Mapping the Ryanodine Receptor FK506-binding Protein Subunit Using Fluorescence Resonance Energy Transfer

The 12-kDa FK506-binding proteins (FKBP12 and FKBP12.6) are regulatory subunits of ryanodine receptor (RyR) Ca²⁺ release channels. To investigate the structural basis of FKBP interactions with the RyR1 and RyR2 isoforms, we used site-directed fluorescent labeling of FKBP12.6, ligand binding measurements, and fluorescence resonance energy transfer (FRET). Single-cysteine substitutions were introduced at five positions distributed over the surface of FKBP12.6. Fluorescent labeling at position 14, 32, 49, or 85 did not affect high affinity binding to the RyR1. By comparison, fluorescent labeling at position 41 reduced the affinity of FKBP12.6 binding by 10-fold. Each of the five fluorescent FKBPs retained the ability to inhibit [³H]ryanodine binding to the RyR1, although the maximal extent of inhibition was reduced by half when the label was attached at position 32. The orientation of FKBP12.6 bound to the RyR1 and RyR2 was examined by measuring FRET from the different labeling positions on FKBP12.6 to an acceptor attached within the RyR calmodulin subunit. FRET was dependent on the position of fluorophore attachment on FKBP12.6; however, for any given position, the distance separating donors and acceptors bound to RyR1 versus RyR2 did not differ significantly. Our results show that FKBP12.6 binds to RyR1 and RyR2 in the same orientation and suggest new insights into the discrete structural domains responsible for channel binding and inhibition. FRET mapping of RyR-bound FKBP12.6 is consistent with the predictions of a previous cryoelectron microscopy study and strongly supports the proposed structural model.     

Generation and evaluation of putative neuroregenerative drugs. Part 2: screening virtual libraries of novel polyketides which possess the binding domain of rapamycin

The use of computational methods to direct engineered biosynthesis toward candidates based on the desired properties of the target compounds has been explored. The objective for this study has been the modification of rapamycin in order to eliminate its immunosuppressive activity and retain its neuroregenerative abilities. We have designed analogues of rapamycin which have truncated effector domains but retain the ability to bind to FKBP proteins, which is a prerequisite for the neuroregenerative abilities of the drugs. The procedures described here consist of the screening of large virtual libraries of molecules which retain the binding domain of rapamycin but in which different substitute ketide units replace the effector domain. These methods have provided analogues of rapamycin that cannot retain the immunosuppressive abilities of rapamycin, have a binding affinity to FKBP12 identical to that of rapamycin (by linear interaction energy calculations), and are suitable for synthesis by modified polyketide synthases.     

The rapamycin-binding domain of the protein kinase mammalian target of rapamycin is a destabilizing domain

Rapamycin is an immunosuppressive drug that binds simultaneously to the 12-kDa FK506- and rapamycin-binding protein (FKBP12, or FKBP) and the FKBP-rapamycin binding (FRB) domain of the mammalian target of rapamycin (mTOR) kinase. The resulting ternary complex has been used to conditionally perturb protein function, and one such method involves perturbation of a protein of interest through its mislocalization. We synthesized two rapamycin derivatives that possess large substituents at the C-16 position within the FRB-binding interface, and these derivatives were screened against a library of FRB mutants using a three-hybrid assay in Saccharomyces cerevisiae. Several FRB mutants responded to one of the rapamycin derivatives, and twenty of these mutants were further characterized in mammalian cells. The mutants most responsive to the ligand were fused to yellow fluorescent protein, and fluorescence levels in the presence and absence of the ligand were measured to determine stability of the fusion proteins. Wild-type and mutant FRB domains were expressed at low levels in the absence of the rapamycin derivative, and expression levels rose up to 10-fold upon treatment with ligand. The synthetic rapamycin derivatives were further analyzed using quantitative mass spectrometry, and one of the compounds was found to contain contaminating rapamycin. Furthermore, uncontaminated analogs retained the ability to inhibit mTOR, although with diminished potency relative to rapamycin. The ligand-dependent stability displayed by wild-type FRB and FRB mutants as well as the inhibitory potential and purity of the rapamycin derivatives should be considered as potentially confounding experimental variables when using these systems.     

