Order out of disorder: working cycle of an intrinsically unfolded chaperone

The redox-regulated chaperone Hsp33 protects organisms against oxidative stress that leads to protein unfolding. Activation of Hsp33 is triggered by the oxidative unfolding of its own redox-sensor domain, making Hsp33 a member of a recently discovered class of chaperones that require partial unfolding for full chaperone activity. Here we address the long-standing question of how chaperones recognize client proteins. We show that Hsp33 uses its own intrinsically disordered regions to discriminate between unfolded and partially structured folding intermediates. Binding to secondary structure elements in client proteins stabilizes Hsp33's intrinsically disordered regions, and this stabilization appears to mediate Hsp33's high affinity for structured folding intermediates. Return to nonstress conditions reduces Hsp33's disulfide bonds, which then significantly destabilizes the bound client proteins and in doing so converts them into less-structured, folding-competent client proteins of ATP-dependent foldases. We propose a model in which energy-independent chaperones use internal order-to-disorder transitions to control substrate binding and release.     

Unique peptide substrate binding properties of 110-kDa heat-shock protein (Hsp110) determine its distinct chaperone activity

The molecular chaperone 70-kDa heat-shock proteins (Hsp70s) play essential roles in maintaining protein homeostasis. Hsp110, an Hsp70 homolog, is highly efficient in preventing protein aggregation but lacks the hallmark folding activity seen in Hsp70s. To understand the mechanistic differences between these two chaperones, we first characterized the distinct peptide substrate binding properties of Hsp110s. In contrast to Hsp70s, Hsp110s prefer aromatic residues in their substrates, and the substrate binding and release exhibit remarkably fast kinetics. Sequence and structure comparison revealed significant differences in the two peptide-binding loops: the length and properties are switched. When we swapped these two loops in an Hsp70, the peptide binding properties of this mutant Hsp70 were converted to Hsp110-like, and more impressively, it functionally behaved like an Hsp110. Thus, the peptide substrate binding properties implemented in the peptide-binding loops may determine the chaperone activity differences between Hsp70s and Hsp110s.     

Interactions between Hsp70 and the hydrophobic core of alpha-synuclein inhibit fibril assembly

Molecular chaperones of the heat shock protein 70 (Hsp70) family counteract protein misfolding in a variety of neurodegenerative disease models. To determine whether human Hsp70 exerts similar effects on the aggregation of alpha-synuclein (alpha-Syn), the key component of insoluble fibrils present in Parkinson's disease, we investigated alpha-Syn fibril assembly in the presence of Hsp70. We found in vitro assembly was efficiently inhibited by substoichiometric concentrations of purified Hsp70 in the absence of cofactors. Experiments using alpha-Syn deletion mutants indicated that interactions between the Hsp70 substrate binding domain and the alpha-Syn core hydrophobic region underlie assembly inhibition. This assembly process was inhibited prior to the elongation stage as we failed to detect any fibrils by electron microscopy. In addition, fluorescence polarization and binding assays suggest that Hsp70 recognizes soluble alpha-Syn species in a highly dynamic and reversible manner. Together, these results provide novel insights into how Hsp70 suppresses alpha-Syn aggregation. Furthermore, our findings suggest that this critical step in Parkinson's disease pathogenesis may be subject to modulation by a common molecular chaperone.     

Molecular basis for interactions of the DnaK chaperone with substrates

Hsp70 chaperones assist a large variety of protein folding processes in the cell by transient association with short peptide segments of proteins. The substrate binding and release cycle is driven by the switching between the low affinity ATP bound state and the high affinity ADP bound state of Hsp70. Considerable progress has been made recently by the identification of in vivo substrates for the Escherichia coli homolog, DnaK, and the molecular mechanisms which govern the DnaK-substrate interactions. Here we review the processes that generate DnaK substrates in vivo and the properties of these substrates, and we describe insights gained from structural and kinetic analysis of DnaK-substrate interaction.     

