer J. D., Jr, Lane W. S., Friedman J., Weissman I., Schreiber S. L. Calcineurin is a common target of cyclophilin-cyclosporin A and FKBP-FK506 complexes. Cell. 1991 Aug 23;66(4):807–815. doi: 10.1016/0092-8674(91)90124-h. [DOI] [PubMed] [Google Scholar]
  19. Schönbrunner E. R., Mayer S., Tropschug M., Fischer G., Takahashi N., Schmid F. X. Catalysis of protein folding by cyclophilins from different species. J Biol Chem. 1991 Feb 25;266(6):3630–3635. [PubMed] [Google Scholar]
  20. Siekierka J. J., Hung S. H., Poe M., Lin C. S., Sigal N. H. A cytosolic binding protein for the immunosuppressant FK506 has peptidyl-prolyl isomerase activity but is distinct from cyclophilin. Nature. 1989 Oct 26;341(6244):755–757. doi: 10.1038/341755a0. [DOI] [PubMed] [Google Scholar]
  21. Stein R. L. Catalysis steps. Nature. 1992 Mar 5;356(6364):23–24. doi: 10.1038/356023c0. [DOI] [PubMed] [Google Scholar]
  22. Swanson S. K., Born T., Zydowsky L. D., Cho H., Chang H. Y., Walsh C. T., Rusnak F. Cyclosporin-mediated inhibition of bovine calcineurin by cyclophilins A and B. Proc Natl Acad Sci U S A. 1992 May 1;89(9):3741–3745. doi: 10.1073/pnas.89.9.3741. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Takahashi N., Hayano T., Suzuki M. Peptidyl-prolyl cis-trans isomerase is the cyclosporin A-binding protein cyclophilin. Nature. 1989 Feb 2;337(6206):473–475. doi: 10.1038/337473a0. [DOI] [PubMed] [Google Scholar]
  24. Tropschug M., Barthelmess I. B., Neupert W. Sensitivity to cyclosporin A is mediated by cyclophilin in Neurospora crassa and Saccharomyces cerevisiae. Nature. 1989 Dec 21;342(6252):953–955. doi: 10.1038/342953a0. [DOI] [PubMed] [Google Scholar]
  25. Van Duyne G. D., Standaert R. F., Karplus P. A., Schreiber S. L., Clardy J. Atomic structure of FKBP-FK506, an immunophilin-immunosuppressant complex. Science. 1991 May 10;252(5007):839–842. doi: 10.1126/science.1709302. [DOI] [PubMed] [Google Scholar]
  26. Weber C., Wider G., von Freyberg B., Traber R., Braun W., Widmer H., Wüthrich K. The NMR structure of cyclosporin A bound to cyclophilin in aqueous solution. Biochemistry. 1991 Jul 2;30(26):6563–6574. doi: 10.1021/bi00240a029. [DOI] [PubMed] [Google Scholar]
  27. Williams J. W., Morrison J. F. The kinetics of reversible tight-binding inhibition. Methods Enzymol. 1979;63:437–467. doi: 10.1016/0076-6879(79)63019-7. [DOI] [PubMed] [Google Scholar]

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