Molecular characterization of a plant FKBP12 that does not mediate action of FK506 and rapamycin.
Immuonosuppressive drugs FK506 and rapamycin block a number of signal transduction pathways in eukaryotic systems. The 12 kDa FK506 binding protein (FKBP12) mediates the action of both FK506 and rapamycin against their functional targets. In this report, we cloned, sequenced and characterized a gene encoding FKBP12 in Vicia faba (VfFKBP12). While VfFKBP12 is highly homologous to animal and yeast FKBP12, it does not mediate the action of FK506 and rapamycin. There are unique features in plant FKBP12 sequences that cause the variation in their function. One lies in the domain that is critical for interaction with calcineurin (CaN), the mammalian and yeast target of FKBP12-FK506 complex. Protein-protein interaction assays revealed a low-affinity and unstable VfFKBP12-FK506-CaN ternary complex. In the genetic assay, VfFKBP12 did not restore the sensitivity of yeast FKBP12 mutant to rapamycin or FK506, supporting that plant FKBP12-ligand complexes are unable to block the function of the drug target. Also unique to plant FKBP12 proteins, a pair of cysteines is spatially adjacent to potentially form disulfide linkage. Treatment of VfFKBP12 with reductant dithiothreitol (DTT) abolished the formation of VfFKBP12-FK506-CaN ternary complex. Site-directed mutagenesis to substitute one of the cysteines, Cys26, with Ser produced a similar effect as DTT treatment. These results indicate that an intramolecular disulfide bond is a novel structural feature required for the low-affinity interaction between plant FKBP12 and CaN. In conclusion, plant FKBP12 proteins have evolved structural changes that modify their protein-protein interacting domains and cause loss of function against the drug targets.
Xu
Q
Q
Department of Plant and Microbial Biology, University of California, Berkeley 94720, USA.
Liang
S
S
Kudla
J
J
Luan
S
S
D000595:Amino Acid Sequence; D019703:Calcineurin / Q000378:metabolism; D003001:Cloning, Molecular; D003545:Cysteine; D004220:Disulfides; D004229:Dithiothreitol; D007887:Fabaceae / Q000235:genetics / Q000378:metabolism*; D017343:Genes, Plant / Q000235:genetics; D005816:Genetic Complementation Test; D020104:Immunophilins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D007166:Immunosuppressive Agents / Q000378:metabolism*; D008969:Molecular Sequence Data; D009154:Mutation; D010946:Plants, Medicinal; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D020123:Sirolimus / Q000378:metabolism*; D013439:Sulfhydryl Reagents; D016559:Tacrolimus / Q000378:metabolism*; D022021:Tacrolimus Binding Proteins
D004220:Disulfides; D007166:Immunosuppressive Agents; D013439:Sulfhydryl Reagents; D019703:Calcineurin; D022021:Tacrolimus Binding Proteins; D020104:Immunophilins; D003545:Cysteine; D004229:Dithiothreitol; D020123:Sirolimus; D016559:Tacrolimus