Prolyl isomerases in a minimal cell. Catalysis of protein folding by trigger factor from Mycoplasma genitalium.

Peptidyl-prolyl cis/trans isomerases (PPIases) catalyze the isomerization of prolyl peptide bonds. Distinct families of this class of enzymes are involved in protein folding in vitro, whereas their significance in free living organisms is not known. Previously, we inspected the smallest known genome of a self-replicating organism and found that Mycoplasma genitalium is devoid of all known PPIases except the trigger factor. Despite the extensive sequence information becoming available, most genes remain hypothetical and enzyme activities in many species have not been assigned to an open reading frame. Therefore, we studied the PPIase activity in crude extracts of M. genitalium. We showed that this is solely attributed to a single enzyme activity, the trigger factor. Characterization of this enzyme revealed that its PPIase activity resides in a central 12-kDa domain. Only the complete trigger factor is able to cis/trans isomerize extended peptide substrates, while the PPIase domain alone can not. The N- and the C-terminal domains of the trigger factor seem to function in binding of proteins as substrates, as demonstrated by protein refolding experiments, in which the complete trigger factor catalyzed protein refolding towards a model protein 500-fold more efficiently than the isolated central PPIase domain. Protein modeling studies suggest that the PPIase domain can fold in a similar way as the PPIase domain of FK506 binding proteins (FKBPs), one class of PPIases, despite only very limited sequence homology. Differences at the active site explain why this enzyme is not inhibited by FK506 in contrast with FKBPs. Trigger factor expressed in Escherichia coli confirms its additional chaperone functions, as shown by its association with chaperones GroEL and GroES after induction of misfolding. In contrast, the isolated PPIase-domain lacks any association with chaperones from E. coli. In summary, trigger factor of M. genitalium is the single folding isomerase of this organism, which harbors an enzymatically active PPIase domain with structural homology to FKBPs. Its additional domains confer its ability to be an efficient catalyst of protein folding. The protein folding machinery is conserved and shows a dual function as a chaperone and a prolyl isomerase.     

Mip protein of Legionella pneumophila exhibits peptidyl-prolyl-cis/trans isomerase (PPIase) activity

Legionella pneumophila is an intracellular parasite which is able to survive and multiply in human monocytes and alveolar macrophages. The Mip (macrophage infectivity potentiator) protein has been shown to be an essential virulence factor. A search of translated nucleic acid data bases has shown that the Mip protein from strain Wadsworth possesses regions homologous to those found in the FK506-binding proteins (FKBPs) of several different eukaryotic organisms. FKBPs are able to bind to the immunosuppressant macrolide FK506 and possess peptidyl-prolyl cis/trans isomerase (PPIase) activity. The gene coding for the Mip protein was cloned from the chromosome of L. pneumophila strain Philadelphia I and sequenced. It was synthesized in Escherichia coli K-12 and after purification it exhibited PPIase activity catalysing the slow cis/trans isomerization of prolyl peptide bonds in oligopeptides. Mip is inhibited by FK506 and fully resistant to cyclosporin A, as was also found for the recently characterized FKBP-type PPIases of eukaryotes. However, the N-terminal extension of Mip and/or the substitutions of the variable amino acids in the C-terminal FKBP core leads to variations, when compared with eukaryotic FKBPs, in substrate specificity with the oligopeptide substrates of type Suc-Ala-Xaa-Pro-Phe-4-nitroanilide. Nevertheless, the Legionella Mip factor represents a bacterial gene product which shares some characteristics normally found in eukaryotic proteins. In view of the activity of PPIases in protein-folding reactions, such prokaryotic FKBP analogues may represent a new class of bacterial pathogenicity factors.     

