Hsp90 functionally interacts with a broad array of client proteins, but in every case examined Hsp90 is accompanied by one or more cochaperones. One class of cochaperone contains a tetratricopeptide repeat domain that targets the cochaperone to the C-terminal region of Hsp90. Within this class are Hsp90-binding peptidylprolyl isomerases, most of which belong to the FK506-binding protein (FKBP) family. Despite the common association of FKBP cochaperones with Hsp90, it is now clear that the client protein influences, and is influenced by, the particular FKBP bound to Hsp90. Examples include Xap2 in aryl hydrocarbon receptor complexes and FKBP52 in steroid receptor complexes. In this chapter, we discuss the known functional roles played by FKBP cochaperones and, where possible, relate distinctive functions to structural differences between FKBP members.

Introduction

Immunophilins are a large, functionally diverse group of proteins that are defined by their ability to bind immunosuppressive ligands. The immunophilins minimally contain a peptidyl-prolyl cis-trans isomerase (PPIase; also termed rotamase) domain to which the immunosuppressive drugs bind. Early investigations into the PPIase enzymatic activity led to the belief that the immunosuppressive drugs elicited their effects by inhibiting the PPIase activity. However, some compounds binding the PPIase active site efficiently inhibit PPIase activity without inducing immunosuppression, so PPIase activity is not critical for immune responses. It is now known that effector domains on the immunosuppressive drugs project from the PPIase pocket. This allows the immunophilin-drug complex to bind tightly to and inhibit calcineurin or target of rapamycin, signal transduction proteins required for immune responses (see reference 1 for a detailed review on the mechanisms by which immunophilins and their ligands suppress immune responses).

Since the initial identification of the immunophilin proteins, multiple family members have been identified in all major branches of life. Some immunophilins are small proteins containing only a single PPIase domain while others are large multidomain proteins that contain one or more PPIase domains, as well as additional functional domains. The immunophilins are divided into two groups based on their ability to bind different immunosuppressive ligands: the FK506 binding proteins (FKBP), which also bind rapamycin, and the cyclosporin-A binding proteins or cyclophilins (CyP). The PPIase domains of FKBP and cyclophilins are structurally distinct and likely evolved independently. On the other hand, some members of either the FKBP or cyclophilin families contain a structurally similar tetratricopeptide repeat (TPR) domain that targets binding to heat shock protein 90 (Hsp90).

Hsp90 is an abundant molecular chaperone that interacts with a broad array of protein clients that regulate numerous important cellular pathways. Among the known Hsp90 clients are transcription factors (e.g., steroid hormone receptors, heat shock transcription factor 1, aryl hydrocarbon receptor), both serine/threonine and tyrosine kinases (e.g., Raf and Src-related kinases), and key regulatory enzymes (e.g., nitric oxide synthase and telomerase). A compilation of known Hsp90 clients maintained by Didier Picard at Univ. of Geneva can be accessed at: http://www.picard.ch/downloads/Hsp90interactors.pdf.

In concert with other chaperone proteins, Hsp90 facilitates client folding and proteolytic stability but can also promote client degradation. In the case of steroid receptors, Hsp90 and it's associated cochaperones also regulate receptor activity. Hsp90 binding to steroid receptors must be preceded by transient receptor interactions with Hsp40, Hsp70, and associated cochaperones. Hsp90, which is recruited as a dimer in the latter stages of complex assembly, binds directly to the receptor ligand binding domain and stabilizes a receptor conformation that is competent for hormone binding. Associated with Hsp90 in the functionally mature receptor complex are p23, a cochaperone that stabilizes Hsp90 binding to receptor, and any one of several TPR cochaperones, including the immunophilin/PPIases FKBP52 (also termed p59, Hsp56, p50, HBI, FKBP59, and FKBP4), FKBP51 (also termed p54, FKBP54, and FKBP5), and CyP40, or the protein phosphatase PP5. As discussed below, receptor activity can vary depending on the particular TPR cochaperone in mature receptor heterocomplexes.

