Practical applications of time-averaged restrained molecular dynamics to ligand-receptor systems: FK506 bound to the Q50R,A95H,K98I triple mutant of FKBP-13

Summary The ability of time-averaged restrained molecular dynamics (TARMD) to escape local low-energy conformations and explore conformational space is compared with conventional simulated-annealing methods. Practical suggestions are offered for performing TARMD calculations with ligand-receptor systems, and are illustrated for the complex of the immunosuppressant FK506 bound to Q50R,A95H,K98I triple mutant FKBP-13. The structure of ¹³C-labeled FK506 bound to triple-mutant FKBP-13 was determined using a set of 87 NOE distance restraints derived from HSQC-NOESY experiments. TARMD was found to be superior to conventional simulated-annealing methods, and produced structures that were conformationally similar to FK506 bound to wild-type FKBP-12. The individual and combined effects of varying the NOE restraint force constant, using an explicit model for the protein binding pocket, and starting the calculations from different ligand conformations were explored in detail.Abbreviations DG distance geometry - dmFKBP-12 double-mutant (R42K,H87V) FKBP-12 - FKBP-12 FK506-binding protein (12 kDa) - FKBP-13 FK506-binding protein (13 kDa) - HSQC heteronuclear single-quantum coherence - K_NOE force constant (penalty) for NOE-derived distance restraints - MD molecular dynamics - NOE nuclear Overhauser effect - SA simulated annealing - TARMD molecular dynamics with time-averaged restraints - tmFKBP-13 triple-mutant (Q50R,A95H,K98I) FKBP-13 - wtFKBP-12 wild-type FKBP-12     

Chromosomal assignment of the human immunophilin FKBP-12 gene

FKBP-12 is the major T cell binding protein for the immunosuppressive drugs FK506 and rapamycin. It is a member of the immunophilin family of proteins which are believed to play a role in immunoregulation and basic cellular processes involving protein folding and trafficking. The chromosomal assignment of the human FKBP-12 gene was determined by using the polymerase chain reaction to amplify an intron-containing region of the gene in purified DNA isolated from 42 human-rodent somatic cell hybrids. The results of this analysis indicated that the FKBP-12 gene resides on human chromosome 20.     

Dynamic NMR studies of ligand-receptor interactions: design and analysis of a rapidly exchanging complex of FKBP-12/FK506 with a 24 kDa calcineurin fragment.

Dynamic NMR methods, such as differential line broadening and transferred NOE spectroscopy, are normally reserved for the study of small molecule ligand interactions with large protein receptors. Using a combination of isotope labeling and isotope edited NMR, we have extended these techniques to characterize interactions of a much larger protein/drug complex, FKBP-12/ FK506 with its receptor protein, calcineurin. In order to examine this multicomponent system by dynamic NMR methods, the 93 kDa, tightly bound FKBP-12/FK506/Cn complex was replaced with a lower affinity, rapidly exchanging system consisting of FKBP-12/FK506 (13 kDa), recombinant calcineurin subunit B (CnB) (20 kDa), and a synthetic peptide (4 kDa) corresponding to the B binding domain (BBD) of calcineurin catalytic subunit A (CnA). Analysis of 1H-13C HSQC data acquired for the FKBP-12/ 13C-FK506 and FKBP-12/13C-FK506/CnB/BBD complexes indicates that FKBP-12/FK506 and CnB/BBD are in fast exchange in the quaternary complex. Comparison of proton line widths shows significant broadening of resonances along the macrocycle backbone at 13-CH, 13-OMe, 15-OMe, 18-CH2, 20-CH, 21-CH, and 25-Me, as well as moderate broadening on the macrocycle backbone at 17-Me, 24-CH, and the pyranose 12-CH2 protons. The tri-substituted olefin and cyclohexyl groups also show moderate broadening at the 27-Me, 28-CH, and 30-CH2 positions, respectively. Unexpectedly, little line broadening was observed for the allyl resonances of FK506 in the quaternary complex, although 13C longitudinal relaxation measurements suggest this group also makes contacts with calcineurin. In addition, intermolecular transfer NOE peaks were observed for the allyl 37-CH2, 21-CH, 30-CH2, 13-OMe, 15-OMe, 17-Me, 25-Me, and 27-Me groups, indicating that these are potential sites on the FK506 molecule that interact with calcineurin.     

Solution structure of FK506 bound to FKBP-12.