FK506-binding protein-type peptidyl-prolyl cis-trans isomerase from a halophilic archaeum, Halobacterium cutirubrum

The halophilic archaeum, Halobacterium cutirubrum, has been shown to have a cyclophilin-type peptidyl-prolyl cis-trans isomerase (PPIase). Because most archaeal genomes studied only have genes for FK506-binding proteins (FKBPs) as a PPIase, it has been unclear whether H. cutirubrum has an FKBP-type PPIase or not. In the present study, a gene encoding an FKBP-type PPIase was cloned from genomic DNA of H. cutirubrum and then sequenced. This FKBP was deduced to be composed of 303 amino acid residues with a molecular mass of 33.3kDa. Alignment of its amino acid sequence with those of other reported FKBPs showed that it contained two insertion sequences in the regions corresponding to the bulge and flap of human FKBP12, which are common to archaeal FKBPs. Its C-terminal amino acid sequence was approximately 130 amino acids longer than the FKBPs of Methanococcus thermolithotrophicus and Thermococcus sp. KS-1. Among the 14 conserved amino acid residues that form the FK506 binding pocket, only three were found in this FKBP. This gene was expressed as a fusion protein with glutathione S-transferase (GST) in Escherichia coli, and the N-terminal GST portion was removed by protease digestion. The purified recombinant FKBP showed a weak PPIase activity with a low sensitivity to FK506. This FKBP suppressed aggregation of the unfolded protein.     

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.     

Neurospora crassa FKBP22 is a novel ER chaperone and functionally cooperates with BiP

FK506 binding proteins (FKBPs) belong to the family of peptidyl prolyl cis-trans isomerases (PPIases) catalyzing the cis/trans isomerisation of Xaa-Pro bonds in oligopeptides and proteins. FKBPs are involved in folding, assembly and trafficking of proteins. However, only limited knowledge is available about the roles of FKBPs in the endoplasmic reticulum (ER) and their interaction with other proteins. Here we show the ER located Neurospora crassa FKBP22 to be a dimeric protein with PPIase and a novel chaperone activity. While the homodimerization of FKBP22 is mediated by its carboxy-terminal domain, the amino-terminal domain is a functional FKBP domain. The chaperone activity is mediated by the FKBP domain but is exhibited only by the full-length protein. We further demonstrate a direct interaction between FKBP22 and BiP, the major Hsp70 chaperone in the ER. The binding to BiP is mediated by the FKBP domain of FKBP22. Interestingly BiP enhances the chaperone activity of FKBP22. Both proteins form a stable complex with an unfolded substrate protein and thereby prevent its aggregation. These results suggest that BiP and FKBP22 form a folding helper complex with a high chaperoning capacity in the ER of Neurospora crassa.     

A nuclear FK506-binding protein is a histone chaperone regulating rDNA silencing

We report a novel chromatin-modulating factor, nuclear FK506-binding protein (FKBP). It is a member of the peptidyl prolyl cis-trans isomerase (PPIase) family, whose members were originally identified as enzymes that assist in the proper folding of polypeptides. The endogenous FKBP gene is required for the in vivo silencing of gene expression at the rDNA locus and FKBP has histone chaperone activity in vitro. Both of these properties depend on the N-terminal non-PPIase domain of the protein. The C-terminal PPIase domain is not essential for the histone chaperone activity in vitro, but it regulates rDNA silencing in vivo. Chromatin immunoprecipitation showed that nuclear FKBP associates with chromatin at rDNA loci in vivo. These in vivo and in vitro findings in nuclear FKBPs reveal a hitherto unsuspected link between PPIases and the alteration of chromatin structure.     

PPIase activities and interaction partners of FK506-binding proteins in the wheat thylakoid

FK506-binding proteins (FKBPs) and cyclophilins, collectively called immunophilins, conserve peptidyl-prolyl cis/trans isomerase (PPIase) active sites, although many lack PPIase activity. The chloroplast thylakoid contains a large proportion of the plant immunophilin family, but their functions within this compartment are unclear. Some lumenal immunophilins are important for assembly of photosynthetic complexes, implicating them in the maintenance and turnover of the photosynthetic apparatus during acclimation processes. In this investigation into the functions of three FKBPs localized to the thylakoid of Triticum aestivum (wheat), we present the first evidence of PPIase activity in the thylakoid of a cereal plant, and also show that PPIase activity is not conserved in all lumenal FKBPs. Using yeast two-hybrid analysis we found that the PPIase-active FKBP13 interacts with the globular domain of the wheat Rieske protein, with potential impact on photosynthetic electron transfer. Specific interaction partners for PPIase-deficient FKBP16-1 and FKBP16-3 link these isoforms to photosystem assembly.     

