Three-dimensional solution structure of an archaeal FKBP with a dual function of peptidyl prolyl cis-trans isomerase and chaperone-like activities

Here we report the solution structure of an archaeal FK506-binding protein (FKBP) from a thermophilic archaeum, Methanococcus thermolithotrophicus (MtFKBP17), which has peptidyl prolyl cis-trans isomerase (PPIase) and chaperone-like activities, to reveal the structural basis for the dual function. In addition to a typical PPIase domain, a newly identified domain is formed in the flap loop by a 48-residue insert that is required for the chaperone-like activity. The new domain, called IF domain (the Insert in the Flap), is a novel-folding motif and exposes a hydrophobic surface, which we consider to play an important role in the chaperone-like activity.     

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.     

Prolyl cis-trans isomerization as a molecular timer

Proline is unique in the realm of amino acids in its ability to adopt completely distinct cis and trans conformations, which allows it to act as a backbone switch that is controlled by prolyl cis-trans isomerization. This intrinsically slow interconversion can be catalyzed by the evolutionarily conserved group of peptidyl prolyl cis-trans isomerase enzymes. These enzymes include cyclophilins and FK506-binding proteins, which are well known for their isomerization-independent role as cellular targets for immunosuppressive drugs. The significance of enzyme-catalyzed prolyl cis-trans isomerization as an important regulatory mechanism in human physiology and pathology was not recognized until the discovery of the phosphorylation-specific prolyl isomerase Pin1. Recent studies indicate that both phosphorylation-dependent and phosphorylation-independent prolyl cis-trans isomerization can act as a novel molecular timer to help control the amplitude and duration of a cellular process, and prolyl cis-trans isomerization might be a new target for therapeutic interventions.     

Crystal structure of Mip, a prolylisomerase from Legionella pneumophila

The human pathogen Legionella pneumophila, the etiological agent of the severe and often fatal Legionnaires' disease, produces a major virulence factor, termed 'macrophage infectivity potentiator protein' (Mip), that is necessary for optimal multiplication of the bacteria within human alveolar macrophages. Mip exhibits a peptidyl prolyl cis-trans isomerase (PPIase) activity, which appears to be important for infection. Here we report the 2.4 A crystal structure of the Mip protein from L. pneumophila Philadelphia 1 and the 3.2 A crystal structure of its complex with the drug FK506. Each monomer of the homodimeric protein consists of an N-terminal dimerization module, a long (65 A) connecting alpha-helix and a C-terminal PPIase domain exhibiting similarity to human FK506-binding protein. In view of the recent significant increase in the number of reported cases of Legionnaires' disease and other intracellular infections, these structural results are of prime interest for the design of new drugs directed against Mip proteins of intracellular pathogens.     

FK506 binding protein from a thermophilic archaeon, Methanococcus thermolithotrophicus, has chaperone-like activity in vitro

The in vitro protein folding activity of an FKBP (FK506 binding protein, abbreviated to MTFK) from a thermophilic archaeon, Methanococcus thermolithotrophicus, was investigated. MTFK exhibited FK506 sensitive PPIase (peptidyl prolyl cis-trans isomerase) activity which accelerated the speed of ribonuclease T1 refolding, which is rate-limited by isomerization of two prolyl peptide bonds. In addition, MTFK suppressed the aggregation of folding intermediates and elevated the final yield of rhodanese refolding. We called this activity of MTFK the chaperone activity. The chaperone activity of MTFK was also inhibited by FK506. Alignment of the amino acid sequences of MTFK with human FKBP12 showed that MTFK has two insertion sequences, consisting of 13 and 44 amino acids, at the N- and C-termini, respectively [Furutani, M., Iida, T., Yamano, S., Kamino, K., and Maruyama, T. (1998) J. Bacteriol. 180, 388-394]. To study the relationship between chaperone and PPIase activities of MTFK, mutant MTFKs with deletions of these insertion sequences or with amino acid substitutions were created. Their PPIase and chaperone activities were measured using a synthetic oligopeptide and denatured rhodanese as the substrates, respectively. The far-UV circular dichroism spectra of the wild type and the mutants were also analyzed. The results suggested that (1) the PPIase activity did not correlate with chaperone activity, (2) both insertion sequences were required for MTFK to take a proper conformation, and (3) the insertion sequence (44 amino acids) in the C-terminus was important for the chaperone activity.     

Chemical shift assignments of the catalytic domain from the yeast proline isomerase Fpr4p

Yeast Fpr4p belongs to the FK506-binding protein (FKBP) class of peptidyl proline isomerases (PPIases), and has been implicated in regulating the cis-trans conversion of proline residues within histone tails. Here we report the (1)H, (13)C and (15)N chemical shift assignments for the bacterially expressed C-terminal PPIase catalytic domain of Fpr4p. Prediction of secondary structure reveals similarity to domains from other members of the FKBP proline isomerases, including yeast Fpr1p and the prototypic PPIase region from human FKBP12.     

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.     

Molecular cloning and characterization of genes encoding FK506-binding proteins (FKBPs) in wheat (<Emphasis Type="Italic">Triticum aestivum</Emphasis> L.)

