The cochaperones described above are involved in maintaining an activatable conformation of Hsp70/Hsp90-dependent "clients", but TPR proteins also function to mediate the degradation of misfolded proteins, indicating a role in quality control (Cyr *et al.*, 2002). Selection of proteins for degradation is mediated by E3 ubiquitin ligases, and CHIP is a member of this enzymatic class (Jiang *et al.*, 2001; Murata *et al.*, 2001). CHIP has an Nterminal TPR domain and a C-terminal U-box domain that mediates its ligase activity, which promotes ubiquitylation of target substrates prior to their degradation by the proteasome.

#### **3.1.6 SGT**

Human SGT binds to viral protein U (Vpu) and Group specific Antigen, 2 proteins associated with human immunodeficiency virus-1, and the rat homologue was identified as an interactor of the non-structural protein NS-1 of the parvovirus H-1. The central TPR domain in SGT is flanked by an N-terminal dimerization domain and a C-terminal glutamine-rich domain involved in association with type 1 glucose transporter (Callahan *et al.*, 1998; Cziepluch *et al.*, 1998; Liou & Wang, 2005).

#### **3.2 Regulation of Hsp70 and Hsp90 ATPases by TPR cochaperones**

Both Hsp70 and Hsp90 require ATP for their functional association with substrates (Pratt & Toft, 2003). In the case of a steroid receptor, Hip binding to the N-terminal ATPase domain of Hsp70, possibly through a unique TPR binding site located within this region (see below), stabilizes the Hsp70-receptor complex (Frydman & Höhfeld, 1997; Höhfeld *et al.*, 1995) in a step that may be important for recognition by Hop and loading of the receptor onto Hsp90 for further processing. Hop contains three distinct TPR domains (TPR1, TPR2a, TPR2b) (Fig. 2), with TPR1 and TPR2a providing anchor points for the C-terminal EEVD peptides of Hsp70 and Hsp90, respectively. These specific interactions, coupled with domain-domain interactions, also involving its TPR domains, allow Hop to play a key role in coordinating the actions of Hsp70 and Hsp90 (Carrigan *et al.*, 2006; Chen *et al.*, 1996b; Chen & Smith, 1998; Odunuga *et al.*, 2003; Prodromou *et al.*, 1999; Ramsey *et al.*, 2009; Scheufler *et al.*, 2000). While the TPR acceptor site for Hop in the C-terminal region of Hsp90 serves to anchor the cochaperone, studies have shown that Sti1, the yeast homologue of Hop, markedly inhibits the ATPase activity of yeast Hsp90 through secondary interactions that block the ATPbinding pocket in the Hsp90 N-terminal domain (Prodromou *et al.*, 1999). By directly competing with Sti1 for binding to Hsp90, the CyP40 yeast homologue Cpr6 can negate the Sti1-mediated blockade of Hsp90 ATPase activity following TPR protein exchange (Prodromou *et al.*, 1999). In contrast, in vitro studies with human Hop determined that the cochaperone had no influence on the weak basal ATPase activity of human Hsp90, but significantly inhibited the increased rate of ATP hydrolysis by Hsp90 in response to interaction with the ligand binding domain of GR, an established Hsp90 client protein (McLaughlin *et al.*, 2002). On the other hand, FKBP52, which like CyP40 binds competitively with Hop to the C-terminal TPR interaction site of Hsp90, was shown to enhance Hsp90 ATPase activity stimulated by GR (McLaughlin *et al.*, 2002). This control over ATP utilization is important for the functional activity of newly synthesized substrates, but ATPase regulation is also required for the degradation of improperly folded substrates. CHIP can bind Hsp70 and inhibit Hsp40-stimulated Hsp70 ATPase activity, and has been reported to deplete cellular GR levels (Ballinger *et al.*, 1999; Connell *et al.*, 2001). Therefore, CHIP can be regarded as a degradatory cochaperone of Hsp70 and Hsp90. SGT negatively regulates Hsp70 such that the chaperone has a reduced ability to refold denatured luciferase (Angeletti *et al.*, 2002).

#### **3.3 Determinants of Hsp70 and Hsp90 interaction with TPR cochaperones**

Deletion studies were the first to demonstrate that TPR domains mediated binding to Hsp90 (Barent *et al.*, 1998; Chen *et al.*, 1996a; Radanyi *et al.*, 1994; Ratajczak & Carrello, 1996). Determination of the TPR domain structure of PP5 revealed that the packing of adjacent TPR units generated an exposed groove capable of accepting a target protein peptide (Das *et al.*, 1998). Although TPR motifs are highly degenerate, they display a consistent pattern of key residues important for structural integrity. The two α-helical sub-domains in each TPR motif are arranged such that the groove is mainly composed of residues from the A helix of each repeat, while B helix residues are buried to form the structural backbone of the superhelix, and this groove forms a critical Hsp recognition surface.

In a PP5 mutagenesis study, Russell and coworkers carefully selected A helix residues with side-chains extended into the groove and identified four basic residues important for PP5- Hsp90 interaction (Russell *et al.*, 1999). These amino acids are highly conserved in other Hsp90-binding TPR proteins, and mutation of aligned residues in CyP40 confirmed their importance in Hsp90 recognition (Ward *et al.*, 2002). The key recognition sequence for the TPR domain in these proteins is the EEVD peptide located at the extreme C-terminus of Hsp90 (Carrello *et al.*, 1999; Chen *et al.*, 1998; Young *et al.*, 1998), which is conserved in Hsp70. Crystallization of individual Hop TPR domains with Hsp70 and Hsp90 N-terminally extended EEVD peptides has defined the mechanism of TPR domain-peptide interaction (Scheufler *et al.*, 2000). The TPR1 domain of Hop binds to Hsp70, while the TPR2a domain mediates Hsp90 recognition (Chen *et al.*, 1996b; Lassle *et al.*, 1997). The groove in each TPR domain accommodates their respective peptide in an extended conformation where the ultimate aspartate residue is tightly held by electrostatic interactions with TPR residue sidechains in a two-carboxylate clamp. Additional EEVD contacts involve hydrogen-bonding, while amino acids upstream of the EEVD enhance the affinity of the peptides for TPR

cochaperone, studies have shown that Sti1, the yeast homologue of Hop, markedly inhibits the ATPase activity of yeast Hsp90 through secondary interactions that block the ATPbinding pocket in the Hsp90 N-terminal domain (Prodromou *et al.*, 1999). By directly competing with Sti1 for binding to Hsp90, the CyP40 yeast homologue Cpr6 can negate the Sti1-mediated blockade of Hsp90 ATPase activity following TPR protein exchange (Prodromou *et al.*, 1999). In contrast, in vitro studies with human Hop determined that the cochaperone had no influence on the weak basal ATPase activity of human Hsp90, but significantly inhibited the increased rate of ATP hydrolysis by Hsp90 in response to interaction with the ligand binding domain of GR, an established Hsp90 client protein (McLaughlin *et al.*, 2002). On the other hand, FKBP52, which like CyP40 binds competitively with Hop to the C-terminal TPR interaction site of Hsp90, was shown to enhance Hsp90 ATPase activity stimulated by GR (McLaughlin *et al.*, 2002). This control over ATP utilization is important for the functional activity of newly synthesized substrates, but ATPase regulation is also required for the degradation of improperly folded substrates. CHIP can bind Hsp70 and inhibit Hsp40-stimulated Hsp70 ATPase activity, and has been reported to deplete cellular GR levels (Ballinger *et al.*, 1999; Connell *et al.*, 2001). Therefore, CHIP can be regarded as a degradatory cochaperone of Hsp70 and Hsp90. SGT negatively regulates Hsp70 such that the chaperone has a reduced ability to refold denatured luciferase

**3.3 Determinants of Hsp70 and Hsp90 interaction with TPR cochaperones**

superhelix, and this groove forms a critical Hsp recognition surface.

Deletion studies were the first to demonstrate that TPR domains mediated binding to Hsp90 (Barent *et al.*, 1998; Chen *et al.*, 1996a; Radanyi *et al.*, 1994; Ratajczak & Carrello, 1996). Determination of the TPR domain structure of PP5 revealed that the packing of adjacent TPR units generated an exposed groove capable of accepting a target protein peptide (Das *et al.*, 1998). Although TPR motifs are highly degenerate, they display a consistent pattern of key residues important for structural integrity. The two α-helical sub-domains in each TPR motif are arranged such that the groove is mainly composed of residues from the A helix of each repeat, while B helix residues are buried to form the structural backbone of the

In a PP5 mutagenesis study, Russell and coworkers carefully selected A helix residues with side-chains extended into the groove and identified four basic residues important for PP5- Hsp90 interaction (Russell *et al.*, 1999). These amino acids are highly conserved in other Hsp90-binding TPR proteins, and mutation of aligned residues in CyP40 confirmed their importance in Hsp90 recognition (Ward *et al.*, 2002). The key recognition sequence for the TPR domain in these proteins is the EEVD peptide located at the extreme C-terminus of Hsp90 (Carrello *et al.*, 1999; Chen *et al.*, 1998; Young *et al.*, 1998), which is conserved in Hsp70. Crystallization of individual Hop TPR domains with Hsp70 and Hsp90 N-terminally extended EEVD peptides has defined the mechanism of TPR domain-peptide interaction (Scheufler *et al.*, 2000). The TPR1 domain of Hop binds to Hsp70, while the TPR2a domain mediates Hsp90 recognition (Chen *et al.*, 1996b; Lassle *et al.*, 1997). The groove in each TPR domain accommodates their respective peptide in an extended conformation where the ultimate aspartate residue is tightly held by electrostatic interactions with TPR residue sidechains in a two-carboxylate clamp. Additional EEVD contacts involve hydrogen-bonding, while amino acids upstream of the EEVD enhance the affinity of the peptides for TPR

(Angeletti *et al.*, 2002).

domains and mediate specificity of Hsp70 and Hsp90 to TPR1 and TPR2a, respectively. Notably, Hop TPR2a provides an example of where an additional sequence within the TPR domain doesn't disrupt the overall structure. TPR2a contains an insertion between units 2 and 3 that extends the helices by a single turn but does not impact Hsp90 peptide recognition (Scheufler *et al.*, 2000).

The Hsp90 dimerization domain, located in the C-terminal region upstream of the MEEVD peptide, contributes to TPR cochaperone recognition (Chen *et al.*, 1998) and contains the putative binding site for novobiocin, a coumarin-based Hsp90 inhibitor (Marcu *et al.*, 2000). *In vitro* studies demonstrated that novobiocin had a differential effect on Hsp90-immunophilin cochaperone interaction, suggesting that the TPR cochaperones modulate Hsp90 function through distinct contacts within the Hsp90 C-terminal domain (Allan *et al.*, 2006).

Although EEVD interactions with the TPR domain groove are critical for Hsp binding, regions outside of the TPR domains are also important in mediating recognition. TPR domains are typically followed by a seventh α-helix that packs against and extends beyond the TPR domain and has been shown to be involved in binding Hsp90 in addition to the TPR domain. FKBP51 and FKBP52 have different affinities for Hsp90 and are assembled differentially with specific receptor complexes, and these differences map in part to sequences C-terminal of their respective TPR domains (Barent *et al.*, 1998; Cheung-Flynn *et al.*, 2003; Pirkl & Buchner, 2001). The charge-Y motif was identified and found to be essential for FKBP-Hsp90 interaction, which was also confirmed for CyP40, but sequences further downstream in FKBP51 and FKBP52 differentially regulated Hsp90 binding (Allan *et al.*, 2006; Cheung-Flynn *et al.*, 2003; Ratajczak & Carrello, 1996). The acidic linker flanking the Nterminus of the CyP40 TPR domain was also shown to be important for efficient interaction (Mok *et al.*, 2006; Ratajczak & Carrello, 1996). Although an interaction partner for Hop TPR2b has yet to be identified, mutations in TPR2b reduced Hop interaction with both Hsp70 and Hsp90, while mutations in the C-terminal DP-rich region inhibited Hop binding to Hsp70 (Chen & Smith, 1998; Nelson *et al.*, 2003).

#### **3.4 Alternative modes of Hsp70 and Hsp90 recognition by TPR cochaperones**

Like Hop, CHIP binds to both Hsp70 and Hsp90 (Ballinger *et al.*, 1999; Connell *et al.*, 2001), but CHIP interacts with either of these major chaperones through a single TPR domain. Recent elucidation of the binding of Hsp90 C-terminal peptide (NH2-DDTSRMEEVD) with the CHIP TPR domain has revealed that the peptide sequence is not accommodated in an extended conformation as for Hop, but turns at the methionine residue and becomes buried within a hydrophobic pocket (Zhang *et al.*, 2005). This pocket can accommodate either the methionine or isoleucine that lies immediately upstream of the EEVD sequence in Hsp90 and Hsp70, respectively, and the peptide is twisted, negating the role of upstream residues in conferring the same specificity seen in binding Hop TPR domains. SGT also recognizes Hsp70 and Hsp90 via its single TPR domain, but possibly through a different mechanism to that described for CHIP as SGT lacks the residues that form the hydrophobic pocket which allows the respective C-terminal peptides in the chaperones to twist (Dutta & Tan, 2008).

Hydrophobic pockets themselves may also be important structural features within TPR domains that confer Hsp specificity, as the crystal structure of Hop TPR2a with the noncognate Hsp70 peptide shows the hydrophobic pocket to be less accommodating for the Ile (-5) residue in the extended Hsp70 peptide than Met (-5) in the extended Hsp90 peptide, with the notable feature of a lack of bending by the Hsp70 peptide, such as with CHIP, to perhaps enhance affinity for TPR2A (Kajander *et al.*, 2009).

General cell UNC-45 (GCUNC-45), a member of the UNC-45/Cro1/She4p (UCS) protein family, is a TPR protein that regulates PR chaperoning by Hsp90 by preventing activation of Hsp90 ATPase activity (Chadli *et al.*, 2006). Hsp90-binding experiments in the presence of Hop revealed a novel GCUNC-45 TPR recognition site in the N-terminal domain of Hsp90, which also bound FKBP52 (Chadli *et al.*, 2008a). Further analysis defined a non-contiguous EEVD-like motif, centered in and around the Hsp90 N-terminal ATP-binding pocket, arranged in a structural conformation that can recognize TPR domains. Nucleotide binding negatively regulates the interaction. These authors also alluded to CyP40 binding to the Nterminal interaction motif, although Onuoha and coworkers have recently confirmed CyP40 interaction only with the C-terminal domain of Hsp90 (Onuoha *et al.*, 2008). GCUNC-45 is the first cochaperone to display a preferential association with Hsp90β over the Hsp90α isoform, resulting in functional Hsp90β-GCUNC-45 interactions that more efficiently block progression of PR chaperoning than seen with Hsp90α-GCUNC-45 complexes (Chadli *et al.*, 2008b). An EEVD-like motif interaction with a TPR domain has also been described for androgen receptor recognition by SGT, where binding is mediated by the first 2 TPR motifs of the SGT TPR domain and the hinge region located between the DNA-binding and ligandbinding domains in the receptor (Buchanan *et al.*, 2007).

