Structure and Activity of the Peptidyl-Prolyl Isomerase Domain from the Histone Chaperone Fpr4 toward Histone H3 Proline Isomerization

The FK506-binding protein (FKBP) family of peptidyl-prolyl isomerases (PPIases) is characterized by a common catalytic domain that binds to the inhibitors FK506 and rapamycin. As one of four FKBPs within the yeast Saccharomyces cerevisiae, Fpr4 has been described as a histone chaperone, and is in addition implicated in epigenetic function in part due to its mediation of cis-trans conversion of proline residues within histone tails. To better understand the molecular details of this activity, we have determined the solution structure of the Fpr4 C-terminal PPIase domain by using NMR spectroscopy. This canonical FKBP domain actively increases the rate of isomerization of three decapeptides derived from the N terminus of yeast histone H3, whereas maintaining intrinsic cis and trans populations. Observation of the uncatalyzed and Fpr4-catalyzed isomerization rates at equilibrium demonstrate Pro¹⁶ and Pro³⁰ of histone H3 as the major proline targets of Fpr4, with little activity shown against Pro³⁸. This alternate ranking of the three target prolines, as compared with affinity determination or the classical chymotrypsin-based fluorescent assay, reveals the mechanistic importance of substrate residues C-terminal to the peptidyl-prolyl bond.     

A novel FK506- and rapamycin-binding protein (FPR3 gene product) in the yeast Saccharomyces cerevisiae is a proline rotamase localized to the nucleolus

The gene (FPR3) encoding a novel type of peptidylpropyl-cis-trans- isomerase (PPIase) was isolated during a search for previously unidentified nuclear proteins in Saccharomyces cerevisiae. PPIases are thought to act in conjunction with protein chaperones because they accelerate the rate of conformational interconversions around proline residues in polypeptides. The FPR3 gene product (Fpr3) is 413 amino acids long. The 111 COOH-terminal residues of Fpr3 share greater than 40% amino acid identity with a particular class of PPIases, termed FK506-binding proteins (FKBPs) because they are the intracellular receptors for two immunosuppressive compounds, rapamycin and FK506. When expressed in and purified from Escherichia coli, both full-length Fpr3 and its isolated COOH-terminal domain exhibit readily detectable PPIase activity. Both fpr3 delta null mutants and cells expressing FPR3 from its own promoter on a multicopy plasmid have no discernible growth phenotype and do not display any alteration in sensitivity to the growth-inhibitory effects of either FK506 or rapamycin. In S. cerevisiae, the gene for a 112-residue cytosolic FKBP (FPR1) and the gene for a 135-residue ER-associated FKBP (FPR2) have been described before. Even fpr1 fpr2 fpr3 triple mutants are viable. However, in cells carrying an fpr1 delta mutation (which confers resistance to rapamycin), overexpression from the GAL1 promoter of the C-terminal domain of Fpr3, but not full-length Fpr3, restored sensitivity to rapamycin. Conversely, overproduction from the GAL1 promoter of full- length Fpr3, but not its COOH-terminal domain, is growth inhibitory in both normal cells and fpr1 delta mutants. In fpr1 delta cells, the toxic effect of Fpr3 overproduction can be reversed by rapamycin. Overproduction of the NH2-terminal domain of Fpr3 is also growth inhibitory in normal cells and fpr1 delta mutants, but this toxicity is not ameliorated in fpr1 delta cells by rapamycin. The NH2-terminal domain of Fpr3 contains long stretches of acidic residues alternating with blocks of basic residues, a structure that resembles sequences found in nucleolar proteins, including S. cerevisiae NSR1 and mammalian nucleolin. Indirect immunofluorescence with polyclonal antibodies raised against either the NH2- or the COOH-terminal segments of Fpr3 expressed in E. coli demonstrated that Fpr3 is located exclusively in the nucleolus.     