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.     

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 ( Vf FKBP12). While Vf FKBP12 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 Vf FKBP12-FK506-CaN ternary complex. In the genetic assay, Vf FKBP12 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 Vf FKBP12 with reductant dithiothreitol (DTT) abolished the formation of Vf FKBP12-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.     

Bifunctional molecules evade cytochrome P(450) metabolism by forming protective complexes with FK506-binding protein

Despite their large size and complexity, the macrolide natural products rapamycin and FK506 have excellent pharmacological characteristics. We hypothesize that these unexpected properties may arise from protective, high affinity interactions with the cellular FK506-binding protein, FKBP. In this model, the drug-FKBP complex might sequester the small molecule and limit its degradation by restricting access to metabolic enzymes. In support of this idea, we found that adding FKBP blocks binding of FK506 to the common cytochrome P(450) enzyme CYP3A4 in vitro. To further test this idea, we have systematically modified a small collection of otherwise unrelated compounds, such that they acquire affinity for FKBP. Strikingly, we found that many of these synthetic derivatives, but not the unmodified parent compounds, are also protected from CYP3A4-mediated metabolism. Depending on the properties of the linker, the bifunctional molecules exhibited up to a 3.5-fold weaker binding to CYP3A4, and this protective effect was observed in the presence of either purified FKBP or FKBP-expressing cells. Together, these results suggest that the surprising pharmacology of rapamycin and FK506 might arise, in part, from binding to their abundant, intracellular target, FKBP. Furthermore, these findings provide a framework by which other small molecules might be systematically modified to impart this protective effect.     

Reactive group-embedded affinity labeling reagent for efficient intracellular protein labeling

Affinity labeling of a target protein is a powerful method for chemical biology studies. However, it is still difficult to label intracellular proteins efficiently in living cells. We propose the novel design strategy of a reactive group-embedded affinity labeling reagent for efficient protein labeling. With FKBP12 as the model target protein, the ligand binding pocket-oriented labeling reagent could label intracellular protein, whereas protein surface-oriented reagent was ineffective for labeling in living cells, partially because of the intracellular protein fluctuation under the macromolecular crowding effects. These results provide new insight for efficient intracellular protein labeling.     

Selective ligand purification using high-performance affinity beads

Since the development of affinity chromatography, affinity purification technology has been applied to many aspects of biological research, becoming an indispensable tool. Efficient strategies for the identification of biologically active compounds based on biochemical specificity have not yet been established, despite widespread interest in identifying chemicals that directly alter biomolecular functions. Here, we report a novel method for purifying chemicals that specifically interact with a target biomolecule using reverse affinity beads, a receptor-immobilized high-performance solid-phase matrix. When FK506-binding protein 12 (FKBP12) immobilized beads were used in this process, FK506 was efficiently purified in one step either from a mixture of chemical compounds or from fermented broth extract. The reverse affinity beads facilitated identification of drug/receptor complex binding proteins by reconstitution of immobilized ligand/receptor complexes on the beads. When FKBP12/FK506 and FKBP12/rapamycin complexes were immobilized, calcineurin and FKBP/rapamycin-associated protein were purified from a crude cell extract, respectively. These data indicate that reverse affinity beads are powerful tools for identification of both specific ligands and proteins that interact with receptor/ligand complexes.     

Generation and evaluation of putative neuroregenerative drugs. Part 1: virtual point mutations to the polyketide rapamycin

This paper explores the use of computational methods to direct engineered biosynthesis based on the desired properties of the target compounds. The immunosuppressive properties of rapamycin are a result of the formation of the complex FKBP12-rapamycin-FRAP. Neuroregenerative properties are exhibited by the complex or complexes of rapamycin with FKBP proteins. Our objective has been to design biosynthetically available analogues of rapamycin that bind tightly to FKBP12 but not to FRAP. This has been carried out by successive single ketide deletions from the effector domain of rapamycin. The approach described here has yielded modified rapamycin analogues (RP2 and RP3) as targets for biosynthesis by modified polyketide synthases. RP2 and RP3 have an identical binding affinity (linear interaction energy calculation) to FKBP12 as rapamycin but little or no affinity for binding to FRAP.     