Hip has similarly been reported to bind the Hsp70 N-terminal ATPase domain via its TPR domain (Höhfeld *et al.*, 1995). Through this interaction, Hip, originally identified in progesterone receptor complex assembly (Prapapanich *et al.*, 1996a; Smith, 1993), can stabilize substrate-Hsp70 binding and competitively counteract the destabilizing effects of the non-TPR cochaperone BAG1 (Bimston *et al.*, 1998; Gebauer *et al.*, 1997; Höhfeld & Jentsch, 1997; Takayama *et al.*, 1997). The Hip-Hsp70 interaction also allows for the simultaneous association of Hip with Hsp70-Hop complexes (Gebauer *et al.*, 1997; Prapapanich *et al.*, 1996a). By analogy with the mode of GCUNC-45 interaction with Hsp90, there is the possibility that Hip targets a similar TPR recognition site in the Nterminal region of Hsp70. However, Hip is unique among the steroid receptor-associated TPR proteins in terms of Hsp recognition in that it binds Hsp70 independently of EEVD interactions (Höhfeld *et al.*, 1995), and that efficient binding may be due to a greater requirement for additional Hsp-interaction determinants, such as the adjacent highly charged region and a C-terminal DP-repeat domain (Prapapanich *et al.*, 1998). It is possible the mechanism of Hsp70 recognition by Hip is not unique, but may be utilized by some of the steroid-receptor TPR cochaperones to interact with binding partners in distinct cellular pathways. Dutta and Tan (2008) reported the SGT TPR domain is sufficient to bind Vpu and identified the sequence 31KILRQ35 in Vpu as being important for this interaction.

#### **4. p23 and Cdc37 interaction with Hsp90**

p23 is an essential component involved in stabilizing mature steroid receptor-Hsp90 complexes and binds to the ATP-bound conformation of a Hsp90 dimer characterised by high affinity for client proteins (Ali *et al.*, 2006; Felts & Toft, 2003; McLaughlin *et al.*, 2006;

(-5) residue in the extended Hsp70 peptide than Met (-5) in the extended Hsp90 peptide, with the notable feature of a lack of bending by the Hsp70 peptide, such as with CHIP, to

General cell UNC-45 (GCUNC-45), a member of the UNC-45/Cro1/She4p (UCS) protein family, is a TPR protein that regulates PR chaperoning by Hsp90 by preventing activation of Hsp90 ATPase activity (Chadli *et al.*, 2006). Hsp90-binding experiments in the presence of Hop revealed a novel GCUNC-45 TPR recognition site in the N-terminal domain of Hsp90, which also bound FKBP52 (Chadli *et al.*, 2008a). Further analysis defined a non-contiguous EEVD-like motif, centered in and around the Hsp90 N-terminal ATP-binding pocket, arranged in a structural conformation that can recognize TPR domains. Nucleotide binding negatively regulates the interaction. These authors also alluded to CyP40 binding to the Nterminal interaction motif, although Onuoha and coworkers have recently confirmed CyP40 interaction only with the C-terminal domain of Hsp90 (Onuoha *et al.*, 2008). GCUNC-45 is the first cochaperone to display a preferential association with Hsp90β over the Hsp90α isoform, resulting in functional Hsp90β-GCUNC-45 interactions that more efficiently block progression of PR chaperoning than seen with Hsp90α-GCUNC-45 complexes (Chadli *et al.*, 2008b). An EEVD-like motif interaction with a TPR domain has also been described for androgen receptor recognition by SGT, where binding is mediated by the first 2 TPR motifs of the SGT TPR domain and the hinge region located between the DNA-binding and ligand-

Hip has similarly been reported to bind the Hsp70 N-terminal ATPase domain via its TPR domain (Höhfeld *et al.*, 1995). Through this interaction, Hip, originally identified in progesterone receptor complex assembly (Prapapanich *et al.*, 1996a; Smith, 1993), can stabilize substrate-Hsp70 binding and competitively counteract the destabilizing effects of the non-TPR cochaperone BAG1 (Bimston *et al.*, 1998; Gebauer *et al.*, 1997; Höhfeld & Jentsch, 1997; Takayama *et al.*, 1997). The Hip-Hsp70 interaction also allows for the simultaneous association of Hip with Hsp70-Hop complexes (Gebauer *et al.*, 1997; Prapapanich *et al.*, 1996a). By analogy with the mode of GCUNC-45 interaction with Hsp90, there is the possibility that Hip targets a similar TPR recognition site in the Nterminal region of Hsp70. However, Hip is unique among the steroid receptor-associated TPR proteins in terms of Hsp recognition in that it binds Hsp70 independently of EEVD interactions (Höhfeld *et al.*, 1995), and that efficient binding may be due to a greater requirement for additional Hsp-interaction determinants, such as the adjacent highly charged region and a C-terminal DP-repeat domain (Prapapanich *et al.*, 1998). It is possible the mechanism of Hsp70 recognition by Hip is not unique, but may be utilized by some of the steroid-receptor TPR cochaperones to interact with binding partners in distinct cellular pathways. Dutta and Tan (2008) reported the SGT TPR domain is sufficient to bind Vpu and identified the sequence 31KILRQ35 in Vpu as being important

p23 is an essential component involved in stabilizing mature steroid receptor-Hsp90 complexes and binds to the ATP-bound conformation of a Hsp90 dimer characterised by high affinity for client proteins (Ali *et al.*, 2006; Felts & Toft, 2003; McLaughlin *et al.*, 2006;

perhaps enhance affinity for TPR2A (Kajander *et al.*, 2009).

binding domains in the receptor (Buchanan *et al.*, 2007).

for this interaction.

**4. p23 and Cdc37 interaction with Hsp90**

Richter *et al.*, 2004). Conformational changes that accompany ATP binding promote dimeric interaction between the N-terminal domains of the Hsp90 C-terminal dimer to form distinct binding surfaces for separate p23 molecules, thus further underpinning the ATP-bound conformation (Ali *et al.*, 2006; Karagöz *et al.*, 2010). In a recent model proposed for the Hsp90 cochaperone cycle, entry of an immunophilin cochaperone into an existing client protein-Hsp90-Sti1/Hop-Hsp70 complex forms an intermediate complex important for cycle progression. Conversion of Hsp90 to the closed conformation on ATP and subsequent p23 binding then favours the release of Sti1/Hop (Li *et al.*, 2011).

Cdc37 serves as an adaptor predominantly facilitating protein kinase interaction with Hsp90, although additional client proteins, including steroid receptors have been identified (MacLean & Picard, 2003). Similar to Hop, Cdc37 arrests the Hsp90 ATPase cycle and functions as an "early" cochaperone for the recruitment of protein kinase clients to the Hsp90 machinery. Hsp90 binding maps to the Cdc37 C-terminal region, while kinase interaction occurs via the N-terminal domain (Roe *et al.*, 2004). Hsp90 ATPase activity is coupled to an opening and closing of a molecular clamp generated by the constitutive Cterminal Hsp90 dimer at one end in combination with the ATP-dependent association of the N-terminal domains at the other (Prodromou *et al.*, 2000). A structural view of the Hsp90- Cdc37 complex shows Cdc37 located as a dimer between the N-terminal domains of the clamp, thus preventing their interaction (Roe *et al.*, 2004). With cycle progression, loss of one Cdc37 monomer leads to the formation of a stable (Hsp90)2-Cdc37-kinase complex (Vaughan *et al.*, 2006; Vaughan *et al.*, 2008).

#### **5. Receptor-cochaperone interactions**

#### **5.1 Cortisol resistance in New World primates; The key role of FKBP51; Structures of FKBP51 and FKBP52**

Analysis of glucocorticoid resistance in New World primates, such as squirrel monkey, has demonstrated that the high circulating cortisol levels result from elevated expression and greatly increased incorporation of FKBP51 into GR-Hsp90 complexes, causing a significant decrease in GR hormone binding affinity (Denny *et al.*, 2000; Reynolds *et al.*, 1999; Scammell *et al.*, 2001). FKBP51 then appears to have a major role in stabilizing an inactive receptor conformation. The FK506 drug-binding pocket of FKBP51 is inaccessible to FK506 in low affinity hormone-binding GR heterocomplexes. However, incubation of receptor cytosols from squirrel monkey lymphocytes with FK506 prevented assembly of FKBP51 with GR-Hsp90 complexes, correlating with a sharp increase in receptor hormone binding and affinity. On the other hand, recognition of FK506 by FKBP52 appeared unaffected by whether the immunophilin exists as a component of mature, high affinity hormone-binding GR complexes or not (Denny *et al.*, 2000; Tai *et al.*, 1992). Furthermore, the immunosuppressant blocks FKBP52-mediated potentiation of GR activity (Riggs *et al.*, 2003). The inhibitory influence of FKBP51 on GR activity requires both FK domains, as well as Hsp90 binding, but is not reliant on FKBP51 PPIase activity (Denny *et al.*, 2005). FK506 may likely serve to sterically hinder receptor LBD interactions with the FK1 domain of FKBP51 and FKBP52 essential for inhibitory and activation effects on receptor, respectively. This differential action of FK506 may arise from distinct domain orientations that have been defined from recent structures of the two immunophilins (Sinars *et al.*, 2003; Wu *et al.*, 2004). Unique interactions between receptor and the FKBP51 and FKBP52 cochaperones have been further highlighted by results showing that deletion of the Asp195, His196, Asp197 insertion within the FK2 domain of FKBP51 compromised assembly of the immunophilin into PR complexes, whereas removal of the corresponding FK2 insertion loop from FKBP52 had no affect on receptor association (Sinars *et al.*, 2003). This raises the possibility that direct interaction of FK2 in FKBP51 with PR might favour the preferred association of FKBP51 over FKBP52 with this receptor.

#### **5.2 Cortisol resistance in the guinea-pig; Do guinea pig GR LBD changes favour FKBP51 binding over FKBP52?**

In contrast to the New World primates, the cause of glucocorticoid resistance in the guinea pig, a New World hystricomorph, has been delineated to an unstructured loop between helix 1 and helix 3 of the guinea pig GR LBD. Five amino acid substitutions in this region differentiate guinea pig GR from the human receptor, with at least four contributing to the low binding affinity phenotype (Fuller *et al.*, 2004). It has been predicted that these crucial residues (Ile538, His539, Ser540, Thr545 and Ser546) lying on the surface of the guinea pig GR LBD, disrupt a contact domain for FKBP52, favouring increased association with FKBP51 and conformational changes that compromise high affinity cortisol binding. Using a yeast-based assay (Riggs *et al.*, 2003) with rat GR substituted in the helix 1 to helix 3 loop with the guinea pig GR-specific residues, we have recently confirmed that FKBP52 can efficiently potentiate the transcriptional activity of the mutated GR, thus discounting a central role of this region in receptor-FKBP52 interaction [Cluning C and Ratajczak T, unpublished observations].

#### **5.3 FKBP52 potentiation of AR, GR and PR**

Direct interaction studies between bacterially expressed FKBP52 and GST-tagged, wild type human GR and C-terminal truncation mutants of the receptor purified from Sf9 cell extracts, identified a 35-amino acid region (hGR 465-500), between the DNA-binding domain and the LBD, to be sufficient for FKBP52 binding, with optimal interaction requiring involvement of the LBD (Silverstein *et al.*, 1999). However, recent demonstration of FKBP52 potentiation of GR activity in association with increased receptor hormone binding affinity has definitively localized the FKBP52 effect to the GR LBD (hGR 521-777) and at the same time pointed to a requirement of FKBP52 PPIase activity residing in the FK1 domain (Riggs *et al.*, 2003). Studies with FKBP52 knockout mouse strains have extended the critical physiological role of FKBP52 to cellular responses controlled by both AR (Cheung-Flynn *et al.*, 2005) and PR (Tranguch *et al.*, 2005; Yang *et al.*, 2006), while similar influences of this immunophilin cochaperone on ERα (Riggs *et al.*, 2003) and MR (Gallo *et al.*, 2007) activity have not been observed, despite the assembly of FKBP52 with Hsp90 complexes containing these receptors.

#### **5.4 Molecular basis of FKBP52 action; Potential interaction of FKBP52 with the BF3 regulatory site**

An initial understanding that FKBP52 potentiation of AR, GR and PR activity was dependent on the FK1-mediated PPIase function of the immunophilin, prompted speculation that FKBP52

defined from recent structures of the two immunophilins (Sinars *et al.*, 2003; Wu *et al.*, 2004). Unique interactions between receptor and the FKBP51 and FKBP52 cochaperones have been further highlighted by results showing that deletion of the Asp195, His196, Asp197 insertion within the FK2 domain of FKBP51 compromised assembly of the immunophilin into PR complexes, whereas removal of the corresponding FK2 insertion loop from FKBP52 had no affect on receptor association (Sinars *et al.*, 2003). This raises the possibility that direct interaction of FK2 in FKBP51 with PR might favour the preferred association of FKBP51

**5.2 Cortisol resistance in the guinea-pig; Do guinea pig GR LBD changes favour**

interaction [Cluning C and Ratajczak T, unpublished observations].

assembly of FKBP52 with Hsp90 complexes containing these receptors.

**5.3 FKBP52 potentiation of AR, GR and PR**

**regulatory site**

In contrast to the New World primates, the cause of glucocorticoid resistance in the guinea pig, a New World hystricomorph, has been delineated to an unstructured loop between helix 1 and helix 3 of the guinea pig GR LBD. Five amino acid substitutions in this region differentiate guinea pig GR from the human receptor, with at least four contributing to the low binding affinity phenotype (Fuller *et al.*, 2004). It has been predicted that these crucial residues (Ile538, His539, Ser540, Thr545 and Ser546) lying on the surface of the guinea pig GR LBD, disrupt a contact domain for FKBP52, favouring increased association with FKBP51 and conformational changes that compromise high affinity cortisol binding. Using a yeast-based assay (Riggs *et al.*, 2003) with rat GR substituted in the helix 1 to helix 3 loop with the guinea pig GR-specific residues, we have recently confirmed that FKBP52 can efficiently potentiate the transcriptional activity of the mutated GR, thus discounting a central role of this region in receptor-FKBP52

Direct interaction studies between bacterially expressed FKBP52 and GST-tagged, wild type human GR and C-terminal truncation mutants of the receptor purified from Sf9 cell extracts, identified a 35-amino acid region (hGR 465-500), between the DNA-binding domain and the LBD, to be sufficient for FKBP52 binding, with optimal interaction requiring involvement of the LBD (Silverstein *et al.*, 1999). However, recent demonstration of FKBP52 potentiation of GR activity in association with increased receptor hormone binding affinity has definitively localized the FKBP52 effect to the GR LBD (hGR 521-777) and at the same time pointed to a requirement of FKBP52 PPIase activity residing in the FK1 domain (Riggs *et al.*, 2003). Studies with FKBP52 knockout mouse strains have extended the critical physiological role of FKBP52 to cellular responses controlled by both AR (Cheung-Flynn *et al.*, 2005) and PR (Tranguch *et al.*, 2005; Yang *et al.*, 2006), while similar influences of this immunophilin cochaperone on ERα (Riggs *et al.*, 2003) and MR (Gallo *et al.*, 2007) activity have not been observed, despite the

**5.4 Molecular basis of FKBP52 action; Potential interaction of FKBP52 with the BF3**

An initial understanding that FKBP52 potentiation of AR, GR and PR activity was dependent on the FK1-mediated PPIase function of the immunophilin, prompted speculation that FKBP52

over FKBP52 with this receptor.

