The full electron structure of the FKBP12/FK506 complex

We present a study of FKBP12/FK506 using an electron structure calculation. These calculations employ a novel technique called eCADD on the protein’s full electron structure along with its hydrophobic pocket and the frontier-orbital-perturbation theory. We first obtain the energy bands and orbital coefficients of protein FKBP12. On this basis, we found that the activity atoms and activity residues of FKBP12 were in good agreement with X-ray crystallography experiments. The results indicate that the interactions occur only between the LUMOs of FKBP12 and the HOMO of FK506, not between the HOMOs of FKBP12 and the LUMO of FK506. In other words, the activity sites of protein FKBP12 are located on its LUMOs, not HOMOs. The electron structures of FKBP12/FK506 give us a clearer understanding of their interaction mechanism and will help us design new ligands of FKBP12.     

用于FK506类小分子化合物筛选的细胞模型的建立

建立FKBP12/Fas双聚体可诱导细胞凋亡生物效应的细胞模型,为FK506类小分子化合药物的初步筛选提供一个可应用的技术平台.首先利用RT-PCR方法钓取人源性Fas胞浆区基因,测序鉴定正确后,插入到载体pC4M-FV2E中,构建pC4M-FV2E/Fas表达质粒.然后将Myr-FKBP12-Fas融合基因从pC4M-FV2E/Fas质粒克隆进pEGFP-N1中.最后将重组质粒pEGFP-N1/FKBP12-Fas转染进HEK293细胞,并利用G418筛选融合基因FKBP12-Fas稳定表达的细胞株.实验结果显示,阳性化合物AP20187作用4～6 h后,稳定转染的靶细胞发生凋亡,基因组呈现典型的“DNA Ladder”,而FKBP12的天然结合蛋白FK506可竞争性地阻断这种阳性药物诱导的细胞凋亡.研究提示：所构建的细胞模型,可用于以FKBP12为靶标、基于FK506空间构型设计合成的FK506类小分子化合药物的初步筛选.     

The human FK506-binding proteins: characterization of human FKBP19

Analysis of the human repertoire of the FK506-binding protein (FKBP) family of peptidyl-prolyl cis/trans isomerases has identified an expansion of genes that code for human FKBPs in the secretory pathway. There are distinct differences in tissue distribution and expression levels of each variant. In this article we describe the characterization of human FKBP19 (Entrez Gene ID: FKBP11), an FK506-binding protein predominantly expressed in vertebrate secretory tissues. The FKBP19 sequence comprises a cleavable N-terminal signal sequence followed by a putative peptidyl-prolyl cis/trans isomerase domain with homology to FKBP12. This domain binds FK506 weakly in vitro. FKBP19 mRNA is abundant in human pancreas and other secretory tissues and high levels of FKBP19 protein are detected in the acinar cells of mouse pancreas.     

Genetic relationships among actinomycetes that produce the immunosuppressant macrolides FK506, FK520/FK523 and rapamycin

Summary A polyphasic taxonomic study was undertaken to establish the genetic and phenotypic relationships among six actinomycetes that produce the immunosuppressant macrolides FK506, FK520/FK523 and rapamycin. Chemotaxonomic studies reveal that all have Type I cell walls. Gas chromatography (GC) of fatty acid methyl esters revealed patterns consistent for strains ofStreptomyces with 160 and 150anteiso predominating. Principal component analysis of GC data revealed distinct profiles for each culture. Reciprocal DNA homology studies atT _m -25 showed the rapamycin-producing strain and one FK506-producing strain to have 38–50% homology with the type strain ofStreptomyces hygroscopicus (ATCC 27438). The remaining strains exhibited 6–17% homology. To further explore the relationships among these strains all were probed for the presence of anO-methyltransferase gene specific to this biosynthetic pathway. Among the strains of interest, onlyStreptomyces hygroscopicus subsp.yakushimaensis, the patent strain for FK520/FK523, failed to hybridize with the probes.     

Mean field analysis of FKBP12 complexes with FK506 and rapamycin: Implications for a role of crystallographic water molecules in molecular recognition and specificity

Mean field analysis of FKBP12 complexes with FK506 and rapamycin has been performed by using structures obtained from molecular docking simulations on a simple, yet robust molecular recognition energy landscape. When crystallographic water molecules are included in the simulations as an extension of the FKBP12 protein surface, there is an appreciable stability gap between the energy of the native FKBP12–FK506 complex and energies of conformations with the “native-like” binding mode. By contrast, the energy spectrum of the FKBP12–rapamycin complex is dense regardless of the presence of the water molecules. The stability gap in the FKBP12–FK506 system is determined by two critical water molecules from the effector region that participate in a network of specific hydrogen bond interactions. This interaction pattern protects the integrity and precision of the composite ligand-protein effector surface in the binary FKBP12–FK506 complex and is preserved in the crystal structure of the FKBP12–FK506–calcineurin ternary complex. These features of the binding energy landscapes provide useful insights into specific and nonspecific aspects of FK506 and rapamycin recognition. Proteins 28:313–324, 1997. © 1997 Wiley-Liss, Inc.     