Molecular cloning of a 25-kDa high affinity rapamycin binding protein, FKBP25.

Two FK506 binding proteins of molecular mass 12 kDa (FKBP12) and 13 kDa (FKBP13) have been identified as common cellular receptors of the immunosuppressants FK506 and rapamycin. Here we report the molecular cloning and overexpression of a 25-kDa rapamycin and FK506 binding protein (termed FKBP25) with peptidylprolyl cis-trans-isomerase (PPIase) activity. The amino acid sequence, predicted from the FKBP25 cDNA, shares identity with FKBP12 (44%) and FKBP13 (47%) in the C-terminal 97 amino acids. Unlike either FKBP12 or FKBP13, the nucleotide sequence of FKBP25 contains a number of putative nuclear localization sequences. The PPIase activity of recombinant FKBP25 was comparable with that of FKBP12. The PPIase activity of FKBP25 was far more sensitive to inhibition by rapamycin (IC50 = 50 nM) than FK506 (IC50 = 400 nM). PPIase activity of 100 nM FKBP25 was almost completely inhibited by 150 nM rapamycin while only 90% inhibition was achieved by 4 microM FK506. These data demonstrate that FKBP25 has a higher affinity for rapamycin than for FK506 and suggest that this cellular receptor may be an important target molecule for immunosuppression by rapamycin.     

Conditionally controlling nuclear trafficking in yeast by chemical-induced protein dimerization

We present here a protocol to conditionally control the nuclear trafficking of target proteins in yeast. In this system, rapamycin is used to heterodimerize two chimeric proteins. One chimera consists of a FK506-binding protein (FKBP12) fused to a cellular 'address' (nuclear localization signal or nuclear export sequence). The second chimera consists of a target protein fused to a fluorescent protein and the FKBP12-rapamycin-binding (FRB) domain from FKBP-12-rapamycin associated protein 1 (FRAP1, also known as mTor). Rapamycin induces dimerization of the FKBP12- and FRB-containing chimeras; these interactions selectively place the target protein under control of the cell address, thereby directing the protein into or out of the nucleus. By chemical-induced dimerization, protein mislocalization is reversible and enables the identification of conditional loss-of-function and gain-of-function phenotypes, in contrast to other systems that require permanent modification of the targeted protein. Yeast strains for this analysis can be constructed in 1 week, and the technique allows protein mislocalization within 15 min after drug treatment.     

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.     

Mutation of FKBP associated protein 48 (FAP48) at proline 219 disrupts the interaction with FKBP12 and FKBP52

Immunophilins are known as intracellular receptors for the immunosuppressant drugs, cyclosporin A, FK506, and rapamycin. They can be divided into two groups, cyclophilins that bind cyclosporin A and FK506 binding proteins (FKBPs) that bind FK506 and rapamycin. Many efforts were made to elucidate the physiological role of the immunophilins. Many of them are involved in intracellular signalling as they bind to calcium channels or to steroid receptor complexes. A yeast two-hybrid screen was used to identify further target proteins that interact with known proteins. Recently, a 48-kDa FKBP associated protein (FAP48) was isolated that binds to FKBP12 and FKBP52. Binding of FAP48 to FKBPs is inhibited by the macrolide FK506 indicating that the binding sites on the immunophilins coincide with the binding site for FK506. A peptidyl-prolyl motif on FAP48 should be responsible for the binding of the protein to FKBPs. We sequentially point mutated proline sites on FAP48 and checked the mutant proteins for interaction with FKBP12 and FKBP52. Mutation of proline 219 to alanine leads to a loss of interaction indicating that a cysteinyl prolyl site might be responsible for the binding of FAP48 to FKBPs. Thus we identified proline 219 being essential for the interaction.     

