Selective Hsp70-Dependent Docking of Hsp104 to Protein Aggregates Protects the Cell from the Toxicity of the Disaggregase

Hsp104 is a yeast chaperone that rescues misfolded proteins from aggregates associated with proteotoxic stress and aging. Hsp104 consists of N-terminal domain, regulatory M-domain and two ATPase domains, assembled into a spiral-shaped hexamer. Protein disaggregation involves polypeptide extraction from an aggregate and its translocation through the central channel. This process relies on Hsp104 cooperation with the Hsp70 chaperone, which also plays important role in regulation of the disaggregase. Although Hsp104 protein-unfolding activity enables cells to survive stress, when uncontrolled, it becomes toxic to the cell.In this work, we investigated the significance of the interaction between Hsp70 and the M-domain of Hsp104 for functioning of the disaggregation system. We identified phenylalanine at position 508 in Hsp104 to be the key site of interaction with Hsp70. Disruption of this site makes Hsp104 unable to bind protein aggregates and to confer tolerance in yeast cells. The use of this Hsp104 variant demonstrates that Hsp70 allows successful initiation of disaggregation only as long as it is able to interact with the disaggregase. As reported previously, this interaction causes release of the M-domain-driven repression of Hsp104. Now we reveal that, apart from this allosteric effect, the interaction between the chaperone partners itself contributes to effective initiation of disaggregation and plays important role in cell protection against Hsp104-induced toxicity. Interaction with Hsp70 shifts Hsp104 substrate specificity from non-aggregated, disordered substrates toward protein aggregates. Accordingly, Hsp70-mediated sequestering of the Hsp104 unfoldase in aggregates makes it less toxic and more productive.     

Regulation of the Hsp104 Middle Domain Activity Is Critical for Yeast Prion Propagation

Molecular chaperones play a significant role in preventing protein misfolding and aggregation. Indeed, some protein conformational disorders have been linked to changes in the chaperone network. Curiously, in yeast, chaperones also play a role in promoting prion maintenance and propagation. While many amyloidogenic proteins are associated with disease in mammals, yeast prion proteins, and their ability to undergo conformational conversion into a prion state, are proposed to play a functional role in yeast biology. The chaperone Hsp104, a AAA+ ATPase, is essential for yeast prion propagation. Hsp104 fragments large prion aggregates to generate a population of smaller oligomers that can more readily convert soluble monomer and be transmitted to daughter cells. Here, we show that the middle (M) domain of Hsp104, and its mobility, plays an integral part in prion propagation. We generated and characterized mutations in the M-domain of Hsp104 that are predicted to stabilize either a repressed or de-repressed conformation of the M-domain (by analogy to ClpB in bacteria). We show that the predicted stabilization of the repressed conformation inhibits general chaperone activity. Mutation to the de-repressed conformation, however, has differential effects on ATP hydrolysis and disaggregation, suggesting that the M-domain is involved in coupling these two activities. Interestingly, we show that changes in the M-domain differentially affect the propagation of different variants of the [PSI+] and [RNQ+] prions, which indicates that some prion variants are more sensitive to changes in the M-domain mobility than others. Thus, we provide evidence that regulation of the M-domain of Hsp104 is critical for efficient prion propagation. This shows the importance of elucidating the function of the M-domain in order to understand the role of Hsp104 in the propagation of different prions and prion variants.     

The Yeast AAA+ Chaperone Hsp104 Is Part of a Network That Links the Actin Cytoskeleton with the Inheritance of Damaged Proteins

