1H, 13C, and 15N resonance assignments of FK506-binding domain of Plasmodium falciparum FKBP35

The immunosuppressant FK506 binds Plasmodium falciparum FK-506 binding protein 35 (PfFKBP35) and shows anti-malarial activity. To understand molecular mechanism of the drug on the parasite, we have done NMR studies. Here, we report the assignment of FK506-binding domain of PfFKBP35.     

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.     

NMR and crystallographic structures of the FK506 binding domain of human malarial parasite Plasmodium vivax FKBP35

The emergence of drug‐resistant malaria parasites is the major threat to effective malaria control, prompting a search for novel compounds with mechanisms of action that are different from the traditionally used drugs. The immunosuppressive drug FK506 shows an antimalarial activity. The mechanism of the drug action involves the molecular interaction with the parasite target proteins PfFKBP35 and PvFKBP35, which are novel FK506 binding protein family (FKBP) members from Plasmodium falciparum and Plasmodium vivax, respectively. Currently, molecular mechanisms of the FKBP family proteins in the parasites still remain elusive. To understand their functions, here we have determined the structures of the FK506 binding domain of Plasmodium vivax (PvFKBD) in unliganded form by NMR spectroscopy and in complex with FK506 by X‐ray crystallography. We found out that PvFKBP35 exhibits a canonical FKBD fold and shares kinetic profiles similar to those of PfFKBP35, the homologous protein in P. falciparum, indicating that the parasite FKBP family members play similar biological roles in their life cycles. Despite the similarity, differences were observed in the ligand binding modes between PvFKBD and HsFKBP12, a human FKBP homolog, which could provide insightful information into designing selective antimalarial drug against the parasites.     

Expression, purification, and molecular characterization of Plasmodium falciparum FK506-binding protein 35 (PfFKBP35)

The immunosuppressive drug FK506 binds its targets FK506-binding protein (FKBP) family and modulates cellular processes. Recent studies demonstrated that FK506 shows anti-malaria effects. Newly identified FK506-binding protein 35 from Plasmodium falciparum (PfFKBP35) is assumed to be the molecular target of FK506 in the parasite. Currently, molecular and structural basis of growth inhibition of the parasite by FK506 remains unclear. In this study, to examine characteristics of PfFKBP35 and also understand its molecular mechanism of the inhibition by FK506, we have cloned, expressed, and purified the full-length PfFKBP35 and its FK506-binding domain (FKBD). We demonstrate that the full-length PfFKBP35 and the FKBD were properly folded, and suitable for biochemical and biophysical studies. PfFKBP35 showed a basal activity in inhibiting the phosphatase activity of calcineurin in the absence of FK506, but the presence of FK506 greatly enhanced its calcineurin-inhibitory activity. Our NMR data indicate that the FKBD binds FK506 with a high affinity.     

FK506-binding protein-like and FK506-binding protein 8 regulate dual leucine zipper kinase degradation and neuronal responses to axon injury

The dual leucine zipper kinase (DLK) is a key regulator of axon regeneration and degeneration in response to neuronal injury; however, regulatory mechanisms of the DLK function via its interacting proteins are largely unknown. To better understand the molecular mechanism of DLK function, we performed yeast two-hybrid screening analysis and identified FK506-binding protein-like (FKBPL, also known as WAF-1/CIP1 stabilizing protein 39) as a DLK-binding protein. FKBPL binds to the kinase domain of DLK and inhibits its kinase activity. In addition, FKBPL induces DLK protein degradation through ubiquitin-dependent pathways. We further assessed other members in the FKBP protein family and found that FK506-binding protein 8 (FKBP8) also induced DLK degradation. We identified the lysine 271 residue in the kinase domain as a major site of DLK ubiquitination and SUMO3 conjugation and was thus responsible for regulating FKBP8-mediated proteasomal degradation that was inhibited by the substitution of the lysine 271 to arginine. FKBP8-mediated degradation of DLK is mediated by autophagy pathway because knockdown of Atg5 inhibited DLK destabilization. We show that in vivo overexpression of FKBP8 delayed the progression of axon degeneration and suppressed neuronal death after axotomy in sciatic and optic nerves. Taken together, this study identified FKBPL and FKBP8 as novel DLK-interacting proteins that regulate DLK stability via the ubiquitin-proteasome and lysosomal protein degradation pathways.     