Ubiquitin regulates TORC1 in yeast Saccharomyces cerevisiae

In the yeast Saccharomyces cerevisiae the TOR complex 1 (TORC1) controls many growth‐related cellular processes and is essential for cell growth and proliferation. Macrolide antibiotic rapamycin, in complex with a cytosol protein named FKBP12, specifically inhibits TORC1, causing growth arrest. The FKBP12‐rapamycin complex interferes with TORC1 function by binding to the FRB domain of the TOR proteins. In an attempt to understand the role of the FRB domain in TOR function, we identified a single point mutation (Tor2^W2041R) in the FRB domain of Tor2 that renders yeast cells rapamycin resistant and temperature sensitive. At the permissive temperature, the Tor2 mutant protein is partially defective for binding with Kog1 and TORC1 is impaired for membrane association. At the restrictive temperature, Kog1 but not the Tor2 mutant protein, is rapidly degraded. Overexpression of ubiquitin stabilizes Kog1 and suppresses the growth defect associated with the tor2 mutant at the nonpremissive temperature. We find that ubiquitin binds non‐covalently to Kog1, prevents Kog1 from degradation and stabilizes TORC1. Our data reveal a unique role for ubiquitin in regulation of TORC1 and suggest that Kog1 requires association with the Tor proteins for stabilization.     

Highly fluorescent protein labeling using dendritic peptide derivatives.

Non-specific fluorescent dyes and photosensitizers are routinely used in clinical practice for the photodetection and photoablation of superficial lesions. Future applications in photomedicine are likely to rely on the selective delivery of photoactive compounds to diseased areas, using specific targeting agents such as antibodies. This fact underlines the need for methods that allow the chemically defined conjugation of several photoactive molecules to a single protein 'vehicle', with full retention of binding affinity. Here, we present methods for the site-specific fluorescent labeling of proteins using dendritic peptides, which had been chemically modified with multiple molecules of fluorescein. Branched peptide derivatives can be stably conjugated to proteins either by reaction with suitable free reactive groups or by using the high-affinity non-covalent interaction between calmodulin and a specific binding peptide. Chemical modification of proteins with one, two or four molecules of fluorescein resulted in a proportional increase in protein fluorescence.     

Rapamycin‐mediated mTORC2 inhibition is determined by the relative expression of FK506‐binding proteins

The mechanism by which the drug rapamycin inhibits the mechanistic target of rapamycin (mTOR) is of intense interest because of its likely relevance in cancer biology, aging, and other age‐related diseases. While rapamycin acutely and directly inhibits mTORC1, only chronic administration of rapamycin can inhibit mTORC2 in some, but not all, cell lines or tissues. The mechanism leading to cell specificity of mTORC2 inhibition by rapamycin is not understood and is especially important because many of the negative metabolic side effects of rapamycin, reported in mouse studies and human clinical trials, have been attributed recently to mTORC2 inhibition. Here, we identify the expression level of different FK506‐binding proteins (FKBPs), primarily FKBP12 and FKBP51, as the key determinants for rapamycin‐mediated inhibition of mTORC2. In support, enforced reduction of FKBP12 completely converts a cell line that is sensitive to mTORC2 inhibition to an insensitive cell line, and increased expression can enhance mTORC2 inhibition. Further reduction of FKBP12 in cell lines with already low FKBP12 levels completely blocks mTORC1 inhibition by rapamycin, indicating that relative FKBP12 levels are critical for both mTORC1 and mTORC2 inhibition, but at different levels. In contrast, reduction of FKBP51 renders cells more sensitive to mTORC2 inhibition. Our findings reveal that the expression of FKBP12 and FKBP51 is the rate limiting factor that determines the responsiveness of a cell line or tissue to rapamycin. These findings have implications for treating specific diseases, including neurodegeneration and cancer, as well as targeting aging in general.     