**FKBP51 binding over FKBP52?**

might target a key proline likely to be conserved among these receptors and that this critical residue would be located on the surface of the LBD, accessible to the cochaperone and in a position where it might influence the shape of the ligand binding pocket (Cheung-Flynn *et al.*, 2005). Although several such candidate prolines exist in the intervening loops between receptor LBD helices, a more extensive mutational analysis of the FK1 catalytic site has excluded a role for the FKBP52 PPIase activity in receptor potentiation (Riggs *et al.*, 2007). Rather, recent evidence has identified a loop overhanging the FK1 catalytic pocket in FKBP52 that is responsible for the functional difference between FKBP52 and FKBP51 relating to AR (and GR/PR) potentiation (Riggs *et al.*, 2007). It is proposed that a critical proline within this loop (human FKBP52 Pro119) allows specific contact with a region of the AR LBD (a structural feature that is also common to GR and PR), thus helping to stabilize an LBD conformation favourable for high affinity hormone binding and leading to efficient transcriptional activation (Riggs *et al.*, 2007). It is speculated that a leucine substitution within the corresponding FK1 sequence of FKBP51 alters the loop conformation sufficiently to disrupt this functionally important contact. The possibility exists that in the hormone-induced transition from inactive to active states of AR-Hsp90 complexes associated with FKBP51 and FKBP52, respectively, Hsp90 orients FKBP52 to achieve unique interactions with the receptor LBD, allowing Hsp90 to facilitate optimal hormone binding and to further fine-tune the hormonal response.

Prior to investigations establishing a noncatalytic involvement of the FKBP52 PPIase domain in the modulation of receptor function, an early attempt to identify the putative proline substrate for FKBP52 isomerase activity within the AR LBD utilized AR-P723S, a proline mutant associated with androgen insensitivity syndrome (Cheung-Flynn *et al.*, 2005). Although predicted to display basal activity, coupled with a lack of response to hormone in the presence of FKBP52, this mutant was characterized by subnormal activity in the absence of FKBP52, showing full restoration to wild type receptor activity levels with the cochaperone on exposure to hormone (Cheung-Flynn *et al.*, 2005). Such a favoured response reflects a greater dependence of the AR-Pro723S mutant on FKBP52 for normal activity. Pro723 lies within the signature sequence conserved among all steroid receptors (Brelivet *et al.*, 2004), close to a region directly involved in ligand binding and is situated in a solvent exposed loop between helices 3 and 4, which combine together with the mobile helix 12 to form the AF2 coactivator binding pocket (He *et al.*, 2004; Matias *et al.*, 2000b). For AR, AF2 initially has a preferred interaction with the AR N-terminal domain, resulting in an intramolecular fold that precedes receptor dimerization and appears critical for AR function (He *et al.*, 2001; He *et al.*, 2004; Schaufele *et al.*, 2005). Pro723 also forms part of the recently identified BF-3 surface that has the ability to allosterically alter the AF2 binding pocket of AR (Estébanez-Perpiñá *et al.*, 2007) (Fig. 4). BF-3 residues altered through natural mutations linked to androgen insensitivity and those associated with prostate cancer, either diminish or enhance AR AF2 activity, respectively, underlining the importance of the BF-3 surface for AR function (Estébanez-Perpiñá *et al.*, 2007). FKBP52 rescue of AR-Pro723S activity might signify FKBP52 influence over some part of the BF-3 allosteric regulatory site leading to conformational changes that allow full recovery of AR activity. Indeed, Cox and coworkers have recently identified small-molecule inhibitors of FKBP52-enhanced AR function in prostate cancer cells that target a region of the AR LBD overlapping the BF3 surface (De Leon *et al.*, 2011) (Fig. 4). Multiple residues that contribute to the FKBP52 sensitivity of AR, some of which form part of the binding site for MJC13, the lead compound, have been identified (De Leon *et al.*, 2011) (Fig. 4). Since MJC13 helps to maintain an intact AR-Hsp90- FKBP52 complex at low hormone concentrations, it is possible that the inhibitor interferes with a critical next step - a hormone-induced, FKBP52-dependent transitory change in AR conformation necessary for nuclear translocation. Sequence comparisons have revealed some conservation of BF-3 residues within the LBDs for AR, GR, MR and PR, suggesting the presence of BF-3-like regulatory domains in each receptor (Estébanez-Perpiñá *et al.*, 2007) (Fig. 4). A very limited conservation of these residues is apparent in ERα, suggesting the formation of a BF-3 type surface that is unique to this receptor (Estébanez-Perpiñá *et al.*, 2007) (Fig. 4). Both ERα and MR behave differently to AR, GR and PR, through their inability to respond to FKBP52. Certain structural differences within their LBDs distinguish these two receptors from the other members of this subfamily (De Leon *et al.*, 2011) (Fig. 4). Since FKBP52 also regulates GR and PR activity, most likely through specific BF3 surfaces, there is the potential for the development of FKBP52-specific inhibitors targeting GR and PR function to treat a range of steroid hormone-based diseases (Moore *et al.*, 2010). The BF-3 pocket is a potential target for second-site modulators that can allosterically block agonistactivated AR function to inhibit prostate cancer cell growth (Joseph *et al.*, 2009).

#### **5.4.1 FKBP51 is an androgen-regulated gene that promotes assembly of mature AR-Hsp90 complexes**

FKBP51 is recognised as a highly sensitive AR-regulated gene that functions as an important component of a feed-forward mechanism linked to the partial reactivation of AR-signalling pathways in the absence of androgens, leading to the outgrowth of androgen-independent tumours (Amler *et al.*, 2000; Febbo *et al.*, 2005; Magee *et al.*, 2006; Mousses *et al.*, 2001; Tomlins *et al.*, 2007). Sanchez and coworkers have confirmed a significantly increased expression of FKBP51, but not that of FKBP52, in most prostate cancer tissues and in androgen-dependent and androgen-independent cell lines (Periyasamy *et al.*, 2010), suggesting that FKBP51 might have a critical role in prostate cancer growth and progression. FKBP51 overexpression was found to increase the AR transcriptional response by facilitating hormone-binding competence through the assembly of the AR LBD with mature FKBP51-Hsp90-p23 complexes (Ni *et al.*, 2010), resulting in higher levels of androgen-liganded receptor and providing a pathway for AR-dependent signalling and growth in a low-androgen environment. The ability of FKBP51 to enhance AR transcription and chaperone complex assembly appears to be dependent on FKBP51 PPIase activity mediated by the FK1 domain and requires Hsp90 binding through its TPR domain (Ni *et al.*, 2010).

#### **6. Receptor LBD contacts with other Hsp90 cochaperones**

#### **6.1 PP5; GCUNC-45; SGT**

The domain structure of the Hsp90 cochaperone, PP5, a serine/threonine protein phosphatase (Chen *et al.*, 1994; Chinkers, 1994), is characterised by a C-terminal phosphatase catalytic domain and an N-terminal TPR domain that competes with FKBP51, FKBP52 and CyP40 for the TPR binding site at the Hsp90 C-terminus during assembly into mature steroid receptor-Hsp90 complexes (Banerjee *et al.*, 2008; Chen *et al.*, 1996a; Hinds Jr & Sanchez, 2008). Through its TPR domain, PP5 has also been shown to bind directly to ERα and ERβ, an interaction that targets the LBDs of these receptors, but does not require the C-terminal region incorporating

identified (De Leon *et al.*, 2011) (Fig. 4). Since MJC13 helps to maintain an intact AR-Hsp90- FKBP52 complex at low hormone concentrations, it is possible that the inhibitor interferes with a critical next step - a hormone-induced, FKBP52-dependent transitory change in AR conformation necessary for nuclear translocation. Sequence comparisons have revealed some conservation of BF-3 residues within the LBDs for AR, GR, MR and PR, suggesting the presence of BF-3-like regulatory domains in each receptor (Estébanez-Perpiñá *et al.*, 2007) (Fig. 4). A very limited conservation of these residues is apparent in ERα, suggesting the formation of a BF-3 type surface that is unique to this receptor (Estébanez-Perpiñá *et al.*, 2007) (Fig. 4). Both ERα and MR behave differently to AR, GR and PR, through their inability to respond to FKBP52. Certain structural differences within their LBDs distinguish these two receptors from the other members of this subfamily (De Leon *et al.*, 2011) (Fig. 4). Since FKBP52 also regulates GR and PR activity, most likely through specific BF3 surfaces, there is the potential for the development of FKBP52-specific inhibitors targeting GR and PR function to treat a range of steroid hormone-based diseases (Moore *et al.*, 2010). The BF-3 pocket is a potential target for second-site modulators that can allosterically block agonist-

activated AR function to inhibit prostate cancer cell growth (Joseph *et al.*, 2009).

**6. Receptor LBD contacts with other Hsp90 cochaperones**

**Hsp90 complexes**

TPR domain (Ni *et al.*, 2010).

**6.1 PP5; GCUNC-45; SGT**

**5.4.1 FKBP51 is an androgen-regulated gene that promotes assembly of mature AR-**

FKBP51 is recognised as a highly sensitive AR-regulated gene that functions as an important component of a feed-forward mechanism linked to the partial reactivation of AR-signalling pathways in the absence of androgens, leading to the outgrowth of androgen-independent tumours (Amler *et al.*, 2000; Febbo *et al.*, 2005; Magee *et al.*, 2006; Mousses *et al.*, 2001; Tomlins *et al.*, 2007). Sanchez and coworkers have confirmed a significantly increased expression of FKBP51, but not that of FKBP52, in most prostate cancer tissues and in androgen-dependent and androgen-independent cell lines (Periyasamy *et al.*, 2010), suggesting that FKBP51 might have a critical role in prostate cancer growth and progression. FKBP51 overexpression was found to increase the AR transcriptional response by facilitating hormone-binding competence through the assembly of the AR LBD with mature FKBP51-Hsp90-p23 complexes (Ni *et al.*, 2010), resulting in higher levels of androgen-liganded receptor and providing a pathway for AR-dependent signalling and growth in a low-androgen environment. The ability of FKBP51 to enhance AR transcription and chaperone complex assembly appears to be dependent on FKBP51 PPIase activity mediated by the FK1 domain and requires Hsp90 binding through its

The domain structure of the Hsp90 cochaperone, PP5, a serine/threonine protein phosphatase (Chen *et al.*, 1994; Chinkers, 1994), is characterised by a C-terminal phosphatase catalytic domain and an N-terminal TPR domain that competes with FKBP51, FKBP52 and CyP40 for the TPR binding site at the Hsp90 C-terminus during assembly into mature steroid receptor-Hsp90 complexes (Banerjee *et al.*, 2008; Chen *et al.*, 1996a; Hinds Jr & Sanchez, 2008). Through its TPR domain, PP5 has also been shown to bind directly to ERα and ERβ, an interaction that targets the LBDs of these receptors, but does not require the C-terminal region incorporating

NCBI accession numbers for receptor sequences are: AR – NP000035, ERα – NP000116, GR – NP001018087, MR – NP000892, PR – NP000917. The ERα sequence has 595 amino acids and is shown terminated at residue 573. LBD helices are based on the structure of AR liganded to R1881 (Matias *et al.*, 2000a) (PDB ID 1E3G). The nuclear receptor signature sequence is indicated (thick black line). Residues that map to the BF-3 allosteric regulatory site defined for AR are highlighted with an asterisk (\*). Multiple residues that contribute to the FKBP52 sensitivity of AR and form the putative binding site for MJC13 (De Leon *et al.*, 2011) are highlighted with a black circle (•). Identical residues are shown white against black; conserved residues (black on grey) are based on the following scheme: (P, G), (M, C), (Y, W, F, H), (L, V, I, A), (K, R), (E, Q, N, D) and (S, T).

Fig. 4. Multiple sequence alignment of human steroid receptor LBDs.

the helix 11-12 loop and helix 12 central to AF2 function (Ikeda *et al.*, 2004). PP5 was found to function as a negative regulator of ERα transcription *in vivo* by inhibiting epidermal growth factor (EGF)-dependent phosphorylation of Ser118 in the receptor N-terminal domain. Although demonstration of a direct PP5-ERα interaction was consistent with a noninvolvement of Hsp90, a role for this major molecular chaperone in the *in vivo* effects of PP5 on ERα function cannot be discounted. Similar observations have been reported for GR with evidence suggesting that PP5-dependent modulation of receptor N-terminal phosphorylation within the GR-Hsp90 apo-receptor complex is mediated through contacts between the phosphatase and receptor LBD (Wang *et al.*, 2007).

A yeast two-hybrid screen, using bait encompassing both the hinge region and LBD of human PR, liganded with the mixed antagonist RU486, identified GCUNC-45 as a PRbinding protein (Chadli *et al.*, 2006). Presence of two LXXLL motifs (similar to NR boxes of known transcriptional coregulatory proteins) within the interacting clone, corresponding to the C-terminal end of GCUNC-45, suggested a mode of interaction similar to that for receptor recognition of transcription coactivators (Ratajczak, 2001), although this remains to be confirmed. Both FKBP52 and CyP40 compete with GCUNC-45 for the N-terminal TPR site, with nucleotides causing a reduction in Hsp90 binding affinity for these cochaperones in this region and favouring their interaction with the Hsp90 C-terminus during progression of receptor to a hormone-binding state (Chadli *et al.*, 2008b). GCUNC-45 therefore, appears to have a role upstream of FKBP52 and CyP40, at an intermediate stage of the receptor activation pathway.

The Hsp70/Hsp90 cochaperone, SGT, has been shown to interact through its TPR domain with the hinge region of human AR, which contains a peptide sequence structurally resembling the EEVD binding site for TPR proteins at the extreme C-terminus of Hsp70 and Hsp90 (Buchanan *et al.*, 2007). It has been proposed that, as a component of AR-Hsp90 complexes, SGT regulates the ligand sensitivity of AR signalling by limiting receptor trafficking to the nucleus at low hormone concentrations and maintaining the receptor within the cytoplasm of the cell.

