Cyclophilin 40 (CyP-40), Mapping of Its hsp90 Binding Domain and Evidence That FKBP52 Competes with CyP-40 for hsp90 Binding

Abstract

The structurally related immunophilins cyclophilin 40 (CyP-40) and FKBP52 have been identified as components of the unactivated estrogen receptor. Both immunophilins have a similar molecular architecture that includes a C-terminal segment with a tetratricopeptide repeat (TPR) domain predicted to mediate protein interaction. hsp90 is a common cellular target for CyP-40 and FKBP52. Deletion mutants of CyP-40 fused to glutathione S-transferase were immobilized on glutathione-agarose and then used in a rapid hsp90 retention assay to define regions of the CyP-40 C terminus that are important for hsp90 binding. Our evidence suggests that the TPR domain is not sufficient for stable association of CyP-40 with hsp90 and requires the participation of flanking acidic and basic residues clustered at the Nand C-terminal ends, respectively. Both microdomains are characterized by ␣-helical structures with segregated hydrophobic and charged residues. Corresponding regions were identified in FKBP52. By preincubating myometrial cytosol with lysates containing bacterially expressed FKBP52, we have shown that FKBP52 competes with CyP-40 for hsp90 binding. Our results raise the possibility of a mutually exclusive association of CyP-40 and FKBP52 with hsp90. This would lead to separate immunophilin-hsp90-receptor complexes and place the estrogen receptor under the control of distinct immunophilin signaling pathways.The immunophilin components of the unactivated estrogen receptor, cyclophilin 40 (CyP-40), 1 and FKBP52, share significant sequence homology in their C-terminal regions (1) and represent separate classes of peptidylprolyl cis-trans-isomerases with binding specificities for the immunosuppressants cyclosporin A and FK506, respectively (2). These immunophilins display a similar structural organization of their functional domains characterized by an N-terminal region with overlapping isomerase and ligand binding domains and a conserved C-terminal segment that incorporates a 3-unit tetratricopeptide repeat (TPR) domain terminated by a potential site for calmodulin binding (1). We have previously speculated that the TPR domain may mediate the protein interaction properties of . This is consistent with evidence that similar repeat units in members of the TPR gene family are involved in functional association with target proteins (3).FKBP52 binds hsp90 within steroid receptor complexes (4) and also exists in association with hsp90 in the absence of receptor (5, 6). The interaction of FKBP52 with hsp90 has been studied extensively (7), and there is recent evidence that the TPR domain, localized in the C-terminal region of FKBP52, is fundamentally important for hsp90 binding (8). The structural similarity between CyP-40 and FKBP52 has led several groups to propose that the immunophilins may have a similar or perhaps competing role in cellular function (8 -10). In this regard, a recent report describes the association of human CyP-40 with hsp90 and provides evidence that the C-terminal, FKBP52-like domain determines this interaction ...

An 11.8 kDa proteolytic fragment of the E. coli trigger factor represents the domain carrying the peptidyl‐prolyl cis/trans isomerase activity

“…There are some larger, multidomain FKBPs and also cyclophilins found as constituents in high molecular mass steroid receptor complexes [31,32]. The FKBP52 (also known as HSP56, p59, FKBP59 or HBI) (reviewed in [33]) contains three domains which are homologous to the FKBP12 [34].…”

A novel chaperone-activity-reducing mechanism of the 90-kDa molecular chaperone HSP90

“…The TPR-containing domain of FKBP52, Cyp40, and protein phosphatase pp5 mediate the binding of these proteins to HSP90 [14][15][16]. Only a specific subset of TPR-containing proteins, with closely related TPR motifs, can bind HSP90.…”

Heat shock cognate protein 70 chaperone-binding site in the co-chaperone murine stress-inducible protein 1 maps to within three consecutive tetratricopeptide repeat motifs

“…Unlike PP5, Hip and the immunophilins [12][13][14]19,20], extensive regions flanking the mSTI1 TPR domains are not imperative for mSTI1-hsp90 and mSTI1-hsc70 interactions. These results might support the classification of TPR proteins involved in chaperone-cochaperone interactions into two different groups ( Figure 5) encompassing those that do not require extensive TPR flanking regions (less than 20 residues) and those that require more extensive flanking regions (more than 20 residues).…”

“…The immunophilins CyP-40, FKBP51 and FKBP52 contain three TPR motifs that mediate their interactions with hsp90 [10][11][12]. Acidic and basic α-helical regions flanking the respective N-and C-terminal ends of the immunophilin TPR domains are Abbreviations used : GST, glutathione S-transferase ; Hip, hsp70-interacting protein ; Hop, hsc70/hsp90-organizing protein ; hsc, heat shock cognate protein ; hsp, heat shock protein ; IPTG, isopropyl β-D-thiogalactoside ; mSTI1, murine stress-inducible protein 1 ; PP5, protein phosphatase 5 ; TPR, tetratricopeptide repeat.…”

“…also necessary for hsp90 binding [12]. In addition, the association of FKBP51 with hsp90 requires a more extensive TPR Cterminal flanking region than FKBP52 [13].…”