The yeast AAA⁺ chaperone Hsp104 is essential for the development of thermotolerance and for the inheritance of prions. Recently, Hsp104, together with the actin cytoskeleton, has been implicated in the asymmetric distribution of carbonylated proteins. Here, we investigated the interplay between Hsp104 and actin by using a dominant-negative variant of Hsp104 (HAP/ClpP) that degrades substrate proteins instead of remodeling them. Coexpression of HAP/ClpP causes defects in morphology and the actin cytoskeleton. Taking a candidate approach, we identified Spa2, a member of the polarisome complex, as an Hsp104 substrate. Furthermore, we provided genetic evidence that links Spa2 and Hsp104 to Hof1, a member of the cytokinesis machinery. Spa2 and Hof1 knockout cells are affected in the asymmetric distribution of damaged proteins, suggesting that Hsp104, Spa2, and Hof1 are members of a network controlling the inheritance of carbonylated proteins.The ensemble of molecular chaperones and proteases constitutes the cellular system that repairs and eliminates misfolded proteins. The activity of this system ensures not only the recovery of cells from protein-damaging stress conditions, but also the maintenance of protein homeostasis under normal growth conditions. The concomitant involvement of members of the Hsp70 and Hsp90 chaperone families in stress-related, regulatory, and housekeeping functions allows the integration of environmental stimuli into regulatory networks (4, 24, 39, 40). However, it has remained unclear whether other chaperones are also involved in regulatory processes.One chaperone which so far has been connected only to stress-related protein quality functions is the oligomeric AAA⁺ chaperone Hsp104 of Saccharomyces cerevisiae. Hsp104 is essential for the development of thermotolerance by reactivating aggregated proteins after severe stress conditions and for prion propagation by severing prion fibrils (31). Yeast cells, when grown at 30°C, harbor approximately 5,000 copies of Hsp104 hexamers per cell, a number that is minor compared to other cytosolic chaperone machineries (e.g., Hsp70 and Hsp90) that are involved in general protein-folding events (10). The known cellular functions of Hsp104, however, cannot provide a rationale for the determined Hsp104 levels, since protein aggregation is hardly detectable in yeast cells at 30°C even in mutant cells lacking Hsp104 function. Furthermore, yeast prions occur de novo at a very low rate of 10⁻⁶ per cell. In consequence, both well-characterized Hsp104 activities are barely required at 30°C, suggesting that Hsp104 has additional, so far unknown housekeeping functions. On the other hand, an S. cerevisiae hsp104 knockout exhibits no obvious phenotype at 30°C (27), giving no clues to a potential involvement of Hsp104 in other cellular processes.Recently, Hsp104 was demonstrated to influence the asymmetric distribution of oxidatively damaged (carbonylated) proteins (8). It remained unclear whether the role of Hsp104 in this process relies on its known activities in protein quality control or on an unknown involvement in other cellular processes. Here, we provide evidence that Hsp104 is part of a network that controls the inheritance of damaged proteins under physiological growth conditions.     

The mammalian disaggregase machinery: Hsp110 synergizes with Hsp70 and Hsp40 to catalyze protein disaggregation and reactivation in a cell-free system

Bacteria, fungi, protozoa, chromista and plants all harbor homologues of Hsp104, a AAA+ ATPase that collaborates with Hsp70 and Hsp40 to promote protein disaggregation and reactivation. Curiously, however, metazoa do not possess an Hsp104 homologue. Thus, whether animal cells renature large protein aggregates has long remained unclear. Here, it is established that mammalian cytosol prepared from different sources possesses a potent, ATP-dependent protein disaggregase and reactivation activity, which can be accelerated and stimulated by Hsp104. This activity did not require the AAA+ ATPase, p97. Rather, mammalian Hsp110 (Apg-2), Hsp70 (Hsc70 or Hsp70) and Hsp40 (Hdj1) were necessary and sufficient to slowly dissolve large disordered aggregates and recover natively folded protein. This slow disaggregase activity was conserved to yeast Hsp110 (Sse1), Hsp70 (Ssa1) and Hsp40 (Sis1 or Ydj1). Hsp110 must engage substrate, engage Hsp70, promote nucleotide exchange on Hsp70, and hydrolyze ATP to promote disaggregation of disordered aggregates. Similarly, Hsp70 must engage substrate and Hsp110, and hydrolyze ATP for protein disaggregation. Hsp40 must harbor a functional J domain to promote protein disaggregation, but the J domain alone is insufficient. Optimal disaggregase activity is achieved when the Hsp40 can stimulate the ATPase activity of Hsp110 and Hsp70. Finally, Hsp110, Hsp70 and Hsp40 fail to rapidly remodel amyloid forms of the yeast prion protein, Sup35, or the Parkinson's disease protein, alpha-synuclein. However, Hsp110, Hsp70 and Hsp40 enhanced the activity of Hsp104 against these amyloid substrates. Taken together, these findings suggest that Hsp110 fulfils a subset of Hsp104 activities in mammals. Moreover, they suggest that Hsp104 can collaborate with the mammalian disaggregase machinery to rapidly remodel amyloid conformers.     