#### **6.2 p23; Cdc37**

Disruption of the p23 gene in mice has revealed that although p23 is not essential for overall perinatal development its absolute requirement for perinatal survival is linked to impaired GR function arising most likely from instability of GR-Hsp90 complexes in the absence of p23 (Grad *et al.*, 2006; Picard, 2006). These findings suggest that GR might be a key molecular target for p23. Overexpression experiments with p23 in tissue culture cells have revealed both positive and negative influences on GR function (Freeman *et al.*, 2000; Wochnik *et al.*, 2004), as well as differential effects on other steroid receptors - increasing PR activity, while decreasing the activities of AR, ERα and MR (Freeman *et al.*, 2000). In yeast, p23 has been shown to be a positive regulator of ERα transcriptional activation, being most effective at low ERα levels and hormone concentrations, consistent with the proposed role for p23 as a component of mature ERα-Hsp90 complexes (Knoblauch & Garabedian, 1999). Ectopic expression of p23 in MCF-7 breast cancer cells increased both hormone-dependent and hormone-independent ERα transcriptional activity (Knoblauch & Garabedian, 1999). Thus, while the major impact of p23 on ERα is likely to be through an Hsp90-dependent effect on estradiol binding, p23 overexpression may also influence receptor activity independent of ligand binding and may participate in the disassembly of receptors at cognate response elements (Freeman *et al.*, 2000; Freeman & Yamamoto, 2001; Freeman & Yamamoto, 2002). It is of interest that although p23 increases AR transcriptional activity in a variety of mammalian cell lines, partly by increasing ligand binding competence of the receptor, Hsp90 inhibitors could not abolish the AR coactivation potential of p23, consistent with an Hsp90-independent role of p23 in AR function (Querol Cano L and Bevan CL, unpublished observations).

Genetic studies in yeast have revealed that Cdc37 plays a role in AR hormone-dependent transactivation through functional interactions with the AR LBD, although the hormonebinding properties of the receptor appear to be unaffected (Fliss *et al.*, 1997). The association with Cdc37 is specific to AR since it does not occur with closely related nuclear receptors such as GR (Rao *et al.*, 2001). Depletion of Cdc37 using RNA interference caused growth arrest in both AR-positive and AR-negative prostate cancer cells, and in the former led to a loss of AR transcriptional activity with a concomitant decrease in androgen-dependent gene expression (Gray *et al.*, 2007). The targeting of Cdc37 in prostate cancer causes growth inhibition that correlates with decreased signalling through multiple pathways - the extracellular signal-regulated kinase (ERK) and Akt kinase cascades, as well as reduced ARdependent signalling (Gray *et al.*, 2008).

#### **7. Conclusions**

88 Protein Interactions

the helix 11-12 loop and helix 12 central to AF2 function (Ikeda *et al.*, 2004). PP5 was found to function as a negative regulator of ERα transcription *in vivo* by inhibiting epidermal growth factor (EGF)-dependent phosphorylation of Ser118 in the receptor N-terminal domain. Although demonstration of a direct PP5-ERα interaction was consistent with a noninvolvement of Hsp90, a role for this major molecular chaperone in the *in vivo* effects of PP5 on ERα function cannot be discounted. Similar observations have been reported for GR with evidence suggesting that PP5-dependent modulation of receptor N-terminal phosphorylation within the GR-Hsp90 apo-receptor complex is mediated through contacts

A yeast two-hybrid screen, using bait encompassing both the hinge region and LBD of human PR, liganded with the mixed antagonist RU486, identified GCUNC-45 as a PRbinding protein (Chadli *et al.*, 2006). Presence of two LXXLL motifs (similar to NR boxes of known transcriptional coregulatory proteins) within the interacting clone, corresponding to the C-terminal end of GCUNC-45, suggested a mode of interaction similar to that for receptor recognition of transcription coactivators (Ratajczak, 2001), although this remains to be confirmed. Both FKBP52 and CyP40 compete with GCUNC-45 for the N-terminal TPR site, with nucleotides causing a reduction in Hsp90 binding affinity for these cochaperones in this region and favouring their interaction with the Hsp90 C-terminus during progression of receptor to a hormone-binding state (Chadli *et al.*, 2008b). GCUNC-45 therefore, appears to have a role upstream of FKBP52 and CyP40, at an intermediate stage of the receptor

The Hsp70/Hsp90 cochaperone, SGT, has been shown to interact through its TPR domain with the hinge region of human AR, which contains a peptide sequence structurally resembling the EEVD binding site for TPR proteins at the extreme C-terminus of Hsp70 and Hsp90 (Buchanan *et al.*, 2007). It has been proposed that, as a component of AR-Hsp90 complexes, SGT regulates the ligand sensitivity of AR signalling by limiting receptor trafficking to the nucleus at low hormone concentrations and maintaining the receptor

Disruption of the p23 gene in mice has revealed that although p23 is not essential for overall perinatal development its absolute requirement for perinatal survival is linked to impaired GR function arising most likely from instability of GR-Hsp90 complexes in the absence of p23 (Grad *et al.*, 2006; Picard, 2006). These findings suggest that GR might be a key molecular target for p23. Overexpression experiments with p23 in tissue culture cells have revealed both positive and negative influences on GR function (Freeman *et al.*, 2000; Wochnik *et al.*, 2004), as well as differential effects on other steroid receptors - increasing PR activity, while decreasing the activities of AR, ERα and MR (Freeman *et al.*, 2000). In yeast, p23 has been shown to be a positive regulator of ERα transcriptional activation, being most effective at low ERα levels and hormone concentrations, consistent with the proposed role for p23 as a component of mature ERα-Hsp90 complexes (Knoblauch & Garabedian, 1999). Ectopic expression of p23 in MCF-7 breast cancer cells increased both hormone-dependent and hormone-independent ERα transcriptional activity (Knoblauch & Garabedian, 1999).

between the phosphatase and receptor LBD (Wang *et al.*, 2007).

activation pathway.

**6.2 p23; Cdc37**

within the cytoplasm of the cell.

We have arrived at a better understanding of the molecular mechanisms that allow the Hsp90 chaperone to modulate steroid receptor function through direct contact with receptor LBDs. Critical to this regulation is the ability of Hsp90 to coordinate and bring to receptor-Hsp90 complexes a selection of cochaperones whose specialized influences target receptor LBDs and combine, at various stages of the receptor activation pathway, to alter receptor hormone-binding status, cellular location and transcriptional activity. A number of these cochaperones may impact on steroid receptor function independently of Hsp90. Substantial gaps still remain, however in our knowledge of how the interplay between Hsp90 and its cochaperones affects receptor function. For example, while it is known the CyP40 yeast homologue, Cpr6, regulates Hsp90 ATPase activity during receptor assembly (Prodromou *et al.*, 1999) and studies of a second yeast homologue, Cpr7, have provided some insight into the role of this immunophilin in Hsp90-dependent signalling by steroid receptors (Duina *et al.*, 1996; Duina *et al.*, 1998), a coherent mechanism at the molecular level has yet to be defined. From the structural similarity between CyP40 and FKBP52, both being characterized by N-terminal PPIase and C-terminal TPR domains, it is tempting to draw parallels for their mechanism of action. Within steroid receptor-Hsp90 complexes it is possible that, as for FKBP52, the CyP40 PPIase domain forms productive interactions with the receptor LBD, serving to modulate receptor conformation and function. This may be of relevance for the function of ERα, purification of which led to the isolation of CyP40 in ERα-Hsp90 complexes (Ratajczak *et al.*, 1993) and for the regulation of AR in prostate cancer where CyP40 appears to be overexpressed (Periyasamy *et al.*, 2010).

Hsp90 is required for the proper function of several key regulatory proteins including multiple tyrosine and serine/threonine kinases and steroid receptors, many of which are involved in promoting malignancy (Calderwood *et al.*, 2006; Pearl, 2005; Whitesell & Lindquist, 2005). The aim of targeting and pharmacological manipulation of the Hsp90 chaperoning system has led to the ongoing development and clinical evaluation of novel Hsp90 and chaperone inhibitors for potential application in therapies against selected malignancies (Donnelly *et al.*, 2010; Kim *et al.*, 2009), syndromes arising from dysfunctional protein folding and neurodegenerative diseases (Jinwal *et al.*, 2010). With growing understanding of the novel mechanisms through which Hsp90 cochaperones modulate the function of specific clients, strategies are now evolving for the targeting of chaperone-client interactions in a wide range of human diseases (De Leon *et al.*, 2011; Gray *et al.*, 2008).

#### **8. Abbreviations**

Hsp, heat shock protein; TPR, tetratricopeptide repeat; PPIase, prolylpeptidyl isomerase; FKBP, FK506-binding protein; CyP40, cyclophilin 40; PP5, serine/threonine protein phosphatase type 5; GCUNC-45, general cell UNC-45; αSGT, small glutamine-rich tetratricopeptide repeat containing protein α; AR, androgen receptor; ERα, estrogen receptor α; ERβ, estrogen receptor β; GR, glucocorticoid receptor; MR, mineralocorticoid receptor; PR, progesterone receptor; LBD, ligand-binding domain; AF2, activation function 2; GST, glutathione S-transferase.

#### **9. Acknowledgments**

The authors wish to acknowledge support from the National Health & Medical Research Council of Australia, the National Breast Cancer Foundation and the Sir Charles Gairdner Hospital Research Fund. The authors also thank colleagues for permitting citation of their data prior to publication.

#### **10. References**

Hsp90 is required for the proper function of several key regulatory proteins including multiple tyrosine and serine/threonine kinases and steroid receptors, many of which are involved in promoting malignancy (Calderwood *et al.*, 2006; Pearl, 2005; Whitesell & Lindquist, 2005). The aim of targeting and pharmacological manipulation of the Hsp90 chaperoning system has led to the ongoing development and clinical evaluation of novel Hsp90 and chaperone inhibitors for potential application in therapies against selected malignancies (Donnelly *et al.*, 2010; Kim *et al.*, 2009), syndromes arising from dysfunctional protein folding and neurodegenerative diseases (Jinwal *et al.*, 2010). With growing understanding of the novel mechanisms through which Hsp90 cochaperones modulate the function of specific clients, strategies are now evolving for the targeting of chaperone-client

interactions in a wide range of human diseases (De Leon *et al.*, 2011; Gray *et al.*, 2008).

Hsp, heat shock protein; TPR, tetratricopeptide repeat; PPIase, prolylpeptidyl isomerase; FKBP, FK506-binding protein; CyP40, cyclophilin 40; PP5, serine/threonine protein phosphatase type 5; GCUNC-45, general cell UNC-45; αSGT, small glutamine-rich tetratricopeptide repeat containing protein α; AR, androgen receptor; ERα, estrogen receptor α; ERβ, estrogen receptor β; GR, glucocorticoid receptor; MR, mineralocorticoid receptor; PR, progesterone receptor; LBD, ligand-binding domain; AF2, activation function 2; GST,

The authors wish to acknowledge support from the National Health & Medical Research Council of Australia, the National Breast Cancer Foundation and the Sir Charles Gairdner Hospital Research Fund. The authors also thank colleagues for permitting citation of their

Ali M. M. U., Roe S. M., Vaughan C. K., Meyer P., Panaretou B., Piper P. W., Prodromou C.

Allan R. K., Mok D., Ward B. K. & Ratajczak T. (2006). Modulation of chaperone function

Amler L. C., Agus D. B., LeDuc C., Sapinoso M. L., Fox W. D., Kern S., Lee D., Wang V.,

Angeletti P. C., Walker D. & Panganiban A. T. (2002). Small glutamine-rich protein/viral

chaperone complex. *Nature*, Vol.440, No.7087, pp. 1013-1017.

model CWR22-R. *Cancer Res*, Vol.60, No.21, pp. 6134-6141.

activity. *Cell Stress Chaperones*, Vol.7, No.3, pp. 258-268

& Pearl L. H. (2006). Crystal structure of an Hsp90-nucleotide-p23/Sba1 closed

and cochaperone interaction by novobiocin in the C-terminal domain of Hsp90: evidence that coumarin antibiotics disrupt Hsp90 dimerization. *J Biol Chem*,

Leysens M., Higgins B., Martin J., Gerald W., Dracopoli N., Cordon-Cardo C., Scher H. I. & Hampton G. M. (2000). Dysregulated expression of androgen-responsive and nonresponsive genes in the androgen-independent prostate cancer xenograft

protein U-binding protein is a novel cochaperone that affects heat shock protein 70

**8. Abbreviations**

glutathione S-transferase.

**9. Acknowledgments**

data prior to publication.

Vol.281, No.11, pp. 7161-7171.

**10. References**

Calderwood S. K., Khaleque M. A., Sawyer D. B. & Ciocca D. R. (2006). Heat shock proteins

Callahan M. A., Handley M. A., Lee Y.-H., Talbot K. J., Harper J. W. & Panganiban A. T.

Carrello A., Ingley E., Minchin R. F., Tsai S. & Ratajczak T. (1999). The common

Carrigan P. E., Sikkink L. A., Smith D. F. & Ramirez-Alvarado M. (2006). Domain:domain

Chadli A., Felts S. J. & Toft D. O. (2008b). GCUNC-45 is the first Hsp90 co-chaperone to show α/β isoform specificity *J Biol Chem*, Vol.283, No.15, pp. 9509-9512. Chadli A., Graham J. D., Abel M. G., Jackson T. A., Gordon D. F., Wood W. M., Felts S. J.,

Chen M. X. & Cohen P. T. W. (1997). Activation of protein phosphatase 5 by limited

Chen M. X., McPartlin A. E., Brown L., Chen Y. H., Barker H. M. & Cohen P. T. W. (1994). A

Chen S., Prapapanich V., Rimerman R. A., Honore B. & Smith D. F. (1996b). Interactions of

Chen S. & Smith D. F. (1998). Hop as an adaptor in the heat shock protein 70 (Hsp70) and Hsp90 chaperone machinery. *J Biol Chem*, Vol.273, No.52, pp. 35194-35200. Chen S., Sullivan W. P., Toft D. O. & Smith D. F. (1998). Differential interactions of p23 and

Cheung-Flynn J., Prapapanich V., Cox M. B., Riggs D. L., Suarez-Quian C. & Smith D. F.

and Hsp70. *Mol Endocrinol*, Vol.10, No.6, pp. 682-693.

mutants. *Cell Stress Chaperones*, Vol.3, No.2, pp. 118-129.

signaling. *Mol Endocrinol*, Vol.19, No.6, pp. 1654-1666.

protein stability and structure. *Protein Sci*, Vol.15, No.3, pp. 522-532. Chadli A., Bruinsma E. S., Stensgard B. & Toft D. (2008a). Analysis of Hsp90 cochaperone

172.

Vol.72, No.6, pp. 5189-5197.

Vol.47, No.9, pp. 2850-2857.

Vol.271, No.50, pp. 32315-32320.

