Structure of TPR domain-peptide complexes: critical elements in the assembly of the Hsp70-Hsp90 multichaperone machine

The adaptor protein Hop mediates the association of the molecular chaperones Hsp70 and Hsp90. The TPR1 domain of Hop specifically recognizes the C-terminal heptapeptide of Hsp70 while the TPR2A domain binds the C-terminal pentapeptide of Hsp90. Both sequences end with the motif EEVD. The crystal structures of the TPR-peptide complexes show the peptides in an extended conformation, spanning a groove in the TPR domains. Peptide binding is mediated by electrostatic interactions with the EEVD motif, with the C-terminal aspartate acting as a two-carboxylate anchor, and by hydrophobic interactions with residues upstream of EEVD. The hydrophobic contacts with the peptide are critical for specificity. These results explain how TPR domains participate in the ordered assembly of Hsp70-Hsp90 multichaperone complexes.     

Domain:domain interactions within Hop, the Hsp70/Hsp90 organizing protein, are required for protein stability and structure

The major heat shock protein (Hsp) chaperones Hsp70 and Hsp90 both bind the co-chaperone Hop (Hsp70/Hsp90 organizing protein), which coordinates Hsp actions in folding protein substrates. Hop contains three tetratricopeptide repeat (TPR) domains that have binding sites for the conserved EEVD C termini of Hsp70 and Hsp90. Crystallographic studies have shown that EEVD interacts with positively charged amino acids in Hop TPR-binding pockets (called carboxylate clamps), and point mutations of these carboxylate clamp positions can disrupt Hsp binding. In this report, we use circular dichroism to assess the effects of point mutations and Hsp70/Hsp90 peptide binding on Hop conformation. Our results show that Hop global conformation is destabilized by single point mutations in carboxylate clamp positions at pH 5, while the structure of individual TPR domains is unaffected. Binding of peptides corresponding to the C termini of Hsp70 and Hsp90 alters the global conformation of wild-type Hop, whereas peptide binding does not alter conformation of individual TPR domains. These results provide biophysical evidence that Hop-binding pockets are directly involved with domain:domain interactions, both influencing Hop global conformation and Hsp binding, and contributing to proper coordination of Hsp70 and Hsp90 interactions with protein substrates.     

Interaction of the Hsp90 cochaperone cyclophilin 40 with Hsc70

The high-affinity ligand-binding form of unactivated steroid receptors exists as a multicomponent complex that includes heat shock protein (Hsp)90; one of the immunophilins cyclophilin 40 (CyP40), FKBP51, or FKBP52; and an additional p23 protein component. Assembly of this heterocomplex is mediated by Hsp70 in association with accessory chaperones Hsp40, Hip, and Hop. A conserved structural element incorporating a tetratricopeptide repeat (TPR) domain mediates the interaction of the immunophilins with Hsp90 by accommodating the C-terminal EEVD peptide of the chaperone through a network of electrostatic and hydrophobic interactions. TPR cochaperones recognize the EEVD structural motif common to both Hsp90 and Hsp70 through a highly conserved clamp domain. In the present study, we investigated in vitro the molecular interactions between CyP40 and FKBP52 and other stress-related components involved in steroid receptor assembly, namely Hsp70 and Hop. Using a binding protein-retention assay with CyP40 fused to glutathione S-transferase immobilized on glutathione-agarose, we have identified the constitutively expressed form of Hsp70, heat shock cognate (Hsc)70, as an additional target for CyP40. Deletion mapping studies showed the binding determinants to be similar to those for CyP40-Hsp90 interaction. Furthermore, a mutational analysis of CyP40 clamp domain residues confirmed the importance of this motif in CyP40-Hsc70 interaction. Additional residues thought to mediate binding specificity through hydrophobic interactions were also important for Hsc70 recognition. CyP40 was shown to have a preference for Hsp90 over Hsc70. Surprisingly, FKBP52 was unable to compete with CyP40 for Hsc70 binding, suggesting that FKBP52 discriminates between the TPR cochaperone-binding sites in Hsp90 and Hsp70. Hop, which contains multiple units of the TPR motif, was shown to be a direct competitor with CyP40 for Hsc70 binding. Similar to Hop, CyP40 was shown not to influence the adenosine triphosphatase activity of Hsc70. Our results suggest that CyP40 may have a modulating role in Hsc70 as well as Hsp90 cellular function.     

