Delineation of the calcineurin-interacting region of cyclophilin B

The immunosuppressant drug cyclosporin A (CsA) inhibits T-cell function by blocking the phosphatase activity of calcineurin. This effect is mediated b y formation of a complex between the drug and cyclophilin (CyP), which crea tes a composite surface able to make high-affinity contacts with calcineuri n. In vitro, the CyPB/CsA complex is more effective in inhibiting calcineur in than the CyPA/CsA and CyPC/CsA complexes, pointing to fine structural di fferences in the calcineurin-binding region. To delineate the calcineurin-b inding region of CyPB, we mutated several amino acids, located in two loops corresponding to CyPA regions known to be involved, as follows: R76A, G77H , D155R, and D158R. Compared to wild-type CyPB, the G77H, D155R, and D158R mutants had intact isomerase and CsA-binding activities, indicating that no major conformational changes had taken place. When complexed to CsA, they all displayed only reduced affinity for calcineurin and much decreased inhi bition of calcineurin phosphatase activity. These results strongly suggest that the three amino acids G77, D155, and D158 are directly involved in the interaction of CyPB/CsA with calcineurin. in agreement with their exposed position. The G77, D155, and D158 residues are not maintained in CyPA and m ight therefore account for the higher affinity of the CyPB/CsA complex for calcineurin.