*Lett*, Vol.400, No.1, pp. 136-140.

pp. 4278-4290.

pp. 2682-2689.

in cancer: chaperones of tumorigenesis. *Trends Biochem Sci*, Vol.31, No.3, pp. 164-

(1998). Functional interaction of human immunodeficiency virus type 1 Vpu and Gag with a novel member of the tetratricopeptide repeat protein family. *J Virol*,

tetratricopeptide repeat acceptor site for steroid receptor-associated immunophilins and Hop is located in the dimerization domain of Hsp90. *J Biol Chem*, Vol.274, No.5,

interactions within Hop, the Hsp70/Hsp90 organizing protein, are required for

interactions reveals a novel mechanism for TPR protein recognition. *Biochemistry*,

Horwitz K. B. & Toft D. (2006). GCUNC-45 is a novel regulator for the progesterone receptor/Hsp90 chaperoning pathway. *Mol Cell Biol*, Vol.26, No.5, pp. 1722-1730. Chen M.-S., Silverstein A. M., Pratt W. B. & Chinkers M. (1996a). The tetratricopeptide

repeat domain of protein phosphatase 5 mediates binding to glucocorticoid receptor heterocomplexes and acts as a dominant negative mutant. *J Biol Chem*,

proteolysis or the binding of polyunsaturated fatty acids to the TPR domain. *FEBS*

novel human protein serine/ threonine phosphatase, which possesses four tetretricopeptide repeat motifs and localizes to the nucleus. *EMBO J*, Vol.13, No.18,

p60, a mediator of progesterone receptor assembly, with heat shock proteins Hsp90

the TPR-containing proteins Hop, Cyp40, FKBP52 and FKBP51 with Hsp90

(2005). Physiological role for the cochaperone FKBP52 in androgen receptor

required to support growth or glucocorticoid receptor activity in *Saccharomyces cerevisiae*. *J Biol Chem*, Vol.273, No.18, pp. 10819-10822.

Dutta S. & Tan Y.-J. (2008). Structural and functional characterization of human SGT and its

Echeverria P. C. & Picard D. (2010). Molecular chaperones, essential partners of steroid

Estébanez-Perpiñá E., Arnold L. A., Nguyen P., Rodrigues E. D., Mar E., Bateman R., Pallai

Fang L., Ricketson D., Getubig L. & Darimont B. (2006). Unliganded and hormone-bound

Febbo P. G., Lowenberg M., Thorner A. R., Brown M., Loda M. & Golub T. R. (2005).

Felts S. J. & Toft D. O. (2003). p23, a simple protein with complex activities. *Cell Stress*

Fliss A. E., Fang Y., Boschelli F. & Caplan A. J. (1997). Differential *in vivo* regulation of

Freeman B. C., Felts S. J., Toft D. O. & Yamamoto K. R. (2000). The p23 molecular chaperones

Freeman B. C. & Yamamoto K. R. (2001). Continuous recycling: a mechanism for modulatory signal transduction. *Trends Biochem Sci*, Vol.26, No.5, pp. 285-290. Freeman B. C. & Yamamoto K. R. (2002). Disassembly of transcriptional regulatory complexes by molecular chaperones. *Science*, Vol.296, No.5576, pp. 2232-2235. Frydman J. & Höhfeld J. (1997). Chaperones get in touch: the Hip-Hop connection. *Trends*

Fuller P. J., Smith B. J. & Rogerson F. M. (2004). Cortisol resistance in the New World

Gallo L. I., Ghini A. A., Piwien Pilipuk G. & Galigniana M. D. (2007). Differential

Gebauer M., Zeiner M. & Gehring U. (1997). Proteins interacting with the molecular

Giannoukos G., Silverstein A. M., Pratt W. B. & Simons Jr S. S. (1999). The seven amino acids

recruitment of tetratricopeptide repeat domain immunophilins to the mineralocorticoid receptor influences both heat-shock protein 90-dependent retrotransport and hormone-dependent transcriptional activity. *Biochemistry*,

chaperone Hsp70/Hsc70: physical associations and effects on refolding activity.

(547-553) of rat glucocorticoid receptor required for steroid and Hsp90 binding

revisited. *Trends Endocrinol Metab*, Vol.15, No.7, pp. 296-299.

*cerevisiae*. *J Biol Chem*, Vol.273, No.18, pp. 10819-10822.

terminal domain. *PNAS*, Vol.103, No.49, pp. 18487-18492.

in prostate cancer. *J Urol*, Vol.173, No.5, pp. 1772-1777.

Vol.47, No.38, pp. 10123-10131.

Vol.1803, No.6, pp. 641-649.

Vol.104, No.41, pp. 16074-16079.

*Chaperones*, Vol.8, No.2, pp. 108-113.

efficacies. *Genes Dev*, Vol.14, 422-434.

*Biochem Sci*, Vol.22, No.3, pp. 87-92.

Vol.46, No.49, pp. 14044-14057.

*FEBS Lett*, Vol.417, No.1, pp. 109-113.

2501-2509.

required to support growth or glucocorticoid receptor activity in *Saccharomyces*

interaction with Vpu of the human immunodeficiency virus type 1. *Biochemistry*,

hormone receptors for activity and mobility. *Biochim Biophys Acta - Mol Cell Res*,

P., Shokat K. M., Baxter J. D., Guy R. K., Webb P. & Fletterick R. J. (2007). A surface on the androgen receptor that allosterically regulates coactivator binding. *PNAS*,

glucocorticoid receptors interact with distinct hydrophobic sites in the Hsp90 C-

Androgen mediated regulation and functional implications of FKBP51 expression

steroid hormone receptor activation by Cdc37p. *Mol Biol Cell*, Vol.8, No.12, pp.

act at a late step in intracellular receptor action to differentially affect ligand

contain a functionally independent LXXLL motif that is critical for steroid binding. *J Biol Chem*, Vol.274, No.51, pp. 36527-36536.

Kallen J., Mikol V., Taylor P. & D.Walkinshaw M. (1998). X-ray structures and analysis of 11

Karagöz G. E., Duarte A. M. S., Ippel H., Uetrecht C., Sinnige T., van Rosmalen M.,

Kauppi B., Jakob C., Farnegardh M., Yang J., Ahola H., Alarcon M., Calles K., Engstrom O.,

Kim Y. S., Alarcon S. V., Lee S., Lee M. J., Giaccone G., Neckers L. & Trepel J. B. (2009).

Knoblauch R. & Garabedian M. J. (1999). Role for Hsp90-associated cochaperone p23 in estrogen receptor signal transduction. *Mol Cell Biol*, Vol.19, No.5, pp. 3748-3759. Kosano H., Stensgard B., Charlesworth M. C., McMahon N. & Toft D. (1998). The assembly

Lassle M., Blatch G. L., Kundra V., Takatori T. & Zetter B. R. (1997). Stress-inducible, murine

Li J., Richter K. & Buchner J. (2011). Mixed Hsp90-cochaperone complexes are important for the progression of the reaction cycle. *Nat Struct Mol Biol*, Vol.18, No.1, pp. 61-66. Liou S.-T. & Wang C. (2005). Small glutamine-rich tetratricopeptide repeat-containing

Louvion J.-F., Warth R. & Picard D. (1996). Two eukaryote-specific regions of Hsp82 are

MacLean M. & Picard D. (2003). Cdc37 goes beyond Hsp90 and kinases. *Cell Stress*

Magee J., Chang L., Stormo G. & Milbrandt J. (2006). Direct, androgen receptor-mediated

Marcu M. G., Chadli A., Bouhouche I., Catelli M. & Neckers L. M. (2000). The heat shock

Matias P. M., Donner P., Coelho R., Thomaz M., Peixoto C., Macedo S., Otto N., Joschko S.,

435-449.

1479-1492.

1884.

No.2, pp. 580-585.

*Chem*, Vol.278, No.25, pp. 22748-22754.

Vol.273, No.49, pp. 32973-32979.

*Biophys*, Vol.435, No.2, pp. 253-263.

*Chaperones*, Vol.8, No.2, pp. 114-119.

Vol.93, No.24, pp. 13937-13942.

No.1, pp. 590-598.

No.47, pp. 37181-37186.

cyclosporin derivatives complexed with cyclophilin A. *J Mol Biol*, Vol.283, No.2, pp.

Hausmann J., Heck A. J. R., Boelens R. & Rüdiger S. G. D. (2010). N-terminal domain of human Hsp90 triggers binding to the cochaperone p23. *PNAS*, Vol.108,

Harlan J., Muchmore S., Ramqvist A.-K., Thorell S., Ohman L., Greer J., Gustafsson J.-A., Carlstedt-Duke J. & Carlquist M. (2003). The three-dimensional structures of antagonistic and agonistic forms of the glucocorticoid receptor ligand-binding domain: RU-486 induces a transconformation that leads to active antagonism. *J Biol*

Update on Hsp90 inhibitors in clinical trial. *Curr Top Med Chem*, Vol.9, No.15, pp.

of progesterone receptor-Hsp90 complexes using purified proteins. *J Biol Chem*,

protein mSTI1. Characterization of binding domains for heat shock proteins and in vitro phosphorylation by different kinases. *J Biol Chem*, Vol.272, No.3, pp. 1876-

protein is composed of three structural units with distinct functions. *Arch Biochem*

dispensable for its viability and signal transduction functions in yeast. *PNAS*,

regulation of the FKBP5 gene via a distal enhancer element. *Endocrinology*, Vol.147,

protein 90 antagonist novobiocin interacts with a previously unrecognized ATPbinding domain in the carboxyl terminus of the chaperone. *J Biol Chem*, Vol.275,

Scholz P., Wegg A., Bäsler S., Schäfer M., Egner U. & Carrondo M. A. (2000a). Structural evidence for ligand specificity in the binding domain of the human androgen receptor: implications for pathogenic gene mutations. *J. Biol. Chem.*, Vol.275, No.34, pp. 26164-26171.

Onuoha S. C., Coulstock E. T., Grossmann J. G. & Jackson S. E. (2008). Structural studies on

Pearl L. H. (2005). Hsp90 and Cdc37 - a chaperone cancer conspiracy. *Curr Opin Genet Dev*,

Periyasamy S., Hinds T., Jr., Shemshedini L., Shou W. & Sanchez E. R. (2010). FKBP51 and

Picard D. (2006). Chaperoning steroid hormone action. *Trends Endocrinol Metab*, Vol.17, No.6,

Picard D., Khursheed B., Garabedian M. J., Fortin M. G., Lindquist S. & Yamamoto K. R.

Pirkl F. & Buchner J. (2001). Functional analysis of the Hsp90-associated human peptidyl

Prapapanich V., Chen S., Nair S. C., Rimerman R. A. & Smith D. F. (1996a). Molecular

and an Hsp70-binding protein. *Mol Endocrinol*, Vol.10, No.4, pp. 420-431. Prapapanich V., Chen S. & Smith D. F. (1998). Mutation of Hip's carboxy-terminal region

Prapapanich V., Chen S., Toran E. J., Rimerman R. A. & Smith D. F. (1996b). Mutational

Pratt W. B. & Toft D. O. (1997). Steroid receptor interactions with heat shock protein and

Pratt W. B. & Toft D. O. (2003). Regulation of signaling protein function and trafficking by

Prodromou C., Panaretou B., Chohan S., Siligardi G., O'Brien R., Ladbury J. E., Roe S. M.,

Prodromou C., Siligardi G., O'Brien R., Woolfson D. N., Regan L., Panaretou B., Ladbury J.

Radanyi C., Chambraud B. & Baulieu E. E. (1994). The ability of the immunophilin FKBP59-

Ramsey A. J. & Chinkers M. (2002). Identification of potential physiological activators of

tetratricopeptide repeat domain. *PNAS*, Vol.91, 11197-11201.

protein phosphatase 5. *Biochemistry*, Vol.41, No.17, pp. 5625-5632.

immunophilin chaperones. *Endocr Rev*, Vol.18, No.3, pp. 306-360.

732-744.

pp. 229-236.

pp. 795-806.

Vol.348, 166-168.

No.2, pp. 944-952.

No.16, pp. 4383-4392.

6207.

111-133.

754-762.

Vol.15, No.1, pp. 55-61.

the co-chaperone Hop and its complexes with Hsp90. *J Mol Biol*, Vol.379, No.4, pp.

Cyp40 are positive regulators of androgen-dependent prostate cancer cell growth and the targets of FK506 and cyclosporin A. *Oncogene*, Vol.29, No.11, pp. 1691-1701.

(1990). Reduced levels of Hsp90 compromise steroid receptor action *in vivo*. *Nature*,

prolyl cis/trans isomerases FKBP51, FKBP52 and CyP40. *J Mol Biol*, Vol.308, No.4,

cloning of human p48, a transient component of progesterone receptor complexes

inhibits a transitional stage of progesterone receptor assembly. *Mol Cell Biol*, Vol.18,

analysis of the hsp70-interacting protein Hip. *Mol Cell Biol*, Vol.16, No.11, pp. 6200-

the Hsp90/Hsp70-based chaperone machinery. *Exp Biol Med*, Vol.228, No.2, pp.

Piper P. W. & Pearl L. H. (2000). The ATPase cycle of Hsp90 drives a molecular 'clamp' via transient dimerization of the N-terminal domains. *EMBO J*, Vol.19,

E., Piper P. W. & Pearl L. H. (1999). Regulation of Hsp90 ATPase activity by tetratricopeptide repeat (TPR)-domain co-chaperones. *EMBO J*, Vol.18, No.3, pp.

HBI to interact with the 90-kDa heat shock protein is encoded by its

resistance in three New World primates. *Gen Comp Endocrinol*, Vol.124, No.2, pp. 152-165.

Schaufele F., Carbonell X., Guerbadot M., Borngraeber S., Chapman M. S., Ma A. A. K.,

Scheufler C., Brinker A., Bourenkov G., Pegoraro S., Moroder L., Bartunik H., Hartl F. U. &

Siligardi G., Hu B., Panaretou B., Piper P. W., Pearl L. H. & Prodromou C. (2004). Co-

Silverstein A. M., Galigniana M. D., Chen M.-S., Owens-Grillo J. K., Chinkers M. & Pratt W.

Silverstein A. M., Galigniana M. D., Kanelakis K. C., Radanyi C., Renoir J.-M. & Pratt W. B.

Sinars C. R., Cheung-Flynn J., Rimerman R. A., Scammell J. G., Smith D. F. & Clardy J.

Skinner J., Sinclair C., Romeo C., Armstrong D., Charbonneau H. & Rossie S. (1997).

Smith D. F. (1993). Dynamics of heat shock protein 90-progesterone receptor binding and the

Smith D. F. (2004). Tetratricopeptide repeat cochaperones in steroid receptor complexes. *Cell*

Smith D. F. & Toft D. O. (2008). Minireview. The intersection of steroid receptors with

Sullivan W. P. & Toft D. O. (1993). Mutational analysis of hsp90 binding to the progesterone

Tai P. K., Albers M. W., Chang H., Faber L. E. & Schreiber S. L. (1992). Association of a 59-

Takayama S., Bimston D. N., Matsuzawa S.-i., Freeman B. C., Aime-Sempe C., Xie Z.,

152-165.

199-210.

868-873.

Vol.102, No.28, pp. 9802-9807.

*Chem*, Vol.279, No.50, pp. 51989-51998.

*Biol Chem*, Vol.272, No.26, pp. 16224-16230.

Vol.274, No.52, pp. 36980-36986.

*Chem*, Vol.272, No.36, pp. 22464-22471.

*Stress Chaperones*, Vol.9, No.2, pp. 109-121.

Vol.256, No.5061, pp. 1315-1318.

receptor. *J Biol Chem*, Vol.268, No.27, pp. 20373-20379.

Hsp70/Hsc70. *EMBO J*, Vol.16, No.16, pp. 4887-4896.

No.11, pp. 1418-1429.

pp. 2229-2240.

resistance in three New World primates. *Gen Comp Endocrinol*, Vol.124, No.2, pp.

