Tamoxifen Enhances the Hsp90 Molecular Chaperone ATPase Activity

Background

Hsp90 is an essential molecular chaperone that is also a novel anti-cancer drug target. There is growing interest in developing new drugs that modulate Hsp90 activity.

Methodology/Principal Findings

Using a virtual screening approach, 4-hydroxytamoxifen, the active metabolite of the anti-estrogen drug tamoxifen, was identified as a putative Hsp90 ligand. Surprisingly, while all drugs targeting Hsp90 inhibit the chaperone ATPase activity, it was found experimentally that 4-hydroxytamoxifen and tamoxifen enhance rather than inhibit Hsp90 ATPase.

Conclusions/Significance

Hence, tamoxifen and its metabolite are the first members of a new pharmacological class of Hsp90 activators.     

Post-translational Regulation of FNIP1 Creates a Rheostat for the Molecular Chaperone Hsp90

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Elucidating the Mechanism of Substrate Recognition by the Bacterial Hsp90 Molecular Chaperone

Hsp90 is a conformationally dynamic molecular chaperone known to promote the folding and activation of a broad array of protein substrates (“clients”). Hsp90 is believed to preferentially interact with partially folded substrates, and it has been hypothesized that the chaperone can significantly alter substrate structure as a mechanism to alter the substrate functional state. However, critically testing the mechanism of substrate recognition and remodeling by Hsp90 has been challenging. Using a partially folded protein as a model system, we find that the bacterial Hsp90 adapts its conformation to the substrate, forming a binding site that spans the middle and C-terminal domains of the chaperone. Cross-linking and NMR measurements indicate that Hsp90 binds to a large partially folded region of the substrate and significantly alters both its local and long-range structure. These findings implicate Hsp90's conformational dynamics in its ability to bind and remodel partially folded proteins. Moreover, native-state hydrogen exchange indicates that Hsp90 can also interact with partially folded states only transiently populated from within a thermodynamically stable, native-state ensemble. These results suggest a general mechanism by which Hsp90 can recognize and remodel native proteins by binding and remodeling partially folded states that are transiently sampled from within the native ensemble.     

Molecular Chaperone Hsp70/Hsp90 Prepares the Mitochondrial Outer Membrane Translocon Receptor Tom71 for Preprotein Loading

