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• W2801645577 abstract "•At physiological concentration, Hsp70 blocks effective protein folding•Hsp90 restarts folding, overcoming the Hsp70-inflicted folding block•The Hsp70-Hsp90 cascade increases folding yields, but does not alter folding kinetics•Short ATP depending chaperone phase is followed by slow Anfinsen folding Protein folding in the cell requires ATP-driven chaperone machines such as the conserved Hsp70 and Hsp90. It is enigmatic how these machines fold proteins. Here, we show that Hsp90 takes a key role in protein folding by breaking an Hsp70-inflicted folding block, empowering protein clients to fold on their own. At physiological concentrations, Hsp70 stalls productive folding by binding hydrophobic, core-forming segments. Hsp90 breaks this deadlock and restarts folding. Remarkably, neither Hsp70 nor Hsp90 alters the folding rate despite ensuring high folding yields. In fact, ATP-dependent chaperoning is restricted to the early folding phase. Thus, the Hsp70-Hsp90 cascade does not fold proteins, but instead prepares them for spontaneous, productive folding. This stop-start mechanism is conserved from bacteria to man, assigning also a general function to bacterial Hsp90, HtpG. We speculate that the decreasing hydrophobicity along the Hsp70-Hsp90 cascade may be crucial for enabling spontaneous folding. Protein folding in the cell requires ATP-driven chaperone machines such as the conserved Hsp70 and Hsp90. It is enigmatic how these machines fold proteins. Here, we show that Hsp90 takes a key role in protein folding by breaking an Hsp70-inflicted folding block, empowering protein clients to fold on their own. At physiological concentrations, Hsp70 stalls productive folding by binding hydrophobic, core-forming segments. Hsp90 breaks this deadlock and restarts folding. Remarkably, neither Hsp70 nor Hsp90 alters the folding rate despite ensuring high folding yields. In fact, ATP-dependent chaperoning is restricted to the early folding phase. Thus, the Hsp70-Hsp90 cascade does not fold proteins, but instead prepares them for spontaneous, productive folding. This stop-start mechanism is conserved from bacteria to man, assigning also a general function to bacterial Hsp90, HtpG. We speculate that the decreasing hydrophobicity along the Hsp70-Hsp90 cascade may be crucial for enabling spontaneous folding. It is the primary sequence of the protein that determines its native fold (Anfinsen, 1973Anfinsen C.B. Principles that govern the folding of protein chains.Science. 1973; 181: 223-230Crossref PubMed Scopus (5137) Google Scholar). Proteins condense around an initial nucleus, the hydrophobic core, and ultimately fold into the same structure each time (Daggett and Fersht, 2003Daggett V. Fersht A. The present view of the mechanism of protein folding.Nat. Rev. Mol. Cell Biol. 2003; 4: 497-502Crossref PubMed Scopus (350) Google Scholar). Recapitulating protein folding in vitro usually requires conditions that are far away from physiological states. In the cell, conserved families of molecular chaperones support folding of proteins in an energy-consuming manner, presumably by repeated cycles of binding and release (Buchberger et al., 2010Buchberger A. Bukau B. Sommer T. Protein quality control in the cytosol and the endoplasmic reticulum: brothers in arms.Mol. Cell. 2010; 40: 238-252Abstract Full Text Full Text PDF PubMed Scopus (377) Google Scholar, Ellis, 1987Ellis J. Proteins as molecular chaperones.Nature. 1987; 328: 378-379Crossref PubMed Scopus (684) Google Scholar, Kim et al., 2013Kim Y.E. Hipp M.S. Bracher A. Hayer-Hartl M. Hartl F.U. Molecular chaperone functions in protein folding and proteostasis.Annu. Rev. Biochem. 