Miner J. N. & Diamond M. I. (2005). The structural basis of androgen receptor activation: intramolecular and intermolecular amino–carboxy interactions. *PNAS*,

Moarefi I. (2000). Structure of TPR domain-peptide complexes: critical elements in the assembly of the Hsp70-Hsp90 multichaperone machine. *Cell*, Vol.101, No.2, pp.

chaperone regulation of conformational switching in the Hsp90 ATPase cycle. *J Biol*

B. (1997). Protein phosphatase 5 is a major component of glucocorticoid receptor·Hsp90 complexes with properties of an FK506-binding immunophilin. *J*

(1999). Different regions of the immunophilin FKBP52 determine its association with the glucocorticoid receptor, Hsp90, and cytoplasmic dynein. *J Biol Chem*,

(2003). Structure of the large FK506-binding protein FKBP51, an Hsp90-binding protein and a component of steroid receptor complexes. *PNAS*, Vol.100, No.3, pp.

Purification of a fatty acid-stimulated protein-serine/threonine phosphatase from bovine brain and its identification as a homolog of protein phosphatase 5. *J Biol*

disactivation loop model for steroid receptor complexes. *Mol Endocrinol*, Vol.7,

molecular chaperones: observations and questions. *Mol Endocrinol*, Vol.22, No.10,

kilodalton immunophilin with the glucocorticoid receptor complex. *Science*,

Morimoto R. I. & Reed J. C. (1997). BAG-1 modulates the chaperone activity of

essential to uterine reproductive physiology controlled by the progesterone receptor A isoform. *Mol Endocrinol*, Vol.20, No.11, pp. 2682-2694.

### **The TPR Motif as a Protein Interaction Module – A Discussion of Structure and Function**

Natalie Zeytuni and Raz Zarivach

*Department of Life Sciences, Ben-Gurion University of the Negev and the National Institute for Biotechnology in the Negev (NIBN) Beer sheva, Israel*

#### **1. Introduction**

102 Protein Interactions

Young E. T., Saario J., Kacherovsky N., Chao A., Sloan J. S. & Dombek K. M. (1998).

receptor A isoform. *Mol Endocrinol*, Vol.20, No.11, pp. 2682-2694.

538.

essential to uterine reproductive physiology controlled by the progesterone

Characterization of a p53-related activation domain in Adr1p that is sufficient for ADR1-dependent gene expression. *J Biol Chem*, Vol.273, No.48, pp. 32080-32087. Zhang M., Windheim M., Roe S. M., Peggie M., Cohen P., Prodromou C. & Pearl L. H.

(2005). Chaperoned ubiquitylation--crystal structures of the CHIP U Box E3 ubiquitin ligase and a CHIP-Ubc13-Uev1a complex. *Mol Cell*, Vol.20, No.4, pp. 525-

> Many biological functions involve the formation of protein-protein complexes. Indeed, protein–protein interactions are considered to be the center of all functional living cells. Proteins can interact with each other by various structural, chemical and physical means. Some of these interactions can be highly specific, accompanied by high affinity, while some proteins are more flexible and bind diverse proteins as ligands. A common group of proteins that participate in protein-protein interactions and serve as multi-protein complex mediators are the tetra-trico-peptide repeat (TPR) proteins. TPR-containing proteins were found to be involved in many diverse processes in eukaryotic cells, including synaptic vesicle fusion (Young et al. 2003), peroxisomal targeting and import (Brocard et al. 2006; Fransen et al. 2008) and mitochondrial and chloroplast import (Baker et al. 2007; Mirus et al. 2009). In addition, TRPs are required for many bacterial pathways, such as outer membrane assembly (Gatsos et al. 2008), bio-mineralization of iron oxides in magnetotactic bacteria (Zeytuni et al. 2011) and pathogenesis ( Edqvist et al. 2006; Tiwari et al. 2009). In addition, mutations in TPR-containing proteins have been linked to a variety of human diseases, such as chronic granulomatous disease and Leber's congenital amaurosis (D'Andrea & Regan 2003).

> To date, more than 5,000 TPR-containing proteins were identified in different organisms by bioinformatics tools. Of these, more than 100 structures have been determined and deposited in the Protein Data Bank. These structures demonstrate the tendency of TPR motifs to exist as an independent fold or as a segment of a fold within a protein. The available structures allow the study of a protein-protein interaction platform at atomic resolution. In general, TPR-containing proteins can serve as a study case for protein interactions as they display great binding variety.

> In this chapter, we describe the basic TPR sequence and structure, as well as several examples of diverse TPR binding properties. These include:

#### **2. TPR sequence and basic structure**

The TPR represents a structural motif consisting of 34 amino acids, sharing a degenerate consensus sequence defined by a pattern of small and large hydrophobic amino acids. In this consensus sequence, there are no completely invariant residual positions. The consensus pattern of conserved residues involves positions 4, 7, 8, 11, 20, 24, 27 and 32, in reference to the single motif N-terminal (Fig. 1A). Residues type is highly conserved only at positions 8 (Gly or Ala), 20 (Ala) and 27 (Ala). The rest of the consensus positions displays a preference for small, large or aromatic amino acids rather than for a specific residue. In addition, important structural characteristic conservation can be found at the turns located between two helical segments which contain helix-breaking residues (D'Andrea & Regan 2003). Nowadays, TPR consensus sequences can be identified by most general sequence analysis programs, such as the Simple Modular Architecture Research Tool (SMART) or the PROSITE dictionary of protein sites and motif patterns. At the same time TPRpred is a specially designated tool that uses the profile representation of the known repeats to detect TPR motifs and other patterns of protein repeats.

The canonical unit of the TPR motif adopts a basic helix-turn-helix fold (Fig. 1B). Adjacent TPR units packed in parallel create a series of repeating anti-parallel α-helices that give rise to an overall super-helix structure. This super-helical twist is affected by the type of residue found between adjacent TPR motifs. The unique super-helix fold forms a pair of concave and convex curved surfaces (Fig. 1C). Concave and convex surfaces display some extent of flexibility, as well as amino acid variety, which permits the binding of diverse ligands, usually via the concave surface.

In today's era of complete genome sequencing, one can predict TPR-containing protein dispersion throughout different organisms. Indeed, TPR proteins were found to be common in all forms of life, namely bacteria, archaea and eukaryotes. In nature, TPR motifs can be found in tandem arrays of 3-16 sequential motifs within a given protein (Fig. 1D). Moreover, Kajander et al. (2007) designed and determined the structure of a non-natural recombinant TPR-containing protein incorporating 20 sequential motifs. By using an increasing number of repeat-containing proteins, these authors found a positive correlation between the number of repeats and protein thermo-stability.

#### **3. Ligand binding diversity**

TPR-containing proteins bind diverse ligands in different binding pockets. These ligands usually do not share sequence or secondary structure similarities. Despite the lack of a set of defined rules, binding is usually highly specific, with TPR-containing proteins able to identify their ligand within the dense cellular environment. To obtain such diverse binding,

4. Protein surface electrostatic potential distribution and the contribution of such

5. Multiple binding pockets which allows TPR-containing proteins to serve as mediators

The TPR represents a structural motif consisting of 34 amino acids, sharing a degenerate consensus sequence defined by a pattern of small and large hydrophobic amino acids. In this consensus sequence, there are no completely invariant residual positions. The consensus pattern of conserved residues involves positions 4, 7, 8, 11, 20, 24, 27 and 32, in reference to the single motif N-terminal (Fig. 1A). Residues type is highly conserved only at positions 8 (Gly or Ala), 20 (Ala) and 27 (Ala). The rest of the consensus positions displays a preference for small, large or aromatic amino acids rather than for a specific residue. In addition, important structural characteristic conservation can be found at the turns located between two helical segments which contain helix-breaking residues (D'Andrea & Regan 2003). Nowadays, TPR consensus sequences can be identified by most general sequence analysis programs, such as the Simple Modular Architecture Research Tool (SMART) or the PROSITE dictionary of protein sites and motif patterns. At the same time TPRpred is a specially designated tool that uses the profile representation of the known repeats to detect

The canonical unit of the TPR motif adopts a basic helix-turn-helix fold (Fig. 1B). Adjacent TPR units packed in parallel create a series of repeating anti-parallel α-helices that give rise to an overall super-helix structure. This super-helical twist is affected by the type of residue found between adjacent TPR motifs. The unique super-helix fold forms a pair of concave and convex curved surfaces (Fig. 1C). Concave and convex surfaces display some extent of flexibility, as well as amino acid variety, which permits the binding of diverse ligands,

In today's era of complete genome sequencing, one can predict TPR-containing protein dispersion throughout different organisms. Indeed, TPR proteins were found to be common in all forms of life, namely bacteria, archaea and eukaryotes. In nature, TPR motifs can be found in tandem arrays of 3-16 sequential motifs within a given protein (Fig. 1D). Moreover, Kajander et al. (2007) designed and determined the structure of a non-natural recombinant TPR-containing protein incorporating 20 sequential motifs. By using an increasing number of repeat-containing proteins, these authors found a positive correlation between the

TPR-containing proteins bind diverse ligands in different binding pockets. These ligands usually do not share sequence or secondary structure similarities. Despite the lack of a set of defined rules, binding is usually highly specific, with TPR-containing proteins able to identify their ligand within the dense cellular environment. To obtain such diverse binding,

distribution to the diverse binding properties of TPRs.

6. The broad distribution of homo-oligomerization states in TPR proteins.

in multi-protein complexes.

7. Ligand computational docking. 8. TPR proteins and biotechnology.

**2. TPR sequence and basic structure**

TPR motifs and other patterns of protein repeats.

number of repeats and protein thermo-stability.

usually via the concave surface.

**3. Ligand binding diversity**

Fig. 1. TPR motif sequence and structure. (A) Schematic representation of the secondary structure arraignment of the 34 amino acids in a TPR motif. Numbers represent the conserved positions of amino acids within the motif. (B) The basic helix-turn-helix fold of a TPR motif canonical unit. (C) Surface representation of a TPR-containing protein displaying concave and convex surfaces. (D) Representative TPR-containing protein structures. From left, the TPR domain of Hop, containing three sequential motifs, MamA, containing five sequential TPR motifs, and the super-helix forming O-linked *N-*acetylglucosamine transferase TPR domain, containing 11 sequential TPR motifs, are shown in side and top view, in blue, green and orange respectively. Images B, C and D were generated by Pymol software (www.pymol.org).

several TPR-distinct folds serve as an interaction platform. This platform can display different surface residues in each binding surface, which later interacts in a specific manner with the ligand. Additionally, residue type influences the electrostatic nature of the binding surface, with, for example, arginine and lysine contributing positive charges, whereas glutamic acid and aspartic acid contribute negative charges. In addition, residues of different hydrophobicity and size can support hydrophobic interactions between the TPR protein and its ligand and therefore enhance protein-ligand specificity. Overall, the available TPR protein structures showing interaction with their ligands show that binding specificity cannot be attributed to a single force and that it is usually achieved by a combination of factors, such as residue type, hydrophobic pockets, charge and electrostatics.

#### **3.1 Ligand sequence diversity**

In this section, we describe two relatively simple TPR ligand interactions to demonstrate the multiple interaction types involved in binding and the correlation between TPR binding surfaces to the amino acid sequence of their ligands.

The first released structures of TPR domains bound to their ligand peptides were the two domains of the Hop protein (Scheufler et al. 2000). Hop is an adaptor protein which mediates the association of the molecular chaperones, Hsp70 and Hsp90. The TPR1 domain of Hop specifically recognizes the C-terminal heptapeptide of Hsp70, whereas the TPR2A domain binds the C-terminal pentapeptide of Hsp90 (Figs. 2A&B). Both C-terminal peptides share EEVD sequence ends and were found to bind in an extended conformation. An extended conformation allows the display of a maximal surface to the TPR domain and facilitates specific recognition of short amino acid stretches with sufficient affinity. Examination of solved crystal structures revealed that all electrostatic contacts between TPR domains and both peptides involve the EEVD sequence motif. These interactions include three classes of hydrogen bonding interaction, namely sequence-independent interactions with the peptide backbone, specific interactions with peptide side chains and the carboxylate of the C -terminal residue interaction with the TPR (Figs. 2C&D). The three strong hydrogen bonds formed between the peptide C-terminal carboxylate and the TPR1 and TPR2A domains conserved residues, Lys 8 (Lys 229), Asn 12 (Asn 233) and Asn 43 (Asn 264) respectively, allows the formation of a two carboxylate clamp. This clamp ensures the proper docking of the peptide ends to the TPR domains. Additional peptide residues located at the N-terminal, relative to EEVD motif, are exclusively engaged in hydrophobic and van der Waal's interactions. These contacts were found to be critical for peptide binding with a physiologically relevant high affinity. Other TPR domains which are known to bind Hsp70/Hsp90 proteins also contains identical residues which form the carboxylate clamp, suggesting that these TPR domains bind to the C-terminal carboxylate via a similar network of electrostatic interactions. The Hop and Hsp70/Hsp90 case studies presented here also demonstrate the importance of sequence conservation between TPR domains and their ligands involved in similar cellular functions.