Crystal structure of the FK506 binding domain of human FKBP25 in complex with FK506

Human FKBP25 (hFKBP25) is a nuclear immunophilin and interacts with several nuclear proteins, hence involving in many nuclear events. Similar to other FKBPs, FK506 binding domain (FKBD) of hFKBP25 also binds to immunosuppressive drugs such as rapamycin and FK506, albeit with a lower affinity for the latter. The molecular basis underlying this difference in affinity could not be addressed due to the lack of the crystal structure of hFKBD25 in complex with FK506. Here, we report the crystal structure of hFKBD25 in complex with FK506 determined at 1.8 Å resolution and its comparison with the hFKBD25–rapamycin complex, bringing out the microheterogeneity in the mode of interaction of these drugs, which could possibly explain the lower affinity for FK506.     

Complete amino acid sequence of the FK506 and rapamycin binding protein, FKBP, isolated from calf thymus

FKBP, an 11.8 kD intracellular protein that binds the immunosuppressants FK506 (K _d=0.4 nM) and rapamycin (K _d=0.2 nM) with high affinity, was purified to homogeneity from calf thymus. The complete amino acid sequence has been determined by automated Edman degradation of the intact molecule and overlapping fragments generated by proteolytic and chemical cleavage. The analysis revealed a 107 amino acid peptide chain with the following sequence: GVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKFDSSRDRNKPFKFVLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPNATLIFDVELLKLE. The molecular weight, calculated from the amino sequence to be 11,778 D, was confirmed by electrospray ionization mass spectrometry. Thus, naturally isolated bovine FKBP does not appear to have any residues modified by glycosylation, phosphorylation, or other post-translational derivatization processes. Bovine FKBP has only three amino acid residues that differ from human FKBP, whose sequence was elucidated by cloning and sequencing complementary DNA (Standaertet al., 1990). The protein has a substantial number of hydrophilic peptide segments with prevalent -strand type of chain fold. Understanding the biological function of FKBP and other members of the immunophilin class and their respective complexes with immunosuppressive drugs may provide insights into cytoplasmic signalling mechanisms, protein folding and translocation, and other cellular processes.     

Crystal structure of the FK506 binding domain of Plasmodium falciparum FKBP35 in complex with FK506

The emergence of multi-drug-resistant strains of Plasmodium parasites has prompted the search for alternative therapeutic strategies for combating malaria. One possible strategy is to exploit existing drugs as lead compounds. FK506 is currently used in the clinic for preventing transplant rejection. It binds to a alpha/beta protein module of approximately 120 amino acids known as the FK506 binding domain (FKBD), which is found in various organisms, including human, yeast, and Plasmodium falciparum (PfFKBD). Antiparasitic effects of FK506 and its analogues devoid of immunosuppressive activities have been demonstrated. We report here the crystallographic structure at 2.35 A resolution of PfFKBD complexed with FK506. Compared to the human FKBP12-FK506 complex reported earlier, the structure reveals structural differences in the beta5-beta6 segment that lines the FK506 binding site. The presence in PfFKBD of Cys-106 and Ser-109 (substituting for His-87 and Ile-90, respectively, in human FKBP12), which are 4-5 A from the nearest atom of the FK506 compound, suggests possible routes for the rational design of analogues of FK506 with specific antiparasitic activity. Upon ligand binding, several conformational changes occur in PfFKBD, including aromatic residues that shape the FK506 binding pocket as shown by NMR studies. A microarray analysis suggests that FK506 and cyclosporine A (CsA) might inhibit parasite development by interfering with the same signaling pathways.     

Leukocyte chemotactic activity of FKBP and inhibition by FK506.

Cyclophilin, the cyclosporin A binding protein and member of the immunophilin family of proteins, demonstrates leukocyte chemotactic activity. In this study we demonstrate that FKBP, the FK506 and rapamycin binding protein, also displays leukocyte chemotactic activity. The chemotactic activity of FKBP is inhibited by FK506, however, FK506 was unable to inhibit cyclophilin-stimulated chemotactic activity. Rapamycin was unable to prevent the chemotactic activity of FKBP, similarly, the CsA analogue Me6Ala-CsA while displaying cyclophilin binding was unable to block cyclophilin-stimulated chemotactic activity. These results suggest that in addition to their intracellular role the immunophilins may also function as chemotactic agents, furthermore this activity is modulated by immunosuppressants.     

