Cloning, expression, and purification of human cyclophilin in Escherichia coli and assessment of the catalytic role of cysteines by site-directed mutagenesis.
The cDNA encoding human cyclophilin from the Jurkat T-cell lymphoma line has been cloned by the expression cassette polymerase chain reaction and sequenced, and an expression vector has been constructed under control of the tac promoter for efficient expression in Escherichia coli. Active cyclophilin is produced at up to 40% of soluble cell protein, facilitating a one-column purification to homogeneity. Wild-type cyclophilin was characterized for binding of the potent immunosuppressant agent cyclosporin A (Kd = 46 nM) by tryptophan fluorescence enhancement and for inhibition (IC50 = 19 nM) of cyclophilin's peptidyl-prolyl cis-trans isomerase (rotamase) activity. With N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide as the substrate, recombinant human cyclophilin has a high catalytic efficiency; kcat/Km is 1.4 X 10(7) M-1.S-1 at 10 degrees C. To test the prior suggestion that a cysteine residue may be essential for catalysis and immunosuppressant binding, the four cysteines at positions 52, 62, 115, and 161 were mutated individually to alanine and the purified mutant proteins were shown to retain full affinity for cyclosporin A and equivalent catalytic efficiency as a rotamase. Clearly the cysteines play no essential role in catalysis or cyclosporin A binding. These results rule out the recently proposed mechanism [Fischer, G., Wittmann-Liebold, B., Lang, K., Kiefhaber, T. & Schmid, F. X. (1989) Nature (London) 337, 476-478)] involving the formation of tetrahedral hemithioorthoamide. Whereas mechanisms that embody other tetrahedral intermediates may be operative, an alternative mechanism is considered that involves distortion of bound substrate with a twisted (90 degrees) peptidyl-prolyl amide bond.
Liu
J
J
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115.
Albers
M W
MW
Chen
C M
CM
Schreiber
S L
SL
Walsh
C T
CT
D000818:Animals; D001483:Base Sequence; D002352:Carrier Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D002460:Cell Line; D003001:Cloning, Molecular; D003524:Cyclosporins / Q000378:metabolism; D003545:Cysteine; D015870:Gene Expression; D005796:Genes; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009154:Mutation; D015345:Oligonucleotide Probes; D019696:Peptidylprolyl Isomerase; D016133:Polymerase Chain Reaction; D011485:Protein Binding; D051381:Rats; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism
D002352:Carrier Proteins; D003524:Cyclosporins; D015345:Oligonucleotide Probes; D011994:Recombinant Proteins; D019696:Peptidylprolyl Isomerase; D003545:Cysteine