Prolyl isomerases show low sequence specificity toward the residue following the proline

Prolyl isomerases catalyze the cis/trans isomerization of peptide bonds preceding proline. Previously, we had determined the specificity toward the residue before the proline for cyclophilin-, FKBP-, and parvulin-type prolyl isomerases by using proline-containing oligopeptides and refolding proteins as model substrates. Here, we report the specificities of members of these three prolyl isomerase families for the residue following the proline, again in short peptide and in refolding protein chains. Human cyclophilin 18 and parvulin 10 from Escherichia coli show high activity, but low specificity, with respect to the residue following the proline. Human FKBP12 prefers hydrophobic residues at this position in the peptide assays and shows a very low activity in the protein folding assays. This activity was strongly improved, and the sequence specificity was virtually eliminated after the insertion of a chaperone domain into the prolyl isomerase domain of human FKBP12.     

A Substrate Preference for the Rough Endoplasmic Reticulum Resident Protein FKBP22 during Collagen Biosynthesis

The biosynthesis of collagens occurs in the rough endoplasmic reticulum and requires a large numbers of molecular chaperones, foldases, and post-translational modification enzymes. Collagens contain a large number of proline residues that are post-translationally modified to 3-hydroxyproline or 4-hydroxyproline, and the rate-limiting step in formation of the triple helix is the cis-trans isomerization of peptidyl-proline bonds. This step is catalyzed by peptidyl-prolyl cis-trans isomerases. There are seven peptidyl-prolyl cis-trans isomerases in the rER, and so far, two of these enzymes, cyclophilin B and FKBP65, have been shown to be involved in collagen biosynthesis. The absence of either cyclophilin B or FKBP65 leads to a recessive form of osteogenesis imperfecta. The absence of FKBP22 leads to a kyphoscoliotic type of Ehlers-Danlos syndrome (EDS), and this type of EDS is classified as EDS type VI, which can also be caused by a deficiency in lysyl-hydroxylase 1. However, the lack of FKBP22 shows a wider spectrum of clinical phenotypes than the absence of lysyl-hydroxylase 1 and additionally includes myopathy, hearing loss, and aortic rupture. Here we show that FKBP22 catalyzes the folding of type III collagen and interacts with type III collagen, type VI collagen, and type X collagen, but not with type I collagen, type II collagen, or type V collagen. These restrictive interactions might help explain the broader phenotype observed in patients that lack FKBP22.     

Clearing the way for tau immunotherapy in Alzheimer's disease

Peptidyl‐prolyl cis/trans isomerases (PPIases), a unique family of molecular chaperones, regulate protein folding at proline residues. These residues are abundant within intrinsically disordered proteins, like the microtubule‐associated protein tau. Tau has been shown to become hyperphosphorylated and accumulate as one of the two main pathological hallmarks in Alzheimer's disease, the other being amyloid beta (Aβ). PPIases, including Pin1, FK506‐binding protein (FKBP) 52, FKBP51, and FKBP12, have been shown to interact with and regulate tau biology. This interaction is particularly important given the numerous proline‐directed phosphorylation sites found on tau and the role phosphorylation has been found to play in pathogenesis. This regulation then affects downstream aggregation and oligomerization of tau. However, many PPIases have yet to be explored for their effects on tau biology, despite the high likelihood of interaction based on proline content. Moreover, Pin1, FKBP12, FKBP52, cyclophilin (Cyp) A, CypB, and CypD have been shown to also regulate Aβ production or the toxicity associated with Aβ pathology. Therefore, PPIases directly and indirectly regulate pathogenic protein multimerization in Alzheimer's disease and represent a family rich in targets for modulating the accumulation and toxicity.

     

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.     

Wheat FKBP73 functions in vitro as a molecular chaperone independently of its peptidyl prolyl cis-trans isomerase activity

Peptidyl-prolyl cis-trans isomerases (PPIases) catalyse protein folding by accelerating the slow step of cis-trans isomerisation of peptidyl-prolyl bonds. Wheat (Triticum aestivum L.) FKBP73 (wFKBP73) is a peptidyl-prolyl cis-trans isomerase belonging to the FK506-binding protein (FKBP) family. It comprises three FKBP12-like domains, tetratricopeptide repeats and a calmodulin-binding domain (CaMbd). In vitro studies indicated that wFKBP73 possesses PPIase activity, binds calmodulin and forms a heterocomplex with mammalian p23 and wheat Hsp90 in wheat-germ lysate. To further study the role of wFKBP73 we have analysed its chaperone properties. Using the thermal unfolding and aggregation of citrate synthase (CS) as a model system, we have shown that the plant wFKBP73 exhibits chaperone activity, being able to suppress CS aggregation independently of its PPIase activity. The wFKBP73 interacts transiently with non-native CS and slows down its inactivation kinetics, whereas the mammalian homologue, hFKBP52 binds tightly to CS and does not affect its rate of inactivation. Hence, the first plant FKBP shown to function as a molecular chaperone has a mode of action different from that of the mammalian FKBP52.     