Real‐time observation of the conformational dynamics of mitochondrial Hsp70 by spFRET

The numerous functions of the important class of molecular chaperones, heat shock proteins 70 (Hsp70), rely on cycles of intricate conformational changes driven by ATP‐hydrolysis and regulated by cochaperones and substrates. Here, we used Förster resonance energy transfer to study the conformational dynamics of individual molecules of Ssc1, a mitochondrial Hsp70, in real time. The intrinsic dynamics of the substrate‐binding domain of Ssc1 was observed to be uncoupled from the dynamic interactions between substrate‐ and nucleotide‐binding domains. Analysis of the fluctuations in the interdomain separation revealed frequent transitions to a nucleotide‐free state. The nucleotide‐exchange factor Mge1 did not induce ADP release, as expected, but rather facilitated binding of ATP. These results indicate that the conformational cycle of Ssc1 is more elaborate than previously thought and provide insight into how the Hsp70s can perform a wide variety of functions.     

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.     

Hsp70 displaces small heat shock proteins from aggregates to initiate protein refolding

Small heat shock proteins (sHsps) are an evolutionary conserved class of ATP-independent chaperones that protect cells against proteotoxic stress. sHsps form assemblies with aggregation-prone misfolded proteins, which facilitates subsequent substrate solubilization and refolding by ATP-dependent Hsp70 and Hsp100 chaperones. Substrate solubilization requires disruption of sHsp association with trapped misfolded proteins. Here, we unravel a specific interplay between Hsp70 and sHsps at the initial step of the solubilization process. We show that Hsp70 displaces surface-bound sHsps from sHsp–substrate assemblies. This Hsp70 activity is unique among chaperones and highly sensitive to alterations in Hsp70 concentrations. The Hsp70 activity is reflected in the organization of sHsp–substrate assemblies, including an outer dynamic sHsp shell that is removed by Hsp70 and a stable core comprised mainly of aggregated substrates. Binding of Hsp70 to the sHsp/substrate core protects the core from aggregation and directs sequestered substrates towards refolding pathway. The sHsp/Hsp70 interplay has major impact on protein homeostasis as it sensitizes substrate release towards cellular Hsp70 availability ensuring efficient refolding of damaged proteins under favourable folding conditions.     

The ClpB homolog Hsp78 is required for the efficient degradation of proteins in the mitochondrial matrix

Molecular chaperones perform vital functions in mitochondrial protein import and folding. In yeast mitochondria, two members of the Clp/Hsp100 chaperone family, Hsp78 and Mcx1, have been identified as homologs of the bacterial proteins ClpB and ClpX, respectively. In this report we employed a novel quantitative assay system to assess the role of Hsp78 and Mcx1 in protein degradation within the matrix. Mitochondria were preloaded with large amounts of two purified recombinant reporter proteins exhibiting different folding stabilities. Proteolysis of the imported substrate proteins depended on the mitochondrial level of ATP and was mediated by the matrix protease Pim1/LON. Degradation rates were found to be independent of the folding stability of the reporter proteins. Mitochondria from hsp78Delta cells exhibited a significant defect in the degradation efficiency of both substrates even at low temperature whereas mcx1Delta mitochondria showed wild-type activity. The proteolysis defect in hsp78Delta mitochondria was independent from the aggregation behavior of the substrate proteins. We conclude that Hsp78 is a genuine component of the mitochondrial proteolysis system required for the efficient degradation of substrate proteins in the matrix.     

Mass spectrometry unravels disulfide bond formation as the mechanism that activates a molecular chaperone

The heat shock protein Hsp33 is a very potent molecular chaperone with a distinctive mode of functional regulation; its activity is redox-regulated. In its reduced form all six cysteinyl residues of Hsp33 are present as thiols, and Hsp33 displays no folding helper activity. Exposure of Hsp33 to oxidizing conditions like H(2)O(2), however, rapidly converts Hsp33 into an efficient molecular chaperone. Activated Hsp33 binds tightly to refolding intermediates of chemically denatured luciferase and suppresses efficiently their aggregation in vitro. Matrix-assisted laser desorption/ionization-mass spectrometry peptide mapping in combination with in vitro and on target protein chemical modification showed that this activation process of Hsp33 is accompanied by the formation of two intramolecular disulfide bonds within Hsp33: Cys(232)-S-S-Cys(234) and Cys(265)-S-S-Cys(268). Cys(141), although not involved in disulfide bond formation, was found highly reactive toward chemical modifications. In contrast, Cys(239) is readily accessible under reducing conditions but becomes poorly accessible though still reduced when Hsp33 is in its active state. This indicates a significant conformational change during the activation process of Hsp33. Mass spectrometry, thus, unraveled a novel molecular mechanism by which alteration of the disulfide bond structure, as a result of changes in the cellular redox potential, results in the activation of a molecular chaperone.     