The domain organization for several TPR cochaperones are compared in Figure 1. These cochaperones compete for a common binding site in the C-terminal region of Hsp90 that includes the highly conserved -MEEVD sequence that terminates Hsp90. Cocrystallographic structures have shown how an MEEVD pentapeptide associates with the TPR binding pocket.^2,3 Although the TPR domains for each of these cochaperones are structurally similar and interact in a similar manner with Hsp90, the client protein bound by Hsp90 can influence the rank order of cochaperone recruitment to Hsp90-client complexes (reviewed in ref. 4). For instance, PP5 and FKBP51 are preferred components in glucocorticoid receptor (GR) complexes, FKBP51 is preferred in progesterone receptor (PR) complexes, and CyP40 is relatively enhanced in estrogen receptor (ER) complexes.^5,6 On the other hand, another TPR-containing FKBP, the hepatitis B virus protein X associated protein 2 (Xap2; also termed AIP, ARA9, and FKBP37) shows little interaction with steroid receptors but is strongly associated with the aryl hydrocarbon receptor-Hsp90 complex.^7,8 The distinctive patterns of preference for cochaperone association in client complexes is one line of evidence that the cochaperones bound to Hsp90 can also interact with the Hsp90-bound client.

Figure 1

Domain organization of representative Hsp90-binding FKBPs. TPR-containing FKBPs from vertebrate, insect, and plant sources were selected for comparison of domain organizations. The proteins are human FKBP52 (accession number NP_002005), human FKBP51 (acc. (more...)

In addition to FKBP52, FKBP51, and XAP2, several other FKBP family members contain TPR domains that are known or likely to bind Hsp90. FKBP6 (also termed FKBP36) is structurally similar to Xap2 but is required for male fertility and homologous chromosome pairing in meiosis.⁹ Drosophila melanogaster express a TPR-containing immunophilin (DmFKBP59) that has high similarity to FKBP52/51 in vertebrates.^10,11 Plants have several FKBP genes that encode TPR domains; for example, in Arabidopsis thaliana there are 4 such genes: AtFKBP42, AtFKBP62, AtFKBP65 and AtFKBP72 (reviewed in refs. 12 and 13). Although prokaryotic and Archaeal genomes also contain FKBP family members,¹⁴ none of these genes encode a TPR domain.

Structure/Function Relationships of Steroid Receptor-Associated FKBPs

Three-dimensional crystal structures have been solved for full-length FKBP51 and for overlapping fragments of FKBP52. Both have a similar TPR domain composed of three tandem repeats of the degenerate 34-amino acid motif that characterizes TPR proteins.¹⁵ Each repeat adopts a helix-turn-helix conformation and adjacent units stack in parallel to form a saddle-shaped domain with a concave binding pocket for Hsp90 (Figs. 2A and 2B). In addition to the TPR domain, both FKBP51 and FKBP52 have two N-terminal domains, each of which is structurally similar to FKBP12. FK506-binding and PPIase activities reside in the most N-terminal domain (FK1), which has a pocket and active site residues similar to FKBP12. Due to several amino acid differences, the second domain (FK2) lacks drug binding and PPIase activity;¹⁶ other functions for FK2 have not been identified.

Figure 2

Three-dimensional structures of FKBP51 and FKBP52. This is a depiction of X-ray crystallographic structures for human FKBP51 (A; protein data bank number 1KT0) and a composite of two partial structures for human FKBP52 (B; protein data bank numbers 1Q1C (more...)

FKBP52 and FKBP51 have distinct functional influences on steroid receptors (see below), but the underlying structure/function relationships are not well understood. FKBP51 and FKBP52 share greater than 60% amino acid sequence similarity, and individual domains do not differ markedly between FKBP51 and FKBP52. The most striking difference in crystal structures relates to apparent domain:domain orientations. The FKBP52 structure shown in (Fig. 2B) is a composite model derived from merging the separate FK1-FK2 and FK2-TPR structures. The composite model suggests that the FKBP52 TPR domain is aligned in a more linear fashion with the FK domains rather than in the kinked conformation seen with FKBP51 (Fig. 2A). In fact, the static orientations shown in crystal structures are likely more dynamic in solution, but the different crystal orientations are perhaps telling. Amino acid side chains unique to FKBP51 form a salt bridge between FK2 and TPR that would stabilize the domain:domain interaction in FKBP51 relative to FKBP52, which lacks this salt bridge. The apparently more flexible structure of FKBP52 might accommodate interactions within the receptor heterocomplex that are constrained in FKBP51.