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.     

FKBP-type peptidyl-prolyl cis–trans isomerase from a sulfur-dependent hyperthermophilic archaeon,Thermococcus sp. KS-1

A gene encoding an FK506 binding protein (FKBP)-type peptidyl-prolyl cis–trans isomerase (PPIase) was cloned from a hyperthermophilic archaeon, Thermococcus sp. KS-1, and sequenced. This gene encoded an FKBP with 159 amino-acid residues with a molecular mass of 17.6 kDa. Two insertion sequences with 13 and 44 amino acids were found in the regions corresponding to the bulge and flap regions of human FKBP-12, respectively. Comparison with other archaeal FKBP sequences obtained from reported genome sequences revealed that the insertion sequences in the bulge and flap regions were common to archaeal FKBPs. It was also revealed that archaeal FKBPs are classified into two groups: one is approx. 17 kDa and the other 27 kDa. This Thermococcus FKBP (TcFK) belonged to the smaller archaeal FKBP. In this TcFK, 9 out of 15 amino acid residues forming the FK506 binding pocket of human FKBP12 were found. This gene was expressed in Escherichia coli and the recombinant protein was purified. The purified protein showed PPIase activity and its activity was inhibited by FK506 with an IC₅₀ of 7 μM. This enzyme showed high kinetic stability with a half-life of 40 min at 100°C. Catalytic efficiency of this recombinant PPIase was 1.2-times higher with the substrate N-succinyl-A-L-P-F-p-nitroanilide than with N-succinyl-A-A-P-F-p-nitroanilide.     

Immunophilins and parvulins. Superfamily of peptidyl prolyl isomerases in Arabidopsis

Immunophilins are defined as receptors for immunosuppressive drugs including cyclosporin A, FK506, and rapamycin. The cyclosporin A receptors are referred to as cyclophilins (CYPs) and FK506- and rapamycin-binding proteins are abbreviated as FKBPs. These two groups of proteins (collectively called immunophilins) share little sequence homology, but both have peptidyl prolyl cis/trans isomerase (PPIase) activity that is involved in protein folding processes. Studies have identified immunophilins in all organisms examined including bacteria, fungi, animals, and plants. Nevertheless, the physiological function of immunophilins is poorly understood in any organism. In this study, we have surveyed the genes encoding immunophilins in Arabidopsis genome. A total of 52 genes have been found to encode putative immunophilins, among which 23 are putative FKBPs and 29 are putative CYPs. This is by far the largest immunophilin family identified in any organism. Both FKBPs and CYPs can be classified into single domain and multiple domain members. The single domain members contain a basic catalytic domain and some of them have signal sequences for targeting to a specific organelle. The multiple domain members contain not only the catalytic domain but also defined modules that are involved in protein-protein interaction or other functions. A striking feature of immunophilins in Arabidopsis is that a large fraction of FKBPs and CYPs are localized in the chloroplast, a possible explanation for why plants have a larger immunophilin family than animals. Parvulins represent another family of PPIases that are unrelated to immunophilins in protein sequences and drug binding properties. Three parvulin genes were found in Arabidopsis genome. The expression of many immunophilin and parvulin genes is ubiquitous except for those encoding chloroplast members that are often detected only in the green tissues. The large number of genes and diversity of structure domains and cellular localization make PPIases a versatile superfamily of proteins that clearly function in many cellular processes in plants.     