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.     

FKBP12 physically and functionally interacts with aspartokinase in Saccharomyces cerevisiae.

The peptidyl-prolyl isomerase FKBP12 was originally identified as the intracellular receptor for the immunosuppressive drugs FK506 (tacrolimus) and rapamycin (sirolimus). Although peptidyl-prolyl isomerases have been implicated in catalyzing protein folding, the cellular functions of FKBP12 in Saccharomyces cerevisiae and other organisms are largely unknown. Using the yeast two-hybrid system, we identified aspartokinase, an enzyme that catalyzes an intermediate step in threonine and methionine biosynthesis, as an in vivo binding target of FKBP12. Aspartokinase also binds FKBP12 in vitro, and drugs that bind the FKBP12 active site, or mutations in FKBP12 surface and active site residues, disrupt the FKBP12-aspartokinase complex in vivo and in vitro.fpr1 mutants lacking FKBP12 are viable, are not threonine or methionine auxotrophs, and express wild-type levels of aspartokinase protein and activity; thus, FKBP12 is not essential for aspartokinase activity. The activity of aspartokinase is regulated by feedback inhibition by product, and genetic analyses reveal that FKBP12 is important for this feedback inhibition, possibly by catalyzing aspartokinase conformational changes in response to product binding.     

Suppression of EGFR autophosphorylation by FKBP12

FK506 binding proteins (FKBPs) represent a subfamily of peptidyl prolyl cis/trans isomerases that can control receptor-mediated intracellular signaling. The prototypic PPIase FKBP12 functionally interacts with EGFR. FKBP12 was shown to inhibit EGF-induced EGFR autophosphorylation with all internal phosphorylation sites equally affected. The inhibition of EGFR catalytic activity is conducted by targeting the EGFR kinase domain. The change of intracellular FKBP12 levels resulted in a change of EGFR autophosphorylation level. Collectively, our results demonstrate that FKBP12 forms an endogenous inhibitor of EGFR phosphorylation directly involved in the control of cellular EGFR activity.     

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.     

The Caenorhabditis elegans parvulin gene subfamily and their expression under cold or heat stress along with the fkb subfamily

Parvulins and FKBPs are members of the peptidyl-prolyl cis/trans isomerases (PPIase) enzyme family whose role is to catalyze the interconversion between the cis trans forms of a peptide bond preceding internal proline residues in a polypeptide substrate. Members of the parvulin subfamily have been found to be involved in a variety of diseases, including Alzheimer's disease and cancer and are also considered possible antiparasitic targets. Genes Y110A2AL.13 (pin-1) and Y48C3A.16 (pin-4) were found in the worm's genome, possibly encoding parvulins. One is homologous to human and fly PIN1 whereas the other is homologous to human and fly PIN4. Both were expressed in Escherichia coli, purified and found to have in vitro PPIase activity. Expression levels of both genes, as well as the fkb genes (that encode FK506-binding proteins) were measured during development and under cold or heat stress conditions. The results revealed a potential role for these genes under temperature-related stress. RNAi silencing was performed for wild type and mutant strain worms under normal and cold or heat stress conditions. A reduced lifespan was observed when pin-4 dsRNA was fed to the fkb-5 deficient worms. Our work presents a first attempt to characterize the Caenorhabditis elegans parvulins and may present an interesting starting point for further experimentation concerning their role, along with the FKBP subfamily, in nematode physiology and their possible use as antiparasitic targets.     