FKBP51 and FKBP52 also differ in the hinge region connecting FK1 and FK2 domains (FK loop). The FK loop of FKBP52 contains a -TEEED- sequence that has been identified as an in vitro substrate for casein kinase II; the corresponding sequence in FKBP51, -FED-, lacks the threonine phosphorylation site. Phosphorylation of FKBP52 is potentially important since the phospho-protein is reported to lose Hsp90 binding.¹⁷ Finally, there are a few amino acid differences in and around the respective FK1 PPIase pockets of FKBP52 and FKBP51. Although these FKBPs have similar PPIase activity measured against a model small peptide substrate,¹⁸ FKBP specificity for prolines in the context of a full-length protein might be distinctive. So, for instance, FKBP52 might recognize a receptor proline site that is poorly accessed by FKBP51.

Cellular and Physiological Functions of Hsp90-Associated FKBPs

Functional Interactions between FKBP52 and FKBP51

Xap2 and FKBP6

Apart from the highly characterized steroid hormone receptor-associated FKBPs, several other TPR-containing FKBPs are present in higher vertebrates. As mentioned in earlier sections of this chapter, Xap2 is a TPR-containing immunophilin that is found almost exclusively in AhR complexes. As the name implies, Xap2 also functionally interacts with the hepatitis B virus protein X.⁶⁰ In addition Xap2 is known to have functional interactions with peroxisome proliferator activated receptor α (PPARα),⁶¹ however these interactions have not been extensively characterized. AhR is a ligand-dependent transcription factor that mediates the physiological response to specific environmental contaminants termed polycyclic aromatic hydrocarbons, the most notorious of which is 2,3,7,8-tetrachlorodibenzo-p-dioxin. Similar to steroid receptors, AhR requires assembly with Hsp90 and p23 to achieve a mature ligand-binding conformation (reviewed in 62), although the AhR ligand binding domain is unrelated to steroid receptor ligand binding domains. AhR complexes also contain an FKBP component, but in this case it is Xap2 rather than FKBP52 or FKBP51.

As with FKBP51 and FKBP52, Xap2 has a C-terminal TPπ™domain that is known to facilitate binding to the MEEVD motif on Hsp90⁶³ (Fig. 1). In addition Xap2 contains one N-terminal FK domain that lacks drug binding and also likely lacks PPIase activity. Although the FK domain is not required for Hsp90 binding, it is required for an interaction with the AhR-Hsp90 complex that functionally influences receptor activity.^63,64 In a cell-free assembly system that lacks Xap2, AhR is capable of assembling with Hsp90 and binding ligand, and upon ligand binding AhR is capable of binding AhR response elements on DNA.⁸ Again, similar to FKBP52 or FKBP51 in steroid receptor complexes, Xap2 is not required for basal maturation of AhR activity, but in both yeast and mammalian systems, Xap2 can modulate AhR-mediated reporter gene expression.^7,8,63,65 By titrating the relative level of Xap2 protein in cells, AhR activity can be enhanced or decreased. For example, when Xap2 is expressed at a level 2- to 3-fold higher than normal, binding of p23 in the AhR-Hsp90 complex is reduced.⁶⁶ Displacement of p23 by high levels of Xap2 would destabilize binding of Hsp90 to AhR and reduce the proportion of AhR in functionally mature complexes. Conversely, there is also evidence that at elevated Xap2 levels AhR is protected from ubiquitination and proteosomal degradation which would increase total AhR levels.^67-71 Finally, several studies suggest that Xap2 facilitates nucleocytoplasmic shuttling of AhR following ligand binding.^71-75