2013; 82: 323-355Crossref PubMed Scopus (973) Google Scholar, Mayer, 2013Mayer M.P. Hsp70 chaperone dynamics and molecular mechanism.Trends Biochem. Sci. 2013; 38: 507-514Abstract Full Text Full Text PDF PubMed Scopus (301) Google Scholar). The non-native polypeptide substrates targeted by chaperones are also known as clients. The molecular determinants of assisted protein folding, however, remain largely enigmatic. The ubiquitous, ATP-dependent Hsp70 chaperone interacts with virtually all unfolded or misfolded proteins and has been demonstrated to refold numerous proteins into their native state. The likewise ATP-dependent Hsp90 chaperone is believed to be more specific, targeting certain proteins in a near-native state (Jakob et al., 1995Jakob U. Lilie H. Meyer I. Buchner J. Transient interaction of Hsp90 with early unfolding intermediates of citrate synthase. Implications for heat shock in vivo.J. Biol. Chem. 1995; 270: 7288-7294Crossref PubMed Scopus (319) Google Scholar, Karagöz and Rüdiger, 2015Karagöz G.E. Rüdiger S.G.D. Hsp90 interaction with clients.Trends Biochem. Sci. 2015; 40: 117-125Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar, Li et al., 2012Li J. Soroka J. Buchner J. The Hsp90 chaperone machinery: conformational dynamics and regulation by co-chaperones.Biochim. Biophys. Acta. 2012; 1823: 624-635Crossref PubMed Scopus (365) Google Scholar, Taipale et al., 2012Taipale M. Krykbaeva I. Koeva M. Kayatekin C. Westover K.D. Karras G.I. Lindquist S. Quantitative analysis of HSP90-client interactions reveals principles of substrate recognition.Cell. 2012; 150: 987-1001Abstract Full Text Full Text PDF PubMed Scopus (591) Google Scholar). Hsp90 acts downstream of Hsp70, but its contribution to protein folding is unclear (Karagöz et al., 2014Karagöz G.E. Duarte A.M.S. Akoury E. Ippel H. Biernat J. Morán Luengo T. Radli M. Didenko T. Nordhues B.A. Veprintsev D.B. et al.Hsp90-Tau complex reveals molecular basis for specificity in chaperone action.Cell. 2014; 156: 963-974Abstract Full Text Full Text PDF PubMed Scopus (201) Google Scholar, Karagöz and Rüdiger, 2015Karagöz G.E. Rüdiger S.G.D. Hsp90 interaction with clients.Trends Biochem. Sci. 2015; 40: 117-125Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar). Hsp70 and Hsp90 work together to promote maturation of steroid receptors in an ATP-dependent manner (Kirschke et al., 2014Kirschke E. Goswami D. Southworth D. Griffin P.R. Agard D.A. Glucocorticoid receptor function regulated by coordinated action of the Hsp90 and Hsp70 chaperone cycles.Cell. 2014; 157: 1685-1697Abstract Full Text Full Text PDF PubMed Scopus (270) Google Scholar, Sanchez et al., 1987Sanchez E.R. Meshinchi S. Tienrungroj W. Schlesinger M.J. Toft D.O. Pratt W.B. Relationship of the 90-kDa murine heat shock protein to the untransformed and transformed states of the L cell glucocorticoid receptor.J. Biol. Chem. 1987; 262: 6986-6991Abstract Full Text PDF PubMed Google Scholar, Smith et al., 1992Smith D.F. Stensgard B.A. Welch W.J. Toft D.O. Assembly of progesterone receptor with heat shock proteins and receptor activation are ATP mediated events.J. Biol. Chem. 1992; 267: 1350-1356PubMed Google Scholar), and the co-chaperone Hop physically links both chaperones, facilitating substrate transfer from Hsp70 to Hsp90 (Wegele et al., 2006Wegele H. Wandinger S.K. Schmid A.B. Reinstein J. Buchner J. Substrate transfer from the chaperone Hsp70 to Hsp90.J. Mol. Biol. 2006; 356: 802-811Crossref PubMed Scopus (123) Google Scholar). Hsp90 is suggested to remodel the client downstream of Hsp70, but its impact on the folding yield is marginal, and its molecular role remains enigmatic (Genest et al., 2011Genest O. Hoskins J.R. Camberg J.L. Doyle S.M. Wickner S. Heat shock protein 90 from Escherichia coli collaborates with the DnaK chaperone system in client protein