MOLECULAR CHARACTERIZATION OF A PLANT FKBP12 THAT DOES NOT MEDIATE ACTION OF FK506 AND RAPAMYCIN

Immunosuppressive drugs FK506 and rapamycin block a number of signal t ransduction pathways in eukaryotic systems. The 12 kDa FK506 binding p rotein (FKBP12) mediates the action of both FK506 and rapamycin agains t their functional targets. In this report, we cloned, sequenced and c haracterized a gene encoding FKBP12 in Vicia faba (VfFKBP12). While Vf FKBP12 is highly homologous to animal and yeast FKBP12, it does not me diate 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 VfFKB P12-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 p air of cysteines is spatially adjacent to potentially form disulfide l inkage. Treatment of VfFKBP12 with reductant dithiothreitol (DTT) abol ished the formation of VfFKBP12-FK506-CaN ternary complex. Site-direct ed mutagenesis to substitute one of the cysteines, Cys(26), with Ser p roduced a similar effect as DTT treatment. These results indicate that an intramolecular disulfide bond is a novel structural feature requir ed for the low-affinity interaction between plant FKBP12 and CaN. In c onclusion, plant FKBP12 proteins have evolved structural changes that modify their protein-protein interacting domains and cause loss of fun ction against the drug targets.