title: Delineation of the calcineurin-interacting region of cyclophilin B.
abstract: The immunosuppressant drug cyclosporin A (CsA) inhibits T-cell function by blocking the phosphatase activity of calcineurin. This effect is mediated by formation of a complex between the drug and cyclophilin (CyP), which creates a composite surface able to make high-affinity contacts with calcineurin. In vitro, the CyPB/CsA complex is more effective in inhibiting calcineurin than the CyPA/CsA and CyPC/CsA complexes, pointing to fine structural differences in the calcineurin-binding region. To delineate the calcineurin-binding 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 inhibition 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 might therefore account for the higher affinity of the CyPB/CsA complex for calcineurin.
authors.affiliation: Laboratoire de Chimie Biologique, Unité Mixte de Recherche No. 8576 du Centre National de la Recherche Scientifique, Université des Sciences et Technologies de Lille, Villeneuve d'Ascq, France.
authors.forename: M
authors.initials: M
authors.lastname: Carpentier
authors.forename: F
authors.initials: F
authors.lastname: Allain
authors.forename: B
authors.initials: B
authors.lastname: Haendler
authors.forename: M C
authors.initials: MC
authors.lastname: Slomianny
authors.forename: G
authors.initials: G
authors.lastname: Spik
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites / Q000235:genetics; D019703:Calcineurin / Q000378:metabolism*; D021983:Cyclophilins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D016572:Cyclosporine / Q000378:metabolism / Q000494:pharmacology; D006801:Humans; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D019696:Peptidylprolyl Isomerase; D010744:Phosphoric Monoester Hydrolases / Q000037:antagonists & inhibitors; D017354:Point Mutation; D011485:Protein Binding / Q000235:genetics
chemical_list: C081786:cyclophilin B; D016572:Cyclosporine; C120817:calcineurin phosphatase; D019703:Calcineurin; D010744:Phosphoric Monoester Hydrolases; D021983:Cyclophilins; D019696:Peptidylprolyl Isomerase