Functional analysis of conserved cis- and trans-elements in the Hsp104 protein disaggregating machine

Hsp104 is a double ring-forming AAA+ ATPase, which harnesses the energy of ATP binding and hydrolysis to rescue proteins from a previously aggregated state. Like other AAA+ machines, Hsp104 features conserved cis- and trans-acting elements, which are hallmarks of AAA+ members and are essential to Hsp104 function. Despite these similarities, it was recently proposed that Hsp104 is an atypical AAA+ ATPase, which markedly differs in 3D structure from other AAA+ machines. Consequently, it was proposed that arginines found in the non-conserved M-domain, but not the predicted Arg-fingers, serve the role of the critical trans-acting element in Hsp104. While the structural discrepancy has been resolved, the role of the Arg-finger residues in Hsp104 remains controversial. Here, we exploited the ability of Hsp104 variants featuring mutations in one ring to retain ATPase and chaperone activities, to elucidate the functional role of the predicted Arg-finger residues. We found that the evolutionarily conserved Arg-fingers are absolutely essential for ATP hydrolysis but are dispensable for hexamer assembly in Hsp104. On the other hand, M-domain arginines are not strictly required for ATP hydrolysis and affect the ATPase and chaperone activities in a complex manner. Our results confirm that Hsp104 is not an atypical AAA+ ATPase, and uses conserved structural elements common to diverse AAA+ machines to drive the mechanical unfolding of aggregated proteins.     

Hsp40s Specify Functions of Hsp104 and Hsp90 Protein Chaperone Machines

Hsp100 family chaperones of microorganisms and plants cooperate with the Hsp70/Hsp40/NEF system to resolubilize and reactivate stress-denatured proteins. In yeast this machinery also promotes propagation of prions by fragmenting prion polymers. We previously showed the bacterial Hsp100 machinery cooperates with the yeast Hsp40 Ydj1 to support yeast thermotolerance and with the yeast Hsp40 Sis1 to propagate [PSI⁺] prions. Here we find these Hsp40s similarly directed specific activities of the yeast Hsp104-based machinery. By assessing the ability of Ydj1-Sis1 hybrid proteins to complement Ydj1 and Sis1 functions we show their C-terminal substrate-binding domains determined distinctions in these and other cellular functions of Ydj1 and Sis1. We find propagation of [URE3] prions was acutely sensitive to alterations in Sis1 activity, while that of [PIN⁺] prions was less sensitive than [URE3], but more sensitive than [PSI⁺]. These findings support the ideas that overexpressing Ydj1 cures [URE3] by competing with Sis1 for interaction with the Hsp104-based disaggregation machine, and that different prions rely differently on activity of this machinery, which can explain the various ways they respond to alterations in chaperone function.     

Hsp104, Hsp70 and Hsp40 interplay regulates formation, growth and elimination of Sup35 prions

Self-templating amyloid forms of Sup35 constitute the yeast prion [PSI(+)]. How the protein-remodelling factor, Hsp104, collaborates with other chaperones to regulate [PSI(+)] inheritance remains poorly delineated. Here, we report how the Ssa and Ssb components of the Hsp70 chaperone system directly affect Sup35 prionogenesis and cooperate with Hsp104. We identify the ribosome-associated Ssb1:Zuo1:Ssz1 complex as a potent antagonist of Sup35 prionogenesis. The Hsp40 chaperones, Sis1 and Ydj1, preferentially interact with Sup35 oligomers and fibres compared with monomers, and facilitate Ssa1 and Ssb1 binding. Various Hsp70:Hsp40 pairs block prion nucleation by disassembling molten oligomers and binding mature oligomers. By binding fibres, Hsp70:Hsp40 pairs occlude prion recognition elements and inhibit seeded assembly. These inhibitory activities are partially relieved by the nucleotide exchange factor, Fes1. Low levels of Hsp104 stimulate prionogenesis and alleviate inhibition by some Hsp70:Hsp40 pairs. At high concentrations, Hsp104 eliminates Sup35 prions. This activity is reduced when Ssa1, or enhanced when Ssb1, is incorporated into nascent prions. These findings illuminate several facets of the chaperone interplay that underpins [PSI(+)] inheritance.     

