MUTATIONS THAT PERTURB CYCLOPHILIN A LIGAND-BINDING POCKET CONFER CYCLOSPORINE-A RESISTANCE IN SACCHAROMYCES-CEREVISIAE

In complex with the peptidyl-prolyl isomerase cyclophilin A, the immun osuppressive antifungal drug cyclosporin A (CsA) inhibits a Ca2+/calmo dulin-dependent protein phosphatase, calcineurin, which regulates sign al 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) alte r single amino acids conserved between yeast and human cyclophilin A, which structural analyses implicate in CsA binding to human cyclophili n A. By Western analysis, all three mutant proteins are expressed in y east, 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 calcineur in phosphatase activity. In contrast, the purified H90Y mutant cycloph ilin is dramatically decreased in prolyl isomerase activity, CsA affin ity, and calcineurin binding and inhibition. These studies identify co nserved cyclophilin A residues that participate in CsA binding and cat alysis.