Multiple domains of the co-chaperone Hop are important for Hsp70 binding

The Hop/Sti1 co-chaperone binds to both Hsp70 and Hsp90. Biochemical and co-crystallographic studies have suggested that the EEVD-containing C terminus of Hsp70 or Hsp90 binds specifically to one of the Hop tetratricopeptide repeat domains, TPR1 or TPR2a, respectively. Mutational analyses of Hsp70 and Hop were undertaken to better characterize interactions between the C terminus of Hsp70 and Hop domains. Surprisingly, truncation of EEVD plus as many as 34 additional amino acids from the Hsp70 C terminus did not reduce the ability of Hsp70 mutants to co-immunoprecipitate with Hop, although further truncation eliminated Hop binding. Hop point mutations targeting a carboxylate clamp position in TPR1 disrupted Hsp70 binding, as was expected; however, similar point mutations in TPR2a or TPR2b also inhibited Hsp70 binding in some settings. Using a yeast-based in vivo assay for Hop function, wild type Hop and TPR2b mutants could fully complement deletion of Sti1p; TPR1 and TPR2a point mutants could partially restore activity. Conformations of Hop and Hop mutants were probed by limited proteolysis. The TPR1 mutant digested in a similar manner to wild type; however, TPR2a and TPR2b mutants each displayed greater resistance to chymotryptic digestion. All point mutants retained an ability to dimerize, and none appeared to be grossly misfolded. These results raise questions about current models for Hop/Hsp70 interaction.     

Tetratricopeptide repeat motif-mediated Hsc70-mSTI1 interaction. Molecular characterization of the critical contacts for successful binding and specificity

Murine stress-inducible protein 1 (mSTI1) is a co-chaperone that is homologous with the human Hsp70/Hsp90-organizing protein (Hop). Guided by Hop structural data and sequence alignment analyses, we have used site-directed mutagenesis, co-precipitation assays, circular dichroism spectroscopy, steady-state fluorescence, and surface plasmon resonance spectroscopy to both qualitatively and quantitatively characterize the contacts necessary for the N-terminal tetratricopeptide repeat domain (TPR1) of mSTI1 to bind to heat shock cognate protein 70 (Hsc70) and to discriminate between Hsc70 and Hsp90. We have shown that substitutions in the first TPR motif of Lys(8) or Asn(12) did not affect binding of mSTI1 to Hsc70, whereas double substitution of these residues abrogated binding. A substitution in the second TPR motif of Asn(43) lowered but did not abrogate binding. Similarly, a deletion in the second TPR motif coupled with a substitution of Lys(8) or Asn(12) reduced but did not abrogate binding. These results suggest that mSTI1-Hsc70 interaction requires a network of interactions not only between charged residues in the TPR1 domain of mSTI1 and the EEVD motif of Hsc70 but also outside the TPR domain. We propose that the electrostatic interactions in the first TPR motif made by Lys(8) or Asn(12) define part of the minimum interactions required for successful mSTI1-Hsc70 interaction. Using a truncated derivative of mSTI1 incapable of binding to Hsp90, we substituted residues on TPR1 potentially involved in hydrophobic contacts with Hsc70. The modified protein had reduced binding to Hsc70 but now showed significant binding capacity for Hsp90. In contrast, topologically equivalent substitutions on a truncated derivative of mSTI1 incapable of binding to Hsc70 did not confer Hsc70 specificity on TPR2A. Our results suggest that binding of Hsc70 to TPR1 is more specific than binding of Hsp90 to TPR2A with serious implications for the mechanisms of mSTI1 interactions with Hsc70 and Hsp90 in vivo.     

