Expression of human cyclophilin-40 and the effect of the His141-->Trp mutation on catalysis and cyclosporin A binding.
The cDNA encoding a human cytosolic 40-kDa cyclophilin (CyP-40) has been inserted into a modified pGEX-3X expression vector and expressed in Escherichia coli to yield recombinant CyP-40 at levels up to 4 mg/l medium. The protein was purified to homogeneity using a cyclosporin affinity matrix and gel filtration. The recombinant CyP-40 showed peptidyl-prolyl cis-trans isomerase activity (kcat/Km = 1.12 x 10(6) M-1.s-1) comparable to that of bovine brain CyP-40. The weak affinity of CyP-40 for cyclosporin A was postulated to arise from a histidine residue that replaces a tryptophan residue critical for cyclosporin A binding and highly conserved in other cyclophilins that have high affinity for cyclosporin A. Site-directed mutagenesis to replace His141 by tryptophan yielded a protein with an approximately 20-fold greater affinity for cyclosporin A (Kdapp 11.5 +/- 2 nM as determined by tryptophan fluorescence measurements). The intrinsic isomerase activity of this mutant protein with succinyl-Ala-Ala-Pro-Phe 4-nitroanilide as substrate was about nine times greater than the value obtained for the nonmutated recombinant CyP-40 and had an activity similar to that of CyP-18. NMR difference spectroscopy and molecular modelling revealed a cyclosporin-A-binding domain that is similar to that of CyP-18.
Hoffmann
K
K
Department of Pharmacology, Yale University School of Medicine, New Haven CT 06520, USA.
Kakalis
L T
LT
Anderson
K S
KS
Armitage
I M
IM
Handschumacher
R E
RE
D000591:Amino Acid Isomerases / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D001665:Binding Sites; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D000081406:Peptidyl-Prolyl Isomerase F; D021983:Cyclophilins; D016572:Cyclosporine / Q000378:metabolism*; D004789:Enzyme Activation; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D019696:Peptidylprolyl Isomerase; D011485:Protein Binding; D011994:Recombinant Proteins / Q000378:metabolism
D002352:Carrier Proteins; D000081406:Peptidyl-Prolyl Isomerase F; D011994:Recombinant Proteins; D016572:Cyclosporine; D000591:Amino Acid Isomerases; D021983:Cyclophilins; D019696:Peptidylprolyl Isomerase