Escherichia coli and other species of the enterobacteriaceae encode a protein similar to the family of Mip-like FK506-binding proteins

A newly identified gene in Escherichia coli, fkpA, encodes a protein with extensive similarity to the macrophage infectivity potentiator (Mip) proteins of Legionella pneumophila and Chlamydia trachomatis. The FkpA protein may be a new member of the family of FK506-binding proteins (FKBPs) because its carboxyl domain includes a sequence that matches the consensus FK506-binding motif in 40 of 48 positions. including those amino acids at the active site that form hydrogen bonds with the drug FK506. The amino acid sequence of the 29kDa FkpA protein is 30–35% identical to the Mip proteins of L. pneumophila, L. micdadei, and C. trachomatis. Of the 270 amino acids of FkpA, 113 (42%) are identical to the sequence of one or another of these Mip proteins. Overexpression of FkpA or deletion of fkpA from the E. coli chromosome had no detrimental effect on bacterial growth, indicating that fkpA is not an essential gene. Hybridization of fkpA-specific DNA probes to genomic blots révealed that similar genes exist in several representatives of the Enterobacteriaceae. Thus, mip-like genes are not found exelusively in bacteria having a predominately intracellular life style, but instead appear to be a new FKBP subfamily that is a common constituent of many bacteria.     

FK-506结合蛋白对钙释放通道的调控

细胞内自由钙作为一种重要的细胞信使广泛地参与细胞生理功能调控.胞内钙库(内质网系和肌浆网系)对调节细胞内自由钙水平起着重要的作用.钙库膜上的钙释放通道(ryanodine受体和三磷酸肌醇受体)受许多因素调控,其中之一就是新近研究得相当多的FK506结合蛋白.免疫抑制剂FK506能特异地结合钙库上一种分子质量为12 ku左右的蛋白,这种FK506结合蛋白与钙释放通道形成一种紧密连接的复合体,在正常生理情况下对钙释放通道起着十分重要的调控作用.     

FKBP133: a novel mouse FK506-binding protein homolog alters growth cone morphology

FK506-binding proteins are the peptidyl prolyl cis-trans isomerases that are involved in various intracellular events. We characterized a novel mouse FK506-binding protein homolog, FKBP133/KIAA0674, in the developing nervous system. FKBP133 contains a domain similar to Wiskott-Aldrich syndrome protein homology region 1 (WH1) and a domain homologous to FK506-binding protein motif. FKBP133 was predominantly expressed in cerebral cortex, hippocampus, and peripheral ganglia at embryonic day 18.5. FKBP133 protein was distributed in the axonal shafts and was partially co-localized with F-actin in the growth cones of dorsal root ganglion neurons (DRG). The number of filopodia was increased in the DRG neurons overexpressing FKBP133. In contrast, the overexpression of a mutant deleted the WH1 domain reduced the growth cone size and the number of filopodia. Furthermore, the neurons overexpressing FKBP133 became significantly resistant to Semaphorin-3A induced collapse response. These results suggest that FKBP133 modulates growth cone behavior with the WH1 domain.     

Solution structure of FK506-binding protein 12 from Aedes aegypti

Dengue remains one of the major public concerns as the virus eludes the immune response. Currently, no vaccines or antiviral therapeutics are available for dengue prevention or treatment. Immunosuppressive drug FK506 shows an antimalarial activity, and its molecular target, FK506‐binding protein (FKBP), was identified in human Plasmodium parasites. Likewise, a conserved FKBP family protein has also been identified in Aedes aegypti (AaFKBP12), which is expected to play a similar role in the life cycle of Aedes aegypti, the primary vector of dengue virus infection. As FKBPs belong to a highly conserved class of immunophilin family and are involved in key biological regulations, they are considered as attractive pharmacological targets. In this study, we have determined the nuclear magnetic resonance solution structure of AaFKBP12, a novel FKBP member from Aedes aegypti, and presented its structural features, which may facilitate the design of potential inhibitory ligands against the dengue‐transmitting mosquitoes. Proteins 2012;. © 2012 Wiley Periodicals, Inc.     