Structural studies on the co-chaperone Hop and its complexes with Hsp90

The tetratricopeptide repeat domain (TPR)-containing co-chaperone Hsp-organising protein (Hop) plays a critical role in mediating interactions between Heat Shock Protein (Hsp)70 and Hsp90 as part of the cellular assembly machine. It also modulates the ATPase activity of both Hsp70 and Hsp90, thus facilitating client protein transfer between the two. Despite structural work on the individual domains of Hop, no structure for the full-length protein exists, nor is it clear exactly how Hop interacts with Hsp90, although it is known that its primary binding site is the C-terminal MEEVD motif. Here, we have undertaken a biophysical analysis of the structure and binding of Hop to Hsp90 using a variety of truncation mutants of both Hop and Hsp90, in addition to mutants of Hsp90 that are thought to modulate the conformation, in particular the N-terminal dimerisation of the chaperone. The results establish that whilst the primary binding site of Hop is the C-terminal MEEVD peptide of Hsp90, binding also occurs at additional sites in the C-terminal and middle domain. In contrast, we show that another TPR-containing co-chaperone, CyP40, binds solely to the C-terminus of Hsp90.Truncation mutants of Hop were generated and used to investigate the dimerisation interface of the protein. In good agreement with recently published data, we find that the TPR2a domain that contains the Hsp90-binding site is also the primary site for dimerisation. However, our results suggest that residues within the TPR2b may play a role. Together, these data along with shape reconstruction analysis from small-angle X-ray scattering measurements are used to generate a solution structure for full-length Hop, which we show has an overall butterfly-like quaternary structure.Studies on the nucleotide dependence of Hop binding to Hsp90 establish that Hop binds to the nucleotide-free, ‘open’ state of Hsp90. However, the Hsp90-Hop complex is weakened by the conformational changes that occur in Hsp90 upon ATP binding. Together, the data are used to propose a detailed model of how Hop may help present the client protein to Hsp90 by aligning the bound client on Hsp70 with the middle domain of Hsp90. It is likely that Hop binds to both monomers of Hsp90 in the form of a clamp, interacting with residues in the middle domain of Hsp90, thus preventing ATP hydrolysis, possibly by the prevention of association of N-terminal and middle domains in individual Hsp90 monomers.     

Endoplasmic Reticulum Protein Quality Control Is Determined by Cooperative Interactions between Hsp/c70 Protein and the CHIP E3 Ligase

The C terminus of Hsp70 interacting protein (CHIP) E3 ligase functions as a key regulator of protein quality control by binding the C-terminal (M/I)EEVD peptide motif of Hsp/c70(90) with its N-terminal tetratricopeptide repeat (TPR) domain and facilitating polyubiquitination of misfolded client proteins via its C-terminal catalytic U-box. Using CFTR as a model client, we recently showed that the duration of the Hsc70-client binding cycle is a primary determinant of stability. However, molecular features that control CHIP recruitment to Hsp/c70, and hence the fate of the Hsp/c70 client, remain unknown. To understand how CHIP recognizes Hsp/c70, we utilized a dominant negative mutant in which loss of a conserved proline in the U-box domain (P269A) eliminates E3 ligase activity. In a cell-free reconstituted ER-associated degradation system, P269A CHIP inhibited Hsc70-dependent CFTR ubiquitination and degradation in a dose-dependent manner. Optimal inhibition required both the TPR and the U-box, indicating cooperativity between the two domains. Neither the wild type nor the P269A mutant changed the extent of Hsc70 association with CFTR nor the dissociation rate of the Hsc70-CFTR complex. However, the U-box mutation stimulated CHIP binding to Hsc70 while promoting CHIP oligomerization. CHIP binding to Hsc70 binding was also stimulated by the presence of an Hsc70 client with a preference for the ADP-bound state. Thus, the Hsp/c70 (M/I)EEVD motif is not a simple anchor for the TPR domain. Rather CHIP recruitment involves reciprocal allosteric interactions between its TPR and U-box domains and the substrate-binding and C-terminal domains of Hsp/c70.     

