FK506-binding protein 52 phosphorylation: a potential mechanism for regulating steroid hormone receptor activity

Functional maturation of steroid hormone receptors requires ordered assembly into a large multichaperone complex consisting of receptor monomer, an Hsp90 dimer, the p23 cochaperone, and an FK506-binding protein (FKBP) family member or alternate peptidylprolyl isomerase-related cochaperone. Previous cellular studies demonstrated that FKBP52 can potentiate receptor function. These results have been confirmed in fkbp4 gene knockout mice in which males are partially androgen insensitive and females display characteristics of progesterone insensitivity. Conversely, FKBP51, which has a high degree of similarity to FKBP52, antagonizes FKBP52-mediated potentiation. Both proteins consist of three domains: two FKBP12-like domains termed FK1 and FK2 and a tetratricopeptide repeat domain that targets binding to Hsp90. To help understand why the two FKBPs behave differently and to gain insight into FKBP52 potentiation activity, we have analyzed the loop structure that links FK1 and FK2. Within the FK linker of FKBP52 is the sequence TEEED, which forms a consensus casein kinase II phosphorylation site; the corresponding sequence in FKBP51 is FED. We demonstrate that the distinct FK linker sequences per se do not account for lack of potentiation activity by FKBP51. However, phosphorylation of the FK linker appears to be an important regulatory determinant of FKBP52-mediated potentiation of steroid receptor activity.     

Crystal Structures of the Free and Ligand-Bound FK1–FK2 Domain Segment of FKBP52 Reveal a Flexible Inter-Domain Hinge

The human Hsp90 co-chaperone FKBP52 belongs to the family of FK506-binding proteins, which act as peptidyl-prolyl isomerases. FKBP52 specifically enhances the signaling of steroid hormone receptors, modulates ion channels and regulates neuronal outgrowth dynamics. In turn, small-molecule ligands of FKBP52 have been suggested as potential neurotrophic or anti-prostate cancer agents. The usefulness of available ligands is however limited by a lack of selectivity. The immunophilin FKBP52 is composed of three domains, an FK506-binding domain with peptidyl-prolyl isomerase activity, an FKBP-like domain of unknown function and a TPR-clamp domain, which recognizes the C-terminal peptide of Hsp90 with high affinity. The herein reported crystal structures of FKBP52 reveal that the short linker connecting the FK506-binding domain and the FKBP-like domain acts as a flexible hinge. This enhanced flexibility and its modulation by phosphorylation might explain some of the functional antagonism between the closely related homologs FKBP51 and FKBP52. We further present two co-crystal structures of FKBP52 in complex with the prototypic ligand FK506 and a synthetic analog thereof. These structures revealed the molecular interactions in great detail, which enabled in-depth comparison with the corresponding complexes of the other cytosolic FKBPs, FKBP51 and FKBP12. The observed subtle differences provide crucial insights for the rational design of ligands with improved selectivity for FKBP52.     

Molecular dynamics simulation,binding free energy calculation and unbinding pathway analysis on selectivity difference between FKBP51 and FKBP52: Insight into the molecular mechanism of isoform selectivity

As co‐chaperones of the 90‐kDa heat shock protein(HSP90), FK506 binding protein 51 (FKBP51) and FK506 binding protein 52 (FKBP52) modulate the maturation of steroid hormone receptor through their specific FK1 domains (FKBP12‐like domain 1). The inhibitors targeting FK1 domains are potential therapies for endocrine‐related physiological disorders. However, the structural conservation of the FK1 domains between FKBP51 and FKBP52 make it difficult to obtain satisfactory selectivity in FK506‐based drug design. Fortunately, a series of iFit ligands synthesized by Hausch et al exhibited excellent selectivity for FKBP51, providing new opportunity for design selective inhibitors. We performed molecular dynamics simulation, binding free energy calculation and unbinding pathway analysis to reveal selective mechanism for the inhibitor iFit4 binding with FKBP51 and FKBP52. The conformational stability evaluation of the “Phe67‐in” and “Phe67‐out” states implies that FKBP51 and FKBP52 have different preferences for “Phe67‐in” and “Phe67‐out” states, which we suggest as the determinant factor for the selectivity for FKBP51. The binding free energy calculations demonstrate that nonpolar interaction is favorable for the inhibitors binding, while the polar interaction and entropy contribution are adverse for the inhibitors binding. According to the results from binding free energy decomposition, the electrostatic difference of residue 85 causes the most significant thermodynamics effects on the binding of iFit4 to FKBP51 and FKBP52. Furthermore, the importance of substructure units on iFit4 were further evaluated by unbinding pathway analysis and residue‐residue contact analysis between iFit4 and the proteins. The results will provide new clues for the design of selective inhibitors for FKBP51.     