The preproteins targeted to the mitochondria are transported through the translocase of the outer membrane complex. Tom70/Tom71 is a major surface receptor of the translocase of the outer membrane complex for mitochondrial preproteins. The preproteins are escorted to Tom70/Tom71 by molecular chaperones Hsp70 and Hsp90. Here we present the high resolution crystal structures of Tom71 and the protein complexes between Tom71 and the Hsp70/Hsp90 C terminus. The crystal structures indicate that Tom70/Tom71 may exhibit two distinct states. In the closed state, the N-terminal domain of Tom70/Tom71 partially blocks the preprotein-binding pocket. In the open state, the N-terminal domain moves away, and the preprotein-binding pocket is fully exposed. The complex formation between the C-terminal EEVD motif of Hsp70/Hsp90 and Tom71 could lock Tom71 in the open state where the preprotein-binding pocket of Tom71 is ready to receive preproteins. The interactions between Hsp70/Hsp90 and Tom71 N-terminal domain generate conformational changes that may increase the volume of the preprotein-binding pocket. The complex formation of Hsp70/Hsp90 and Tom71 also generates significant domain rearrangement within Tom71, which may position the preprotein-binding pocket closer to Hsp70/Hsp90 to facilitate the preprotein transfer from the molecular chaperone to Tom71. Therefore, molecular chaperone Hsp70/Hsp90 may function to prepare the mitochondrial outer membrane receptor Tom71 for preprotein loading.The mitochondrion plays important roles in cell physiology. The mitochondrion functions as the “cellular power house” by generating most of the supply of ATP for the cell. In addition, the mitochondrion is involved in a number of critical cellular processes including the synthesis of metabolites, lipid metabolism, free radical production, and metal ion homeostasis. The mitochondrion consists of four compartments, the outer membrane, the inner membrane, the intermembrane space, and the mitochondrial matrix. The mitochondrion contains a large number of proteins (1), but only a few of these are translated within the mitochondrion (2). Therefore, the majority of the mitochondrial proteins are synthesized in the cytosol and translocated into the mitochondrion.The mitochondrial preproteins contain specific targeting signals to reach the correct compartments within the mitochondria. The mitochondrial matrix preproteins contain N-terminal targeting sequences that form the short amphipathic helices (2–6). On the other hand, some mitochondrial proteins of the inner and outer membrane contain internal targeting signals within the mature proteins (7). The mitochondrion has developed a set of delicate translocons to transport the preproteins into the mitochondrial compartments, one translocase of the outer membrane (TOM)² and two translocases of the inner membrane (TIM23 and TIM22) (4, 5, 8). The TOM complex has two surface receptors, Tom20 and Tom70 (9, 10). Tom20 recognizes the N-terminal mitochondrial targeting signals from the preproteins, whereas Tom70 binds to internal targeting sequences of preproteins such as the multi-transmembrane carrier proteins residing in the mitochondrial membranes (9–12). The crystal structure of Saccharomyces cerevisiae Tom70 revealed that Tom70 contained 11 TPR motifs, and the TPR motifs were clustered into two domains. The three TPR motifs in the N-terminal domain of Tom70p form a peptide-binding groove for the C-terminal EEVD motif of Hsp70/Hsp90, whereas the C-terminal domain of Tom70p contains a large preprotein-binding pocket (13).Molecular chaperones Hsp70 and Hsp90 play important roles in targeting the preproteins to TOM complex (14). Hsp70 and Hsp90 can protect these preproteins from aggregation in the cytosol (15). The C-terminal EEVD motifs of Hsp70/Hsp90 may interact directly with the N-terminal domain of Tom70p to target the preproteins to TOM complex (13, 14, 16). The C-terminal EEVD motif of Hsp70/Hsp90 has been indicated to bind several proteins containing TPR motifs including Hop and CHIP. The complex structures for the Hsp70/Hsp90 EEVD motif and Hop and CHIP TPR regions have been determined (17–21).Tom71 (also known as Tom72) was identified as a homologue with Tom70 with high amino acid sequence identity (>50%) (22). Tom71 shares overlapping functions with Tom70 to transfer the preproteins and maintain the mitochondrial morphology (23, 24). In this study, we have determined the crystal structures of S. cerevisiae Tom71 and the complexes of Tom71 and Hsp70/Hsp90 C-terminal EEVD motifs. These structures suggest that the Hsp70/Hsp90 binding to Tom70/Tom71 may keep Tom70/Tom71 in the open state for receiving preproteins. The Hsp70/Hsp90 interactions may also increase the volume of the preprotein-binding pocket of Tom70/Tom71 and prepare Tom70/Tom71 for preprotein loading.     

The Molecular Mechanism of Hsp100 Chaperone Inhibition by the Prion Curing Agent Guanidinium Chloride

The Hsp100 chaperones ClpB and Hsp104 utilize the energy from ATP hydrolysis to reactivate aggregated proteins in concert with the DnaK/Hsp70 chaperone system, thereby playing an important role in protein quality control. They belong to the family of AAA+ proteins (ATPases associated with various cellular activities), possess two nucleotide binding domains per monomer (NBD1 and NBD2), and oligomerize into hexameric ring complexes. Furthermore, Hsp104 is involved in yeast prion propagation and inheritance. It is well established that low concentrations of guanidinium chloride (GdmCl) inhibit the ATPase activity of Hsp104, leading to so called “prion curing,” the loss of prion-related phenotypes. Here, we present mechanistic details about the Hsp100 chaperone inhibition by GdmCl using the Hsp104 homolog ClpB from Thermus thermophilus. Initially, we demonstrate that NBD1 of ClpB, which was previously considered inactive as a separately expressed construct, is a fully active ATPase on its own. Next, we show that only NBD1, but not NBD2, is affected by GdmCl. We present a crystal structure of ClpB NBD1 in complex with GdmCl and ADP, showing that the Gdm⁺ ion binds specifically to the active site of NBD1. A conserved essential glutamate residue is involved in this interaction. Additionally, Gdm⁺ interacts directly with the nucleotide, thereby increasing the nucleotide binding affinity of NBD1. We propose that both the interference with the essential glutamate and the modulation of nucleotide binding properties in NBD1 is responsible for the GdmCl-specific inhibition of Hsp100 chaperones.     

