Xu Q, Liang S, Kudla J, Luan S
Department of Plant and Microbial Biology, University of California, Berkeley 94720, USA.
Plant J. 1998 Aug;15(4):511-9. doi: 10.1046/j.1365-313x.1998.00232.x.
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.
免疫抑制药物FK506和雷帕霉素可阻断真核系统中的多种信号转导途径。12 kDa的FK506结合蛋白（FKBP12）介导FK506和雷帕霉素对其功能靶点的作用。在本报告中，我们克隆、测序并鉴定了蚕豆中编码FKBP12的基因（VfFKBP12）。虽然VfFKBP12与动物和酵母的FKBP12高度同源，但它并不介导FK506和雷帕霉素的作用。植物FKBP12序列具有独特特征，导致其功能发生变化。其中一个特征在于与钙调神经磷酸酶（CaN）相互作用至关重要的结构域，CaN是FKBP12 - FK506复合物在哺乳动物和酵母中的靶点。蛋白质 - 蛋白质相互作用分析显示VfFKBP12 - FK506 - CaN三元复合物亲和力低且不稳定。在基因分析中，VfFKBP12不能恢复酵母FKBP12突变体对雷帕霉素或FK506的敏感性，这支持了植物FKBP12 - 配体复合物无法阻断药物靶点功能的观点。植物FKBP12蛋白的另一个独特之处在于，一对半胱氨酸在空间上相邻，可能形成二硫键。用还原剂二硫苏糖醇（DTT）处理VfFKBP12可消除VfFKBP12 - FK506 - CaN三元复合物的形成。将其中一个半胱氨酸Cys26定点突变为丝氨酸产生了与DTT处理类似的效果。这些结果表明，分子内二硫键是植物FKBP12与CaN之间低亲和力相互作用所需的一种新的结构特征。总之，植物FKBP12蛋白发生了结构变化，改变了其蛋白质 - 蛋白质相互作用结构域，并导致对药物靶点的功能丧失。