remodeling.Proc. Natl. Acad. Sci. USA. 2011; 108: 8206-8211Crossref PubMed Scopus (92) Google Scholar, Genest et al., 2015Genest O. Hoskins J.R. Kravats A.N. Doyle S.M. Wickner S. Hsp70 and Hsp90 of E. coli Directly Interact for Collaboration in Protein Remodeling.J. Mol. Biol. 2015; 427: 3877-3889Crossref PubMed Scopus (47) Google Scholar). Indeed, Hsp70 can refold proteins in the absence of Hsp90 (Schröder et al., 1993Schröder H. Langer T. Hartl F.U. Bukau B. DnaK, DnaJ and GrpE form a cellular chaperone machinery capable of repairing heat-induced protein damage.EMBO J. 1993; 12: 4137-4144Crossref PubMed Scopus (498) Google Scholar). However, two issues appeared paradoxical to us. First, Hsp70 promotes protein folding while it binds to short, very hydrophobic stretches, which are required to form the hydrophobic core of the protein (Rüdiger et al., 1997Rüdiger S. Germeroth L. Schneider-Mergener J. Bukau B. Substrate specificity of the DnaK chaperone determined by screening cellulose-bound peptide libraries.EMBO J. 1997; 16: 1501-1507Crossref PubMed Scopus (660) Google Scholar, Karagöz et al., 2014Karagöz G.E. Duarte A.M.S. Akoury E. Ippel H. Biernat J. Morán Luengo T. Radli M. Didenko T. Nordhues B.A. Veprintsev D.B. et al.Hsp90-Tau complex reveals molecular basis for specificity in chaperone action.Cell. 2014; 156: 963-974Abstract Full Text Full Text PDF PubMed Scopus (201) Google Scholar). Second, Hsp70 is also required to bind with high association rate, within seconds, to outcompete their high aggregation propensity (Mayer et al., 2000Mayer M.P. Schröder H. Rüdiger S. Paal K. Laufen T. Bukau B. Multistep mechanism of substrate binding determines chaperone activity of Hsp70.Nat. Struct. Biol. 2000; 7: 586-593Crossref PubMed Scopus (308) Google Scholar, Mayer and Bukau, 2005Mayer M.P. Bukau B. Hsp70 chaperones: cellular functions and molecular mechanism.Cell. Mol. Life Sci. 2005; 62: 670-684Crossref PubMed Scopus (2055) Google Scholar). How can hydrophobic cores form when Hsp70 binds to the very stretches that are required for folding, and how do complex proteins form their hydrophobic core and ultimately reach the native state in the presence of fast-rebinding Hsp70? Here, we propose that the chaperone machines Hsp70 and Hsp90 form a conserved cascade that promotes spontaneous protein folding by a stop-start mechanism. Instead of refolding proteins, Hsp70 blocks folding when present at physiological concentrations. It is the transfer of the client to Hsp90 that is crucial to break the deadlock and to allow the protein to start a productive folding trajectory. Our findings describe the mode of action of the Hsp70-Hsp90 cascade and thus provide molecular understanding of chaperone-assisted protein folding. Protein folding activity of Hsp70 chaperones was established both in vivo and in vitro using luciferase as a paradigmatic client, nota bene in the absence of Hsp90 (Schröder et al., 1993Schröder H. Langer T. Hartl F.U. Bukau B. DnaK, DnaJ and GrpE form a cellular chaperone machinery capable of repairing heat-induced protein damage.EMBO J. 1993; 12: 4137-4144Crossref PubMed Scopus (498) Google Scholar). To understand the chaperone activity of Hsp70, we first revisited refolding of luciferase by the E. coli Hsp70 system. This consists of the Hsp70 DnaK, the ATPase-stimulating J-protein DnaJ, and the nucleotide exchange factor GrpE. We confirmed the seminal findings of the Bukau laboratory that luciferase refolds in the presence of the Hsp70 system. Substrate proteins bind to DnaK at high rates, within seconds, but folding of, for example, luciferase takes around 30 min (Hu et al., 2006Hu B. Mayer M.P. Tomita M. Modeling Hsp70-mediated protein folding.Biophys. J. 2006; 91: 496-507Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar, Kityk et al., 2015Kityk R. Vogel M. Schlecht R. Bukau B. Mayer M.P. Pathways of allosteric regulation in Hsp70 chaperones.Nat. Commun. 