Crystallographic structure of the tetratricopeptide repeat domain of Plasmodium falciparum FKBP35 and its molecular interaction with Hsp90 C‐terminal pentapeptide

Plasmodium falciparum FK506‐binding protein 35 (PfFKBP35) that binds to FK506 contains a conserved tetratricopeptide repeat (TPR) domain. Several known TPR domains such as Hop, PPP5, CHIP, and FKBP52 are structurally conserved and are able to interact with molecular chaperones such as Hsp70/Hsp90. Here, we present the crystal structure of PfFKBP35‐TPR and demonstrate its interaction with Hsp90 C‐terminal pentapeptide (MEEVD) by surface plasmon resonance and nuclear magnetic resonance spectroscopy‐based binding studies. Our sequence and structural analyses reveal that PfFKBP35 is similar to Hop and PPP5 in possessing all the conserved residues which are important for carboxylate clamping with Hsp90. Mutational studies were carried out on positively charged clamp residues that are crucial for binding to carboxylate groups of aspartate, showing that all the mutated residues are important for Hsp90 binding. Molecular docking and electrostatic calculations demonstrated that the MEEVD peptide of Hsp90 can form aspartate clamp unlike FKBP52. Our results provide insightful information and structural basis about the molecular interaction between PfFKBP35‐TPR and Hsp90.     

The architecture of functional modules in the Hsp90 co-chaperone Sti1/Hop

Sti1/Hop is a modular protein required for the transfer of client proteins from the Hsp70 to the Hsp90 chaperone system in eukaryotes. It binds Hsp70 and Hsp90 simultaneously via TPR (tetratricopeptide repeat) domains. Sti1/Hop contains three TPR domains (TPR1, TPR2A and TPR2B) and two domains of unknown structure (DP1 and DP2). We show that TPR2A is the high affinity Hsp90-binding site and TPR1 and TPR2B bind Hsp70 with moderate affinity. The DP domains exhibit highly homologous α-helical folds as determined by NMR. These, and especially DP2, are important for client activation in vivo. The core module of Sti1 for Hsp90 inhibition is the TPR2A-TPR2B segment. In the crystal structure, the two TPR domains are connected via a rigid linker orienting their peptide-binding sites in opposite directions and allowing the simultaneous binding of TPR2A to the Hsp90 C-terminal domain and of TPR2B to Hsp70. Both domains also interact with the Hsp90 middle domain. The accessory TPR1-DP1 module may serve as an Hsp70-client delivery system for the TPR2A-TPR2B-DP2 segment, which is required for client activation in vivo.     

Functional coevolutionary networks of the Hsp70-Hop-Hsp90 system revealed through computational analyses

Currently, the identification of groups of amino acid residues that are important in the function, structure, or interaction of a protein can be both costly and prohibitively complex, involving vast numbers of mutagenesis experiments. Here, we present the application of a novel computational method, which identifies the presence of coevolution in a data set, thereby enabling the a priori identification of amino acid residues that play an important role in protein function. We have applied this method to the heat shock protein (Hsp) protein-folding system, studying the network between Hsp70, Hsp90, and Hop (heat shock-organizing protein). Our analysis has identified functional residues within the tetratricopeptide repeat (TPR) 1 and 2A domains in Hop, previously shown to be interacting with Hsp70 and Hsp90, respectively. Further, we have identified significant residues elsewhere in Hop within domains that have been recently proposed as being important for Hop interaction with Hsp70 and/or Hsp90. In addition, several amino acid sites present in groups of coevolution were identified as 3-dimensionally or linearly proximal to functionally important sites or domains. Based on our results, we also investigate a further functional domain within Hop, between TPR1 and TPR2A, which we suggest as being functionally important in the interaction of Hop with both Hsp70 and Hsp90 whether directly or otherwise. Our method has identified all the previously characterized functionally important regions in this system, thereby indicating the power of this method in the a priori identification of important regions for site-directed mutagenesis studies.     