The rough endoplasmic reticulum-resident FK506-binding protein FKBP65 is a molecular chaperone that interacts with collagens

The rough endoplasmic reticulum-resident FK-506-binding protein FKBP65 can be isolated from chick embryos on a gelatin-Sepharose column, indicating some involvement in the biosynthesis of procollagens. The peptidylprolyl cis-trans-isomerase activity of FKBP65 was previously shown to have only marginal effects on the rate of triple helix formation (Zeng, B., MacDonald, J. R., Bann, J. G., Beck, K., Gambee, J. E., Boswell, B. A., and B?chinger, H. P. (1998) Biochem. J. 330, 109-114). Here we show that FKBP65 is a monomer in solution and acts as a chaperone molecule when tested with two classic chaperone assays: FKBP65 inhibits the thermal aggregation of citrate synthase and is active in the denatured rhodanese refolding and aggregation assay. The chaperone activity is comparable to that of protein-disulfide isomerase, a well characterized chaperone. FKBP65 delays the in vitro fibril formation of type I collagen, indicating that FKBP65 is also able to interact with triple helical collagen, and acts as a collagen chaperone.     

FK506-binding protein (FKBP12) regulates ryanodine receptor-evoked Ca2+ release in colonic but not aortic smooth muscle

In smooth muscle, the ryanodine receptor (RyR) mediates Ca(2+) release from the sarcoplasmic reticulum (SR) Ca(2+) store. Release may be regulated by the RyR accessory FK506-binding protein (FKBP12) either directly, as a result of FKBP12 binding to RyR, or indirectly via modulation of the activity of the phosphatase calcineurin or kinase mTOR. Here we report that RyR-mediated Ca(2+) release is modulated by FKBP12 in colonic but not aortic myocytes. Neither calcineurin nor mTOR are required for FKBP12 modulation of Ca(2+) release in colonic myocytes to occur. In colonic myocytes, co-immunoprecipitation techniques established that FKBP12 and calcineurin each associated with the RyR2 receptor isoform (the main isoform in this tissue). Single colonic myocytes were voltage clamped in the whole cell configuration and cytoplasmic Ca(2+) concentration ([Ca(2+)](c)) increases evoked by the RyR activator caffeine. Under these conditions FK506, which displaces FKBP12 (to inhibit calcineurin) and rapamycin, which displaces FKBP12 (to inhibit mTOR), each increased the [Ca(2+)](c) rise evoked by caffeine. Notwithstanding, neither mTOR nor calcineurin are required to potentiate caffeine-evoked Ca(2+) increases evoked by each drug. Thus, the mTOR and phosphatidylinositol 3-kinase inhibitor, LY294002, which directly inhibits mTOR without removing FKBP12 from RyR, did not alter caffeine-evoked [Ca(2+)](c) transients. Nor did inhibition of calcineurin by cypermethrin, okadaic acid or calcineurin inhibitory peptide block the FK506-induced increase in RyR-mediated Ca(2+) release. In aorta, although RyR3 (the main isoform), FKBP12 and calcineurin were each present, RyR-mediated Ca(2+) release was unaffected by either FK506, rapamycin or the calcineurin inhibitors cypermethrin and okadaic acid in single voltage clamped aortic myocytes. Presumably failure of FKBP12 to associate with RyR3 resulted in the immunosuppressant drugs (FK506 and rapamycin) being unable to alter the activity of RyR. The effects of these drugs are therefore, apparently dependent on an association of FKBP12 with RyR. Together, removal of FKBP12 from RyR augmented Ca(2+) release via the channel in colonic myocytes. Neither calcineurin nor mTOR are required for the FK506- or rapamycin-induced potentiation of RyR Ca(2+) release to occur. The results indicate that FKBP12 directly inhibits RyR channel activity in colonic myocytes but not in aorta.     

Antinociceptive action of FK506 in mice

Immunophilins are abundantly present in the brain as compared to the immune system. Immunophilin-binding agents like FK506 are known to inactivate neuronal nitric oxide synthase (nNOS) by inhibiting calcineurin and decrease the production of nitric oxide. Nitric oxide is involved in the mediation of nociception at the spinal level. In the present study, the effect of FK506 on the tail flick response in mice and the possible involvement of NO-L-arginine pathway in this paradigm was evaluated. FK506 (0.5, 1 and 3 mg/kg, ip) produced a significant antinociception in the tail flick test. Nitric oxide synthase (NOS) inhibitor L-NAME significantly and dose dependently (10-40 mg/kg, ip) potentiated the FK506 (0.5 mg/kg)-induced antinociception. On the other hand, NOS substrate L-arginine (100, 200 and 400 mg/kg) inhibited the FK506-induced antinociception in a dose-dependent manner. Concomitant administration of L-NAME (20 and 40 mg/kg) with L-arginine (200 mg/kg) blocked the inhibition exerted by L-arginine on the FK506-induced antinociception. Thus, it was concluded that NO- L-arginine pathway may be involved in the FK506-induced antinociception in tail flick test.     

cDNA cloning of a human 25 kDa FK506 and rapamycin binding protein.