The Assembly and Intermolecular Properties of the Hsp70-Tomm34-Hsp90 Molecular Chaperone Complex

Maintenance of protein homeostasis by molecular chaperones Hsp70 and Hsp90 requires their spatial and functional coordination. The cooperation of Hsp70 and Hsp90 is influenced by their interaction with the network of co-chaperone proteins, some of which contain tetratricopeptide repeat (TPR) domains. Critical to these interactions are TPR domains that target co-chaperone binding to the EEVD-COOH motif that terminates Hsp70/Hsp90. Recently, the two-TPR domain-containing protein, Tomm34, was reported to bind both Hsp70 and Hsp90. Here we characterize the structural basis of Tomm34-Hsp70/Hsp90 interactions. Using multiple methods, including pull-down assays, fluorescence polarization, hydrogen/deuterium exchange, and site-directed mutagenesis, we defined the binding activities and specificities of Tomm34 TPR domains toward Hsp70 and Hsp90. We found that Tomm34 TPR1 domain specifically binds Hsp70. This interaction is partly mediated by a non-canonical TPR1 two-carboxylate clamp and is strengthened by so far unidentified additional intermolecular contacts. The two-carboxylate clamp of the isolated TPR2 domain has affinity for both chaperones, but as part of the full-length Tomm34 protein, the TPR2 domain binds specifically Hsp90. These binding properties of Tomm34 TPR domains thus enable simultaneous binding of Hsp70 and Hsp90. Importantly, we provide evidence for the existence of an Hsp70-Tomm34-Hsp90 tripartite complex. In addition, we defined the basic conformational demands of the Tomm34-Hsp90 interaction. These results suggest that Tomm34 represents a novel scaffolding co-chaperone of Hsp70 and Hsp90, which may facilitate Hsp70/Hsp90 cooperation during protein folding.     

Modeling Signal Propagation Mechanisms and Ligand-Based Conformational Dynamics of the Hsp90 Molecular Chaperone Full-Length Dimer

Hsp90 is a molecular chaperone essential for protein folding and activation in normal homeostasis and stress response. ATP binding and hydrolysis facilitate Hsp90 conformational changes required for client activation. Hsp90 plays an important role in disease states, particularly in cancer, where chaperoning of the mutated and overexpressed oncoproteins is important for function. Recent studies have illuminated mechanisms related to the chaperone function. However, an atomic resolution view of Hsp90 conformational dynamics, determined by the presence of different binding partners, is critical to define communication pathways between remote residues in different domains intimately affecting the chaperone cycle. Here, we present a computational analysis of signal propagation and long-range communication pathways in Hsp90. We carried out molecular dynamics simulations of the full-length Hsp90 dimer, combined with essential dynamics, correlation analysis, and a signal propagation model. All-atom MD simulations with timescales of 70 ns have been performed for complexes with the natural substrates ATP and ADP and for the unliganded dimer. We elucidate the mechanisms of signal propagation and determine “hot spots” involved in interdomain communication pathways from the nucleotide-binding site to the C-terminal domain interface. A comprehensive computational analysis of the Hsp90 communication pathways and dynamics at atomic resolution has revealed the role of the nucleotide in effecting conformational changes, elucidating the mechanisms of signal propagation. Functionally important residues and secondary structure elements emerge as effective mediators of communication between the nucleotide-binding site and the C-terminal interface. Furthermore, we show that specific interdomain signal propagation pathways may be activated as a function of the ligand. Our results support a “conformational selection model” of the Hsp90 mechanism, whereby the protein may exist in a dynamic equilibrium between different conformational states available on the energy landscape and binding of a specific partner can bias the equilibrium toward functionally relevant complexes.     

