CRYSTAL-STRUCTURES OF CYCLOPHILIN-A COMPLEXED WITH CYCLOSPORINE-A ANDN-METHYL-4-[(E)-2-BUTENYL]-4,4-DIMETHYLTHREONINE CYCLOSPORINE-A

Background: Cyclophilin (CyP) is a ubiquitous intracellular protein th at binds the immunosuppressive drug cyclosporin A. (CsA). CyP-CsA form s a ternary complex with calcineurin and thereby inhibits T-cell activ ation. CyP also has enzymatic activity, catalyzing the cis-trans isome rization of peptidyl-prolyl amide bonds. Results: We have determined t he structure of human cyclophilin A (CyPA) complexed with CsA to 2.1 A ngstrom resolution. We also report here the structure of CyPA complexe d with an analog of CsA, N-methyl-4-[(E)-2-butenyl]-4,4-dimethylthreon ine CsA (MeBm(2)tl-CsA), which binds less well to CyPA, but has increa sed immunosuppressive activity. Comparison of these structures with pr eviously determined structures of unligated CyPA and CyPA complexed wi th a candidate substrate for the isomerase activity, the dipeptide Ala Pro, reveals that subtle conformational changes occur in both CsA and CyPA on complex formation. Conclusions: MeBm(2)tl-CsA binds to CyPA in an essentially similar manner to CsA. The 100-fold weaker affinity of its binding may be attributable to the close contact between MeBmtl a nd the active site residue Ala103 of CyPA, which causes small conforma tional changes in both protein and drug. One change, the slight moveme nt of MeLeu6 in CsA relative to MeBm(2)tl-CsA, may be at least partial ly responsible for the higher affinity of the CyPA-MeBm(2)tl-CsA compl ex for calcineurin. Our comparison between CyPA-CsA and CyPA-AlaPro su ggests that CsA is probably not an analog of the natural substrate, co nfirming that the catalytic activity of CyPA is not related to its rol e in immunosuppression either structurally or functionally.