Mutations that perturb cyclophilin A ligand binding pocket confer cyclosporin A resistance in Saccharomyces cerevisiae.
In complex with the peptidyl-prolyl isomerase cyclophilin A, 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.
Cardenas
M E
ME
Department of Genetics, Howard Hughes Medical Institute, Duke University Medical Center, Durham, North Carolina 27710, USA.
Lim
E
E
Heitman
J
J
D000591:Amino Acid Isomerases / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D001483:Base Sequence; D019703:Calcineurin; D002148:Calmodulin-Binding Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D017124:Conserved Sequence; D016572:Cyclosporine / Q000737:chemistry / Q000494:pharmacology*; D004271:DNA, Fungal; D004352:Drug Resistance, Microbial / Q000235:genetics; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D019696:Peptidylprolyl Isomerase; D010749:Phosphoprotein Phosphatases / Q000037:antagonists & inhibitors / Q000378:metabolism; D011485:Protein Binding; D012441:Saccharomyces cerevisiae / Q000187:drug effects* / Q000235:genetics; D017386:Sequence Homology, Amino Acid
D002148:Calmodulin-Binding Proteins; D002352:Carrier Proteins; D004271:DNA, Fungal; D016572:Cyclosporine; D019703:Calcineurin; D010749:Phosphoprotein Phosphatases; D000591:Amino Acid Isomerases; D019696:Peptidylprolyl Isomerase