Conformational dynamics of the molecular chaperone Hsp90

The ubiquitous molecular chaperone Hsp90 makes up 1-2% of cytosolic proteins and is required for viability in eukaryotes. Hsp90 affects the folding and activation of a wide variety of substrate proteins including many involved in signaling and regulatory processes. Some of these substrates are implicated in cancer and other diseases, making Hsp90 an attractive drug target. Structural analyses have shown that Hsp90 is a highly dynamic and flexible molecule that can adopt a wide variety of structurally distinct states. One driving force for these rearrangements is the intrinsic ATPase activity of Hsp90, as seen with other chaperones. However, unlike other chaperones, studies have shown that the ATPase cycle of Hsp90 is not conformationally deterministic. That is, rather than dictating the conformational state, ATP binding and hydrolysis only shift the equilibria between a pre-existing set of conformational states. For bacterial, yeast and human Hsp90, there is a conserved three-state (apo-ATP-ADP) conformational cycle; however; the equilibria between states are species specific. In eukaryotes, cytosolic co-chaperones regulate the in vivo dynamic behavior of Hsp90 by shifting conformational equilibria and affecting the kinetics of structural changes and ATP hydrolysis. In this review, we discuss the structural and biochemical studies leading to our current understanding of the conformational dynamics of Hsp90, as well as the roles that nucleotide, co-chaperones, post-translational modification and substrates play. This view of Hsp90's conformational dynamics was enabled by the use of multiple complementary structural methods including, crystallography, small-angle X-ray scattering (SAXS), electron microscopy, F?rster resonance energy transfer (FRET) and NMR. Finally, we discuss the effects of Hsp90 inhibitors on conformation and the potential for developing small molecules that inhibit Hsp90 by disrupting the conformational dynamics.     

Dihydroxylphenyl amides as inhibitors of the Hsp90 molecular chaperone

Information from X-ray crystal structures were used to optimize the potency of a HTS hit in a Hsp90 competitive binding assay. A class of novel and potent small molecule Hsp90 inhibitors were thereby identified. Enantio-pure compounds 31 and 33 were potent in PGA-based competitive binding assay and inhibited proliferation of various human cancer cell lines in vitro, with IC(50) values averaging 20 nM.     

Modulating molecular chaperone Hsp90 functions through reversible acetylation

The molecular chaperone protein Hsp90 is a key regulator of approximately 100 'client' proteins crucial for numerous cell signaling processes. Consequently, understanding the molecular underpinnings that regulate Hsp90 activity is an important biological endeavor. Exciting new results now suggest that, at least for nuclear receptor activity, Hsp90 function is directly regulated by histone deacetylase 6 (HDAC6). These observations have consequences for various biological processes and potentially important implications for the development of cancer therapeutics.     

The Hsp90 chaperone complex regulates GDI-dependent Rab recycling

Rab GTPase regulated hubs provide a framework for an integrated coding system, the membrome network, that controls the dynamics of the specialized exocytic and endocytic membrane architectures found in eukaryotic cells. Herein, we report that Rab recycling in the early exocytic pathways involves the heat-shock protein (Hsp)90 chaperone system. We find that Hsp90 forms a complex with guanine nucleotide dissociation inhibitor (GDI) to direct recycling of the client substrate Rab1 required for endoplasmic reticulum (ER)-to-Golgi transport. ER-to-Golgi traffic is inhibited by the Hsp90-specific inhibitors geldanamycin (GA), 17-(dimethylaminoethylamino)-17-demethoxygeldanamycin (17-DMAG), and radicicol. Hsp90 activity is required to form a functional GDI complex to retrieve Rab1 from the membrane. Moreover, we find that Hsp90 is essential for Rab1-dependent Golgi assembly. The observation that the highly divergent Rab GTPases Rab1 involved in ER-to-Golgi transport and Rab3A involved in synaptic vesicle fusion require Hsp90 for retrieval from membranes lead us to now propose that the Hsp90 chaperone system may function as a general regulator for Rab GTPase recycling in exocytic and endocytic trafficking pathways involved in cell signaling and proliferation.     

