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.     

NMR solution structure and dynamics of the peptidyl-prolyl cis-trans isomerase domain of the trigger factor from Mycoplasma genitalium compared to FK506-binding protein

We have solved the solution structure of the peptidyl-prolyl cis-trans isomerase (PPIase) domain of the trigger factor from Mycoplasma genitalium by homo- and heteronuclear NMR spectroscopy. Our results lead to a well-defined structure with a backbone rmsd of 0.23 A. As predicted, the PPIase domain of the trigger factor adopts the FK506 binding protein (FKBP) fold. Furthermore, our NMR relaxation data indicate that the dynamic behavior of the trigger factor PPIase domain and of FKBP are similar. Structural variations when compared to FKBP exist in the flap region and within the bulges of strand 5 of the beta sheet. Although the active-site crevice is similar to that of FKBP, subtle steric variations in this region can explain why FK506 does not bind to the trigger factor. Sequence variability (27% identity) between trigger factor and FKBP results in significant differences in surface charge distribution and the absence of the first strand of the central beta sheet. Our data indicate, however, that this strand may be partially structured as "nascent" beta strand. This makes the trigger factor PPIase domain the most minimal representative of the FKBP like protein family of PPIases.     

The cytosolic-binding protein for the immunosuppressant FK-506 is both a ubiquitous and highly conserved peptidyl-prolyl cis-trans isomerase.

We have recently isolated an abundant cytosolic protein from human T-cells which specifically binds the immunosuppressive agent, FK-506. The FK-506-binding protein (FKBP) is a member of a novel class of proteins possessing peptidyl-prolyl cis-trans isomerase activity. These proteins are believed to play an important role in accelerating the rate at which proteins fold into their native conformations. In the present study, we demonstrate that FKBP is not a lymphoid-specific protein, but is widely distributed and phylogenically conserved. FKBP, purified from three sources (a human T-lymphocyte cell line JURKAT, bovine calf thymus, and Saccharomyces cerevisiae) exhibit identical molecular weights, immunological cross-reactivities, and a high degree of NH2-terminal amino acid sequence homology. In addition, FKBP from all sources possesses peptidyl-prolyl cis-trans isomerase activity which can be specifically inhibited by FK-506. We conclude that FKBP may serve an important biological function in all eukaryotic cells.     

The CYP2 gene of Saccharomyces cerevisiae encodes a cyclosporin A-sensitive peptidyl-prolyl cis-trans isomerase with an N-terminal signal sequence.

Cells of Saccharomyces cerevisiae contain a major cytosolic cyclophilin (Cyp)-related peptidyl-prolyl cis-trans isomerase (PPIase) which is the target for cyclosporin A (CsA) cytotoxicity and which is encoded by the CYP1 gene [Haendler et al., Gene 83 (1989) 39-46]. We recently identified a second Cyp-related gene in yeast, CYP2 [Koser et al., Nucleic Acids Res. 18 (1990) 1643] which predicts a protein with a hydrophobic leader sequence. A sequence lacking 33 codons from the 5'-end of the CYP2 open reading frame was generated by the polymerase chain reaction and engineered for expression in Escherichia coli. The corresponding recombinant truncated protein was purified and found to exhibit PPIase activity which was inhibited by CsA. The CYP2 gene is genetically unlinked to CYP1. As with CYP1, genomic disruption of CYP2 had no effect on haploid cell viability. Disruption of all three of the known yeast PPIase-encoding genes [CYP1, CYP2, and RBP1 for rapamycin-binding protein; Koltin et al., Mol. Cell. Biol. 11 (1991) 1718-1723] in the same haploid cell also resulted in no apparent cellular phenotype, suggesting either that none of these enzymes have an essential function or that additional PPIases can compensate for their specific absence. Whereas cells containing a genomic disruption of CYP1 exhibited a CsA-resistant phenotype, genomic disruption of CYP2 had no effect on CsA sensitivity. This suggests that the CYP1 gene product is the primary cellular target for CsA toxicity in yeast. Since both purified Cyps display CsA sensitivity in vitro, our data suggest that Cyp1 and Cyp2 differ in terms of their cellular function and/or localization.     

Chloroplast cyclophilin is a target protein of thioredoxin. Thiol modulation of the peptidyl-prolyl cis-trans isomerase activity

Chloroplast cyclophilin has been identified as a potential candidate of enzymes in chloroplasts that are regulated by thioredoxin (Motohashi, K., Kondoh, A., Stumpp, M. T., and Hisabori, T. (2001) Proc. Natl. Acad. Sci. U. S. A. 98, 11224-11229). In the present study we found that the peptidyl-prolyl cis-trans isomerase activity of cyclophilin is fully inactivated in the oxidized form. Reduction of cyclophilin by thioredoxin-m recovered the isomerase activity. Two crucial disulfide bonds were determined by disulfide-linked peptide mapping. The relevance of these cysteines for isomerase activity was confirmed by the mutagenesis studies. Because four cysteine residues in Arabidopsis thaliana cyclophilin were conserved in the isoforms from several organisms, it appears that this redox regulation must be one of the common regulation systems of cyclophilin.     