The complex of the immunosuppressant FK506 bound to FKBP-12 has been studied in solution using 1H and inverse-detected 13C NMR methods. The resonances of bound, 13C-labelled FK506 were assigned and a set of 66 intraligand NOE distance restraints were used to calculate the structure of the bound ligand by distance geometry and restrained molecular dynamics methods. The structure of bound FK506 in solution closely resembles that seen in the X-ray structure [17], except for the allyl region. The differences reflect the influence of intermolecular crystal contacts and have implications for interpretation of the interaction of the FK506/FKBP complex with its putative biological receptor.     

Streptomyces chrysomallus FKBP-33 is a novel immunophilin consisting of two FK506 binding domains; its gene is transcriptionally coupled to the FKBP-12 gene.

The nucleotide sequence of the region 5' to the fkbA gene, encoding the Streptomyces chrysomallus FK506 binding protein (FKBP-12), revealed an open reading frame (fkbB) encoding a protein of 312 amino acids, with an M(r) of approximately 33,000. FkbB and fkbA appear to be co-transcribed under the control of a promoter upstream of fkbB. The presumptive protein encoded by fkbB would be an FKBP (designated FKBP-33) consisting of two FK506 binding domains with 43 and 32% sequence identity to FKBP-12 and a signal peptide sequence characteristic of bacterial membrane lipoproteins. The portion of the gene comprising the two FKBP domains, as well as each individual domain, were expressed as fusion proteins in Escherichia coli and purified. Each expressed domain, as well as FKBP-33 itself, possesses peptidyl-prolyl cis-trans isomerase activity, though with much lower specific activities than FKBP-12. FKBP-33 is located in the cell membrane of S.chrysomallus and of other streptomycetes, as predicted from the presence of the signal peptide sequence. Pulse-chase experiments with radioactive palmitate in whole cells revealed significant labelling of FKBP-33, which probably carries palmitate at its N-terminus and an additional diacylglycerol residue attached to the N-terminal cysteine in thioether linkage. The two domains of FKBP-33 showed considerable homology with numerous eukaryotic and prokaryotic FKB domains. Calculations of phylogenetic relationships indicate with high probability that the two domains of the protein have arisen by a double gene duplication of fkbA lying in tandem to fkbB.     

Multimer formation by FKBP-12: roles for cysteine 23 and phenylalanine 36.

FKBP-12 mediates the immunosuppressive actions of FK506 and rapamycin, and modulates the activities of the ryanodine, IP3 and type 1 TGF-ss receptors. Additionally, FKBP-12 possesses cis-trans peptidylprolyl isomerase (rotamase) activity. We have discovered that recombinant FKBP-12 readily forms a dimer and a small amount of trimer under nonreducing conditions. A mutant with substitution at the sole cysteine residue of FKBP-12 (C23S) did not form dimers or trimers. Using mutants with 5% or less rotamase activity, the formation of dimers was independent of enzymatic activity. The formation of trimers was abrogated by a F36Y substitution, even though dimer formation was preserved. Dimers were also observed with native FKBP-12 that was detached from rabbit skeletal muscle ryanodine receptors using FK590. The multimers of FKBP-12 could interact with molecular targets distinctly from the FKBP-12 monomer, for example, by facilitating the assembly of multimeric receptors or coordinating the activity of receptor subunits.     

Exon organization of the human FKBP-12 gene: correlation with structural and functional protein domains.

FKBP-12, the major T-cell binding protein for the immunosuppressive agents FK506 and rapamycin, catalyzes the interconversion of the cis and trans rotamers of the peptidyl-prolyl amide bond of peptide and protein substrates. The function of rotamase activity in cells and the role of FKBP-12 in immunoregulation is uncertain. In this paper we report the cloning and characterization of the human chromosomal FKBP-12 gene and four processed FKBP-12 pseudogenes. The FKBP-12 gene is 24 kilobases in length and contains five exons. The protein-coding region of the gene is divided into four exon modules that correlate with the structural and functional domains of the protein. The novel structure of FKBP-12 resulting from the topology of the antiparallel beta-sheet is the topological crossing of two loops that are encoded by separate exons. Separate exons also encode the antiparallel beta-sheet and alpha-helical region that define the drug-binding pocket and enzyme activity site of FKBP-12. The exon organization of the FKBP-12 gene also provided insight into the genetic evolution of the immunophilin family. Knowledge of the FKBP-12 gene structure will enable inactivation of this gene by homologous recombination in cells to provide a model to study the role of FKBP-12 in immunoregulation and normal cellular processes.     