A directed approach for engineering conditional protein stability using biologically silent small molecules

The ability to regulate the function of specific proteins using cell-permeable molecules can be a powerful method for interrogating biological systems. To bring this type of "chemical genetic" control to a wide range of proteins, we recently developed an experimental system in which the stability of a small protein domain expressed in mammalian cells depends on the presence of a high affinity ligand. This ligand-dependent stability is conferred to any fused partner protein. The FK506- and rapamycin-binding protein (FKBP12) has been the subject of extensive biophysical analyses, including both kinetic and thermodynamic studies of the wild-type protein as well as dozens of mutants. The goal of this study was to determine if the thermodynamic stabilities (DeltaDeltaG(U-F)) of various amino acid substitutions within a given protein are predictive for engineering additional ligand-dependent destabilizing domains. We used FKBP12 as a model system and found that in vitro thermodynamic stability correlates weakly with intracellular degradation rates of the mutants and that the ability of a given mutation to destabilize the protein is context-dependent. We evaluated several new FKBP12 ligands for their ability to stabilize these mutants and found that a cell-permeable molecule called Shield-1 is the most effective stabilizing ligand. We then performed an unbiased microarray analysis of NIH3T3 cells treated with various concentrations of Shield-1. These studies show that Shield-1 does not elicit appreciable cellular responses.     

Bimolecular Fluorescence Complementation Analysis of Inducible Protein Interactions: Effects of Factors Affecting Protein Folding on Fluorescent Protein Fragment Association

Bimolecular fluorescence complementation (BiFC) analysis enables visualization of the subcellular locations of protein interactions in living cells. Using fragments of different fluorescent proteins, we investigated the temporal resolution and the quantitative accuracy of BiFC analysis. We determined the kinetics of BiFC complex formation in response to the rapamycin-inducible interaction between the FK506 binding protein (FKBP) and the FKBP-rapamycin binding domain (FRB). Fragments of yellow fluorescent protein fused to FKBP and FRB produced detectable BiFC complex fluorescence 10 min after the addition of rapamycin and a 10-fold increase in the mean fluorescence intensity in 8 h. The N-terminal fragment of the Venus fluorescent protein fused to FKBP produced constitutive BiFC complexes with several C-terminal fragments fused to FRB. A chimeric N-terminal fragment containing residues from Venus and yellow fluorescent protein produced either constitutive or inducible BiFC complexes depending on the temperature at which the cells were cultured. The concentrations of inducers required for half-maximal induction of BiFC complex formation by all fluorescent protein fragments tested were consistent with the affinities of the inducers for unmodified FKBP and FRB. Treatment with the FK506 inhibitor of FKBP-FRB interaction prevented the formation of BiFC complexes by FKBP and FRB fusions, but did not disrupt existing BiFC complexes. Proteins synthesized before the addition of rapamycin formed BiFC complexes with the same efficiency as did newly synthesized proteins. Inhibitors of protein synthesis attenuated BiFC complex formation independent of their effects on fusion protein synthesis. The kinetics at which they inhibited BiFC complex formation suggests that they prevented association of the fluorescent protein fragments, but not the slow maturation of BiFC complex fluorescence. Agents that induce the unfolded protein response also reduced formation of BiFC complexes. The effects of these agents were suppressed by cellular adaptation to protein folding stress. In summary, BiFC analysis enables detection of protein interactions within minutes after complex formation in living cells, but does not allow detection of complex dissociation. Conditional BiFC complex formation depends on the folding efficiencies of fluorescent protein fragments and can be affected by the cellular protein folding environment.     

A sol-gel-integrated protein array system for affinity analysis of aptamer-target protein interaction

A sol-gel microarray system was developed for a protein interaction assay with high activity. Comparing to 2-dimensional microarray surfaces, sol-gel can offer a more dynamic and broad range for proteins. In the present study, this sol-gel-integrated protein array was used in binding affinity analysis for aptamers. Six RNA aptamers and their target protein, yeast TBP (TATA-binding protein), were used to evaluate this method. A TBP-containing sol-gel mixture was spotted using a dispensing workstation under high-humidity conditions and each Cy-3-labeled aptamer was incubated. The dissociation constants (K(d)) were calculated by plotting the fluorescent intensity of the bound aptamers as a function of the TBP concentrations. The K(d) value of the control aptamer was found to be 8?nM, which agrees well with the values obtained using the conventional method, electric mobility shift assay. The sol-gel-based binding affinity measurements fit well with conventional binding affinity measurements, suggesting their possible use as an alternative to the conventional method. In addition, aptamer affinity measurements by the sol-gel-integrated protein chip make it possible to develop a simple high-throughput affinity method for screening high-affinity aptamers.