Xenopus FK 506-binding protein, a novel immunophilin expressed during early development

FK 506-binding proteins (FKBPs) are a family of cytosolic proteins identified by virtue of their ability to bind the immunosuppressants FK 506 and rapamycin. While their function has been extensively studied in the immune system, little is known about their role during early embryonic development. Here we describe the cloning and expression of a new Xenopus FKBP (xFKBP). xFKBP encodes a 63-kDa protein that shares high sequence homology with mouse FKBP65. It is expressed maternally and becomes restricted after the gastrula stage to dorsal mesoderm and notochord. At the tailbud stage expression persists in the notochord and begins to accumulate in epidermis, branchial arches and developing somites. In adults, xFKBP mRNA is confined to the testis.     

Genomic organization of mouse and human 65 kDa FK506-binding protein genes and evolution of the FKBP multigene family

FK506-binding proteins (FKBPs) are peptidyl-prolyl cis/trans isomerases PPIases) that bind the immunosuppressive drug FK506. Of the many eukaryotic FKBPs that have been identified, FKBP65 is an endoplasmic reticulum-localized protein that associates with tropoelastin in the secretory pathway. Unlike any other FKBP characterized so far, FKBP65 is developmentally regulated and may be intimately involved in organogenesis. Here, we report the isolation, sequencing, and genomic organization of the mouse FKBP65 gene (Fkbp10) and provide a comparison with the human ortholog. Mouse Fkbp10 contains 10 exons and 9 introns encompassing 8.5 kb. The exon-intron organization of Fkbp10 displays a pattern of repetition that reflects the coding sequence of the four PPIase, or FK506-binding, domains present in the mature protein. The exon organization of the PPIase domains differs from that of the other FKBP family members. The evolution of the FKBP65 gene and other members of the FKBP multigene family were therefore investigated from a taxonomically diverse array of prokaryotic and eukaryotic taxa. These analyses suggest that the FKBP multigene family emerged early in the evolutionary history of eukaryotes, and during that time some members, including the FKBP65 gene, have experienced gene elongation by means of PPIase domain duplication.     

FK506-binding protein 51 interacts with Clostridium botulinum C2 toxin and FK506 inhibits membrane translocation of the toxin in mammalian cells

The binary Clostridium botulinum C2 toxin consists of the binding/translocation component C2IIa and the separate enzyme component C2I. C2IIa delivers C2I into the cytosol of eukaryotic target cells where C2I ADP-ribosylates actin. After receptor-mediated endocytosis of the C2IIa/C2I complex, C2IIa forms pores in membranes of acidified early endosomes and unfolded C2I translocates through the pores into the cytosol. Membrane translocation of C2I is facilitated by the activities of host cell chaperone Hsp90 and the peptidyl-prolyl cis/trans isomerase (PPIase) cyclophilin A. Here, we demonstrated that Hsp90 co-precipitates with C2I from lysates of C2 toxin-treated cells and identified the FK506-binding protein (FKBP) 51 as a novel interaction partner of C2I in vitro and in intact mammalian cells. Prompted by this finding, we used the specific pharmacological inhibitor FK506 to investigate whether the PPIase activity of FKBPs plays a role during membrane translocation of C2 toxin. Treatment of cells with FK506 protected cultured cells from intoxication with C2 toxin. Moreover, FK506 inhibited the pH-dependent translocation of C2I across membranes into the cytosol but did not interfere with the enzyme activity of C2I or binding of C2 toxin to cells. Furthermore, FK506 treatment delayed intoxication with the related binary actin ADP-ribosylating toxins from Clostridium perfringens (iota toxin) and Clostridium difficile (CDT) but not with the Rho-glucosylating Clostridium difficile toxin A (TcdA). In conclusion, our results support the hypothesis that clostridial binary actin-ADP-ribosylating toxins share a specific FKBP-dependent translocation mechanism during their uptake into mammalian cells.     