High Throughput Scintillation Proximity Assay for the Identification of FKBP-12 Ligands

A high throughput scintillation proximity assay (SPA) was developed to identify novel ligands of FKBP-12, an immunophilin with peptidyl prolyl isomerase (rotamase) activity. Recombinant histidine-tagged FKBP-12 was expressed in Escherichia coli, purified by metal ion affinity chromatography, and immobilized to SPA beads by an antibody that recognizes the histidine tag of the recombinant protein. Using 1 nM [3H] FK506, a well-known macrolid ligand of FKBP-12, specific binding was saturable and accounted for 95% of total binding. Analysis of saturation and homologous displacement isotherms indicated the existence of a single binding site with a Kd value of 1.6 nM. The specificity of [3H] FK506 binding was demonstrated in displacement experiments and showed that rapamycin, another macrolid, was as active as FK506 (IC50 of 3.5 and 3.2 nM, respectively), whereas GPI-1046, a prototype of small molecular compounds with neurotrophic properties and affinity for FKBP-type immunophilins, was more than 1000-fold less active. The high signal-to-noise ratio of 30, together with small standard deviations, makes this novel assay well suited for automated high throughput screening.     

FK506 and the role of immunophilins in nerve regeneration

FK506 is a new FDA-approved immunosuppressant used for prevention of allograft rejection in, for example, liver and kidney transplantations. FK506 is inactive by itself and requires binding to an FK506 binding protein-12 (FKBP-12), or immunophilin, for activation. In this regard, FK506 is analogous to cyclosporin A, which must bind to its immunophilin (cyclophilin A) to display activity. This FK506-FKBP complex inhibits the activity of the serine/threonine protein phosphatase 2B (calcineurin), the basis for the immunosuppressant action of FK506. The discovery that immunophilins are also present in the nervous system introduces a new level of complexity in the regulation of neuronal function. Two important calcineurin targets in brain are the growth-associated protein GAP-43 and nitric oxide (NO) synthase (NOS). This review focuses on studies showing that systemic administration of FK506 dose-dependently speeds nerve regeneration and functional recovery in rats following a sciatic-nerve crush injury. The effect appears to result from an increased rate of axonal regeneration. The nerve regenerative property of this class of agents is separate from their immunosuppressant action because FK506-related compounds that bind to FKBP-12 but do not inhibit calcineurin are also able to increase nerve regeneration. Thus, FK506's ability to increase nerve regeneration arises via a calcineurin-independent mechanism (i.e., one not involving an increase in GAP-43 phosphorylation). Possible mechanisms of action are discussed in relation to known actions of FKBPs: the interaction of FKBP-12 with two Ca²⁺ release-channels (the ryanodine and inositol 1,4,5-triphosphate receptors) which is disrupted by FK506, thereby increasing Ca²⁺ flux; the type 1 receptor for the transforming growth factor-β (TGF-β1), which stimulates nerve growth factor (NGF) synthesis by glial cells, and is a natural ligand for FKBP-12; and the immunophilin FKBP-52/FKBP-59, which has also been identified as a heat-shock protein (HSP-56) and is a component of the nontransformed glucocorticoid receptor. Taken together, studies of FK506 indicate broad functional roles for the immunophilins in the nervous system. Both calcineurin-dependent (e.g., neuroprotection via reduced NO formation) and calcineurin-independent mechanisms (i.e., nerve regeneration) need to be invoked to explain the many different neuronal effects of FK506. This suggests that multiple immunophilins mediate FK506's neuronal effects. Novel, nonimmunosuppressant ligands for FKBPs may represent important new drugs for the treatment of a variety of neurological disorders.     

A rapamycin-selective 25-kDa immunophilin.

FKBP25, a previously uncharacterized 25-kDa FK506- and rapamycin-binding protein, was purified to homogeneity from calf thymus, brain, and spleen, and the sequence of a 215 amino acid (aa) 24-kDa C-terminal peptide was established. The N-terminal domain (101 aa) is unrelated to any known protein, is hydrophilic, and is predicted by circular dichroism spectroscopy to be largely alpha-helix. The C-terminal domain (114 aa) is homologous to FKBP12 and other FKBPs but has a potential nuclear targeting sequence and a unique insertion of seven amino acids in one of its loops. FKBP25 displays the rotamase activity characteristic of FKBPs; the activity is inhibited by the immunosuppressants rapamycin (Ki = 0.9 nM) and FK506 (Ki = 160 nM), but not cyclosporin A. The protein, its rapamycin selectivity, and the potential nuclear targeting sequence are discussed in terms of the structure of hFKBP12.     

Cloning and sequence analysis of a rapamycin-binding protein-encoding gene (RBP1) from Candida albicans.