Synthesis of novel fluorescent probes for the molecular chaperone Hsp90

Heat shock protein 90 (Hsp90) is a molecular chaperone necessary for maintaining oncogenic transformation. There is substantial interest in developing novel agents that bind to the N-terminal of the chaperone. Here we report the synthesis and characterization of two fluorescent Hsp90 inhibitors and probe their use in an Hsp90 fluorescent polarization assay.     

Conformational switching of the molecular chaperone Hsp90 via regulated phosphorylation

Hsp90 is an essential molecular chaperone in the eukaryotic cytosol. Its function is modulated by cochaperones and posttranslational modifications. Importantly, the phosphatase Ppt1 is a dedicated regulator of the Hsp90 chaperone system. Little is known about Ppt1-dependent phosphorylation sites and how these affect Hsp90 activity. Here, we identified the major phosphorylation sites of yeast Hsp90 in its middle or the C-terminal domain and determined the subset regulated by Ppt1. In general, phosphorylation decelerates the Hsp90 machinery, reduces chaperone function in vivo, sensitizes yeast cells to Hsp90 inhibition and affects DNA repair processes. Modification of one particular site (S485) is lethal, whereas others modulate Hsp90 activity via distinct mechanisms affecting the ATPase activity, cochaperone binding and manipulating conformational transitions in Hsp90. Our mechanistic analysis reveals that phosphorylation of Hsp90 permits a regulation of the conformational cycle at distinct steps by targeting switch points for the communication of remote regions within Hsp90.     

Substrate binding drives large-scale conformational changes in the Hsp90 molecular chaperone

Hsp90 is a ubiquitous molecular chaperone. Previous structural analysis demonstrated that Hsp90 can adopt a large number of structurally distinct conformations; however, the functional role of this flexibility is not understood. Here we investigate the structural consequences of substrate binding with a model system in which Hsp90 interacts with a partially folded protein (Δ131Δ), a well-studied fragment of staphylococcal nuclease. SAXS measurements reveal that under apo conditions, Hsp90 partially closes around Δ131Δ, and in the presence of AMPPNP, Δ131Δ binds with increased affinity to Hsp90's fully closed state. FRET measurements show that Δ131Δ accelerates the nucleotide-driven open/closed transition and stimulates ATP hydrolysis by Hsp90. NMR measurements reveal that Hsp90 binds to a specific, highly structured region of Δ131Δ. These results suggest that Hsp90 preferentially binds a locally structured region in a globally unfolded protein, and this binding drives functional changes in the chaperone by lowering a rate-limiting conformational barrier.     

Osmolyte‐induced conformational changes in the Hsp90 molecular chaperone

Osmolytes are small molecules that play a central role in cellular homeostasis and the stress response by maintaining protein thermodynamic stability at controlled levels. The underlying physical chemistry that describes how different osmolytes impact folding free energy is well understood, however little is known about their influence on other crucial aspects of protein behavior, such as native‐state conformational changes. Here we investigate this issue with the Hsp90 molecular chaperone, a large dimeric protein that populates a complex conformational equilibrium. Using small angle X‐ray scattering we observe dramatic osmolyte‐dependent structural changes within the native ensemble. The degree to which different osmolytes affect the Hsp90 conformation strongly correlates with thermodynamic metrics of their influence on stability. This observation suggests that the well‐established osmolyte principles that govern stability also apply to large‐scale conformational changes, a proposition that is corroborated by structure‐based fitting of the scattering data, surface area comparisons and m‐value analysis. This approach shows how osmolytes affect a highly cooperative open/closed structural transition between two conformations that differ by a domain‐domain interaction. Hsp90 adopts an additional ligand‐specific conformation in the presence of ATP and we find that osmolytes do not significantly affect this conformational change. Together, these results extend the scope of osmolytes by suggesting that they can maintain protein conformational heterogeneity at controlled levels using similar underlying principles that allow them to maintain protein stability; however the relative impact of osmolytes on different structural states can vary significantly.     