Functional interactions between Hsp90 and the co-chaperones Cns1 and Cpr7 in Saccharomyces cerevisiae

Hsp90 complexes contain a class of co-chaperones characterized by a tetratricopeptide repeat (TPR) domain, which mediates binding to a carboxyl-terminal EEVD region in Hsp90. Among Hsp90 TPR co-chaperones in Saccharomyces cerevisiae, only Cns1 is essential. The amino terminus of Cns1, which harbors the TPR domain, is sufficient for viability when overexpressed. In a screen for temperature-sensitive alleles of CNS1, we identified mutations resulting in substitutions of conserved residues in the TPR domain. Mutations in CNS1 disrupt in vitro and in vivo interaction with Hsp90 and reduce Hsp90 function, indicating that Cns1 is a bona fide co-chaperone. Genetic interactions between CNS1 and another Hsp90 co-chaperone, CPR7, suggest that the two co-chaperones share an essential role in the cell. Although both the TPR and the isomerase domains of the cyclophilin Cpr7 are required for viability of cns1 mutant cells, this requirement does not depend on the catalytic function of the isomerase domain. Instead, hydrophilic residues on the surface of this domain appear to be important for the common Cns1.Cpr7 function. Although both co-chaperones interact with Hsp90 primarily through the carboxyl terminus (EEVD), Cns1 and Cpr7 are mostly found in complexes distinct from Hsp90. EEVD is required for normal growth in cns1 mutant cells, demonstrating for the first time in vivo requirement for this conserved region of Hsp90. Overall, our findings reveal a considerable degree of complexity in the interactions not only between Hsp90 and its co-chaperones, but also among the co-chaperones themselves.     

Interactions of S100A2 and S100A6 with the tetratricopeptide repeat proteins, Hsp90/Hsp70-organizing protein and kinesin light chain

S100A2 and S100A6 interact with several target proteins in a Ca2+-regulated manner. However, the exact intracellular roles of the S100 proteins are unclear. In this study we identified Hsp70/Hsp90-organizing protein (Hop) and kinesin light chain (KLC) as novel targets of S100A2 and S100A6. Hop directly associates with Hsp70 and Hsp90 through the tetratricopeptide (TPR) domains and regulates Hop-Hsp70 and Hop-Hsp90 complex formation. We have found that S100A2 and S100A6 bind to the TPR domain of Hop, resulting in inhibition of the Hop-Hsp70 and Hop-Hsp90 interactions in vitro. Although endogenous Hsp70 and Hsp90 interact with Hop in resting Cos-7 cells, but not with S100A6, stimulation of these cells with ionomycin caused a Hop-S100A6 interaction, resulting in the dissociation of Hsp70 and Hsp90 from Hop. Similarly, glutathione S-transferase pulldown and co-immunoprecipitation experiments revealed that S100A6 binds to the TPR domain of KLC, resulting in inhibition of the KLC-c-Jun N-terminal kinase (JNK)-interacting protein 1 (JIP-1) interaction in vitro. The transiently expressed JIP-1 interacts with KLC in resting Cos-7 cells but not with S100A6. Stimulation of these cells with ionomycin also caused a KLC-S100A6 interaction, resulting in dissociation of JIP-1 from KLC. These results strongly suggest that the S100 proteins modulate Hsp70-Hop-Hsp90 multichaperone complex formation and KLC-cargo interaction via Ca2+-dependent S100 protein-TPR protein complex formation in vivo as well as in vitro. Moreover, we have shown that S100A2 and S100A6 interact with another TPR protein Tom70 and regulate the Tom70-ligand interaction in vitro. Thus, our findings suggest a new intracellular Ca2+-signaling pathway via S100 proteins-TPR motif interactions.     