Other example for the importance of TPR-ligand sequence conservation can be found in the peroxin 5 (PEX5) protein from the protozoan parasite, *Trypanasomas brucei*, the agent of human African trypanosomiasis (sleeping sickness). PEX5 is a cytosolic receptor which promotes cargo translocation across the glycosomic membrane, with the glycosome being a peroxisomelike organelle which hosts the metabolic reactions of the parasite. Two domains comprise PEX5, with the C-terminal domain consisting of 7 TPR motifs. The PEX5 C-terminal domain binds either the type 1 (PTS1) or type 2 (PTS2) peroxisomal targeting signal. The more common PTS1 sequence is a C-terminal tripeptide with the SKL amino acid sequence, or variants thereof, such as AKL or SHL. Additional residues upstream to the SKL sequence have also been implicated in binding to PEX5. The crystal structure of the C-terminal domain of PEX5 with five different PST1 fragments revealed that the protein does not fold as a sequential TPR protein with classic super-helical fold, but rather as two distinct sub-domains (Sampathkumar et al. 2008). The N-terminal sub-domain comprises TPR motifs 1-3 while the C-terminal sub-domain comprises TPR motifs 5-7. The fourth TPR motif that serves to interconnect the two sub-domains is only partial ordered and cannot be seen in the electron density resulted from the X-ray determination (Fig. 3A). The disordered nature of the fourth TPR motif implies its flexible tendencies and involvement in ligand-induced conformational

In this section, we describe two relatively simple TPR ligand interactions to demonstrate the multiple interaction types involved in binding and the correlation between TPR binding

The first released structures of TPR domains bound to their ligand peptides were the two domains of the Hop protein (Scheufler et al. 2000). Hop is an adaptor protein which mediates the association of the molecular chaperones, Hsp70 and Hsp90. The TPR1 domain of Hop specifically recognizes the C-terminal heptapeptide of Hsp70, whereas the TPR2A domain binds the C-terminal pentapeptide of Hsp90 (Figs. 2A&B). Both C-terminal peptides share EEVD sequence ends and were found to bind in an extended conformation. An extended conformation allows the display of a maximal surface to the TPR domain and facilitates specific recognition of short amino acid stretches with sufficient affinity. Examination of solved crystal structures revealed that all electrostatic contacts between TPR domains and both peptides involve the EEVD sequence motif. These interactions include three classes of hydrogen bonding interaction, namely sequence-independent interactions with the peptide backbone, specific interactions with peptide side chains and the carboxylate of the C -terminal residue interaction with the TPR (Figs. 2C&D). The three strong hydrogen bonds formed between the peptide C-terminal carboxylate and the TPR1 and TPR2A domains conserved residues, Lys 8 (Lys 229), Asn 12 (Asn 233) and Asn 43 (Asn 264) respectively, allows the formation of a two carboxylate clamp. This clamp ensures the proper docking of the peptide ends to the TPR domains. Additional peptide residues located at the N-terminal, relative to EEVD motif, are exclusively engaged in hydrophobic and van der Waal's interactions. These contacts were found to be critical for peptide binding with a physiologically relevant high affinity. Other TPR domains which are known to bind Hsp70/Hsp90 proteins also contains identical residues which form the carboxylate clamp, suggesting that these TPR domains bind to the C-terminal carboxylate via a similar network of electrostatic interactions. The Hop and Hsp70/Hsp90 case studies presented here also demonstrate the importance of sequence conservation between TPR domains and their

Other example for the importance of TPR-ligand sequence conservation can be found in the peroxin 5 (PEX5) protein from the protozoan parasite, *Trypanasomas brucei*, the agent of human African trypanosomiasis (sleeping sickness). PEX5 is a cytosolic receptor which promotes cargo translocation across the glycosomic membrane, with the glycosome being a peroxisomelike organelle which hosts the metabolic reactions of the parasite. Two domains comprise PEX5, with the C-terminal domain consisting of 7 TPR motifs. The PEX5 C-terminal domain binds either the type 1 (PTS1) or type 2 (PTS2) peroxisomal targeting signal. The more common PTS1 sequence is a C-terminal tripeptide with the SKL amino acid sequence, or variants thereof, such as AKL or SHL. Additional residues upstream to the SKL sequence have also been implicated in binding to PEX5. The crystal structure of the C-terminal domain of PEX5 with five different PST1 fragments revealed that the protein does not fold as a sequential TPR protein with classic super-helical fold, but rather as two distinct sub-domains (Sampathkumar et al. 2008). The N-terminal sub-domain comprises TPR motifs 1-3 while the C-terminal sub-domain comprises TPR motifs 5-7. The fourth TPR motif that serves to interconnect the two sub-domains is only partial ordered and cannot be seen in the electron density resulted from the X-ray determination (Fig. 3A). The disordered nature of the fourth TPR motif implies its flexible tendencies and involvement in ligand-induced conformational

**3.1 Ligand sequence diversity**

surfaces to the amino acid sequence of their ligands.

ligands involved in similar cellular functions.

changes that can promote cargo translocation. The PTS1 peptide is bound within the cavity found between the two sub-domains and interacts with residues from both, although the major binding contribution is attributed to the C-terminal sub-domain. The five PEX5 structures bound to five different peptides containing PTS1 sequences indicate that the ligand recognition mechanism involves three critical factors. The first is recognition of the C-terminal

Fig. 2. TPR domains of the Hop protein and their interacting Hsp70/Hsp90 partner peptides. (A) The TPR2A domain of the Hop protein in complex with a Hsp90-derived peptide. (B) The TPR1 domain of the Hop protein in complex with a Hsp70-derived peptide. (C) Schematic representation of the TPR2A-hsp90 peptide interaction. (D) Schematic representation of the TPR1-Hsp70 peptide interaction. Images A and B were generated using Pymol software (www.pymol.org). Images C and D were generated using LigPlot software (Wallace et al. 1995).

Fig. 3. TPR domains of PEX5 protein and their interacting PST1 partner peptides. (A) TPR domains of PEX5 in complex with a PST1 peptide (NFNELSHLC). PEX5 is represented as a cartoon, whereas the N-terminal domain, the fourth TPR motif and C- terminal domain are colored in blue, green and orange, respectively. The PST1 peptide is represented as sticks in yellow. (B) Schematic representation of the PEX5-PST1 peptide interactions. (C) Superposition of five PST1 peptides bound to PEX5, in green, blue, pink, yellow and light pink. Images A and C were generated using Pymol software (www.pymol.org). Image B was generated using LigPlot software (Wallace et al. 1995).

PST1 carboxylate in a similar manner to the Hop and Hsp70/Hsp90 complex by two PEX5 Asn residues and a single Arg residue, the second is hydrophobic embedding of the PTS1 Cterminal residue side chain and the third is multiple PTS1 backbone interactions with PEX5 Asn side chains (Fig. 3B). Overall, PTS1 peptides are bound in a similar manner despite substantial differences in amino acid composition. In addition, the spatial positions of the five Asn residues and a single Arg residue of PEX5 involved in backbone binding of PTS1 peptides are similar, emphasizing their significant function in diverse ligand binding (Fig. 3C).

#### **3.2 Ligand secondary structure and length diversity**

108 Protein Interactions

Fig. 3. TPR domains of PEX5 protein and their interacting PST1 partner peptides. (A) TPR domains of PEX5 in complex with a PST1 peptide (NFNELSHLC). PEX5 is represented as a cartoon, whereas the N-terminal domain, the fourth TPR motif and

yellow and light pink. Images A and C were generated using Pymol software

C- terminal domain are colored in blue, green and orange, respectively. The PST1 peptide is represented as sticks in yellow. (B) Schematic representation of the PEX5-PST1 peptide interactions. (C) Superposition of five PST1 peptides bound to PEX5, in green, blue, pink,

(www.pymol.org). Image B was generated using LigPlot software (Wallace et al. 1995).

Secondary structure of a TPR-bound ligand varies between the coiled extended conformation to an α-helix or both. As mentioned previously, an extended conformation maximizes the ligand surface to the TPR domain and facilitates specific recognition of short amino acid stretches with sufficient affinity. Two examples of extended conformation peptide binding are Hop bound to Hsp70/Hsp90 peptides (Fig. 4A) and PEX5 bound to PTS1 peptides. Three TPRcontaining proteins bound to their ligands display binding of long peptides that adopt both helical and extended conformations. The first structure of such binding is PscG-PscE in complex with the PscF peptide (Quinaud et al. 2007). PscG-PscE–PscF proteins are members of the bacterial Type III secretion system and play a specific role in formation of the needle that transports virulence effectors into target cell cytoplasm. PscG displays a three TPR motif fold with an additional C-terminal helix, even though its TPR fold could not have been predicted from its sequence. PscG also interacts with PscE through a hydrophobic platform formed by the N-terminal TPR motif of PscG. PscF is composed of two sub-domains, an extended coil (13 amino acids-long) and a C-terminal helix (17 amino acids-long). PscG and PscE fold into a cupped-hand form, whereas the amphipathic C-terminal helix of PscF is bound to the concave surface of PscG. The N-terminal of PscF is bound to the PscG convex surface (Fig. 4B).

The second structure displaying binding of a long peptide which adopts both helical and extended conformations is the TPR-containing protein, APC6, in complex with CDC26 (Wang et al. 2009). APC6 and CDC26 are both members of the multi-subunit anaphasepromoting complex (APC), an essential cell-cycle regulator. The crystallized APC6 TPR domain contains eight full TPR motifs and an additional C-terminal helix. APC6 adopts a solenoid-like structure, wrapping around the entire length of N-terminal region of CDC26 (26 amino acids). The bound CDC26 forms a rod-like structure with the first 12 amino acids adopting an extended conformation and the last 14 amino acids forming a helix (Fig. 4C). Interestingly, as the CDC26 peptide interacts with APC6, an additional non-TPR C-terminal helix with geometry that mimics two helices in a TPR motif appears. This inter-molecular TPR mimic continues the sinuous form of the overall structure and packs against the eight TPR motifs of APC6 to form a four-helix bundle.

A third structure displaying binding of two helices connected by a loop to a TPR-containing protein is Fis1 in complex with Caf4 (Zhang et al. 2007). Both Fis1 and Caf4 participate in the formation of yeast mitochondrial fission complex that controls the shape and physiology of the mitochondria. The Fis1 protein core is composed of two TPR motifs with two additional helices at each motif end and another N-terminal helix arm packed against the hydrophobic groove formed by the protein core. The Caf4 peptide adopts a U-fold with two helices formed at each end connected by a loop. The unique U-fold of Caf4 allows large scale interactions between the peptide and both the Fis1 core at the concave and convex surfaces (Fig. 4D).

Although these are only a few examples of TPR binding modes reflecting secondary structure conformations and peptides lengths, they describe well the diverse nature of ligand types that theTPR platform can bind.

Fig. 4. TPR ligand secondary structure and length diversity. (A) TPR2A domain of Hop is shown in surface representation with secondary structure indicated in light pink, bound to Hsp90, in green. (B) A surface representation of a PscG-PscE dimer shown with secondary structure indicated in light blue and purple, respectively. PscF, in pink, is bound to the PscG-PscE dimer. (C) APC6 is shown in surface representation with secondary structure indicated in light green, bound to CDC26, in red. (D) Caf4 is shown in surface representation with secondary structure indicated in pink, bound to Fis1, in dark blue. Images were generated using Pymol software (www.pymol.org).

#### **3.3 Curvature angle role and diversity**

110 Protein Interactions

Although these are only a few examples of TPR binding modes reflecting secondary structure conformations and peptides lengths, they describe well the diverse nature of

Fig. 4. TPR ligand secondary structure and length diversity. (A) TPR2A domain of Hop is shown in surface representation with secondary structure indicated in light pink, bound to Hsp90, in green. (B) A surface representation of a PscG-PscE dimer shown with secondary structure indicated in light blue and purple, respectively. PscF, in pink, is bound to the PscG-PscE dimer. (C) APC6 is shown in surface representation with secondary structure

representation with secondary structure indicated in pink, bound to Fis1, in dark blue.

indicated in light green, bound to CDC26, in red. (D) Caf4 is shown in surface

Images were generated using Pymol software (www.pymol.org).

ligand types that theTPR platform can bind.

Major diversity can be seen in the overall shape and curvature angle displayed by TPRcontaining proteins. There are many factors that determine the overall shape and curvatures angle of a protein. Among these are the number of repeating motifs, their arrangement as sequential motifs or separation into sub-domains, the presence of helix-breaking residues within turns between motifs, as well as residues type, protein function and others. Despite the apparent significance, no extensive study addressing the overall shape and curvature angle displayed by TPR-containing proteins has been reported. Indeed, articles presenting new TPR protein structures hardly refer to this property. The majority of TPR protein structures were determined by X-ray crystallography, a method that favors ordered proteins and can only tolerate a low extant of protein flexibly within the crystal. Since proteins and protein interactions are usually flexible and require the partners to be rigid, the use of X-ray crystallography to study protein interactions can be quite challenging. To overcome this challenge, structural biologists often use a relatively short peptide from the whole binding partner that participates directly in recognition and binding in their experiments. By using a small portion of the binding partner, the overall flexibility is reduced throughout the crystal, a trait that later improves crystallization and crystal quality. Due to method limitations, we usually obtain a close look at the detailed interaction and the forces participating in such interactions but cannot see the complete interaction or the spatial positions of the partners, nor detect remote stabilizing interactions. This incomplete observation of interactions between partners creates substantial difficulty in determining the exact role and importance of the curvature angle. Perhaps in the future, when new structure determination techniques emerge, a better understanding of the role of the curvature angle in TPR protein recognition and binding will be achieved.

#### **4. TPR homo-oligomerization**

TPR-containing proteins serve as mediators in the formation of multi-protein complexes. Within these complexes, TPR proteins or domains can be found as homo-oligomers, with oligomerization serving as a crucial factor for realizing proper protein function in the cell. TPR proteins displays a broad range of oligomerization states, such as monomers (Scheufler et al. 2000; Sampathkumar et al. 2008) and dimers (Lunelli et al. 2009; Z. Zhang et al. 2010). It is of note that a complex containing 24-26 TPR protein monomers has been described (Zeytuni et al. 2011). The protein surfaces involved in oligomer formation are diverse, as are the inter-molecule interaction types seen between monomers. In the following section, three interesting protein oligomerization forms will be presented.

The first TPR-containing protein displaying a dimer formation to be considered is invasion plasmid gene C (IpgC), a chaperone that binds two essential virulence factors of the pathogenic bacteria, *Shigella* (Lunelli et al. 2009). IpgC binds the invasion plasmid antigens, IpaB and IpaC which are responsible for epithelial cell invasion, membrane lysis of the phagocytic vacuole, contact hemolysis and macrophage cell death. The IpgC chaperone contains three TPR motifs, with two additional helices at the protein N-terminal and another helix at the protein C-terminal. The protein functional unit that allows efficient substrate binding is a dimer, with dimerization occuring in an asymmetric manner. Such asymmetric dimerization presents an interesting binding mode in which the first helix of one of the monomers binds on the convex surface presented by the other monomer (Fig. 5A). While the

Fig. 5. Homo-oligomerization by -ontaining proteins. (A) Cylinder representation of a IpgC dimer, in green and purple, where one monomer is bound to an IpgB peptide, in pink. (B) The N-terminal domain of Cut9 promotes dimerization. Cylinder representation of the N-terminal domain of each monomer, shown in cyan and light pink. C-terminal domains are in blue and light purple. Hcn1 peptides are colored in green and yellow. (C) Transmission electron microscopy images of negatively stained MamA protein complexes. Images A, B were generated using Pymol software (www.pymol.org). The images shown 4C were taken by Zeytuni N. as described in Zeytuni et al. (2011).

Fig. 5. Homo-oligomerization by -ontaining proteins. (A) Cylinder representation of a IpgC dimer, in green and purple, where one monomer is bound to an IpgB peptide, in pink. (B) The N-terminal domain of Cut9 promotes dimerization. Cylinder representation of the N-terminal domain of each monomer, shown in cyan and light pink. C-terminal domains

Transmission electron microscopy images of negatively stained MamA protein complexes. Images A, B were generated using Pymol software (www.pymol.org). The images shown 4C

are in blue and light purple. Hcn1 peptides are colored in green and yellow. (C)

were taken by Zeytuni N. as described in Zeytuni et al. (2011).

first helix of one of the monomers displays inter-molecule binding, the first helix of the bound monomer appears to be found in a more packed, unbound conformation. The formation of a dimer interface at the TPR concave surface allows the binding of the IpaB peptide at the concave surface in an extended conformation. Moreover, the mode of IpgC dimerization demonstrates the significance of a convex surface as an additional protein interaction platform.