The physiological relevance of Xap2 interactions with AhR complexes has not been examined in a whole animal model, but Xap2 could potentially influence any of several physiological and pathological pathways mediated by AhR. Mice that are homozygous for a disrupted AhR gene have many physiological and developmental defects; among these are immune system impairment, hepatic fibrosis, cardiac hypertrophy, impaired insulin regulation, and defects in ovarian and vascular development.^76-79 In addition, many of the toxic and teratogenic effects produced by AhR ligands require an intact AhR signaling pathway.^80,81 For example, dioxin induced defects in prostate development are absent in AhR knockout mice.⁸² Development of a mouse strain lacking Xap2 could be very helpful in determining the role Xap2 plays in these processes and might validate Xap2 as a potential target for therapeutic intervention.

In addition to Xap2, vertebrates contain FKBP36 (gene name FKBP6 in humans), another TPR-containing FKBP that is structurally similar to Xap2 yet functionally distinct. FKBP36 has a single N-terminal FK domain and a C-terminal TPR domain. In vitro studies show that FKBP36 binds Hsp90 and can assemble with steroid receptor complexes (unpublished observation), but there is currently no evidence that FKBP36 alters receptor activity. FKBP36 mRNA is broadly expressed in vertebrate tissues with an exceptionally high level observed in the testis; male FKBP6 knockout mice lack sperm and FKBP36 was shown to be a critical component in meiotic synaptonemal complexes.⁹ Patients with Williams syndrome, which is characterized by congenital cardiovascular defects, dysmorphic facial features, mental retardation, growth defects, azoospermia, and hypercalcemia, are typically haploinsufficient for FKBP6;⁸³ however, the contribution of FKBP6 deletion in this syndrome is not clear since several contiguous genes on chromosome 11, including genes for elastin and LIM-Kinase 1, are also deleted in these patients and clearly contribute to some phenotypic aspects.

Plant FKBPs

Hsp90-binding TPR immunophilins have been identified in all eukaryotes examined. A few examples of plant TPR-containing FKBPs are shown in Figure 1. The TPR domain of each FKBP is very similar in amino acid sequence to that of vertebrate proteins; these are presumed to bind Hsp90, but that has not been determined in all cases. The plant and insect FKBPs contain one or more PPIase-related domain and can contain other functional domains. For example, AtFKBP42 contains a C-terminal transmembrane domain that localizes the protein to the inner plasma membrane and the vacuolar membrane.^84-86

There is ample evidence to suggest that the plant and insect FKBPs are physiologically important. Mutations in AtFKBP42 cause the severe developmental phenotypes termed twisted dwarf 1 (TWD)⁸⁵ and ultracurvata (UCU2).⁸⁷ The mechanism by which these phenotypes occur likely involves impairment of membrane transport as AtFKBP42 is known to interact with several ATP-binding cassette transporters on the plasma and vacuolar membranes.^85,86,88 Mutations in AtFKBP72 result in a class of mutants termed pasticcino or pas mutants, which are characterized by a wide variety of developmental defects.⁸⁹ Two Hsp90-binding TPR FKBPs in wheat, wFKBP72 and the heat shock-inducible wFKBP77, have been shown in transgenic plants to distinctively influence developmental patterns.⁹⁰

Summary

In addressing the physiological importance of PPIases, Heitman and colleagues⁹¹ generated an S. cerevisiae strain that lacked all 12 PPIase genes in the FKBP and cyclophilin families; the pluri-mutant strain displayed some growth abnormalities but was viable, thus demonstrating that these genes collectively are nonessential in yeast. Nonetheless, it has become increasingly clear that the Hsp90-binding PPIases, through interactions with steroid receptors, kinases, and other cellular factors, play important physiological and potentially pathological roles in mammals. Elucidation of these roles, definition of underlying molecular mechanisms, and identification of specific inhibitors will likely quicken in the coming few years and lead to therapeutic targeting of individual PPIases.

Acknowledgements

Studies in the authors' laboratory were supported by NIH R01-DK48218 and the Mayo Foundation.

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