Rab-alphaGDI activity is regulated by a Hsp90 chaperone complex

The Rab-specific alphaGDP-dissociation inhibitor (alphaGDI) regulates the recycling of Rab GTPases. We have now identified a novel alphaGDI complex from synaptic membranes that contains three chaperone components: Hsp90, Hsc70 and cysteine string protein (CSP). We find that the alphaGDI-chaperone complex is dissociated in response to Ca(2+)-induced neurotransmitter release, that chaperone complex dissociation is sensitive to the Hsp90 inhibitor geldanamycin (GA) and that GA inhibits the ability of alphaGDI to recycle Rab3A during neurotransmitter release. We propose that alphaGDI interacts with a specialized membrane-associated Rab recycling Hsp90 chaperone system on the vesicle membrane to coordinate the Ca(2+)-dependent events triggering Rab-GTP hydrolysis with retrieval of Rab-GDP to the cytosol.     

Development of a fluorescence polarization assay for the molecular chaperone Hsp90

Heat shock protein 90 (Hsp90) is a molecular chaperone with essential functions in maintaining transformation, and there is increasing interest in developing Hsp90 inhibitors as cancer therapeutics. In this study, the authors describe the development and optimization of a novel assay for the identification of Hsp90 inhibitors using fluorescence polarization. The assay is based on the competition of fluorescently (BODIPY) labeled geldanamycin (GM) for binding to purified recombinant Hsp90alpha (GM is a natural product that binds to the ATP/ADP pocket in the amino terminal of Hsp90). The authors show that GM-BODIPY binds Hsp90alpha with high affinity. Even at low Hsp90alpha concentrations (30 nM), the measured polarization value is close to the maximum assay range of 160 mP, making measurements very sensitive. Its performance, as judged by signal-to-noise ratios (> 10) and Z and Z' values (> 0.5), suggests that this is a robust and reliable assay. GM, PU24FCl, ADP, and ATP, all known to bind to the Hsp90 pocket, compete with GM-BODIPY for binding to Hsp90alpha with EC(50)s in agreement with reported values. These data demonstrate that the Hsp90-FP-based assay can be used for high-throughput screening in aiding the identification of novel Hsp90 inhibitors.     

Regulation of PIDD auto-proteolysis and activity by the molecular chaperone Hsp90

In response to DNA damage, p53-induced protein with a death domain (PIDD) forms a complex called the PIDDosome, which either consists of PIDD, RIP-associated protein with a death domain and caspase-2, forming a platform for the activation of caspase-2, or contains PIDD, RIP1 and NEMO, important for NF-κB activation. PIDDosome activation is dependent on auto-processing of PIDD at two different sites, generating the fragments PIDD-C and PIDD-CC. Despite constitutive cleavage, endogenous PIDD remains inactive. In this study, we screened for novel PIDD regulators and identified heat shock protein 90 (Hsp90) as a major effector in both PIDD protein maturation and activation. Hsp90, together with p23, binds PIDD and inhibition of Hsp90 activity with geldanamycin efficiently disrupts this association and impairs PIDD auto-processing. Consequently, both PIDD-mediated NF-κB and caspase-2 activation are abrogated. Interestingly, PIDDosome formation itself is associated with Hsp90 release. Characterisation of cytoplasmic and nuclear pools of PIDD showed that active PIDD accumulates in the nucleus and that only cytoplasmic PIDD is bound to Hsp90. Finally, heat shock induces Hsp90 release from PIDD and PIDD nuclear translocation. Thus, Hsp90 has a major role in controlling PIDD functional activity.     

S100A1 is a novel molecular chaperone and a member of the Hsp70/Hsp90 multichaperone complex