The FK506-binding protein, Fpr4, is an acidic histone chaperone

Fpr4, a FK506-binding protein (FKBP), is a recently identified novel histone chaperone. How it interacts with histones and facilitates their deposition onto DNA, however, are not understood. Here, we report a functional analysis that shows Fpr4 forms complexes with histones and facilitates nucleosome assembly like previously characterized acidic histone chaperones. We also show that the chaperone activity of Fpr4 resides solely in an acidic domain, while the peptidylprolyl isomerase domain conserved among all FKBPs inhibits the chaperone activity. These observations argue that Fpr4, while unique structurally, deposits histones onto DNA for nucleosome assembly through the well-established mechanism shared by other chaperones.     

Role of FK506-binding protein 12 in development of the chick embryonic heart

A cDNA encoding chicken FK506-binding protein 12 (FKBP12) was isolated and sequenced. The predicted amino acid sequence of the chicken protein shows high homology to those of FKBP12 proteins of other species ranging from human to frog. The possible role of FKBP12 in chick embryonic cardiac development was examined. Northern blot analysis revealed that FKBP12 mRNA is distributed widely in chick embryos, being especially abundant in the heart; the amount of FKBP12 mRNA in the embryonic heart decreased with time. Administration of FK506 to chick embryos at 7 to 9 days resulted in marked cardiac enlargement. FK506 also reduced the expression of myosin, induced a more elongated cell morphology, and impaired network formation in cultured chick embryonic cardiomyocytes. These results suggest that FKBP12 is important in the regulation of contractile function and phenotypic expression in chick cardiomyocytes during embryonic development.     

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.     

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.     

Whole genome identification and analysis of FK506-binding protein family genes in grapevine (Vitis vinifera L.)

In plant and animal species FK506-binding protein (FKBP) family genes are important conserved genes and it is defined as the receptors of FK506 and rapamycin, where they work as PPIase and protein folding chaperones. FKBP have been isolated from Arabidopsis thaliana, Oryza sativa, and Zea mays. In grape, twenty-three genes containing the FK506-binding domain (FKBP_C) were first time identified by HMMER and blast research, they were classified into three groups and 17 out of the 23 genes were located on 11 chromosomes (Chr1, 3, 5, 7, 8, 14, 15, 16, 17, 18, and 19). The predicted gene expression pattern and semi-quantitative RT-PCR results revealed that five VvFKBPs were expressed in all tissues, while seven VvFKBPs were expressed only in some of the tissues, and the remaining VvFKBPs were not expressed in leaf, stem, inflorescences, flowers, and a mixture of fruit tissues (small, medium and big-sized fruits). Most of the VvFKBPs in grapevine ‘Summer Black’ were similar to those predicted one in ‘Pinot Noir’ except for VvFKBP16-4 and VvFKBPa. VvFKBP12, FaFKBP12 and PpFKBP12 were cloned from ‘Summer Black’, ‘Sweet Charlie’ and ‘Xiahui 6’. Protein structure analysis confirmed that homologous genes have some differences during the process of protein structure construction. In this study, we characterized and verified 23 FKBP family genes in grapevine (Vitis vinifera L.) as well as their sub-cellular and chromosome location. The successful cloning of CDS regions and protein structural analysis of VvFKBP12, FaFKBP12, and PpFKBP12 can provide useful information for further study.     

Genome-wide analysis of genes encoding FK506-binding proteins in rice

The FK506-binding proteins (FKBPs) are a class of peptidyl-prolyl cis/trans isomerase enzymes, some of which can also operate as molecular chaperones. FKBPs comprise a large ubiquitous family, found in virtually every part of the cell and involved in diverse processes from protein folding to stress response. Higher plant genomes typically encode about 20 FKBPs, half of these found in the chloroplast thylakoid lumen. Several FKBPs in plants are regulators of hormone signalling pathways, with important roles in seed germination, plant growth and stress response. Some FKBP isoforms exists as homologous duplicates operating in finely tuned mechanisms to cope with abiotic stress. In order to understand the roles of the plant FKBPs, especially in view of the warming environment, we have identified and analysed the gene families encoding these proteins in rice using computational approaches. The work has led to identification of all FKBPs from the rice genome, including novel high molecular weight forms. The rice FKBP family appears to have evolved by duplications of FKBP genes, which may be a strategy for increased stress tolerance.     

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.