FKBP immunophilins and Alzheimer’s disease: A chaperoned affair

The FK506-binding protein (FKBP) family of immunophilins consists of proteins with a variety of protein–protein interaction domains and versatile cellular functions. Analysis of the functions of immunophilins has been the focus of studies in recent years and has led to the identification of various molecular pathways in which FKBPs play an active role. All FKBPs contain a domain with prolyl cis/trans isomerase (PPIase) activity. Binding of the immunosuppressant molecule FK506 to this domain inhibits their PPIase activity while mediating immune suppression through inhibition of calcineurin. The larger members, FKBP51 and FKBP52, interact with Hsp90 and exhibit chaperone activity that is shown to regulate steroid hormone signalling. From these studies it is clear that FKBP proteins are expressed ubiquitously but show relatively high levels of expression in the nervous system. Consistent with this expression, FKBPs have been implicated with both neuroprotection and neurodegeneration. This review will focus on recent studies involving FKBP immunophilins in Alzheimer’s-disease-related pathways.     

The Hsp90-binding peptidylprolyl isomerase FKBP52 potentiates glucocorticoid signaling in vivo

Hsp90 is required for the normal activity of steroid receptors, and in steroid receptor complexes it is typically bound to one of the immunophilin-related co-chaperones: the peptidylprolyl isomerases FKBP51, FKBP52 or CyP40, or the protein phosphatase PP5. The physiological roles of the immunophilins in regulating steroid receptor function have not been well defined, and so we examined in vivo the influences of immunophilins on hormone-dependent gene activation in the Saccharomyces cerevisiae model for glucocorticoid receptor (GR) function. FKBP52 selectively potentiates hormone-dependent reporter gene activation by as much as 20-fold at limiting hormone concentrations, and this potentiation is readily blocked by co-expression of the closely related FKBP51. The mechanism for potentiation is an increase in GR hormone-binding affinity that requires both the Hsp90-binding ability and the prolyl isomerase activity of FKBP52.     

FKBP51, a novel T-cell-specific immunophilin capable of calcineurin inhibition.

The immunosuppressive drugs FK506 and cyclosporin A block T-lymphocyte proliferation by inhibiting calcineurin, a critical signaling molecule for activation. Multiple intracellular receptors (immunophilins) for these drugs that specifically bind either FK506 and rapamycin (FK506-binding proteins [FKBPs]) or cyclosporin A (cyclophilins) have been identified. We report the cloning and characterization of a new 51-kDa member of the FKBP family from murine T cells. The novel immunophilin, FKBP51, is distinct from the previously isolated and sequenced 52-kDa murine FKBP, demonstrating 53% identity overall. Importantly, Western blot (immunoblot) analysis showed that unlike all other FKBPs characterized to date, FKBP51 expression was largely restricted to T cells. Drug binding to recombinant FKBP51 was demonstrated by inhibition of peptidyl prolyl isomerase activity. As judged from peptidyl prolyl isomerase activity, FKBP51 had a slightly higher affinity for rapamycin than for FK520, an FK506 analog. FKBP51, when complexed with FK520, was capable of inhibiting calcineurin phosphatase activity in an in vitro assay system. Inhibition of calcineurin phosphatase activity has been implicated both in the mechanism of immunosuppression and in the observed toxic side effects of FK506 in nonlymphoid cells. Identification of a new FKBP that can mediate calcineurin inhibition and is restricted in its expression to T cells suggests that new immunosuppressive drugs may be identified that, by virtue of their specific interaction with FKBP51, would be targeted in their site of action.     