The family of FK506-binding proteins (FKBPs) consists of several members, which show peptidyl prolyl cis–trans isomerase (PPIase) activity. PPIases facilitate the conversion of peptidyl prolyl bonds from cis to trans conformation, a rate-limiting step in protein folding. In the present study, we carried out cloning of cDNAs encoding three different wheat FKBPs viz., TaFKBP20-1, TaFKBP16-1 and TaFKBP15-1. In silico analysis suggested their likely localization to nucleus, cytosol and endoplasmic reticulum, respectively. Biochemical analyses demonstrated that none of the three purified FKBP proteins possesses detectable PPIase activity. Several putative interacting partners of TaFKBP20-1, TaFKBP16-1 and TaFKBP15-1were identified using online software tools. The results of this study provide further evidence that PPIase activity in plant FKBPs is not conserved, and these proteins may be playing important roles in the cell through interaction with target proteins.     

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.     

The FKBP families of higher plants: Exploring the structures and functions of protein interaction specialists

The FK506-binding proteins (FKBPs) are known both as the receptors for immunosuppressant drugs and as prolyl isomerase (PPIase) enzymes that catalyse rotation of prolyl bonds. FKBPs are characterised by the inclusion of at least one FK506-binding domain (FKBd), the receptor site for proline and the active site for PPIase catalysis. The FKBPs form large and diverse families in most organisms, with the largest FKBP families occurring in higher plants. Plant FKBPs are molecular chaperones that interact with specific protein partners to regulate a diversity of cellular processes. Recent studies have found that plant FKBPs operate in intricate and coordinated mechanisms for regulating stress response and development processes, and discoveries of new interaction partners expand their cellular influences to gene expression and photosynthetic adaptations. This review presents an examination of the molecular and structural features and functional roles of the higher plant FKBP family within the context of these recent findings, and discusses the significance of domain conservation and variation for the development of a diverse, versatile and complex chaperone family.     

Biochemical and Genetic Characterization of an FK506-Sensitive Peptidyl Prolyl cis-trans Isomerase from a Thermophilic Archaeon, Methanococcus thermolithotrophicus

A peptidyl prolyl cis-trans isomerase (PPIase) was purified from a thermophilic methanogen, Methanococcus thermolithotrophicus. The PPIase activity was inhibited by FK506 but not by cyclosporine. The molecular mass of the purified enzyme was estimated to be 16 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 42 kDa by gel filtration. The enzyme was thermostable, with the half-lives of its activity at 90 and 100°C being 90 and 30 min, respectively. The catalytic efficiencies (k_cat/K_m) measured at 15°C for the peptidyl substrates, N-succinyl-Ala-Leu-Pro-Phe-p-nitroanilide and N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide, were 0.35 and 0.20 μM⁻¹ s⁻¹, respectively, in chymotrypsin-coupled assays. The purified enzyme was sensitive to FK506 and therefore was called MTFK (M. thermolithotrophicus FK506-binding protein). The MTFK gene (462 bp) was cloned from an M. thermolithotrophicus genomic library. The comparison of the amino acid sequence of MTFK with those of other FK506-binding PPIases revealed that MTFK has a 13-amino-acid insertion in the N-terminal region that is unique to thermophilic archaea. The relationship between the thermostable nature of MTFK and its structure is discussed.     

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.     

Inhibition and Substrate Specificity Properties of FKBP22 from a Psychrotrophic Bacterium, <Emphasis Type="Italic">Shewanella</Emphasis> sp. SIB1

SIB1 FKBP22 is a peptidyl prolyl cis–trans isomerase (PPIase) member from a psychrotrophic bacterium, Shewanella sp. SIB1, consisting of N- and C-domains responsible for dimerization and catalytic PPIase activity, respectively. This protein was assumed to be involved in cold adaptation of SIB1 cells through its dual activity of PPIase activity and chaperone like-function. Nevertheless, the catalytic inhibition by FK506 and its substrate specificity remain unknown. Besides, ability of SIB1 FKBP22 to inhibit phosphatase activity of calcinuerin is also interesting to be studied since it may reflect wider cellular functions of SIB1 FKBP22. In this study, we found that wild type (WT) SIB1 FKBP22 bound to FK506 with IC₅₀ of 77.55 nM. This value is comparable to that of monomeric mutants (NNC-FKBP22, C-domain+ and V37R/L41R mutants), yet significantly higher than that of active site mutant (R142A). In addition, WT SIB1 FKBP22 and monomeric variants were found to prefer hydrophobic residues preceding proline. Meanwhile, R142A mutant has wider preferences on bulkier hydrophobic residues due to increasing hydrophobicity and binding pocket space. Surprisingly, in the absence of FK506, SIB1 FKBP22 and its variants inhibited, with the exception of N-domain, calcineurin phosphatase activity, albeit low. The inhibition of SIB1 FKBP22 by FK506 is dramatically increased in the presence of FK506. Altogether, we proposed that local structure at substrate binding pocket of C-domain plays crucial role for the binding of FK506 and peptide substrate preferences. In addition, C-domain is essential for inhibition, while dimerization state is important for optimum inhibition through efficient binding to calcineurin.     

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.     

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.     

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.     

植物FKBP基因家族的结构及生物学功能

FK506结合蛋白(FK506 binding protein, FKBP)是一种在生物体中广泛存在、进化上高度保守的组成型蛋白质。除了作为免疫抑制剂FK506受体以外, FKBP还具有肽脯氨酰顺反异构酶(PPIase)的活性。FKBP在植物中是个大家族, 可作为分子伴侣与一些蛋白相互作用从而调控不同的生化过程。研究表明, 该家族基因在植物的响应胁迫和不同生长发育过程中都扮演着重要角色。最近许多新的FKBP互作蛋白的发现和鉴定表明, FKBP在调控基因表达和光合适应性方面具有广泛的生物学功能。文章对植物FKBP家族的结构特点、分类以及最新的功能研究进展进行了详细的综述。