Structure of human cyclophilin and its binding site for cyclosporin A determined by X-ray crystallography and NMR spectroscopy

Abstract

The protein cyclophilin is the major intracellular receptor for the immunosuppressive drug cyclosporin A. Cyclosporin A acts as an inhibitor of T-cell activation and can prevent graft rejection in organ and bone marrow transplantation. Cyclophilin may be responsible for mediating this immunosuppressive response. Cyclophilin also catalyses the interconversion of the cis and trans isomers of the peptidyl-prolyl amide bonds of peptide and protein substrates. Here we report the X-ray crystal structure of human recombinant cyclophilin complexed with a tetrapeptide and the identification, by nuclear magnetic resonance spectroscopy, of the specific binding site for cyclosporin A. Cyclophilin has an eight-stranded antiparallel beta-barrel structure. The prolyl isomerase substrate-binding site is coincident with the cyclosporine-binding site. These results may help to provide a structural basis for rationalizing the immunosuppressive function of the cyclosporin-cyclophilin system and will also be important in the design of improved immunosuppressant drugs.

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

“…IB). This pocket has been implicated in PPIase catalysis by the location of bound N-acetyl-AAPA-amidomethyl-coumarin (Kallen et al, 1991) and by the recent evidence that a CsA analogue is a competitive inhibitor of tetrapeptide substrates (Kofron et al, 1991). To evaluate structural predictions, we determined PPIase activity and CsA recognition and ultimately determined the activity of the mutants for calcineurin inhibition.…”

“…For example mutagenesis of W121 to F121 reduced CsA affinity by 75-fold, whereas mutagenesis of F112 in Escherichia coli CyP to W 112 enhanced drug sensitivity 23-fold (Liu et al, 1991a). Also, isotopeedited NMR studies on I3C-labeled CsA reveal nuclear Overhauser effects to this tryptophan (Kallen et al, 1991;Neri et al, 1991). Within the binding cleft identified by X-ray crystallography are the side chains of H54, R55, F60, 4111, F113, W121, and H126, which, we have noted (Ke et al, 1991), are highly conserved amongst several CyPs.…”

“…Within the binding cleft identified by X-ray crystallography are the side chains of H54, R55, F60, 4111, F113, W121, and H126, which, we have noted (Ke et al, 1991), are highly conserved amongst several CyPs. The structure of hCyPA bound to tetrapeptide (Kallen et al, 1991) shows that the guanidinium side chain of R55 and the imidazole of H126 are in close proximity to the prolyl ring of the bound substrate. CsA competitively inhibits tetrapeptide Xaa-Pro isomerization, indicating that it binds in the PPIase active site (Kofron et al, 1991).…”

“…Bound FK506 is likewise distinct from its unbound conformation (Tanaka et al, 1987;Van Duyne et al, 1991). Recently, two reports of the X-ray structure of recombinant human T-cell CyPA have appeared, one unliganded (Ke et al, 1991>, the other with a bound tetrapeptide substrate (Kallen et al, 1991).…”

Unfolding of CPR3 Gets Initiated at the Active Site and Proceeds via Two Intermediates

“…Cyclophilin, a member of the immunophilins found in all the organisms, including prokaryotes and eukaryotes, is known to be the intracellular receptor of the immunosuppressive drug cyclosporine A (CsA) (2)(3)(4). In humans, seven major cyclophilins (from a total of 16 cyclophilins) are expressed, viz., CypA (18 kDa), CypB (22 kDa), CypC, CypD, CypE, Cyp40 (40 kDa), and CypNK (first identified from human natural killer cells).…”