FKBP51 employs both scaffold and isomerase functions to promote NF-κB activation in melanoma

Melanoma is the most aggressive skin cancer; its prognosis, particularly in advanced stages, is disappointing largely due to the resistance to conventional anticancer treatments and high metastatic potential. NF-κB constitutive activation is a major factor for the apoptosis resistance of melanoma. Several studies suggest a role for the immunophilin FKBP51 in NF-κB activation, but the underlying mechanism is still unknown. In the present study, we demonstrate that FKBP51 physically interacts with IKK subunits, and facilitates IKK complex assembly. FKBP51-knockdown inhibits the binding of IKKγ to the IKK catalytic subunits, IKK-α and -β, and attenuates the IKK catalytic activity. Using FK506, an inhibitor of the FKBP51 isomerase activity, we found that the IKK-regulatory role of FKBP51 involves both its scaffold function and its isomerase activity. Moreover, FKBP51 also interacts with TRAF2, an upstream mediator of IKK activation. Interestingly, both FKBP51 TPR and PPIase domains are required for its interaction with TRAF2 and IKKγ, whereas only the TPR domain is involved in interactions with IKKα and β. Collectively, these results suggest that FKBP51 promotes NF-κB activation by serving as an IKK scaffold as well as an isomerase. Our findings have profound implications for designing novel melanoma therapies based on modulation of FKBP51.     

Differential control of glucocorticoid receptor hormone-binding function by tetratricopeptide repeat (TPR) proteins and the immunosuppressive ligand FK506

Many laboratories have documented the existence of tetratricopeptide repeat (TPR) proteins (also known as immunophilins) in hormone-free steroid receptor complexes. Yet, the distinct roles of these proteins in steroid receptor action are poorly understood. In this work, we have investigated the effects of four TPR proteins (FKBP52, FKBP51, Cyp40, and PP5) on hormone-binding function of glucocorticoid receptor (GR) endogenously expressed in mammalian L929 cells. As a first step, we treated L929 cells with select immunophilin ligands [FK506, rapamycin, cyclosporin A (CsA), and cyclosporin H (CsH)], which are commonly thought to increase the GR response to hormone by inhibiting membrane-based steroid exporters. As expected, all four immunophilin ligands increased both the intracellular concentration of dexamethasone and GR activity at the MMTV-CAT reporter. To determine whether these ligands could target GR function independent of steroid export mechanisms, we performed GR reporter gene assays under conditions of immunophilin ligand and dexamethasone treatment that yielded equal intracellular hormone concentrations. FK506 was found to stimulate GR transactivity beyond the effect of this ligand on hormone retention. In contrast, CsA only affected the GR through upregulation of hormone retention. By Scatchard analysis, FK506 was found to increase GR hormone-binding affinity while decreasing total binding sites for hormone. This result correlated with loss of GR-associated FKBP51 and replacement with PP5. Interestingly, no GR-associated Cyp40 was found in these cells, consistent with the ability of CsA ligand to only affect GR through the hormone export mechanism. To test the role of FKBP52 independent of FK506, FKBP52 was placed under the control of a tetracycline-inducible promoter. Upregulation of FKBP52 caused an increase in both GR hormone-binding affinity and transactivity, even in the absence of FK506. These results show that immunosuppressive ligands can alter GR hormone-binding function by changing the TPR protein composition of receptor complexes and that TPR proteins exert a hierarchical effect on this GR function in the following order: FKBP52 > PP5 > FKBP51.     

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.     