The molecular chaperone Hsp70 family members function by a bidirectional heterotrophic allosteric mechanism

The Hsp70 family is one of the most important and conserved molecular chaperone families. It is well documented that Hsp70 family members assist many cellular processes involving protein quality control, as follows: protein folding, transport through membranes, protein degradation, escape from aggregation, intracellular signaling, among several others. The Hsp70 proteins act as a cellular pivot capable of receiving and distributing substrates among the other molecular chaperone families. Despite the high identity of the Hsp70 proteins, there are several homologue Hsp70 members that do not have the same role in the cell, which allow them to develop and participate in such large number of activities. The Hsp70 proteins are composed of two main domains: one that binds ATP and hydrolyses it to ADP and another which directly interacts with substrates. These domains present bidirectional heterotrophic allosteric regulation allowing a fine regulated cycle of substrate binding and release. The general mechanism of the Hsp70s cycle is under the control of ATP hydrolysis that modulates the low (ATP-bound state) and high (ADP-bound state) affinity states of Hsp70 for substrates. An important feature of the Hsp70s cycle is that they have several co-chaperones that modulate their cycle and that can also interact and select substrates. Here, we review some known details of the bidirectional heterotrophic allosteric mechanism and other important features for Hsp70s regulating cycle and function.     

Thermodynamics and kinetics of Hsp70 association with A + U-rich mRNA-destabilizing sequences

Rapid mRNA degradation directed by A + U-rich elements (AREs) is mediated by the interaction of specific RNA-binding proteins to these sequences. The protein chaperone Hsp70 has been identified in a cellular complex containing the ARE-binding protein AUF1 and has also been detected in direct contact with A + U-rich RNA substrates, indicating that Hsp70 may be involved in the regulation of ARE-directed mRNA turnover. By using gel mobility shift and fluorescence anisotropy assays, we have determined that Hsp70 directly and specifically associates with U-rich RNA substrates in solution. With the ARE from tumor necrosis factor alpha (TNFalpha) mRNA, Hsp70 forms a dynamic complex consistent with a 1:1 association of protein:RNA but demonstrates cooperative binding behavior on polyuridylate substrates. Unlike AUF1, the RNA binding activity of Hsp70 is not regulated by ion-dependent folding of the TNFalpha ARE, suggesting that AUF1 and Hsp70 recognize distinct binding determinants on this RNA substrate. Binding of Hsp70 to the TNFalpha ARE is driven entirely by enthalpy at physiological temperatures, indicating that burial of hydrophobic surfaces is likely the principal mechanism stabilizing the Hsp70.RNA complex. Potential roles for the interaction of Hsp70 with ARE-containing mRNAs in the regulation of mRNA turnover and/or translational efficiency are discussed.     

Calreticulin functions in vitro as a molecular chaperone for both glycosylated and non-glycosylated proteins

Calreticulin (CRT) is thought to be a molecular chaperone that interacts with glycoproteins exclusively through a lectin site specific for monoglucosylated oligosaccharides. However, this chaperone function has never been directly demonstrated nor is it clear how lectin-oligosaccharide interactions facilitate glycoprotein folding. Using purified components, we show that CRT suppresses the aggregation not only of a glycoprotein bearing monoglucosylated oligosaccharides but also that of non-glycosylated proteins. Furthermore, CRT forms stable complexes with unfolded, non-glycosylated substrates but does not associate with native proteins. ATP and Zn(2+) enhance CRT's ability to suppress aggregation of non- glycoproteins, whereas engagement of its lectin site with purified oligosaccharide attenuates this function. CRT also confers protection against thermal inactivation and maintains substrates in a folding-competent state. We conclude that in addition to being a lectin CRT possesses a polypeptide binding capacity capable of discriminating between protein conformational states and that it functions in vitro as a classical molecular chaperone.     