The Non-canonical Hop Protein from Caenorhabditis elegans Exerts Essential Functions and Forms Binary Complexes with Either Hsc70 or Hsp90

Heat shock protein (Hsp) 70/Hsp90-organizing proteins (Hop/Sti1) are thought to function as adaptor proteins to link the two chaperone machineries Hsp70 and Hsp90 during the processing of substrate proteins in eukaryotes. Hop (Hsp70/Hsp90-organizing protein) is composed of three tetratricopeptide repeat (TPR) domains, of which the first (TPR1) binds to Hsp70, the second (TPR2A) binds to Hsp90, and the third (TPR2B) is of unknown function. Contrary to most other eukaryotes, the homologue closest to the Caenorhabditis elegans Hop homologue R09E12.3 (CeHop) lacks the TPR1 domain and the short linker region connecting it to TPR2A, questioning the reported function as an Hsp90/Hsp70 adaptor in vitro and in vivo. We observed high constitutive expression levels of CeHop and detected significant phenotypes upon knockdown, linking the protein to functions in gonad development. Interestingly, we observed physical interactions with both chaperones Hsp70 and Hsp90, albeit only the interaction with Hsp90 is strong and inhibition of the Hsp90 ATPase activity can be observed upon binding of CeHop. However, the formation of ternary complexes with both chaperone machineries is impaired, as Hsp70 and Hsp90 compete for CeHop interaction sites, in particular as Hsp90 binds to both TPR domains simultaneously and requires both TPR domains for ATPase regulation. These results imply that, at least in C. elegans, essential functions of Hop exist which apparently do not depend on the simultaneous binding of Hsp90 and Hsp70 to Hop.     

Crystal structure of yeast Sis1 peptide-binding fragment and Hsp70 Ssa1 C-terminal complex

Heat shock protein (Hsp) 40 facilitates the critical role of Hsp70 in a number of cellular processes such as protein folding, assembly, degradation and translocation in vivo. Hsp40 and Hsp70 stay in close contact to achieve these diverse functions. The conserved C-terminal EEVD motif in Hsp70 has been shown to regulate Hsp40-Hsp70 interaction by an unknown mechanism. Here, we provide a structural basis for this regulation by determining the crystal structure of yeast Hsp40 Sis1 peptide-binding fragment complexed with the Hsp70 Ssa1 C-terminal. The Ssa1 extreme C-terminal eight residues, G634PTVEEVD641, form a beta-strand with the domain I of Sis1 peptide-binding fragment. Surprisingly, the Ssa1 C-terminal binds Sis1 at the site where Sis1 interacts with the non-native polypeptides. The negatively charged residues within the EEVD motif in Ssa1 C-terminal form extensive charge-charge interactions with the positively charged residues in Sis1. The structure-based mutagenesis data support the structural observations.     

C-terminal sequences outside the tetratricopeptide repeat domain of FKBP51 and FKBP52 cause differential binding to Hsp90

Hsp90 assembles with steroid receptors and other client proteins in association with one or more Hsp90-binding cochaperones, some of which contain a common tetratricopeptide repeat (TPR) domain. Included in the TPR cochaperones are the Hsp70-Hsp90-organizing protein Hop, the FK506-binding immunophilins FKBP52 and FKBP51, the cyclosporin A-binding immunophilin CyP40, and protein phosphatase PP5. The TPR domains from these proteins have similar x-ray crystallographic structures and target cochaperone binding to the MEEVD sequence that terminates Hsp90. However, despite these similarities, the TPR cochaperones have distinctive properties for binding Hsp90 and assembling with Hsp90.steroid receptor complexes. To identify structural features that differentiate binding of FKBP51 and FKBP52 to Hsp90, we generated an assortment of truncation mutants and chimeras that were compared for coimmunoprecipitation with Hsp90. Although the core TPR domain (approximately amino acids 260-400) of FKBP51 and FKBP52 is required for Hsp90 binding, the C-terminal 60 amino acids (approximately 400-end) also influence Hsp90 binding. More specifically, we find that amino acids 400-420 play a critical role for Hsp90 binding by either FKBP. Within this 20-amino acid region, we have identified a consensus sequence motif that is also present in some other TPR cochaperones. Additionally, the final 30 amino acids of FKBP51 enhance binding to Hsp90, whereas the corresponding region of FKBP52 moderates binding to Hsp90. Taking into account the x-ray crystal structure for FKBP51, we conclude that the C-terminal regions of FKBP51 and FKBP52 outside the core TPR domains are likely to assume alternative conformations that significantly impact Hsp90 binding.     