A second TPR-containing protein displaying dimer formation is *Schizosaccharomyces pombe* Cut9, the yeast homolog of human APC6. The Cut9 structure was determined while bound to Hcn1, the yeast homolog of human CDC26 in a similar binding conformation as APC6 in complex with CDC26 (Z. Zhang et al. 2010). Cut9 is composed of 14 TPR motifs forming a contiguous super-helix divided into two functionally and structurally distinct domains, namely an N-terminal domain comprising the first seven TPR motifs and a C-terminal domain comprising the last seven TPR motifs. The Cut9 subunits homo-dimerize through their N-terminal domains to generate a shallow 'V'-shaped molecule (Fig. 5B). The more globular N-terminal dimerization module forms the apex of the 'V'-shape, with narrower Cterminal TPR domains projecting away from the dimer interface. The majority of Cut9 interactions with Hcn1 involve the C-terminal TPR-containing domain. Dimerization of the N-terminal domains forms a tight interface in which the concave surface of each N-terminal domain encircles its dimer counterpart in an inter-lock clasp-like arrangement. In these interactions, the first two TPR motifs of a single monomer interact with residues lining the inner groove formed by the seven TPR motifs of the opposite Cut9 N-terminal domain.

An additional example involving dimerization is the TPR-containing protein, MamA, from *Magnetospirillium* magnetotactic bacteria species. MamA forms large homo-oligomeric complex of 24-26 monomers. This complex is presumed to serve as a wide platform for protein interaction during the iron-oxide bio-mineralization by the magnetotactic bacteria (Zeytuni et al. 2011). MamA contains five TPR motifs with an additional N-terminal putative TPR motif that was found to be responsible for oligomerization and complex formation. Through binding of the first helix of a single monomer to a binding surface displayed on a different monomer, a round-shaped complex of 14-20 nm in diameter with a central pore cavity is formed (Fig. 5C). The structural details of the monomer-monomer interaction remain unclear, since crystallization trials of MamA in complex with peptides of the first and/or second helices proved unsuccessful.

Overall, these three examples of homo-oligomrization by TPR-containing proteins describe additional TPR diversity and further establish the TPR motif as a broad platform for protein interactions.

#### **5. Predicting TPR-ligand interactions**

Today, available bioinformatics tools and the well-defined TPR profile allow us to identify TPR-containing proteins with great accuracy through analysis of amino acid sequence. However, these tools still cannot predict TPR-interacting partners and/or the region of interaction. As discussed in section 3.3, the majority of TPR-containing protein structures were determined by X-ray crystallography, a methodology in which the crystallization probability of protein complexes is significantly lower than the probability of crystallization of a single protein. Therefore, the majority of determined TPR proteins were crystallized in a non-complex form, not bound to their interacting partner, even if that partner was previously identified. In order to predict ligand binding, one should discriminate between two cases, the first involving an unidentified partner protein and the second involving an unknown binding region. In this section, we discuss these two cases and provide several examples of each.

#### **5.1 Interacting protein prediction**

A genomic approach for TPR-interacting protein identification was first presented by D'Andrea and Regan (2003). In their study, the authors generated a list of all TPR-containing proteins predicted from the *Saccharomyces cerevisiae* genome. Later, they used the generated list of 22 predicted TPR-containing proteins in protein-protein interaction databases to identify potential binding partners. Their search revealed about 80 potential interacting proteins, some of which are known to participate in multi-protein complex formation. Nevertheless, these authors could not exclude the possibility that these interacting proteins might not all interact directly with TPR domains within the multi-protein complexes.

Another approach uses information derived from the structures of unbound TPR-containing proteins. Certain properties of the binding partner can be deduced from a simple examination of a TPR protein structure, especially from its concave binding pocket, specifically its dimensions, residue composition and electrostatic potential. Although the concave surface serves as the common binding area, the convex surface can also participate in binding and should not be excluded from predictions.

Our first example is derived from the structure of the super-helical TPR domain of O-linked *N-*acetylglucosamine transferase (OGT). The TPR domain of OGT contains 11 motifs with an additional C-terminal helix and forms a homo-dimer through interactions at the convex surface. The inner surface of the elongated super-helix is highly conserved and contains an asparagine ladder. This asparagine ladder bears marked similarity to the array of conserved asparagines in the ARM-repeat importin α- and β-catenin proteins. In both ARM-repeat proteins, the asparagine side chains contribute to binding of the target peptide by forming hydrogen bonds with the peptide backbone. This structural similarity suggests that a similar binding mechanism for the OGT protein. In addition, the extensive surface generated by OGT is likely to represent several overlapping binding pockets which can accommodate multiple substrates (Fig. 6A). Furthermore, partner binding can rely on a mechanism similar to the mode of binding described earlier, in the case of CDC26 bound to APC6 (see Fig. 4C) (Wang et al. 2009).

Another example of TPR partner prediction is derived from the bacterial YrrB protein structure. YrrB is a *Bacillus subtilis* protein containing five TPR motifs with an additional Cterminal helix. The YrrB structure reveals a unique, highly negatively charged deep concave surface containing an aspartic acid array that can accommodate the binding of positively charged residues (Fig 6B). In order to discover new details on the role of the YrrB protein, functional gene cluster localization analysis was performed. Such analysis suggested that YrrB plays a role in mediating complex formation among RNA sulfuration components, RNA processing components and aminoacyl-tRNA synthetases. An opposite charge distribution to the YrrB protein was found in the TPR-containing protein, MamA, a protein that displays a highly positive concave binding surface, implying the electrostatic charge

non-complex form, not bound to their interacting partner, even if that partner was previously identified. In order to predict ligand binding, one should discriminate between two cases, the first involving an unidentified partner protein and the second involving an unknown binding region. In this section, we discuss these two cases and provide several

A genomic approach for TPR-interacting protein identification was first presented by D'Andrea and Regan (2003). In their study, the authors generated a list of all TPR-containing proteins predicted from the *Saccharomyces cerevisiae* genome. Later, they used the generated list of 22 predicted TPR-containing proteins in protein-protein interaction databases to identify potential binding partners. Their search revealed about 80 potential interacting proteins, some of which are known to participate in multi-protein complex formation. Nevertheless, these authors could not exclude the possibility that these interacting proteins

might not all interact directly with TPR domains within the multi-protein complexes.

in binding and should not be excluded from predictions.

Another approach uses information derived from the structures of unbound TPR-containing proteins. Certain properties of the binding partner can be deduced from a simple examination of a TPR protein structure, especially from its concave binding pocket, specifically its dimensions, residue composition and electrostatic potential. Although the concave surface serves as the common binding area, the convex surface can also participate

Our first example is derived from the structure of the super-helical TPR domain of O-linked *N-*acetylglucosamine transferase (OGT). The TPR domain of OGT contains 11 motifs with an additional C-terminal helix and forms a homo-dimer through interactions at the convex surface. The inner surface of the elongated super-helix is highly conserved and contains an asparagine ladder. This asparagine ladder bears marked similarity to the array of conserved asparagines in the ARM-repeat importin α- and β-catenin proteins. In both ARM-repeat proteins, the asparagine side chains contribute to binding of the target peptide by forming hydrogen bonds with the peptide backbone. This structural similarity suggests that a similar binding mechanism for the OGT protein. In addition, the extensive surface generated by OGT is likely to represent several overlapping binding pockets which can accommodate multiple substrates (Fig. 6A). Furthermore, partner binding can rely on a mechanism similar to the mode of binding described earlier, in the case of CDC26 bound to APC6 (see Fig. 4C)

Another example of TPR partner prediction is derived from the bacterial YrrB protein structure. YrrB is a *Bacillus subtilis* protein containing five TPR motifs with an additional Cterminal helix. The YrrB structure reveals a unique, highly negatively charged deep concave surface containing an aspartic acid array that can accommodate the binding of positively charged residues (Fig 6B). In order to discover new details on the role of the YrrB protein, functional gene cluster localization analysis was performed. Such analysis suggested that YrrB plays a role in mediating complex formation among RNA sulfuration components, RNA processing components and aminoacyl-tRNA synthetases. An opposite charge distribution to the YrrB protein was found in the TPR-containing protein, MamA, a protein that displays a highly positive concave binding surface, implying the electrostatic charge

examples of each.

(Wang et al. 2009).

**5.1 Interacting protein prediction**

nature of ligand bindng (Fig. 6C). Although sharing a similar fold, MamA and YrrB demonstrate yet another variation among TPR-containing proteins, namely charge distribution. In general, binding partner identification is not straightforward and can be challenging, as the information obtained from the genetic approach can point to indirect interactions. Indeed, the structural approach can only provide clues for identifying the specific region of the partner involved in binding.

Fig. 6. Structural analysis of concave binding surface. (A) The TPR domain of O-linked *N*acetylglucosamine transferase is presented as a grey cartoon, while the asparagine ladder within the inner surface of the super-helix is presented as yellow sticks. (B) *left* - YrrB in light orange cartoon. The aspartic acid ladder is presented as green sticks. *right* –Electrostatic surface potential representation, where blue and red represent positive and negative electrostatic potentials, respectively. The YrrB concave surface displays a highly negative potential distribution. (C) *Left* - MamA in light pink cartoon representation. *Right* – Electrostatic surface potential representation, where blue and red represent positive and negative potentials, respectively. The MamA concave surface displays a highly positive potential distribution. Images were generated using Pymol software (www.pymol.org). Surface electrostatic potential calculations were performed using the APBS plug-in (www.poissonboltzmann.org/apbs).

#### **5.2 Interaction region prediction**

Interaction region prediction requires at least two components, the TPR protein structure and the identified binding sequence of the partner. Over the past few years, several attempts to dock ligand peptides onto TPR-containing proteins have been made. These attempts mainly included manual docking of the ligand peptide onto the available TPR protein structure. The resulting models did not, however, consider side chain flexibility and conformational changes due to peptide binding and used limited energy minimization tools (Gatto et al. 2000; Kim et al. 2006). However, with the remarkable development of computational docking servers, more accurate models can be generated for TPR protein–peptide interactions. To date, however, no study has employed these advanced servers to demonstrate TPR-peptide docking, although successful docking have been recorded with other proteins involved in protein-protein interactions, such as PDZ domains and others (London et al. 2010; Raveh et al. 2010; Raveh et al. 2011). Overall, the prediction of interaction region is considered to be more accurate than binding partner prediction, since the former uses algorithms that consider chemical restrains, as well as energy minimization of the final model. In the near future, these tools might aid in overcoming challenges associated with crystallizing proteins with their binding partners and could provide important insight for molecular understanding of binding and recognition.

#### **6. TPR design and biotechnology**

The basic TPR fold resulting from its consensus sequence can be considered as a protein scaffold. Redesigning this stable basic scaffold by grafting functional residues involved in binding recognition and specificity enables the introduction of novel binding activities. TPR design includes three major steps. The first stage includes stable consensus scaffold generation by an alignment of natural TPR motifs (D'Andrea et al. 2003). Later, the minimal number of repeating motifs required for thermodynamic stability are determined (Kajander et al. 2007) and finally, functional residues are grafted onto the generated scaffold. An example for a successful designing process was the designed TPR module that binds the Cterminal peptide of Hsp90. This module has been designed by grafting Hsp90-binding residues from natural TPR proteins onto a consensus TPR scaffold to bind Hsp90 with greater affinity and specificity than natural co-chaperones. Introduction of this designed protein into breast cancer cells inhibited Hsp90 activity, presumably by out-competing the interaction of Hsp90 with its natural co-chaperones. Hsp90 inhibition resulted in misfolding and degradation of Hsp90-dependent proteins, such as HER2, and led to cancer cell death (Cortajarena et al. 2008).

TPR binding properties can also be used for specific identification of tagged proteins and can act as a functional substitute for antibodies in a wide range of applications (Jackrel et al. 2009). Conjugation of a peptide ligand sequence to the N or C termini of a desirable protein allows its identification by a TPR-containing protein that can bind the ligand peptide with sufficient affinity. This TPR-containing protein can be conjugated to a reporter protein, such as horseradish peroxidase, biotin or green fluorescent protein and can be later used in one or two-step western blot detection systems, replacing any requirement for antibodies. In addition, conjugation of a TPR-containing protein or domain directly to resin or beads can be used for affinity purification. Immobilizing a TPR-containing protein onto a resin permits the specific binding and enrichment of desirable proteins conjugated to a peptide ligand. Interaction dissociation at the elution step does not require extremely harsh pH conditions as may be needed with the use of antibodies.

#### **7. Concluding remarks**

116 Protein Interactions

attempts to dock ligand peptides onto TPR-containing proteins have been made. These attempts mainly included manual docking of the ligand peptide onto the available TPR protein structure. The resulting models did not, however, consider side chain flexibility and conformational changes due to peptide binding and used limited energy minimization tools (Gatto et al. 2000; Kim et al. 2006). However, with the remarkable development of computational docking servers, more accurate models can be generated for TPR protein–peptide interactions. To date, however, no study has employed these advanced servers to demonstrate TPR-peptide docking, although successful docking have been recorded with other proteins involved in protein-protein interactions, such as PDZ domains and others (London et al. 2010; Raveh et al. 2010; Raveh et al. 2011). Overall, the prediction of interaction region is considered to be more accurate than binding partner prediction, since the former uses algorithms that consider chemical restrains, as well as energy minimization of the final model. In the near future, these tools might aid in overcoming challenges associated with crystallizing proteins with their binding partners and could provide important insight for molecular understanding of binding and

The basic TPR fold resulting from its consensus sequence can be considered as a protein scaffold. Redesigning this stable basic scaffold by grafting functional residues involved in binding recognition and specificity enables the introduction of novel binding activities. TPR design includes three major steps. The first stage includes stable consensus scaffold generation by an alignment of natural TPR motifs (D'Andrea et al. 2003). Later, the minimal number of repeating motifs required for thermodynamic stability are determined (Kajander et al. 2007) and finally, functional residues are grafted onto the generated scaffold. An example for a successful designing process was the designed TPR module that binds the Cterminal peptide of Hsp90. This module has been designed by grafting Hsp90-binding residues from natural TPR proteins onto a consensus TPR scaffold to bind Hsp90 with greater affinity and specificity than natural co-chaperones. Introduction of this designed protein into breast cancer cells inhibited Hsp90 activity, presumably by out-competing the interaction of Hsp90 with its natural co-chaperones. Hsp90 inhibition resulted in misfolding and degradation of Hsp90-dependent proteins, such as HER2, and led to cancer cell death

TPR binding properties can also be used for specific identification of tagged proteins and can act as a functional substitute for antibodies in a wide range of applications (Jackrel et al. 2009). Conjugation of a peptide ligand sequence to the N or C termini of a desirable protein allows its identification by a TPR-containing protein that can bind the ligand peptide with sufficient affinity. This TPR-containing protein can be conjugated to a reporter protein, such as horseradish peroxidase, biotin or green fluorescent protein and can be later used in one or two-step western blot detection systems, replacing any requirement for antibodies. In addition, conjugation of a TPR-containing protein or domain directly to resin or beads can be used for affinity purification. Immobilizing a TPR-containing protein onto a resin permits the specific binding and enrichment of desirable proteins conjugated to a peptide ligand. Interaction dissociation at the elution step does not require extremely harsh pH conditions

recognition.

(Cortajarena et al. 2008).

as may be needed with the use of antibodies.

**6. TPR design and biotechnology**