Distinct but overlapping functions of Hsp70, Hsp90, and an Hsp70 nucleotide exchange factor during protein biogenesis in yeast

Hsp70 and Hsp90 molecular chaperones play essential roles in protein expression and maturation, and while catalyzing protein folding they can "decide" to target mis-folded substrates for degradation. In this report, we show for the first time distinct but partially overlapping requirements for Hsp90, Hsp70, and an Hsp70 nucleotide exchange factor (NEF) at different steps during the biogenesis of a model substrate, firefly luciferase (FFLux), in yeast. By examining the inducible expression of FFLux in wild type cells and in specific yeast mutants, we find that the Fes1p NEF is required for efficient FFLux folding, whereas the Hsp70, Ssa1p, is required for both protein folding and stability, and to maintain maximal FFLux mRNA levels. In contrast, Hsp90 function was primarily necessary to express the FFLux-encoding gene from an inducible promoter. Together, these data indicate previously unknown roles for these proteins and point to the complexity with which chaperones and cochaperones function in the cell.     

Hsp104 facilitates the endoplasmic‐reticulum–associated degradation of disease‐associated and aggregation‐prone substrates

Misfolded proteins in the endoplasmic reticulum (ER) are selected for ER‐associated degradation (ERAD). More than 60 disease‐associated proteins are substrates for the ERAD pathway due to the presence of missense or nonsense mutations. In yeast, the Hsp104 molecular chaperone disaggregates detergent‐insoluble ERAD substrates, but the spectrum of disease‐associated ERAD substrates that may be aggregation prone is unknown. To determine if Hsp104 recognizes aggregation‐prone ERAD substrates associated with human diseases, we developed yeast expression systems for a hydrophobic lipid‐binding protein, apolipoprotein B (ApoB), along with a chimeric protein harboring a nucleotide‐binding domain from the cystic fibrosis transmembrane conductance regulator (CFTR) into which disease‐causing mutations were introduced. We discovered that Hsp104 facilitates the degradation of ER‐associated ApoB as well as a truncated CFTR chimera in which a premature stop codon corresponds to a disease‐causing mutation. Chimeras containing a wild‐type version of the CFTR domain or a different mutation were stable and thus Hsp104 independent. We also discovered that the detergent solubility of the unstable chimera was lower than the stable chimeras, and Hsp104 helped retrotranslocate the unstable chimera from the ER, consistent with disaggregase activity. To determine why the truncated chimera was unstable, we next performed molecular dynamics simulations and noted significant unraveling of the CFTR nucleotide‐binding domain. Because human cells lack Hsp104, these data indicate that an alternate disaggregase or mechanism facilitates the removal of aggregation‐prone, disease‐causing ERAD substrates in their native environments.     

Electrostatic interactions play a critical role in Mycobacterium tuberculosis Hsp16.3 binding of substrate proteins

Mycobacterium tuberculosis Hsp16.3, a member of a small heat shock protein family, has chaperone-like activity in vitro and suppresses thermally or chemically induced aggregation of proteins. The nature of the interactions between Hsp16.3 and the denatured substrate proteins was investigated. A dramatic enhancement of chaperone-like activity of Hsp16.3 upon increasing temperature was accompanied by decreased ANS-detectable surface hydrophobicity. Hsp16.3 exhibited significantly enhanced chaperone-like activity after preincubation at 100°C with almost unchanged surface hydrophobicity. The interaction between Hsp16.3 and dithiothreitol-treated insulin B chains was markedly weakened in the presence of NaCl but greatly enhanced by the addition of a low-polarity alcohol, accompanied by significantly increased and decreased surface hydrophobicity, respectively. A working model for Hsp16.3 binding to its substrate proteins is proposed.     

Identification of a regulatory motif in Hsp70 that affects ATPase activity, substrate binding and interaction with HDJ-1.

The Hsp70 family of molecular chaperones has an essential role in the synthesis, folding and translocation of the nascent peptide chain. While the general features of these activities are well documented, less is understood about the regulation of these activities. The ATPase rate is stimulated by non-native proteins, furthermore, interaction with ATP leads to the release of protein substrate concurrent with a conformational change in Hsp70. One interpretation of these data is that the two domains of Hsp70 interact. In the process of mapping the carboxyl-terminal boundary of the substrate binding domain for human Hsp70, we identified a regulatory motif, EEVD, which is conserved at the extreme carboxyl terminus among nearly all cloned cytosolic eukaryotic Hsp70s. Deletion or mutation of EEVD affects the ATPase activity, the ability to interact with substrates, and interferes with the ability of the mutant Hsp70 to interact with HDJ-1 in the refolding of denatured firefly luciferase. Examination of the biophysical properties of the mutant Hsp70s reveals a change in the overall shape and conformation of the protein consistent with reduced interactions between the two domains. These data suggest that the EEVD motif is involved in the intramolecular regulation of Hsp70 function and intermolecular interactions with HDJ-1.     