The abilities of FK506 and rapamycin to block distinct signal transduction pathways are mediated by soluble binding proteins. Previously, a family of these receptors has been recognized that includes a 25 kDa protein, FKBP25. We now report the isolation of a cDNA for FKBP25 from a human hippocampal cDNA library by oligonucleotide screening. The nucleotide sequence reveals an open reading frame that encodes a 224 amino acid polypeptide. Human FKBP25 shows 97% amino acid identity with bovine FKBP25 and 62% homology with human FKBP12.     

Establishment of a cell model based on FKBP12 dimerization for screening of FK506-like neurotrophic small molecular compounds

FK506 is an efficient immunosuppressive agent with an increasing number of clinical applications. It has been approved to prevent rejection in transplant patients and be efficacious in several autoimmune diseases. Its immunosuppressive activity results from binding to receptor proteins designated as immunophilins (i.e., FKBP12, FK506 binding protein). Recent studies have suggested that FK506 can promote neurite outgrowth as a 2nd activity. Furthermore, it has been shown that the neurotrophic property of FK506 is independent of its immunosuppressive action. Although the mechanism of its neurotrophic activity has not yet been well elucidated, FKBP12 is identified as a drug target, and much effort has been directed toward the design of FKBP12-binding molecules, which are neurotrophic but non-immunosuppressive, for clinical use. In this present study, the authors constructed a stable cell line, which underwent apoptosis upon treatment by AP20187, a wholly synthesized, cell-permeable dimeric FK506 derivative, based on FKBP12-mBax dimerization. This AP20187-mediated apoptosis was rapidly reversed by the addition of an FKBP12-binding competitor molecule (FK506 or rapamycin), indicating that this cell line might be used to screen FK506 derivatives. Using the screening model, hundreds of synthetic FK506 analogs were analyzed. A promising compound, named N308, was obtained. The results showed that N308 could inhibit AP20187-induced gene-modified target cell apoptosis and elicit augmentation of neurite extension from both cultured PC-12 cells and chicken dorsal root ganglia cultures.     

The 12 kD FK 506 binding protein FKBP12 is released in the male reproductive tract and stimulates sperm motility.

BACKGROUND: The 12 kD FK506 binding protein FKBP12 is a cytosolic receptor for the immunosuppressant drugs FK506 and rapamycin. In addition to its critical role in drug-induced T-cell immunosuppression, FKBP12 associates physiologically with ryanodine and inositol 1,4,5-trisphosphate (IP3) receptors, regulating their ability to flux calcium. We investigated a role for FKBP12 in male reproductive physiology on the basis of our identification of extremely high levels of [3H]FK506 binding in male reproductive tissues. MATERIALS AND METHODS: [3H]FK506 binding studies were performed to identify tissues enriched with FK506 binding sites. The abundant [3H]FK506 binding sites identified in the male reproductive tract were localized by [3H]FK506 autoradiography. FK506 affinity chromatography was employed to purify FKBP from epididymal fluid. Anti-FKBP12 Western analysis was used to confirm the identity of the purified FKBP. The binding of exogenous FKBP12 to sperm was evaluated by [32P]FKBP12 binding studies and [33P]FKBP12 autoradiography. The effect of recombinant FKBP12 on sperm motility was investigated using a Hamilton Thorne motility analyzer. RESULTS: Male reproductive tissues contained high levels of [3H]FK506 binding. The localization of [3H]FK506 binding sites to the tubular epithelium of the caput epididymis and the lumen of the cauda and vas deferens suggested that FKBP is released in the male reproductive tract. FKBP12 was purified from epididymal plasma by FK506 affinity chromatography. Radiolabeled FKBP12 specifically bound to immature but not mature sperm. In sperm motility studies, FKBP12-treated caput sperm exhibited double the curvilinear velocity of untreated controls. CONCLUSIONS: High levels of FKBP12 are released in the male reproductive tract and specifically associate with maturing sperm. Recombinant FKBP12 enhances the curvilinear velocity of immature sperm, suggesting a role for FKBP12 in motility initiation. The highest concentrations of soluble FKBP12 in the male reproductive tract occur in the lumen of the vas deferens, a site of sperm storage and the conduit for ejaculated sperm. Preservation of mammalian sperm for reproductive technologies may be optimized by supplementing incubation or storage media with FKBP12.