2015; 6: 8308Crossref PubMed Scopus (87) Google Scholar, Schröder et al., 1993Schröder H. Langer T. Hartl F.U. Bukau B. DnaK, DnaJ and GrpE form a cellular chaperone machinery capable of repairing heat-induced protein damage.EMBO J. 1993; 12: 4137-4144Crossref PubMed Scopus (498) Google Scholar). Increasing Hsp70 levels should further favor the association of Hsp70 with an unfolding protein, so that chaperone activity may possibly compete with productive folding. We therefore chemically denatured luciferase and monitored the refolding rate in the presence of the Hsp70 system (Figure 1A). We kept the concentration of luciferase, DnaJ, and GrpE constant and titrated DnaK. We observed that independent of chaperone levels, the activity of refolded luciferase reaches a plateau after around 30 min (Figure 1B). The refolding yield, however, depended strongly on the concentration of Hsp70. Refolded luciferase increased to a maximum of 82% refolded luciferase at 2 μM DnaK. Strikingly, however, increasing Hsp70 levels further successively reduced the yield, dropping eventually to background levels at 10 μM DnaK (Figure 1C). Thus, Hsp70 is not only a promoter but also an effective inhibitor of folding of luciferase. This phenomenon depends only on the levels of Hsp70 itself and not on the relative ratio of chaperone to co-chaperones. A similar picture was revealed when titrating DnaK, DnaJ, and GrpE together: a maximal refolding yield of 83.5% was reached at 5 μM DnaK, subsequently falling to background levels at 10 μM (Figures 2A and 2B ). The physiological concentration of E. coli Hsp70 is even higher (∼27 μM at 30°C) and doubles upon heat shock (Mogk et al., 1999Mogk A. Tomoyasu T. Goloubinoff P. Rüdiger S. Röder D. Langen H. Bukau B. Identification of thermolabile Escherichia coli proteins: prevention and reversion of aggregation by DnaK and ClpB.EMBO J. 1999; 18: 6934-6949Crossref PubMed Scopus (516) Google Scholar). A 10-fold increase in luciferase levels did not release the Hsp70 block (Figure 2C). The Hsp70 block thus depends on the absolute Hsp70 levels, not on the ratio of Hsp70 to substrate. Together, these results suggest that Hsp70 requires an additional factor for effective refolding at physiological concentrations. Given that Hsp90 acts downstream of Hsp70, we considered whether E. coli Hsp90 (HtpG) would restore the folding activity at physiological Hsp70 levels (Karagöz and Rüdiger, 2015Karagöz G.E. Rüdiger S.G.D. Hsp90 interaction with clients.Trends Biochem. Sci. 2015; 40: 117-125Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar). Hsp90 acts on steroid receptors downstream of Hsp70, and E. coli Hsp90, HtpG, was found to have a mild effect on DnaK-dependent luciferase refolding (Genest et al., 2011Genest O. Hoskins J.R. Camberg J.L. Doyle S.M. Wickner S. Heat shock protein 90 from Escherichia coli collaborates with the DnaK chaperone system in client protein remodeling.Proc. Natl. Acad. Sci. USA. 2011; 108: 8206-8211Crossref PubMed Scopus (92) Google Scholar, Genest et al., 2015Genest O. Hoskins J.R. Kravats A.N. Doyle S.M. Wickner S. Hsp70 and Hsp90 of E. coli Directly Interact for Collaboration in Protein Remodeling.J. Mol. Biol. 2015; 427: 3877-3889Crossref PubMed Scopus (47) Google Scholar, Kirschke et al., 2014Kirschke E. Goswami D. Southworth D. Griffin P.R. Agard D.A. Glucocorticoid receptor function regulated by coordinated action of the Hsp90 and Hsp70 chaperone cycles.Cell. 2014; 157: 1685-1697Abstract Full Text Full Text PDF PubMed Scopus (270) Google Scholar, Sanchez et al., 1987Sanchez E.R. Meshinchi S. Tienrungroj W. Schlesinger M.J. Toft D.O. Pratt W.B. Relationship of the 90-kDa murine