Rapamycin (Rm) and FK506 are macrolide antifungal agents that exhibit potent immunosuppressive properties in higher eukaryotes which are mediated through interaction with specific receptor proteins (FKBPs or RBPs, for FK506- and Rm-binding proteins, respectively). These proteins possess peptidyl-prolyl cis-trans isomerase (PPIase) activity in vitro which is inhibited by the binding of Rm and FK506. We previously isolated a gene encoding an RBP from Saccharomyces cerevisiae, and demonstrated that null mutations in this gene (called RBP1) result in a recessive Rm-resistant (RmR) phenotype. We now have cloned the Candida albicans RBP1 gene via complementation of the RmR phenotype in S. cerevisiae. The predicted C. albicans RBP exhibits 61%, 52% and 49% amino acid (aa) sequence identity with RBPs (FKBPs) from S. cerevisiae, Neurospora crassa and human cells (FKBP-12), respectively. Furthermore, several of the aa residues identified as being important for drug binding in human FKBP-12 are conserved within the C. albicans RBP.     

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.     

Sequence diversification of the FK506-binding proteins in several different genomes.

Sequences of FK506-binding proteins (FKBPs) from four genomes of the following organisms were compared: the prokaryote Escherichia coli, the lower eukaryote Saccharomyces cerevisiae, the plant Arabidopsis thaliana, the nematode Caenorhabditis elegans and a composite of 14 unique FKBPs from two mammalian organisms Homo sapiens (man) and Mus musculus (domestic mouse). A singular FK506-like binding domain (FKBD) has about 12 kDa and occurs in the form of archetypal FKBP-12 and as a part of different proteins ranging in size from 13 to 135 kDa. Some organisms may contain a variable number of proteins which consist from two to four consecutively fused FKBDs. In the 12-kDa subgroup of archetypal FKBPs sequence identity (ID) varies from 100 to 83% (mammalian FKBPs-12), 75-50% in mammalian vs. invertebrate FKBPs-12, and fall to about 30% for pairwise sequence comparisons of mammalian and bacterial FKBPs-12 which suggests that their sequences are divergent. Multiple sequence alignment of FKBPs from the four genomes and a set of unique mammalian FKBPs does not contain any explicit consensus sequence but certain sequence positions have conserved physico-chemical characteristics. Variations of hydrophobicity and bulkiness in the multiple sequence alignment are nonsymmetrical because the physico-chemical properties of the aligned sequences changed during evolution. These variations at the sequence positions which are crucial for binding the immunosuppressive macrolide FK506 and peptidyl-prolyl cis/trans isomerase (PPIase) activity are small.     

The effects of rapamycin in murine peripheral nerve isografts and allografts

The FKBP-12-binding ligand FK506 has been successfully used to stimulate nerve regeneration and prevent the rejection of peripheral nerve allografts. The immunosuppressant rapamycin, another FKBP-12-binding ligand, stimulates axonal regeneration in vitro, but its influence on nerve regeneration in peripheral nerve isografts or allografts has not been studied. Sixty female inbred BALB/cJ mice were randomized into six tibial nerve transplant groups, including three isograft and three allograft (C57BL/6J) groups. Grafts were left untreated (groups I and II), treated with FK506 (groups III and IV), or treated with rapamycin (groups V and VI). Nerve regeneration was quantified in terms of histomorphometry and functional recovery, and immunosuppression was confirmed with mixed lymphocyte reactivity assays. Animals treated with FK506 and rapamycin were immunosuppressed and demonstrated significantly less immune cell proliferation relative to untreated recipient animals. Although every animal demonstrated some functional recovery during the study, animals receiving an untreated peripheral nerve allograft were slowest to recover. Isografts treated with FK506 but not rapamycin demonstrated significantly increased nerve regeneration. Nerve allografts in animals treated with FK506, and to a lesser extent rapamycin, however, both demonstrated significantly more nerve regeneration and increased nerve fiber widths relative to untreated controls. The authors suggest that rapamycin can facilitate regeneration through peripheral nerve allografts, but it is not a neuroregenerative agent in this in vivo model. Nerve regeneration in FK506-treated peripheral nerve isografts and allografts was superior to that found in rapamycin-treated animals. Rapamycin may have a role in the treatment of peripheral nerve allografts when used in combination with other medications, or in the setting of renal failure that often precludes the use of calcineurin inhibitors such as FK506.     

Charged surface residues of FKBP12 participate in formation of the FKBP12-FK506-calcineurin complex.

