title: A single Trp121 to Ala121 mutation in human cyclophilin alters cyclosporin A affinity and peptidyl-prolyl isomerase activity.
abstract: Fluorescence and NMR spectral data have suggested an interaction between the single tryptophan in cyclophilin (CyP) and its high affinity ligand cyclosporin A (CsA). To study this interaction, a site mutation of Trp121 to Ala was introduced into human cyclophilin (CyP) and the encoded protein was expressed in E. coli. The Ala121 mutant was shown to catalyze the peptidyl-prolyl cis-trans isomerase (rotomase) reaction with several peptide substrates, albeit at less than ten percent the rate of the purified recombinant human CyP. Values for the apparent inhibition constant (Ki,app) of cyclosporin A with the human CyP and the Ala121 mutant were determined to be 1.6 +/- 0.4 nM and 640 +/- 90 nM, respectively by tight-binding inhibition analysis. The greater loss of affinity for CsA binding (400-fold) than for rotomase catalysis (20 fold) suggests that the catalytic and CsA binding properties associated with CyP can be decoupled as has been observed with an homologous protein found in E. coli (Liu, J. & Walsh, C.T. (1990) Proc. Natl. Acad. Sci. USA 87, 4028-4032).
authors.affiliation: Department of Medicinal Chemistry SmithKline Beecham Pharmaceuticals, King of Prussia, Pennsylvania 19406.
authors.forename: M J
authors.initials: MJ
authors.lastname: Bossard
authors.forename: P L
authors.initials: PL
authors.lastname: Koser
authors.forename: M
authors.initials: M
authors.lastname: Brandt
authors.forename: D J
authors.initials: DJ
authors.lastname: Bergsma
authors.forename: M A
authors.initials: MA
authors.lastname: Levy
mesh_terms: D000409:Alanine; D000591:Amino Acid Isomerases / Q000235:genetics* / Q000378:metabolism; D000595:Amino Acid Sequence; D001483:Base Sequence; D002352:Carrier Proteins / Q000235:genetics* / Q000378:metabolism; D003001:Cloning, Molecular; D003524:Cyclosporins / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D015345:Oligonucleotide Probes; D019696:Peptidylprolyl Isomerase; D011994:Recombinant Proteins / Q000378:metabolism; D015183:Restriction Mapping; D013379:Substrate Specificity; D014364:Tryptophan
chemical_list: D002352:Carrier Proteins; D003524:Cyclosporins; D015345:Oligonucleotide Probes; D011994:Recombinant Proteins; D014364:Tryptophan; D000591:Amino Acid Isomerases; D019696:Peptidylprolyl Isomerase; D000409:Alanine