Cyclophilin 40: An Hsp90-cochaperone associated with apo-steroid receptors

Cyclophilin 40, a divergent loop cyclophilin first identified in association with the estrogen receptor α, contains a C-terminal tetratricopeptide repeat domain through which it shares structural identity with FK506-binding protein 52 (FKBP52) and other partner cochaperones in steroid receptor-heat shock protein 90 (Hsp90) complexes. By dynamically competing for Hsp90 interaction, the cochaperones allow the receptors to establish distinct Hsp90-chaperone complexes, with the potential to exert tissue-specific control over receptor activity. Cyclophilin 40 regulates Hsp90 ATPase activity during receptor-Hsp90 assembly. Functional deletion of the cyclophilin 40 yeast homologue, Cpr7, adversely affected estrogen receptor α and glucocorticoid receptor activity that could be fully restored, either with wild type Cpr7 or Cpr7 with a cyclophilin domain lacking isomerase activity. We draw parallels with the mechanism already established for FKBP52 and propose that the cyclophilin 40 divergent loop interfaces with a contact surface on the steroid receptor ligand-binding domain to achieve an optimal orientation for receptor activity.     

Molecular cloning of human FKBP51 and comparisons of immunophilin interactions with Hsp90 and progesterone receptor.

A cDNA for human FKBP51 has been cloned and sequenced, and protein products have been expressed in both in vitro and bacterial systems. The deduced amino acid sequence for human FKBP51 is 90% identical to sequences of recently described murine proteins and is 55% identical to the sequence of human FKBP52. Human FKBP51 mRNA is expressed in a wide range of tissues, and the protein has peptidylprolyl isomerase activity that is inhibited by FK506 but not cyclosporine. FKBP51 is the same as a previously described progesterone receptor-associated immunophilin that, similar to FKBP52 and cyclophilin 40, is an Hsp90-binding protein and appears in functionally mature steroid receptor complexes along with Hsp90 and p23. Each of the three receptor-associated immunophilins displays interactions with progesterone receptor that are more dynamic than Hsp90-receptor interactions. Whereas FKBP52 and FKBP51 compete about equally well for binding to Hsp90 in a purified system, FKBP51 accumulates preferentially in progesterone receptor complexes assembled in a cell-free system. This observation provides a precedent for differential interactions between Hsp90-associated immunophilins and target proteins such as steroid receptors.     

Different regions of the immunophilin FKBP52 determine its association with the glucocorticoid receptor, hsp90, and cytoplasmic dynein

FKBP52 is a high molecular mass immunophilin possessing peptidylprolyl isomerase (PPIase) activity that is inhibited by the immunosuppressant drug FK506. FKBP52 is a component of steroid receptor.hsp90 heterocomplexes, and it binds to hsp90 via a region containing three tetratricopeptide repeats (TPRs). Here we demonstrate by cross-linking of the purified proteins that there is one binding site for FKBP52/dimer of hsp90. This accounts for the common heterotetrameric structure of native receptor heterocomplexes being 1 molecule of receptor, 2 molecules of hsp90, and 1 molecule of a TPR domain protein. Immunoadsorption of FKBP52 from reticulocyte lysate also yields co-immunoadsorption of cytoplasmic dynein, and we show that co-immunoadsorption of dynein is competed by a fragment of FKBP52 containing its PPIase domain, but not by a TPR domain fragment that blocks FKBP52 binding to hsp90. Using purified proteins, we also show that FKBP52 binds directly to the hsp90-free glucocorticoid receptor. Because neither the PPIase fragment nor the TPR fragment affects the binding of FKBP52 to the glucocorticoid receptor under conditions in which they block FKBP52 binding to dynein or hsp90, respectively, different regions of FKBP52 must determine its association with these three proteins.     