Molecular cloning and characterization of the genes encoding the immunoreactive major cell-surface proteins of Porphyromonas gingivalis

Abstract The Mip ('macrophage infectivity potentiator') protein of Legionella pneumophila has been shown to be an essential virulence factor, exhibiting peptidyl-prolyl cis/trans isomerase (PPIase) activity that can be inhibited by the immunosuppressant FK506. The cloning and sequencing of mip genes from three different L. pneumophila strains revealed a single amino acid substitution which did not affect the isomerase property of the enzyme. Mip proteins isolated from two wild-type L. pneumophila strains and from two corresponding Escherichia coli K-12 recombinant clones derived from these strains exhibited identical enzymatic properties and the precursor proteins are processed at identical cleavage sites. The mature Mip proteins exist in an oligomeric form. Site-directed mutagenesis demonstrated that a substitution of an Asp residue at position 142 by a Leu residue affects PPIase activity of Mip.     

The PPIase active site of Legionella pneumophila Mip protein is involved in the infection of eukaryotic host cells

We analysed eight monoclonal antibodies (mAbs) directed against the Mip (macrophage infectivity potentiator) protein, a virulence factor of the intracellular pathogen Legionella pneumophila. Mip belongs to the FK506-binding proteins (FKBPs) and exhibits peptidyl prolyl cis/trans isomerase (PPIase) activity. Five of the mAbs recognised epitopes in the C-terminal, FKBP-homologous domain of Mip, which is highly conserved among all Legionella species. Upon immunological binding to Mip, all but one of these mAbs caused inhibition of the PPIase activity in vitro. mAb binding to the N-terminal domain of Mip did not influence its enzymatic activity. All but one of the PPIase inhibiting mAbs were able to significantly inhibit the early establishment and initiation of an intracellular infection of the bacteria in Acanthamoeba castellanii, the natural host, and in the human phagocytic cell line U937. These data demonstrate for the first time that for the virulence-enhancing property of the L. pneumophila Mip protein, an intact active site of the enzyme is an essential requirement.     

The binding of FKBP23 to BiP modulates BiP's ATPase activity with its PPIase activity

Peptidyl-prolyl cis-trans-isomerases (PPIases) are enzymes that can cis-trans-isomerize a Xaa-Pro peptide bond. Three families of PPIases are known: cyclophilins, FKBPs, and parvulins. The physiological functions of the PPIases are only poorly understood. In previous work, we reported that the mouse FK506-binding protein 23 (mFKBP23), which comprises an N-terminal PPIase domain and a C-terminal domain with Ca(2+)-binding sites, binds to mBiP in the endoplasmic reticulum (ER) and this binding is affected by the Ca(2+) concentration. In this study, we demonstrate the ability of mFKBP23 to modulate the ATPase activity of BiP, and that the bound mFKBP23, but not the free mFKBP23, can suppress the ATPase activity of mBiP through its PPIase activity.     

FK506 binding protein from the hyperthermophilic archaeon Pyrococcus horikoshii suppresses the aggregation of proteins in Escherichia coli

The 29-kDa FK506 binding protein (FKBP) gene is the only peptidyl-prolyl cis-trans isomerase (PPIase) gene in the genome of Pyrococcus horikoshii. We characterized the function of this FKBP (PhFKBP29) and used it to increase the production yield of soluble recombinant protein in Escherichia coli. The PPIase activity (k(cat)/K(m)) of PhFKBP29 was found to be much lower than that of other archaeal 16- to 18-kDa FKBPs by a chymotrypsin-coupled assay of the oligo-peptidyl substrate at 15 degrees C. Besides this low PPIase activity, PhFKBP29 showed chaperone-like protein folding activity which enhanced the refolding yield of chemically unfolded rhodanese in vitro. In addition, it suppressed thermal protein aggregation in a temperature range of 45 to 100 degrees C. When the PhFKBP29 gene was coexpressed with the recombinant Fab fragment gene of the anti-hen egg lysozyme antibody in the cytoplasm of E. coli, whose expressed product tended to form an inactive aggregate in E. coli, it improved the yield of the soluble Fab fragments with antibody specificity. PhFKBP29 exerted protein folding and aggregation suppression in E. coli cells.     

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.     