Effect of mutation of the tetratricopeptide repeat and asparatate-proline 2 domains of Sti1 on Hsp90 signaling and interaction in Saccharomyces cerevisiae

Through simultaneous interactions with Hsp70 and Hsp90 via separate tetratricopeptide repeat (TPR) domains, the cochaperone protein Hop/Sti1 has been proposed to play a critical role in the transfer of client proteins from Hsp70 to Hsp90. However, no prior mutational analysis demonstrating a critical in vivo role for the TPR domains of Sti1 has been reported. We used site-directed mutagenesis of the TPR domains combined with a genetic screen to isolate mutations that disrupt Sti1 function. A single amino acid alteration in TPR2A disrupted Hsp90 interaction in vivo but did not significantly affect function. However, deletion of a conserved residue in TPR2A or mutations in the carboxy-terminal DP2 domain completely disrupted Sti1 function. Surprisingly, mutations in TPR1, previously shown to interact with Hsp70, were not sufficient to disrupt in vivo functions unless combined with mutations in TPR2B, suggesting that TPR1 and TPR2B have redundant or overlapping in vivo functions. We further examined the genetic and physical interaction of Sti1 with a mutant form of Hsp90, providing insight into the importance of the TPR2A domain of Sti1 in regulating Hsp90 function.     

Propagation of Saccharomyces cerevisiae [PSI+] prion is impaired by factors that regulate Hsp70 substrate binding

The Saccharomyces cerevisiae [PSI(+)] prion is believed to be a self-propagating cytoplasmic amyloid. Earlier characterization of HSP70 (SSA1) mutations suggested that [PSI(+)] propagation is impaired by alterations that enhance Ssa1p's substrate binding. This impairment is overcome by second-site mutations in Ssa1p's conserved C-terminal motif (GPTVEEVD), which mediates interactions with tetratricopeptide repeat (TPR) cochaperones. Sti1p, a TPR cochaperone homolog of mammalian Hop1 (Hsp70/90 organizing protein), activates Ssa1p ATPase, which promotes substrate binding by Ssa1p. Here we find that in SSA1-21 cells depletion of Sti1p improved [PSI(+)] propagation, while excess Sti1p weakened it. In contrast, depletion of Fes1p, a nucleotide exchange factor for Ssa1p that facilitates substrate release, weakened [PSI(+)] propagation, while overproducing Fes1p improved it. Therefore, alterations of Hsp70 cochaperones that promote or prolong Hsp70 substrate binding impair [PSI(+)] propagation. We also find that the GPTVEEVD motif is important for physical interaction with Hsp40 (Ydj1p), another Hsp70 cochaperone that promotes substrate binding but is dispensable for viability. We further find that depleting Cpr7p, an Hsp90 TPR cochaperone and CyP-40 cyclophilin homolog, improved [PSI(+)] propagation in SSA1 mutants. Although Cpr7p and Sti1p are Hsp90 cochaperones, we provide evidence that Hsp90 is not involved in [PSI(+)] propagation, suggesting that Sti1p and Cpr7p functionally interact with Hsp70 independently of Hsp90.     