A novel role for the immunophilin FKBP52 in copper transport

FK506-binding protein 52 (FKBP52) is an immunophilin that possesses peptidylprolyl cis/trans-isomerase (PPIase) activity and is a component of a subclass of steroid hormone receptor complexes. Several recent studies indicate that immunophilins can regulate neuronal survival and nerve regeneration although the molecular mechanisms are poorly understood. To investigate the function of FKBP52 in the nervous system, we employed a yeast two-hybrid strategy using the PPIase domain (domain I) as bait to screen a neonatal rat dorsal root ganglia cDNA expression library. We identified an interaction between FKBP52 domain I and Atox1, a copper-binding metallochaperone. Atox1 interacts with Menkes disease protein and Wilson disease protein (WD) and functions in copper efflux. The interaction between FKBP52 and Atox1 was observed in both glutathione S-transferase pull-down experiments and when proteins were ectopically expressed in human embryonic kidney (HEK) 293T cells and was sensitive to FK506. Interestingly, the FKBP52/Atox1 interaction was enhanced when HEK 293T cells were cultured in copper-supplemented medium and decreased in the presence of the copper chelator, bathocuproine disulfate, suggesting that the interaction is regulated in part by intracellular copper. Overexpression of FKBP52 increased rapid copper efflux in (64)Cu-loaded cells, as did the overexpression of WD transporter. Taken together, our present findings suggest that FKBP52 is a component of the copper efflux machinery, and in so, may also promote neuroprotection from copper toxicity.     

Characterization of Arabidopsis thaliana AtFKBP42 that is membrane-bound and interacts with Hsp90

The twisted dwarf1 (twd1) mutant from Arabidopsis thaliana was identified in a screen for plant architecture mutants. The TWD1 gene encodes a 42 kDa FK506-binding protein (AtFKBP42) that possesses similarity to multidomain PPIases such as mammalian FKBP51 and FKBP52, which are known to be components of mammalian steroid hormone receptor complexes. We report here for the first time the stoichiometry and dissociation constant of a protein complex from Arabidopsis that consists of AtHsp90 and AtFKBP42. Recombinant AtFKBP42 prevents aggregation of citrate synthase in almost equimolar concentrations, and can be cross-linked to calmodulin. In comparison to one active and one inactive FKBP domain in FKBP52, AtFKBP42 lacks the PPIase active FKBP domain. While FKBP52 is found in the cytosol and translocates to the nucleus, AtFKBP42 was predicted to be membrane-localized, as shown by electron microscopy.     

Extended Impact of Pin1 Catalytic Loop Phosphorylation Revealed by S71E Phosphomimetic

Pin1 is a two-domain human protein that catalyzes the cis–trans isomerization of phospho-Ser/Thr–Pro (pS/T–P) motifs in numerous cell-cycle regulatory proteins. These pS/T–P motifs bind to Pin1's peptidyl-prolyl isomerase (PPIase) domain in a catalytic pocket, between an extended catalytic loop and the PPIase domain core. Previous studies showed that post-translational phosphorylation of S71 in the catalytic loop decreases substrate binding affinity and isomerase activity. To define the origins for these effects, we investigated a phosphomimetic Pin1 mutant, S71E-Pin1, using solution NMR. We find that S71E perturbs not only its host loop but also the nearby PPIase core. The perturbations identify a local network of hydrogen bonds and salt bridges that is more extended than previously thought, and includes interactions between the catalytic loop and the α2/α3 turn in the PPIase core. Explicit-solvent molecular dynamics simulations and phylogenetic analysis suggest that these interactions act as conserved “latches” between the loop and PPIase core that enhance binding of phosphorylated substrates, as they are absent in PPIases lacking pS/T–P specificity. Our results suggest that S71 is a hub residue within an electrostatic network primed for phosphorylation, and may illustrate a common mechanism of phosphorylation-mediated allostery.     

FKBP52

The large molecular-weight immunophilin, FKBP52, is a known target of the immunosuppressive drug FK506. FKBP52 exhibits peptidyl-prolyl cis-trans isomerase (PPIase) activity, which is inhibited by the binding of FK506--properties that it shares with the smaller but better-studied immunophilin, FKBP12. Unlike FKBP12, however, FKBP52 does not mediate the immunosuppressive actions of FK506 and, due to its larger size, contains additional numerous functional domains. One such structure is a series of tetratricopeptide repeat (TPR) domains, which serve as binding sites for the ubiquitous and abundant molecular chaperone, Hsp90. It is this property as a TPR protein that best characterizes the known cellular roles of FKBP52. Here, we review the structural features of FKBP52 and relate them to the evolving and diverse functions of this protein. Although the most recognized role of FKBP52 is in regulation of steroid receptor signaling, other less well-known functions are also discussed.