Influence of Hsp70s and their regulators on yeast prion propagation

Propagation of yeast prions requires normal abundance and activity of many protein chaperones. Central among them is Hsp70, a ubiquitous and essential chaperone involved in many diverse cellular processes that helps promote proper protein folding and acts as a critical component of several chaperone machines. Hsp70 is regulated by a large cohort of co-chaperones, whose effects on prions are likely mediated through Hsp70. Hsp104 is another chaperone, absent from mammalian cells, that resolubilizes proteins from aggregates. This activity, which minimally requires Hsp70 and its co-chaperone Hsp40, is essential for yeast prion replication. Although much is known about how yeast prions can be affected by altering protein chaperones, mechanistic explanations for these effects are uncertain. We discuss the variety of effects Hsp70 and its regulators have on different prions and how the effects might be due to the many ways chaperones interact with each other and with amyloid.Key words: Hsp70, Hsp40, chaperone, prion, yeast     

Substrate transfer from the chaperone Hsp70 to Hsp90

Hsp90 is an essential chaperone protein in the cytosol of eukaryotic cells. It cooperates with the chaperone Hsp70 in defined complexes mediated by the adaptor protein Hop (Sti1 in yeast). These Hsp70/Hsp90 chaperone complexes play a major role in the folding and maturation of key regulatory proteins in eukaryotes. Understanding how non-native client proteins are transferred from one chaperone to the other in these complexes is of central importance. Here, we analyzed the molecular mechanism of this reaction using luciferase as a substrate protein. Our experiments define a pathway for luciferase folding in the Hsp70/Hsp90 chaperone system. They demonstrate that Hsp70 is a potent capture device for unfolded protein while Hsp90 is not very efficient in this reaction. When Hsp90 is absent, in contrast to the in vivo situation, Hsp70 together with the two effector proteins Ydj1 and Sti1 exhibits chaperone activity towards luciferase. In the presence of the complete chaperone system, Hsp90 exhibits a specific positive effect only in the presence of Ydj1. If this factor is absent, the transferred luciferase is trapped on Hsp90 in an inactive conformation. Interestingly, identical results were observed for the yeast and the human chaperone systems although the regulatory function of human Hop is completely different from that of yeast Sti1.     

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.     

Substrate threading through the central pore of the Hsp104 chaperone as a common mechanism for protein disaggregation and prion propagation

The oligomeric AAA+ chaperone Hsp104 is essential for thermotolerance development and prion propagation in yeast. Thermotolerance relies on the ability of Hsp104 to cooperate with the Hsp70 chaperone system in the reactivation of heat-aggregated proteins. Prion propagation requires the Hsp104-dependent fragmentation of prion fibrils to create infectious seeds. It remained elusive whether both processes rely on common or different activities of Hsp104. Specifically, protein reactivation has been suggested to require a substrate threading activity of Hsp104 whereas fibril fragmentation may be mediated by a crowbar activity. Here we engineered an Hsp104 variant, HAP, which cooperates with the bacterial peptidase ClpP to form a novel proteolytic system. HAP threads aggregated model substrates as well as the yeast prion Sup35 through its central pore into associated ClpP. HAP variants that harbour a reduced threading activity were affected in both protein disaggregation and prion propagation, demonstrating that substrate threading represents the common mechanism for the processing of both substrate classes.     

The ClpB/Hsp104 molecular chaperone-a protein disaggregating machine

ClpB and Hsp104 (ClpB/Hsp104) are essential proteins of the heat-shock response and belong to the class 1 family of Clp/Hsp100 AAA+ ATPases. Members of this family form large ring structures and contain two AAA+ modules, which consist of a RecA-like nucleotide-binding domain (NBD) and an alpha-helical domain. Furthermore, ClpB/Hsp104 has a longer middle region, the ClpB/Hsp104-linker, which is essential for chaperone activity. Unlike other Clp/Hsp100 proteins, however, ClpB/Hsp104 neither associates with a cellular protease nor directs the degradation of its substrate proteins. Rather, ClpB/Hsp104 is a bona fide molecular chaperone, which has the remarkable ability to rescue proteins from an aggregated state. The full recovery of these proteins requires the assistance of the cognate DnaK/Hsp70 chaperone system. The mechanism of this "bi-chaperone" network, however, remains elusive. Here we review the current understanding of the structure-function relationship of the ClpB/Hsp104 molecular chaperone and its role in protein disaggregation.     

Chaperone networks in protein disaggregation and prion propagation

The oligomeric AAA+ chaperones Escherichia coli ClpB and Saccharomyces cerevisiae Hsp104 cooperate with cognate Hsp70/Hsp40 chaperone machineries in the reactivation of aggregated proteins in E. coli and S. cerevisiae. In addition, Hsp104 and Hsp70/Hsp40 are crucial for the maintenance of prion aggregates in yeast cells. While the bichaperone system efficiently solubilizes stress-generated amorphous aggregates, structurally highly ordered prion fibrils are only partially processed, resulting in the generation of fragmented prion seeds that can be transmitted to daughter cells for stable inheritance. Here, we describe and discuss the most recent mechanistic findings on yeast Hsp104 and Hsp70/Hsp40 cooperation in the remodeling of both types of aggregates, emphasizing similarities in the mechanism but also differences in the sensitivities towards chaperone activities.     