Functional comparison of human and Drosophila Hop reveals novel role in steroid receptor maturation

Hsp70/Hsp90 organizing protein (Hop) coordinates Hsp70 and Hsp90 interactions during assembly of steroid receptor complexes. Hop is composed of three tetratricopeptide repeat (TPR) domains (TPR1, TPR2a, and TPR2b) and two DP repeat domains (DP1 and DP2); Hsp70 interacts directly with TPR1 and Hsp90 with TPR2a, but the function of other domains is less clear. Human Hop and the Saccharomyces cerevisiae ortholog Sti1p, which share a common domain arrangement, are functionally interchangeable in a yeast growth assay and in supporting the efficient maturation of glucocorticoid receptor (GR) function. To gain a better understanding of Hop structure/function relationships, we have extended comparisons to the Hop ortholog from Drosophila melanogaster (dHop), which lacks DP1. Although dHop binds Hsp70 and Hsp90 and can rescue the growth defect in yeast lacking Sti1p, dHop failed to support GR function in yeast, which suggests a novel role for Hop in GR maturation that goes beyond Hsp binding. Chimeric Hop constructs combining human and Drosophila domains demonstrate that the C-terminal domain DP2 is critical for this previously unrecognized role in steroid receptor function.     

Differential interactions of p23 and the TPR-containing proteins Hop, Cyp40, FKBP52 and FKBP51 with Hsp90 mutants

Hsp90 is required for the normal function of steroid receptors, but its binding to steroid receptors is mediated by Hsc70 and several hsp-associated accessory proteins. An assortment of Hsp90 mutants were tested for their abilities to interact with each of the following accessories: Hop, Cyp40, FKBP52, FKBP51, and p23. Of the 11 Hsp90 mutants tested, all were defective to some extent in associating with progestin (PR) complexes. In every case, however, reduced PR binding correlated with a defect in binding of one or more accessories. Co-precipitation of mutant Hsp90 forms with individual accessories was used to map Hsp90 sequences required for accessory protein interactions. Mutation of Hsp90's highly conserved C-terminal EEVD to AAVD resulted in diminished interactions with several accessory proteins, most particularly with Hop. Deletion of amino acids 661–677 resulted in loss of Hsp90 dimerization and also caused diminished interactions with all accessory proteins. Binding of p23 mapped most strongly to the N-terminal ATP-binding domain of Hsp90 while binding of TPR proteins mapped to the C-terminal half of Hsp90. These results and others further suggest that the N- and C-terminal regions of Hsp90 maintain important conformational links through intramolecular interactions and/or intermolecular influences in homodimers.     

Hsp104 interacts with Hsp90 cochaperones in respiring yeast

The highly abundant molecular chaperone Hsp90 functions with assistance from auxiliary factors, collectively referred to as Hsp90 cochaperones, and the Hsp70 system. Hsp104, a molecular chaperone required for stress tolerance and for maintenance of [psi(+)] prions in the budding yeast Saccharomyces cerevisiae, appears to collaborate only with the Hsp70 system. We now report that several cochaperones previously thought to be dedicated to Hsp90 are shared with Hsp104. We show that the Hsp90 cochaperones Sti1, Cpr7, and Cns1, which utilize tetratricopeptide repeat (TPR) domains to interact with a common surface on Hsp90, form complexes with Hsp104 in vivo and that Sti1 and Cpr7 interact with Hsp104 directly in vitro. The interaction is Hsp90 independent, as further emphasized by the fact that two distinct TPR domains of Sti1 are required for binding Hsp90 and Hsp104. In a striking parallel to the sequence requirements of Hsp90 for binding TPR proteins, binding of Sti1 to Hsp104 requires a related acidic sequence at the C-terminal tail of Hsp104. While Hsp90 efficiently sequesters the cochaperones during fermentative growth, respiratory conditions induce the interaction of a fraction of Hsp90 cochaperones with Hsp104. This suggests that cochaperone sharing may favor adaptation to altered metabolic conditions.