Mutations that perturb cyclophilin A ligand binding pocket confer cyclosporin A resistance in Saccharomyces cerevisiae

• Published in: The Journal of biological chemistry 1995-09, Vol.270 (36), p.20997-21002
• Main Authors: Cardenas, M.E. (Duke University Medical Center, Durham, NC.), Lim, E, Heitman, J
• Format: Article
• Language: English
• Subjects: ACTIVIDAD ENZIMATICA; ACTIVITE ENZYMATIQUE; Amino Acid Isomerases - genetics; Amino Acid Isomerases - metabolism; Amino Acid Sequence; Base Sequence; Calcineurin; Calmodulin-Binding Proteins - antagonists & inhibitors; Calmodulin-Binding Proteins - metabolism; Carrier Proteins - genetics; Carrier Proteins - metabolism; COMPOSICION QUIMICA; COMPOSITION CHIMIQUE; Conserved Sequence; Cyclosporine - chemistry; Cyclosporine - pharmacology; DNA, Fungal; Drug Resistance, Microbial - genetics; ESTERASAS; ESTERASE; Humans; ISOMERASAS; ISOMERASE; MEDICAMENT; MEDICAMENTOS; Models, Molecular; Molecular Sequence Data; MUTACION INDUCIDA; MUTANT; MUTANTES; Mutation; MUTATION PROVOQUEE; Peptidylprolyl Isomerase; Phosphoprotein Phosphatases - antagonists & inhibitors; Phosphoprotein Phosphatases - metabolism; Protein Binding; PROTEINAS AGLUTINANTES; PROTEINE DE LIAISON; RESISTANCE AUX PRODUITS CHIMIQUES; RESISTENCIA A PRODUCTOS QUIMICOS; SACCHAROMYCES CEREVISIAE; Saccharomyces cerevisiae - drug effects; Saccharomyces cerevisiae - genetics; Sequence Homology, Amino Acid
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.270.36.20997

In complex with the peptidyl-prolyl isomerase cyclophilin 4 the immunosuppressive antifungal drug cyclosporin A (CsA) inhibits a Ca2+/calmodulin-dependent protein phosphatase, calcineurin, which regulates signal transduction. We isolated and characterized cyclophilin A mutations that confer CsA resistance in a Saccharomyces cerevisiae strain whose growth is CsA-sensitive. Three mutations (G70S, H90Y, and G102A) alter single amino acids conserved between yeast and human cyclophilin A, which structural analyses implicate in CsA binding to human cyclophilin A. By Western analysis, all three mutant proteins are expressed in yeast. In vitro, two purified mutant cyclophilins (G70S, G102A) retain prolyl isomerase activity and have moderately reduced affinity for CsA and calcineurin but, when bound to CsA, do bind and inhibit calcineurin phosphatase activity. In contrast, the purified H90Y mutant cyclophilin is dramatically decreased in prolyl isomerase activity, CsA affinity, and calcineurin binding and inhibition. These studies identify conserved cyclophilin A residues that participate in CsA binding and catalysis