Crystal structure of the three FK506 binding protein domains of wheat FKBP73: evidence for a unique wFK73_2 domain

Here we describe the crystal structure of the N-terminal domain of the FK506-binding protein (FKBP) from wheat (wFKBP73), which is the first structure presenting three FK domains (wFK73_1, wFK73_2 and wFK73_3). The crystal model includes wFK73_2 and wFK73_3 domains and only part of the wFK73_1 domain. The wFK73_1 domain is responsible for binding FK506 and for peptidyl prolyl cis/trans isomerase (PPIase) activity, while the wFK73_2 and wFK73_3 domains lack these activities. A structure-based sequence comparison demonstrated that the absence of a large enough hydrophobic pocket important for PPIase activity, and of the conserved residues necessary for drug binding in the wFK73_2 and wFK73_3 domains explains the lack of these activities in these domains. Sequence and structural comparison between the three wFKBP73 domains suggest that the wFK73_2 domain is the most divergent. A structural comparison of the FK domains of wFKBP73 with other FKBPs containing more than one FK domain, revealed that while the overall architecture of each of the three FK domains displays a typical FKBP fold, their relative arrangement in space is unique and may have important functional implications. We suggest that the existence of FKBPs with three FK domains offers additional interactive options for these plant proteins enlarging the overall regulatory functions of these proteins.     

Comparative analysis of different peptidyl-prolyl isomerases reveals FK506-binding protein 12 as the most potent enhancer of alpha-synuclein aggregation

FK506-binding proteins (FKBPs) are members of the immunophilins, enzymes that assist protein folding with their peptidyl-prolyl isomerase (PPIase) activity. Some non-immunosuppressive inhibitors of these enzymes have neuroregenerative and neuroprotective properties with an unknown mechanism of action. We have previously shown that FKBPs accelerate the aggregation of α-synuclein (α-SYN) in vitro and in a neuronal cell culture model for synucleinopathy. In this study we investigated whether acceleration of α-SYN aggregation is specific for the FKBP or even the PPIase family. Therefore, we studied the effect of several physiologically relevant PPIases, namely FKBP12, FKBP38, FKBP52, FKBP65, Pin1, and cyclophilin A, on α-SYN aggregation in vitro and in neuronal cell culture. Among all PPIases tested in vitro, FKBP12 accelerated α-SYN aggregation the most. Furthermore, only FKBP12 accelerated α-SYN fibril formation at subnanomolar concentrations, pointing toward an enzymatic effect. Although stable overexpression of various FKBPs enhanced the aggregation of α-SYN and cell death in cell culture, they were less potent than FKBP12. When FKBP38, FKBP52, and FKBP65 were overexpressed in a stable FKBP12 knockdown cell line, they could not fully restore the number of α-SYN inclusion-positive cells. Both in vitro and cell culture data provide strong evidence that FKBP12 is the most important PPIase modulating α-SYN aggregation and validate the protein as an interesting drug target for Parkinson disease.     

Peptidylprolylisomerases,Protein Folders,or Scaffolders? The Example of FKBP51 and FKBP52

Peptidylprolyl-isomerases (PPIases) comprise of the protein families of FK506 binding proteins (FKBPs), cyclophilins, and parvulins. Their common feature is their ability to expedite the transition of peptidylprolyl bonds between the cis and the trans conformation. Thus, it seemed highly plausible that PPIase enzymatic activity is crucial for protein folding. However, this has been difficult to prove over the decades since their discovery. In parallel, more and more studies have discovered scaffolding functions of PPIases. This essay discusses the hypothesis that PPIase enzymatic activity might be the consequence of binding to peptidylprolyl protein motifs. The main focus of this paper is the large immunophilins FKBP51 and FKBP52, but other PPIases such as cyclophilin A and Pin1 are also described. From the hypothesis, it follows that the PPIase activity of these proteins might be less relevant, if at all, than the organization of protein complexes through versatile protein binding. Also see the video abstract here https://youtu.be/A33la0dx5LE .