Extracellular Roles for the Molecular Chaperone,HSP90

Heat shock proteins (hsps) are versatile molecular chaperones that are responsiblefor many cellular functions including proper folding, oligomeric assembly, activation,and transport of proteins. Most of the known roles for hsps involve intracellular proteinsand processes. Mounting evidence suggests that hsps are present and function in theextracellular space. Hsp90 alpha was recently found on the surface and in conditionedmedia of HT-1080 fibrosarcoma cells. Here it acts as a molecular chaperone that assistsin the activation of matrix metalloproteinase-2 (MMP2), leading to increased tumorinvasiveness. Few other extracellular substrates of hsp90 have been identified, butseveral independent observations of extracellular hsp90 suggest that this protein may beimportant for both normal physiology and disease states. Hsp90 typically works in acomplex of associated proteins, and some of these proteins have also been observedextracellularly. Here we show that some of these components, including hsp90organizing protein (hop) and p23, are also found in HT-1080 conditioned mediasupporting the notion that hsp90 complexes function in invasiveness. These findingssuggest a wide-ranging phenomenon of extracellular molecular chaperoning that couldhave implications for biological processes and disease.     

Different Poses for Ligand and Chaperone in Inhibitor-Bound Hsp90 and GRP94: Implications for Paralog-Specific Drug Design

Hsp90 chaperones contain an N-terminal ATP binding site that has been effectively targeted by competitive inhibitors. Despite the myriad of inhibitors, none to date have been designed to bind specifically to just one of the four mammalian Hsp90 paralogs, which are cytoplasmic Hsp90α and β, endoplasmic reticulum GRP94, and mitochondrial Trap-1. Given that each of the Hsp90 paralogs is responsible for chaperoning a distinct set of client proteins, specific targeting of one Hsp90 paralog may result in higher efficacy and therapeutic control. Specific inhibitors may also help elucidate the biochemical roles of each Hsp90 paralog. Here, we present side-by-side comparisons of the structures of yeast Hsp90 and mammalian GRP94, bound to the pan-Hsp90 inhibitors geldanamycin (Gdm) and radamide. These structures reveal paralog-specific differences in the Hsp90 and GRP94 conformations in response to Gdm binding. We also report significant variation in the pose and disparate binding affinities for the Gdm-radicicol chimera radamide when bound to the two paralogs, which may be exploited in the design of paralog-specific inhibitors.     

Resolving Hot Spots in the C-Terminal Dimerization Domain that Determine the Stability of the Molecular Chaperone Hsp90

Human heat shock protein of 90 kDa (hHsp90) is a homodimer that has an essential role in facilitating malignant transformation at the molecular level. Inhibiting hHsp90 function is a validated approach for treating different types of tumors. Inhibiting the dimerization of hHsp90 via its C-terminal domain (CTD) should provide a novel way to therapeutically interfere with hHsp90 function. Here, we predicted hot spot residues that cluster in the CTD dimerization interface by a structural decomposition of the effective energy of binding computed by the MM-GBSA approach and confirmed these predictions using in silico alanine scanning with DrugScore^PPI. Mutation of these residues to alanine caused a significant decrease in the melting temperature according to differential scanning fluorimetry experiments, indicating a reduced stability of the mutant hHsp90 complexes. Size exclusion chromatography and multi-angle light scattering studies demonstrate that the reduced stability of the mutant hHsp90 correlates with a lower complex stoichiometry due to the disruption of the dimerization interface. These results suggest that the identified hot spot residues can be used as a pharmacophoric template for identifying and designing small-molecule inhibitors of hHsp90 dimerization.     

Modulation of the Hsp90 Chaperone Cycle by a Stringent Client Protein

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Disease Variants of FGFR3 Reveal Molecular Basis for the Recognition and Additional Roles for Cdc37 in Hsp90 Chaperone System

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