heat shock protein to the untransformed and transformed states of the L cell glucocorticoid receptor.J. Biol. Chem. 1987; 262: 6986-6991Abstract Full Text PDF PubMed Google Scholar). Here, we found that at high levels of the Hsp70 system, Hsp90 dramatically restored the folding capacity to maximum levels, increasing the folding yield from less than 10% to more than 80%, even when substoichiometric to Hsp70 (1 μM HtpG restores folding in the presence of 10 μM DnaK; Figures 1D and 2D). Importantly, and in contrast to Hsp70, high levels of Hsp90 (15 μM) were not detrimental to folding. Thus, Hsp90 becomes essential for folding at high Hsp70 levels, without adverse side effects at physiological concentrations. Since Hsp90 is essential for folding at high Hsp70 levels, we explored whether Hsp90 functions as a safeguard, making the Hsp70 system robust to fluctuation in free chaperone levels, as naturally occurs upon and after cell stress. In the presence of Hsp90 (1 μM HtpG), we titrated the E. coli Hsp70 system up to physiological levels (27.4 μM DnaK/1 μM DnaJ/6.2 μM GrpE). The yield of refolded protein reached a plateau above 5 μM DnaK (Figure 2E). Thus, Hsp90 ensures that folding efficiency is independent of the levels of free Hsp70 and its co-chaperones. In the absence of Hsp70, however, Hsp90 alone does not refold luciferase (Figure 2E). Hsp90 activity is linked to ATP hydrolysis, which is required to take over the client from Hsp70 (Kirschke et al., 2014Kirschke E. Goswami D. Southworth D. Griffin P.R. Agard D.A. Glucocorticoid receptor function regulated by coordinated action of the Hsp90 and Hsp70 chaperone cycles.Cell. 2014; 157: 1685-1697Abstract Full Text Full Text PDF PubMed Scopus (270) Google Scholar, Prodromou et al., 1997Prodromou C. Roe S.M. O’Brien R. Ladbury J.E. Piper P.W. Pearl L.H. Identification and structural characterization of the ATP/ADP-binding site in the Hsp90 molecular chaperone.Cell. 1997; 90: 65-75Abstract Full Text Full Text PDF PubMed Scopus (1118) Google Scholar). To determine the timing of ATPase action of Hsp90, we added the Hsp90-specific, ATP-competitive inhibitor radicicol to the assay. Radicicol blocked Hsp90 folding activity, consistent with earlier findings (Genest et al., 2011Genest O. Hoskins J.R. Camberg J.L. Doyle S.M. Wickner S. Heat shock protein 90 from Escherichia coli collaborates with the DnaK chaperone system in client protein remodeling.Proc. Natl. Acad. Sci. USA. 2011; 108: 8206-8211Crossref PubMed Scopus (92) Google Scholar) (Figure 2F). Remarkably, radicicol prevented substrate takeover by Hsp90 only when pre-incubated with the chaperone. As radicicol binds quickly to Hsp90 (Phillips et al., 2007Phillips J.J. Yao Z.P. Zhang W. McLaughlin S. Laue E.D. Robinson C.V. Jackson S.E. Conformational dynamics of the molecular chaperone Hsp90 in complexes with a co-chaperone and anticancer drugs.J. Mol. Biol. 2007; 372: 1189-1203Crossref PubMed Scopus (39) Google Scholar), these findings imply that the Hsp90 ATPase is only relevant in the earliest phase on the folding path. As the folding of luciferase continues for a further 30 min, this indicates that there is a short, initial period of chaperone action, after which folding of luciferase is independent of the ATPase activity of Hsp90. As Hsp90 acts downstream of Hsp70, this finding suggests that chaperoning is restricted to the first few seconds of the folding path. The largest part is free of ATP-dependent chaperone activity and thus chaperone-free. To elucidate whether the interplay between the bacterial Hsp70 and Hsp90 systems is a conserved process, we repeated the experiments with the human chaperones. We confirmed that, assisted by the J-protein Hdj1 and the nucleotide exchange factor Apg2, human Hsp70 (HSPA1A) is able to refold luciferase (Figure 3A) (Freeman and Morimoto, 1996Freeman B.C. Morimoto R.I. The human cytosolic molecular chaperones hsp90, hsp70 (hsc70) and hdj-1 have distinct roles in recognition of a non-native protein and protein refolding.EMBO J. 1996; 15: 2969-2979Crossref PubMed Scopus (381) Google Scholar, Rampelt et al., 2012Rampelt H. Kirstein-Miles J. Nillegoda N.B. Chi K. Scholz S.R. Morimoto R.I. Bukau B. Metazoan Hsp70 machines use Hsp110 to power protein disaggregation.EMBO J. 2012; 31: 4221-4235Crossref PubMed Scopus (217) Google Scholar). We monitored luciferase refolding in the presence of increasing concentrations of all three members of the Hsp70 system (Figures 3A and 3B). At 2 μM Hsp70, refolding of luciferase was maximal (75%). Further increase of Hsp70 concentration subsequently reduced refolding efficiency until basal refolding levels were reached at 12 μM Hsp70. Notably, the concentration of Hsp70 in eukaryotic cells is around 18 μM (Geiger et al., 2012Geiger T. Wehner A. Schaab C. Cox J. Mann M. Comparative proteomic analysis of eleven common cell lines reveals ubiquitous but varying expression of most proteins.Mol. Cell. Proteomics. 2012; 11 (M111.014050)Crossref Scopus (577) Google Scholar, Stankiewicz et al., 2010Stankiewicz M. Nikolay R. Rybin V. Mayer M.P. CHIP participates in protein triage decisions by preferentially ubiquitinating Hsp70-bound substrates.FEBS J. 2010; 277: 3353-3367Crossref PubMed Scopus (78) Google Scholar) (Table S1). However, addition of Hsp90 and the Hsp70-Hsp90 adaptor protein Hop at high Hsp70 concentrations restored folding of luciferase in a substoichiometric manner (Figure 3C). This indicates that the function of Hsp90 chaperones to counter the Hsp70-inflicted folding block is conserved between bacteria and man. We wondered whether the chaperones affected the folding rate of luciferase. In fact, the association rate of DnaK is much higher than the folding rate of the client. The association rate (kON) for peptides to DnaK in the ATP state is ∼106 M−1 s−1 (Mayer et al., 2000Mayer M.P. Schröder H. Rüdiger S. Paal K. Laufen T. Bukau B. Multistep mechanism of substrate binding determines chaperone activity of Hsp70.Nat. Struct. Biol. 2000; 7: 586-593Crossref PubMed Scopus (308) Google Scholar, Schmid et al., 1994Schmid D. Baici A. Gehring H. Christen P. Kinetics of molecular chaperone action.Science. 1994; 263: 971-973Crossref PubMed Scopus (423) Google Scholar), and the T1/2 for folding of luciferase with the DnaK system 6–8 min (∼400 s). Thus, at the concentrations of our assay, DnaK is constantly binding to luciferase (∼1 s−1 at the lowest concentration and 10 s−1 at the highest). During folding, DnaK can re-bind luciferase multiple times and presumably in different sites before it reaches the native state, which at high DnaK concentration translates into inhibition of the folding process. Therefore, we developed an equation system (see STAR Methods) to take the dynamic nature and the fast and multiple binding of Hsp70 to the client into account and analyzed the luciferase refolding curves. Remarkably, in all experiments, the folding rate of luciferase always followed first-order kinetics (averaged rates 5 ± 2 × 10−4 s−1 for 0–10 μM Hsp70 [Figure 2A] and 6 ± 4 × 10−4 s−1 for 0–8 μM Hsp90 [Figure 2D]). First-order kinetics indicate that the reaction depends on the concentration of the folding protein itself; however, neither presence nor concentration of any chaperone tested in this study significantly influenced the folding rate (Figures 1, 2, and 3). We concluded that although the chaperones dramatically improved the refolding yield, they did not change the refolding rate and thus not the transition state on the folding path. This is consistent with Anfinsen’s thermodynamic hypothesis for protein folding (Anfinsen, 1973Anfinsen C.B. Principles that govern the folding of protein chains.Science. 