Asymmetric deceleration of ClpB or Hsp104 ATPase activity unleashes protein-remodeling activity

Two members of the AAA+ superfamily, ClpB and Hsp104, collaborate with Hsp70 and Hsp40 to rescue aggregated proteins. However, the mechanisms that elicit and underlie their protein-remodeling activities remain unclear. We report that for both Hsp104 and ClpB, mixtures of ATP and ATP-gammaS unexpectedly unleash activation, disaggregation and unfolding activities independent of cochaperones. Mutations reveal how remodeling activities are elicited by impaired hydrolysis at individual nucleotide-binding domains. However, for some substrates, mixtures of ATP and ATP-gammaS abolish remodeling, whereas for others, ATP binding without hydrolysis is sufficient. Remodeling of different substrates necessitates a diverse balance of polypeptide 'holding' (which requires ATP binding but not hydrolysis) and unfolding (which requires ATP hydrolysis). We suggest that this versatility in reaction mechanism enables ClpB and Hsp104 to reactivate the entire aggregated proteome after stress and enables Hsp104 to control prion inheritance.     

Processing of proteins by the molecular chaperone Hsp104

The molecular chaperone Hsp104 is an AAA+ ATPase (ATPase associated with a variety of cellular activities) from yeast that catalyzes protein disaggregation. Using mutagenesis, we impaired nucleotide binding or hydrolysis in the two nucleotide-binding domains (NBD) of Hsp104 and analyzed the consequences for chaperone function by monitoring ATP hydrolysis, polypeptide binding, polypeptide processing, and disaggregation. Our results reveal that ATP binding to NBD1 serves as a central regulatory switch for the chaperone; it triggers binding of polypeptides, and stimulates ATP hydrolysis in the C-terminal NBD2 by more than two orders of magnitude, implying that ATP hydrolysis in this domain is important for disaggregation. Moreover, we show that Hsp104 actively unfolds its polypeptide substrates during processing, demonstrating that AAA+ proteins involved in disaggregation share a common threading mechanism with AAA+ proteins mediating protein unfolding/degradation.     

The molecular chaperone Hsp104--a molecular machine for protein disaggregation

At the Cold Spring Harbor Meeting on 'Molecular Chaperones and the Heat Shock Response' in May 1996, Susan Lindquist presented evidence that a chaperone of yeast termed Hsp104, which her group had been investigating for several years, is able to dissolve protein aggregates (Glover, J.R., Lindquist, S., 1998. Hsp104, Hsp70, and Hsp40: a novel chaperone system that rescues previously aggregated proteins. Cell 94, 73-82). Among many of the participants this news stimulated reactions reaching from decided skepticism to utter disbelief because protein aggregation was widely considered to be an irreversible process. Several years and publications later, it is undeniable that Susan had been right. Hsp104 is an ATP dependent molecular machine that-in cooperation with Hsp70 and Hsp40-extracts polypeptide chains from protein aggregates and facilitates their refolding, although the molecular details of this process are still poorly understood. Meanwhile, close homologues of Hsp104 have been identified in bacteria (ClpB), in mitochondria (Hsp78), and in the cytosol of plants (Hsp101), but intriguingly not in the cytosol of animal cells (Mosser, D.D., Ho, S., Glover, J.R., 2004. Saccharomyces cerevisiae Hsp104 enhances the chaperone capacity of human cells and inhibits heat stress-induced proapoptotic signaling. Biochemistry 43, 8107-8115). Observations that Hsp104 plays an essential role in the maintenance of yeast prions (see review by James Shorter in this issue) have attracted even more attention to the molecular mechanism of this ATP dependent chaperone (Chernoff, Y.O., Lindquist, S.L., Ono, B., Inge-Vechtomov, S.G., Liebman, S.W., 1995. Role of the chaperone protein Hsp104 in propagation of the yeast prion-like factor [PSI+]. Science 268, 880-884).     