The 2.2 A crystal structure of Hsp33: a heat shock protein with redox-regulated chaperone activity

BACKGROUND: One strategy that cells employ to respond to environmental stresses (temperature, oxidation, and pathogens) is to increase the expression of heat shock proteins necessary to maintain viability. Several heat shock proteins function as molecular chaperones by binding unfolded polypeptides and preventing their irreversible aggregation. Hsp33, a highly conserved bacterial heat shock protein, is a redox-regulated molecular chaperone that appears to protect cells against the lethal effects of oxidative stress. RESULTS: The 2.2 A crystal structure of a truncated E. coli Hsp33 (residues 1-255) reveals a domain-swapped dimer. The core domain of each monomer (1-178) folds with a central helix that is sandwiched between two beta sheets. The carboxyl-terminal region (179-235), which lacks the intact Zn binding domain of Hsp33, folds into three helices that pack on the other subunit. The interface between the two core domains is comprised of conserved residues, including a rare Glu-Glu hydrogen bond across the dyad axis. Two potential polypeptide binding sites that span the dimer are observed: a long groove containing pockets of conserved and hydrophobic residues, and an intersubunit 10-stranded beta sheet "saddle" with a largely uncharged or hydrophobic surface. CONCLUSIONS: Hsp33 is a dimer in the crystal structure. Solution studies confirmed that this dimer reflects the structural changes that occur upon activation of Hsp33 as a molecular chaperone. Patterns of conserved residues and surface charges suggest that two grooves might be potential binding sites for protein folding intermediates.     

Primate chaperones Hsc70 (constitutive) and Hsp70 (induced) differ functionally in supporting growth and prion propagation in Saccharomyces cerevisiae

Hsp70's are highly conserved essential protein chaperones that assist protein folding and prevent protein aggregation. They have modular structures consisting of ATPase, substrate-binding, and C-terminal domains. Substrate binding and release is regulated by ATP hydrolysis and nucleotide exchange, which in turn are regulated by cochaperones. Eukaryotes have constitutive (Hsc70) and stress-inducible (iHsp70) isoforms, but their functions have not been systematically compared. Using a yeast system to evaluate heterologous Hsp70's we find that primate Hsc70 supported growth but iHsp70 did not. Plant Hsc70 and iHsp70 counterparts behaved similarly, implying evolutionary conservation of this distinction. Swapping yeast and primate Hsp70 domains showed that (i) the Hsc70-iHsp70 distinction resided in the ATPase domain, (ii) substrate-binding domains of Hsp70's within and across species functioned similarly regarding growth, (iii) C-terminal domain function was important for growth, and (iv) Hsp70 functions important for cell growth and prion propagation were separable. Enzymatic analysis uncovered a correlation between substrate affinity and prion phenotype and showed that ATPase and protein-folding activities were generally similar. Our data support a view that intrinsic activities of Hsp70 isoforms are comparable, and functional differences in vivo lie mainly in complex interactions of Hsp70 with cochaperones.     

Energy landscape remodeling mechanism of Hsp70-chaperone-accelerated protein folding

Hsp70 chaperone is one of the key protein machines responsible for the quality control of protein production in cells. Facilitating in vivo protein folding by counteracting misfolding and aggregation is the essence of its biological function. Although the allosteric cycle during its functional actions has been well characterized both experimentally and computationally, the mechanism by which Hsp70 assists protein folding is still not fully understood. In this work, we studied the Hsp70-mediated folding of model proteins with rugged energy landscape by using molecular simulations. Different from the canonical scenario of Hsp70 functioning, which assumes that folding of substrate proteins occurs spontaneously after releasing from chaperones, our results showed that the substrate protein remains in contacts with the chaperone during its folding process. The direct chaperone-substrate interactions in the open conformation of Hsp70 tend to shield the substrate sites prone to form non-native contacts, which therefore avoids the frustrated folding pathway, leading to a higher folding rate and less probability of misfolding. Our results suggest that in addition to the unfoldase and holdase functions widely addressed in previous studies, Hsp70 can facilitate the folding of its substrate proteins by remodeling the folding energy landscape and directing the folding processes, demonstrating the foldase scenario. These findings add new, to our knowledge, insights into the general molecular mechanisms of chaperone-mediated protein folding.