The FK506 binding protein Fpr3 counteracts protein phosphatase 1 to maintain meiotic recombination checkpoint activity

The meiotic recombination checkpoint delays gamete precursors in G2 until DNA breaks created during recombination are repaired and chromosome structure has been restored. Here, we show that the FK506 binding protein Fpr3 prevents premature adaptation to damage and thus serves to maintain recombination checkpoint activity. Impaired checkpoint function is observed both in cells lacking FPR3 and in cells treated with rapamycin, a small molecule inhibitor that binds to the proline isomerase (PPIase) domain of Fpr3. FPR3 functions in the checkpoint through controlling protein phosphatase 1 (PP1). Fpr3 interacts with PP1 through its PPIase domain, regulates PP1 localization, and counteracts the activity of PP1 in vivo. Our findings define a branch of the recombination checkpoint involved in the adaptation to persistent chromosomal damage and a critical function for FK506 binding proteins during meiosis.     

Substrate specificities of the peptidyl prolyl cis-trans isomerase activities of cyclophilin and FK-506 binding protein: evidence for the existence of a family of distinct enzymes.

Substrate specificities, as reflected in kc/Km, were determined for the peptidyl prolyl cis-trans isomerase activities of cyclophilin and the FK-506 binding protein (FKBP). The substrates investigated were peptides of the general structure Suc-Ala-Xaa-Pro-Phe-p-nitroanilide, where Xaa = Gly, Ala, Val, Leu, Phe, His, Lys, on Glu. While kc/Km for cyclophilin-catalyzed isomerization shows little dependence on Xaa, kc/Km values for FKBP-catalyzed isomerization display a marked dependence on Xaa and vary over 3 orders of magnitude. An important outcome of this work is the discovery that Suc-Ala-Leu-Pro-Phe-pNA is a reactive substrate for FKBP (kc/Km = 640,000 M-1 s-1). This substrate can be used with FKBP concentrations that are low enough to allow, for the first time, accurate determinations of Ki values for tight-binding inhibitors of FKBP. Using this new assay, we found that FK-506 inhibits FKBP with Ki = 1.7 +/- 0.6 nM. The results of this work support the hypothesis that cyclophilin and FKBP are members of a family of peptidyl prolyl cis-trans isomerases and that the members of this family possess distinct substrate specificities that allow them to play diverse physiologic roles.     

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.     

A novel type of FKBP in the secretory pathway of Neurospora crassa

FKBPs define a subfamily of peptidyl-prolyl cis/trans isomerases (PPIases). PPIases are known to play roles in cellular protein folding, protein interactions and signal transduction. Here we describe NcFKBP22 from Neurospora crassa, a novel type of FKBP. NcFKBP22 is synthesized as a precursor protein with a cleavable signal sequence. In addition to a typical FKBP domain in the amino-terminal part mature NcFKBP22 contains a novel second domain which is unique amongst all known FKBPs. The amino acid composition of this carboxy-terminal domain is highly biased. Secondary structure predictions suggest that this domain may form an amphipathic α-helix. The carboxy-terminus of NcFKBP22 is –HNEL, a potential endoplasmic reticulum (ER) retention signal, suggesting that NcFKBP22 is a resident protein of the ER.     

Bcl-2 regulator FKBP38 is activated by Ca2+/calmodulin

FKBP-type peptidyl prolyl cis/trans isomerases (PPIases) are folding helper enzymes involved in the control of functional regrowth of damaged sciatic, cortical cholinergic, dopaminergic and 5-HT neurones. Here, we show that the constitutively inactive human FK506-binding protein 38 (FKBP38) is capable of responding directly to intracellular Ca2+ rise through formation of a heterodimeric Ca2+/calmodulin/FKBP38 complex. Only complex formation creates an enzymatically active FKBP, displaying affinity for Bcl-2 mediated through the PPIase site. Association between Bcl-2 and the active site of Ca2+/calmodulin/FKBP38 regulates Bcl-2 function and thereby participates in the promotion of apoptosis in neuronal tissues. FKBP38 proapoptotic function mediated by this interaction is abolished by either potent inhibitors of the PPIase activity of the Ca2+/calmodulin/FKBP38 complex or RNA interference-mediated depletion of FKBP38, promoting neuronal cell survival.