The tetratricopeptide repeat (TPR) motif is one of many repeat motifs that form structural domains in proteins that can act as interaction scaffolds in the formation of multi-protein complexes involved in numerous cellular processes such as transcription, the cell cycle, protein translocation, protein degradation and host defence against invading pathogens. The crystal structures of many TPR domain-containing proteins have been determined, showing TPR motifs as two anti-parallel α-helices packed in tandem arrays to form a structure with an amphipathic groove which can bind a target peptide. This is however not the only mode of target recognition by TPR domains, with short amino acid insertions and alternative TPR motif conformations also shown to contribute to protein interactions, highlighting diversity in TPR domains and the versatility of this structure in mediating biological events.

The C-terminal domain of Hsp90 displays independent chaperone activity, mediates dimerization, and contains the MEEVD motif essential for interaction with tetratricopeptide repeat-containing immunophilin cochaperones assembled in mature steroid receptor complexes. An ␣-helical region, upstream of the MEEVD peptide, helps form the dimerization interface and includes a hydrophobic microdomain that contributes to the Hsp90 interaction with the immunophilin cochaperones and corresponds to the binding site for novobiocin, a coumarin-related Hsp90 inhibitor. Mutation of selected residues within the hydrophobic microdomain significantly impacted the chaperone function of a recombinant C-terminal Hsp90 fragment and novobiocin inhibited wild-type chaperone activity. Prior incubation of the Hsp90 fragment with novobiocin led to a direct blockade of immunophilin cochaperone binding. However, the drug had little influence on the pre-formed Hsp90-immunophilin complex, suggesting that bound cochaperones mask the novobiocin-binding site. We observed a differential effect of the drug on Hsp90-immunophilin interaction, suggesting that the immunophilins make distinct contacts within the C-terminal domain to specifically modulate Hsp90 function. Novobiocin also precluded the interaction of full-length Hsp90 with the p50 There is now increasing evidence that receptor function is critically dependent on the selection of immunophilin within steroid receptor complexes (2-4). This may be governed, in part, by the selective preference of receptors for specific immunophilins. PP5 has been reported to have a modulating role in glucocorticoid signaling (5), and there are strong indications that FKBP51 inhibits glucocorticoid receptor function. Elevated expression of FKBP51, resulting in greatly increased incorporation of FKBP51 into glucocorticoid receptor complexes, reduces hormone-binding affinity and promotes glucocorticoid resistance in primates (6, 7). FKBP51 also sequesters glucocorticoid receptor within the cytoplasm (8, 9), but hormone binding induces a functional exchange of FKBP52 for FKBP51 in receptor complexes allowing translocation of the complex to the nucleus (8). In a yeast model, FKBP52 was shown to dramatically potentiate glucocorticoid-dependent reporter gene activity through a mechanism that results in increased receptor hormone-binding affinity (10). Consistent with previous findings, coexpression of FKBP51 blocked the potentiating effects of FKBP52. These potentiating properties require FKBP52 catalytic activity as well as a functional interaction of the immunophilin with Hsp90. Receptor function, then, can be directly influenced by the prolyl isomerase activity of a TPR immunophilin. The Smith laboratory has now extended the study of FKBP52 function to a FKBP52 knock-out mouse model (11-13). Male mice exhibit many features in common with partial androgen insensitivity, reflecting loss of FKBP52-mediated potentiation of androgen receptor function (11). Female mice display a maternal defect linked to progesterone ins...

Cyclophilin 40 (CyP40) is a tetratricopeptide repeat (TPR)-containing immunophilin and a modulator of steroid receptor function through its binding to heat shock protein 90 (Hsp90). Critical to this binding are the carboxyl-terminal MEEVD motif of Hsp90 and the TPR domain of CyP40. Two different models of the CyP40-MEEVD peptide interaction were used as the basis for a comprehensive mutational analysis of the Hsp90-interacting domain of CyP40. Using a carboxyl-terminal CyP40 construct as template, 24 amino acids from the TPR and flanking acidic and basic domains were individually mutated by site-directed mutagenesis, and the mutants were coexpressed in yeast with a carboxyl-terminal Hsp90␤ construct and qualitatively assessed for binding using a ␤-galactosidase filter assay. For quantitative assessment, mutants were expressed as glutathione S-transferase fusion proteins and assayed for binding to carboxyl-terminal Hsp90␤ using conventional pulldown and enzyme-linked immunosorbent assay microtiter plate assays. Collectively, the models predict that the following TPR residues help define a binding groove for the MEEVD peptide: Lys-227, Asn-231, Phe-234, Ser-274, Asn-278, Lys-308, and Arg-312. Mutational analysis identified five of these residues (Lys-227, Asn-231, Asn-278, Lys-308, and Arg-312) as essential for Hsp90 binding. The other two residues (Phe-234 and Ser-274) and another three TPR domain residues not definitively associated with the binding groove (Leu-284, Lys-285, and Asp-329) are required for efficient Hsp90 binding. These data confirm the critical importance of the MEEVD binding groove in CyP40 for Hsp90 recognition and reveal that additional charged and hydrophobic residues within the CyP40 TPR domain are required for Hsp90 binding.In the absence of hormone, estrogen receptor ␣ is found predominantly in the nucleus as part of a multicomponent complex with Hsp90 1 (1). This abundant molecular chaperone plays an essential role in maintaining steroid receptors in a hormone-activable state, and the association of receptor with Hsp90 is a prerequisite for efficient signal transduction (1). In addition to dimeric Hsp90 and the receptor subunit, mature steroid receptor complexes contain p23 and one of a group of modulating proteins that include the immunophilins cyclophilin 40 (CyP40), FKBP51, and FKBP52 (1). CyP40 was first isolated as a component of estrogen receptor ␣ heterocomplexes (2). This novel cyclophilin binds the immunosuppressant cyclosporin A and shares structural similarity with its partner co-chaperones, FKBP51 and FKBP52, both immunophilins of the FK506-binding class (1). The structure of these large immunophilins is characterized by an amino-terminal immunophilin-like domain that is targeted by immunosuppressant drugs and overlaps a peptidylprolyl isomerase function and a carboxyl-terminal protein interaction domain incorporating 3 units of the tetratricopeptide repeat (TPR) motif (3). These immunophilins have been implicated in steroid receptor function through their association with Hsp...