Although calmodulin is known to be a component of the Hsp70/Hsp90 multichaperone complex, the functional role of the protein remains uncertain. In this study, we have identified S100A1, but not calmodulin or other S100 proteins, as a potent molecular chaperone and a new member of the multichaperone complex. Glutathione S-transferase pull-down assays and co-immunoprecipitation experiments indicated the formation of stable complexes between S100A1 and Hsp90, Hsp70, FKBP52, and CyP40 both in vitro and in mammalian cells. S100A1 potently protected citrate synthase, aldolase, glyceraldehyde-3-phosphate dehydrogenase, and rhodanese from heat-induced aggregation and suppressed the aggregation of chemically denatured rhodanese and citrate synthase during the refolding pathway. In addition, S100A1 suppressed the heat-induced inactivation of citrate synthase activity, similar to that for Hsp90 and p23. The chaperone activity of S100A1 was antagonized by calmodulin antagonists, such as fluphenazine and prenylamine, that is, indeed an intrinsic function of the protein. The overexpression of S100A1 in COS-7 cells protected transiently expressed firefly luciferase and Escherichia coli beta-galactosidase from inactivation during heat shock. The results demonstrate a novel physiological function for S100A1 and bring us closer to a comprehensive understanding of the molecular mechanisms of the Hsp70/Hsp90 multichaperone complex.     

Hop: more than an Hsp70/Hsp90 adaptor protein

Molecular chaperones facilitate the correct folding of other proteins under physiological and stress conditions. Recently it has become evident that various co-chaperone proteins regulate the cellular functions of these chaperones, particularly Hsp70 and Hsp90. Hop is one of the most extensively studied co-chaperones that is able to directly associate with both Hsp70 and Hsp90. The current dogma proposes that Hop functions primarily as an adaptor that directs Hsp90 to Hsp70-client protein complexes in the cytoplasm. However, recent evidence suggests that Hop can also modulate the chaperone activities of these Hsps, and that it is not dedicated to Hsp70 and Hsp90. While the co-chaperone function of Hop within the cytoplasm has been extensively studied, its association with nuclear complexes and prion proteins remains to be elucidated. This article will review the structural features of Hop, and the evidence that its biological function is considerably broader than previously envisaged.     

A comprehensive study on the immunological reactivity of the Hsp90 molecular chaperone

Periodontitis is a chronic infectious disease, Porphyromonas gingivalis being the most implicated pathogen. In the present study, we investigated the role of P. gingivalis HtpG (PgHtpG), a bacterial ortholog of mammalian Hsp90, in the growth of P. gingivalis and also assessed the immunological cross-reactivity of the members of the Hsp90 family. Antiserum against rat liver Hsp90 potently reacted with yeast Hsp90, called Hsc82, and also weakly with human Hsp90 (hHsp90) and human mitochondrial paralog Trap1, but did not react with PgHtpG, Escherichia coli HtpG, or human endoplasmic reticulum paralog Grp94. Moreover, among 19 monoclonal antibodies raised against hHsp90, nine cross-reacted with yeast Hsc82, and one with human Grp94, but none bound to PgHtpG or E. coli HtpG. Among them, three mAbs that strongly reacted with yeast Hsc82 recognized Asn(291)-Ile(304), a conserved region of the family protein. The polyclonal antibody raised against a peptide, Met(315)-Glu(328), of human Grp94, which corresponded to the conserved region of hHsp90, cross-reacted with hHsp90, but not with other Hsp90-family members. Thus, although mammalian Hsp90 shares some immunological reactivity with yeast Hsc82, human Grp94, and human Trap1, it is considerably distinct from its bacterial ortholog, HtpG. Disruption of the P. gingivalis htpG gene neither affected bacterial survival nor altered the sensitivity of P. gingivalis to various forms of stress.     

Diverse cellular functions of the Hsp90 molecular chaperone uncovered using systems approaches

A comprehensive understanding of the cellular functions of the Hsp90 molecular chaperone has remained elusive. Although Hsp90 is essential, highly abundant under normal conditions, and further induced by environmental stress, only a limited number of Hsp90 "clients" have been identified. To define Hsp90 function, a panel of genome-wide chemical-genetic screens in Saccharomyces cerevisiae were combined with bioinformatic analyses. This approach identified several unanticipated functions of Hsp90 under normal conditions and in response to stress. Under normal growth conditions, Hsp90 plays a major role in various aspects of the secretory pathway and cellular transport; during environmental stress, Hsp90 is required for the cell cycle, meiosis, and cytokinesis. Importantly, biochemical and cell biological analyses validated several of these Hsp90-dependent functions, highlighting the potential of our integrated global approach to uncover chaperone functions in the cell.