1973; 181: 223-230Crossref PubMed Scopus (5137) Google Scholar). Thus, neither Hsp70 nor Hsp90 actively contribute to the folding process of luciferase. Therefore, the Hsp70-Hsp90 cascade lacks foldase activity, despite being required to generate high yields of refolded protein. A key reason for Hsp70 to inhibit the folding of luciferase could be multiple simultaneous binding events. This is consistent with earlier findings that potential binding sites for Hsp70 exist on average every 30–40 residues (Rüdiger et al., 1997Rüdiger S. Germeroth L. Schneider-Mergener J. Bukau B. Substrate specificity of the DnaK chaperone determined by screening cellulose-bound peptide libraries.EMBO J. 1997; 16: 1501-1507Crossref PubMed Scopus (660) Google Scholar). Since an average protein in E. coli has about 360 residues, and an average eukaryotic protein has about 500 residues, these proteins have on average 10–15 sites Hsp70 can bind. Our data are consistent with several Hsp70 molecules binding simultaneously a single client polypeptide chain (see Scheme 1, STAR Methods), as had been shown by molecular simulations and NMR experiments (Kellner et al., 2014Kellner R. Hofmann H. Barducci A. Wunderlich B. Nettels D. Schuler B. Single-molecule spectroscopy reveals chaperone-mediated expansion of substrate protein.Proc. Natl. Acad. Sci. USA. 2014; 111: 13355-13360Crossref PubMed Scopus (86) Google Scholar, Rosenzweig et al., 2017Rosenzweig R. Sekhar A. Nagesh J. Kay L.E. Promiscuous binding by Hsp70 results in conformational heterogeneity and fuzzy chaperone-substrate ensembles.eLife. 2017; 6: e28030Crossref PubMed Scopus (58) Google Scholar). Since Hsp70 dissociation is a stochastic and concentration-independent process, whereas Hsp70 binding to the client is concentration dependent, above a certain Hsp70 concentration, rebinding will occur at higher rates than dissociation. Folding of the client, however, would require all hydrophobic sites to be simultaneously available for the formation of the hydrophobic core. Thus, Hsp70 rebinding may prevent folding of the protein. Notably, we could fit the Hsp70-dependent folding block when assuming that up to three Hsp70 may bind to the client, converting it into an irreversible state, e.g., when re-association of Hsp70 occurs at higher rates than dissociation (Figures 1B and 3B). This led us to test whether the mechanisms established here using luciferase could be confirmed for a classical Hsp90 in vivo and in vitro client, the ligand binding domain of the glucocorticoid receptor (GR-LBD) (Kirschke et al., 2014Kirschke E. Goswami D. Southworth D. Griffin P.R. Agard D.A. Glucocorticoid receptor function regulated by coordinated action of the Hsp90 and Hsp70 chaperone cycles.Cell. 2014; 157: 1685-1697Abstract Full Text Full Text PDF PubMed Scopus (270) Google Scholar, Lorenz et al., 2014Lorenz O.R. Freiburger L. Rutz D.A. Krause M. Zierer B.K. Alvira S. Cuéllar J. Valpuesta J.M. Madl T. Sattler M. Buchner J. Modulation of the Hsp90 chaperone cycle by a stringent client protein.Mol. Cell. 2014; 53: 941-953Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar, Sanchez et al., 1987Sanchez E.R. Meshinchi S. Tienrungroj W. Schlesinger M.J. Toft D.O. Pratt W.B. Relationship of the 90-kDa murine heat shock protein to the untransformed and transformed states of the L cell glucocorticoid receptor.J. Biol. Chem. 1987; 262: 6986-6991Abstract Full Text PDF PubMed Google Scholar). Folding of GR-LBD can be monitored by binding to a fluorescent-labeled hormone derivative (Kirschke et al., 2014Kirschke E. Goswami D. Southworth D. Griffin P.R. Agard D.A. Glucocorticoid receptor function regulated by coordinated action of the Hsp90 and Hsp70 