PPIase catalysis by human FK506-binding protein proceeds through a conformational twist mechanism.

FK506-binding protein (FKBP) catalyzes the cis-trans isomerization of the peptidyl-prolyl amide bond (the PPIase reaction) and is the major intracellular receptor for the immunosuppressive drugs FK506 and rapamycin. One mechanism proposed for catalysis of the PPIase reaction requires attack of an enzyme nucleophile on the carbonyl carbon of the isomerized peptide bond. An alternative mechanism requires conformational distortion of the peptide bond with or without assistance by an enzyme hydrogen bond donor. We have determined the kinetic parameters of the human FKBP-catalyzed PPIase reaction. At 5 degrees C, the isomerization of Suc-Ala-Leu-Pro-Phe-pNA proceeds in 2.5% trifluorethanol with kcat = 600 s-1, Km = 0.5 mM and kcat/Km = 1.2 x 10(6) M-1s-1. The kcat/Km shows little pH dependence between 5 and 10. A normal secondary deuterium isotope effect is observed on both kcat and kcat/Km. To investigate dependence on enzyme nucleophiles and proton donors, we have replaced eight potential catalytic residues with alanine by site-directed mutagenesis. Each FKBP variant efficiently catalyzes the PPIase reaction. Taken together, these data support an unassisted conformational twist mechanism with rate enhancement due in part to desolvation of the peptide bond at the active site. Fluorescence quenching of the buried tryptophan 59 residue by peptide substrate suggests that isomerization occurs in a hydrophobic environment.     

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.     

Secretion by Trypanosoma cruzi of a peptidyl-prolyl cis-trans isomerase involved in cell infection.

Macrophage infectivity potentiators are membrane proteins described as virulence factors in bacterial intracellular parasites, such as Legionella and Chlamydia. These factors share amino acid homology to eukaryotic peptidyl-prolyl cis-trans isomerases that are inhibited by FK506, an inhibitor of signal transduction in mammalian cells with potent immunosuppressor activity. We report here the characterization of a protein released into the culture medium by the infective stage of the protozoan intracellular parasite Trypanosoma cruzi. The protein possesses a peptidyl-prolyl cis-trans isomerase activity that is inhibited by FK506 and its non-immunosuppressing derivative L-685,818. The corresponding gene presents sequence homology with bacterial macrophage infectivity potentiators. The addition of the protein, produced heterologously in Escherichia coli, to cultures of trypomastigotes and simian epithelial or HeLa cells enhances invasion of the mammalian cells by the parasites. Antibodies raised in mice against the T.cruzi isomerase greatly reduce infectivity. A similar reduction of infectivity is obtained by addition to the cultures of FK506 and L-685,818. We concluded that the T.cruzi isomerase is involved in cell invasion.     

Requirements for peptidyl-prolyl isomerization activity: a comprehensive mutational analysis of the substrate-binding cavity of FK506-binding protein 12

Peptidyl-prolyl isomerase (PPIase) activity is exhibited by many proteins belonging to the PPIase family. However, the catalytic mechanism of this activity remains to be completely elucidated. Here, we selected human FK506-binding protein 12 (FKBP12) as the model PPIase and investigated the nature of amino acid residues essential for the activity. The crystal structures of several complexes of PPIase with short peptides revealed that the residues Asp37, Arg42, Phe46, Val55, Trp59, and Tyr82 in the substrate-binding cavity of FKBP12 appear to play key roles in the PPIase activity. Each of these six residues was substituted by 20 common amino acid residues. The activity of each mutant protein was measured using a peptide analog by the chymotrypsin digestion assay and then compared with wild-type FKBP12. It was found that site-specific interactions by the side chains of amino acid residues constituting the substrate-binding cavity were not essential for the PPIase activity, although the 37th, 55th, and 82nd amino acid residues significantly contributed to the activity. This suggests that the PPIase activity requires only the hydrophobic cavity that captures the Pro-containing peptide.     

Rapamycin inhibits didemnin B-induced apoptosis in human HL-60 cells: evidence for the possible involvement of FK506-binding protein 25.

In the present paper we show that the immunosuppressant rapamycin inhibits the induction of apoptosis by didemnin B in human promyeloid HL-60 cells. The mechanism of this inhibition is investigated using FK506, which competes with rapamycin for binding to their common target FK506-binding protein (FKBP)12. The lack of competition for rapamycin-mediated inhibition of didemnin B-induced apoptosis by FK506 suggests that rapamycin inhibits apoptosis through some mechanism other than inhibition of p70 S6 kinase activation. The lack of inhibition of didemnin B-induced apoptosis by inhibitors of phosphatidylinositol 3-kinase and mitogen-activated protein (MAP) kinase kinase further supports the conclusion that rapamycin does not inhibit didemnin B-induced apoptosis through inhibition of the MAP kinase pathway. Furthermore, didemnin B-induced apoptosis is not inhibited by the inhibitors of cyclin-dependent kinase, roscovitine and olomoucine. This indicates that rapamycin does not act through inhibition of cyclin-dependent kinases. Together with the lack of competition for the effect of rapamycin by FK506, our data suggest the possible involvement of the FK506-binding protein, FKBP25, which is localized in the nucleus. This interpretation of our data gains support from the fact that didemnin B does not induce apoptosis in enucleated HL-60 cells, which supports the possible involvement of FKBP25 in the inhibition of apoptosis by rapamycin.     