The elusive middle domain of Hsp104 and ClpB: location and function

Hsp104 in yeast and ClpB in bacteria are homologous, hexameric AAA+ proteins and Hsp100 chaperones, which function in the stress response as ring-translocases that drive protein disaggregation and reactivation. Both Hsp104 and ClpB contain a distinctive coiled-coil middle domain (MD) inserted in the first AAA+ domain, which distinguishes them from other AAA+ proteins and Hsp100 family members. Here, we focus on recent developments concerning the location and function of the MD in these hexameric molecular machines, which remains an outstanding question. While the atomic structure of the hexameric assembly of Hsp104 and ClpB remains uncertain, recent advances have illuminated that the MD is critical for the intrinsic disaggregase activity of the hexamer and mediates key functional interactions with the Hsp70 chaperone system (Hsp70 and Hsp40) that empower protein disaggregation.     

Influence of Hsp70s and their regulators on yeast prion propagation

Propagation of yeast prions requires normal abundance and activity of many protein chaperones. Central among them is Hsp70, a ubiquitous and essential chaperone involved in many diverse cellular processes that helps promote proper protein folding and acts as a critical component of several chaperone machines. Hsp70 is regulated by a large cohort of co-chaperones, whose effects on prions are likely mediated through Hsp70. Hsp104 is another chaperone, absent from mammalian cells, that resolubilizes proteins from aggregates. This activity, which minimally requires Hsp70 and its co-chaperone Hsp40, is essential for yeast prion replication. Although much is known about how yeast prions can be affected by altering protein chaperones, mechanistic explanations for these effects are uncertain. We discuss the variety of effects Hsp70 and its regulators have on different prions and how the effects might be due to the many ways chaperones interact with each other and with amyloid.     

Suramin Inhibits Hsp104 ATPase and Disaggregase Activity

Hsp104 is a hexameric AAA+ protein that utilizes energy from ATP hydrolysis to dissolve disordered protein aggregates as well as amyloid fibers. Interestingly, Hsp104 orthologues are found in all kingdoms of life except animals. Thus, Hsp104 could represent an interesting drug target. Specific inhibition of Hsp104 activity might antagonize non-metazoan parasites that depend on a potent heat shock response, while producing little or no side effects to the host. However, no small molecule inhibitors of Hsp104 are known except guanidinium chloride. Here, we screen over 16,000 small molecules and identify 16 novel inhibitors of Hsp104 ATPase activity. Excluding compounds that inhibited Hsp104 activity by non-specific colloidal effects, we defined Suramin as an inhibitor of Hsp104 ATPase activity. Suramin is a polysulphonated naphthylurea and is used as an antiprotozoal drug for African Trypanosomiasis. Suramin also interfered with Hsp104 disaggregase, unfoldase, and translocase activities, and the inhibitory effect of Suramin was not rescued by Hsp70 and Hsp40. Suramin does not disrupt Hsp104 hexamers and does not effectively inhibit ClpB, the E. coli homolog of Hsp104, establishing yet another key difference between Hsp104 and ClpB behavior. Intriguingly, a potentiated Hsp104 variant, Hsp104^A503V, is more sensitive to Suramin than wild-type Hsp104. By contrast, Hsp104 variants bearing inactivating sensor-1 mutations in nucleotide-binding domain (NBD) 1 or 2 are more resistant to Suramin. Thus, Suramin depends upon ATPase events at both NBDs to exert its maximal effect. Suramin could develop into an important mechanistic probe to study Hsp104 structure and function.     

Substrate binding to the molecular chaperone Hsp104 and its regulation by nucleotides

The Hsp104 protein from Saccharomyces cerevisiae is a member of the Hsp100/Clp family of molecular chaperones. It mediates the solubilization of aggregated proteins in an ATP-dependent process assisted by the Hsp70/40 system. Although the principal function of Hsp104 is well established, the mechanistic details of this catalyzed disaggregation are poorly understood. In this work, we have investigated the interaction of Hsp104 with reduced, carboxymethylated alpha-lactalbumin (RCMLa), a permanently unfolded model substrate. Our results demonstrate that the affinity of Hsp104 toward polypeptides is regulated by nucleotides. In the presence of ATP or adenosine-5' -O-(3-thiotriphosphate), the chaperone formed complexes with RCMLa, whereas no binding was observed in the presence of ADP. In particular, the occupation of the N-terminally located nucleotide-binding domain with ATP seems to be crucial for substrate interaction. When ATP binding to this domain was impaired by mutation, Hsp104 lost its ability to interact with RCMLa. Our results also indicate that upon association with a polypeptide, a conformational change occurs within Hsp104 that strongly reduces the dynamics of nucleotide exchange and commits the bound polypeptide to ATP hydrolysis.