The mechanism of FK506 immunosuppression has been proposed to proceed by formation of a tight-binding complex with the intracellular 12-kDa FK506-binding protein (FKBP12). The FK506-FKBP12 complex then acts as a specific high-affinity inhibitor of the intracellular protein phosphatase PP2B (calcineurin), interrupting downstream dephosphorylation events required for T-cell activation. Site-directed mutagenesis of many of the surface residues of FKBP12 has no significant effect on its affinity for calcineurin. We have identified, however, three FKBP12 surface residues (Asp-37, Arg-42, and His-87) proximal to a solvent-exposed segment of bound FK506 that may be direct contacts in the calcineurin complex. Site-directed mutagenesis of two of these residues decreases the affinity of FKBP12-FK506 for calcineurin (Ki) from 6 nM for wild-type FKBP12 to 3.7 microM for a R42K/H87V double mutant, without affecting the peptidylprolyl isomerase activity or FK506 affinity of the mutant protein. These FKBP12 mutations along with several substitutions on FK506 known to affect calcineurin binding form a roughly 100-A2 region of the FKBP12-FK506 complex surface that is likely to be within the calcineurin binding site.     

Mammalian FKBP-25 and its associated proteins

Soluble proteins from porcine brain were divided into two packs: (1) proteins which pass freely through CM52-cellulose, and (2) proteins retained on CM52. Each of these two packs of proteins was fractionated on preparative flat-bed isoelectrofocusing gel in the range of pH 2-12. Native FKBP-25 and its truncated forms were found among other proteins retained on CM52-cellulose. Immunoblotting with anti-FKBP-25 showed two bands in the range 27-30 kDa, one due to unmodified FKBP-25 and other due to FKBP-25 mixed with high-mobility group II protein (HMG-II). Selective immunostaining with anti-FKBP-25 antibodies of proteins which were not retained on CM52-cellulose showed several bands within the range of pI 7-5 and mass of 23 +/- 2 kDa. These fractions of proteins were next resolved on two-dimensional gels and immunostained with anti-FKBP-25 antibodies. Six proteins in the pI range 7-5 were detected. Edman degradation of alpha-chymotrypsin digests of the major spot suggests that it contains the GTP-binding protein Rab5 co-migrating with guanylyl kinase, whereas MALDI-TOF showed that a residual content of FKBP-25 may be also associated with these two proteins. A residual quantity of FKBP-25 was also associated with the phosphatidylethanolamine-binding protein which is abundant in the brain.     

Neural roles of immunophilins and their ligands

The immunophilins are a family of proteins that are receptors for immunosuppressant drugs, such as cyclosporin A, FK506, and rapamycin. The occur in two classes, the FK506-binding proteins (FKBPs), which bind FK506 and rapamycin, and the cyclophilins, which bind cyclosporin A. Immunosuppressant actions of cyclosporin A and FK506 derive from the drug-immunophilin complex binding to and inhibiting the phosphatase calcineurin. Rapamycin binds to FKBP and the complex binds toRapamycinAnd FKBP-12Target (RAFT). RAFT affects protein translation by phosphorylating p70-S6 kinase, which phosphorylates the ribosomal S6 protein, and 4E-BP1, a repressor of protein translation initiation. Immunophilin levels are much higher in the brain than in immune tissues, and levels of FKBP12 increase in regenerating neurons in parallel with GAP-43. Immunophilin ligands, including nonimmunosuppressants that do not inhibit calcineurin, stimulate regrowth of damaged peripheral and central neurons, including dopamine, serotonin, and cholinergic neurons in intact animals. FKPB12 is physiologically associated with the ryanodine and inositol 1,4,5-trisphosphate (IP₃) receptors and regulates their calcium flux. By influencing phosphorylation of nruronal nitric oxide synthase, FKBP12 regulates nitric oxide formation, which is reduced by FK506.     

Isolation of a human cDNA encoding a 25 kDa FK-506 and rapamycin binding protein.

Recently, the nearly complete peptide sequence of a 25 kDa rapamycin and FK-506 binding protein that had been isolated from calf thymus, brain, and spleen was reported (1). Based upon the amino acid sequence of this bovine protein, bFKBP25, we have isolated from a JURKAT cDNA library the cDNA encoding the human homolog, hFKBP25. Translation of the open reading frame contained within this cDNA clone yields a sequence that, in its C-terminal half, is 41% identical to the major human FK-506 binding protein, hFKBP12, and 43% identical to hFKBP13. The N-terminal half of hFKBP25 is unrelated to any known protein.