The immunosuppressant FK506 inhibits amino acid import in Saccharomyces cerevisiae.

The immunosuppressants cyclosporin A, FK506, and rapamycin inhibit growth of unicellular eukaryotic microorganisms and also block activation of T lymphocytes from multicellular eukaryotes. In vitro, these compounds bind and inhibit two different types of peptidyl-prolyl cis-trans isomerases. Cyclosporin A binds cyclophilins, whereas FK506 and rapamycin bind FK506-binding proteins (FKBPs). Cyclophilins and FKBPs are ubiquitous, abundant, and targeted to multiple cellular compartments, and they may fold proteins in vivo. Previously, a 12-kDa cytoplasmic FKBP was shown to be only one of at least two FK506-sensitive targets in the yeast Saccharomyces cerevisiae. We find that a second FK506-sensitive target is required for amino acid import. Amino acid-auxotrophic yeast strains (trp1 his4 leu2) are FK506 sensitive, whereas prototrophic strains (TRP1 his4 leu2, trp1 HIS4 leu2, and trp1 his4 LEU2) are FK506 resistant. Amino acids added exogenously to the growth medium mitigate FK506 toxicity. FK506 induces GCN4 expression, which is normally induced by amino acid starvation. FK506 inhibits transport of tryptophan, histidine, and leucine into yeast cells. Lastly, several genes encoding proteins involved in amino acid import or biosynthesis confer FK506 resistance. These findings demonstrate that FK506 inhibits amino acid import in yeast cells, most likely by inhibiting amino acid transporters. Amino acid transporters are integral membrane proteins which import extracellular amino acids and constitute a protein family sharing 30 to 35% identity, including eight invariant prolines. Thus, the second FK506-sensitive target in yeast cells may be a proline isomerase that plays a role in folding amino acid transporters during transit through the secretory pathway.     

Rectification of skeletal muscle ryanodine receptor mediated by FK506 binding protein.

The cytosolic receptor for immunosuppressant drugs, FK506 binding protein (FKBP12), maintains a tight association with ryanodine receptors of sarcoplasmic reticulum (SR) membrane in skeletal muscle. The interaction between FKBP12 and ryanodine receptors resulted in distinct rectification of the Ca release channel. The endogenous FKBP-bound Ca release channel conducted current unidirectionally from SR lumen to myoplasm; in the opposite direction, the channel deactivated with fast kinetics. The binding of FKBP12 is likely to alter subunit interactions within the ryanodine receptor complex, as revealed by changes in conductance states of the channel. Both on- and off-rates of FKBP12 binding to the ryanodine receptor showed clear dependence on the membrane potential, suggesting that the binding sites of FKBP12 reside in or near the conduction pore of the Ca release channel. Rectification of the Ca release channel would prevent counter-current flow during the rapid release of Ca from SR membrane, and thus may serve as a negative feedback mechanism that participates in the process of muscle excitation-contraction coupling.     

The rough endoplasmic reticulum-resident FK506-binding protein FKBP65 is a molecular chaperone that interacts with collagens

The rough endoplasmic reticulum-resident FK-506-binding protein FKBP65 can be isolated from chick embryos on a gelatin-Sepharose column, indicating some involvement in the biosynthesis of procollagens. The peptidylprolyl cis-trans-isomerase activity of FKBP65 was previously shown to have only marginal effects on the rate of triple helix formation (Zeng, B., MacDonald, J. R., Bann, J. G., Beck, K., Gambee, J. E., Boswell, B. A., and B?chinger, H. P. (1998) Biochem. J. 330, 109-114). Here we show that FKBP65 is a monomer in solution and acts as a chaperone molecule when tested with two classic chaperone assays: FKBP65 inhibits the thermal aggregation of citrate synthase and is active in the denatured rhodanese refolding and aggregation assay. The chaperone activity is comparable to that of protein-disulfide isomerase, a well characterized chaperone. FKBP65 delays the in vitro fibril formation of type I collagen, indicating that FKBP65 is also able to interact with triple helical collagen, and acts as a collagen chaperone.     

Glucose-induced cAMP signaling in Saccharomyces cerevisiae is mediated by the CDC25 protein

Functional mapping of the cell cycle START gene CDC25 has revealed two domains which are dispensable for viability (germination and growth in glucose media), but are essential for sporulation and differentially involved in glucose-induced cAMP signaling. The transient rise of cAMP is completely prevented by various deletions within the amino-terminal half (alpha domain) of the CDC25 gene product. In contrast, the deletion of the carboxy-terminal 38 residues (beta 2 domain) results in a rapid, but persisting, rise of cAMP. Our data suggest that the alpha domain of the CDC25 protein is involved in glucose signal transduction, whereas the beta 2 domain is required for downregulating the cAMP control chain.