chaperone cycles.Cell. 2014; 157: 1685-1697Abstract Full Text Full Text PDF PubMed Scopus (270) Google Scholar, Lorenz et al., 2014Lorenz O.R. Freiburger L. Rutz D.A. Krause M. Zierer B.K. Alvira S. Cuéllar J. Valpuesta J.M. Madl T. Sattler M. Buchner J. Modulation of the Hsp90 chaperone cycle by a stringent client protein.Mol. Cell. 2014; 53: 941-953Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar). After thermally unfolding GR-LBD, we monitored refolding at permissive temperature in the presence of Hsp70 and Hsp90. At a high concentration of Hsp70 (15 μM) and in line with previous findings, Hsp90 (together with its co-chaperone Hop) strictly controlled refolding (Kirschke et al., 2014Kirschke E. Goswami D. Southworth D. Griffin P.R. Agard D.A. Glucocorticoid receptor function regulated by coordinated action of the Hsp90 and Hsp70 chaperone cycles.Cell. 2014; 157: 1685-1697Abstract Full Text Full Text PDF PubMed Scopus (270) Google Scholar, Sanchez et al., 1987Sanchez E.R. Meshinchi S. Tienrungroj W. Schlesinger M.J. Toft D.O. Pratt W.B. Relationship of the 90-kDa murine heat shock protein to the untransformed and transformed states of the L cell glucocorticoid receptor.J. Biol. Chem. 1987; 262: 6986-6991Abstract Full Text PDF PubMed Google Scholar). At a low Hsp70 concentration (5 μM), however, Hsp90 was not essential for GR-LBD folding (Figure 3D). We conclude that the mechanistic interplay of Hsp70 and Hsp90 is also valid for folding of this paradigmatic steroid hormone receptor, which requires Hsp90 for maturation in the cell. Here, we identify the key of the function of Hsp90 in protein folding as resolving the Hsp70-imposed folding deadlock. This block is intrinsically linked to the specificity of Hsp70 for core-forming polypeptide segments. Hsp90 binding enables proteins to complete the early nucleation phase and to re-start the stalled folding trajectory, which ultimately leads to the native state. We find this to be a conserved and general function generating an Hsp70-Hsp90 cascade robust against fluctuations in chaperone levels and co-chaperone ratios. Remarkably, neither Hsp70 nor Hsp90 alter the folding rate, despite dramatically increasing the folding yield. Together, our findings suggest a molecular mechanism through which Hsp90 improves the folding efficiency of the Hsp70 machine. Hsp90 can act downstream of Hsp70 (Genest et al., 2011Genest O. Hoskins J.R. Camberg J.L. Doyle S.M. Wickner S. Heat shock protein 90 from Escherichia coli collaborates with the DnaK chaperone system in client protein remodeling.Proc. Natl. Acad. Sci. USA. 2011; 108: 8206-8211Crossref PubMed Scopus (92) Google Scholar, Genest et al., 2015Genest O. Hoskins J.R. Kravats A.N. Doyle S.M. Wickner S. Hsp70 and Hsp90 of E. coli Directly Interact for Collaboration in Protein Remodeling.J. Mol. Biol. 2015; 427: 3877-3889Crossref PubMed Scopus (47) Google Scholar, Nakamoto et al., 2014Nakamoto H. Fujita K. Ohtaki A. Watanabe S. Narumi S. Maruyama T. Suenaga E. Misono T.S. Kumar P.K. Goloubinoff P. Yoshikawa H. Physical interaction between bacterial heat shock protein (Hsp) 90 and Hsp70 chaperones mediates their cooperative action to refold denatured proteins.J. Biol. Chem. 2014; 289: 6110-6119Crossref PubMed Scopus (56) Google Scholar, Pratt and Toft, 2003Pratt W.B. Toft D.O. Regulation of si" @default.
• W2801645577 created "2018-05-17" @default.
• W2801645577 creator A5008889456 @default.
• W2801645577 creator A5013840879 @default.
• W2801645577 creator A5050938065 @default.
• W2801645577 creator A5078715883 @default.
• W2801645577 date "2018-05-01" @default.
• W2801645577 modified "2023-10-18" @default.
• W2801645577 title "Hsp90 Breaks the Deadlock of the Hsp70 Chaperone System" @default.
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