The crystal structure of the reduced, Zn2+-bound form of the B. subtilis Hsp33 chaperone and its implications for the activation mechanism

The bacterial heat shock protein Hsp33 is a redox-regulated chaperone activated by oxidative stress. In response to oxidation, four cysteines within a Zn2+ binding C-terminal domain form two disulfide bonds with concomitant release of the metal. This leads to the formation of the biologically active Hsp33 dimer. The crystal structure of the N-terminal domain of the E. coli protein has been reported, but neither the structure of the Zn2+ binding motif nor the nature of its regulatory interaction with the rest of the protein are known. Here we report the crystal structure of the full-length B. subtilis Hsp33 in the reduced form. The structure of the N-terminal, dimerization domain is similar to that of the E. coli protein, although there is no domain swapping. The Zn2+ binding domain is clearly resolved showing the details of the tetrahedral coordination of Zn2+ by four thiolates. We propose a structure-based activation pathway for Hsp33.     

Chaperone activity with a redox switch

Hsp33, a member of a newly discovered heat shock protein family, was found to be a very potent molecular chaperone. Hsp33 is distinguished from all other known molecular chaperones by its mode of functional regulation. Its activity is redox regulated. Hsp33 is a cytoplasmically localized protein with highly reactive cysteines that respond quickly to changes in the redox environment. Oxidizing conditions like H2O2 cause disulfide bonds to form in Hsp33, a process that leads to the activation of its chaperone function. In vitro and in vivo experiments suggest that Hsp33 protects cells from oxidants, leading us to conclude that we have found a protein family that plays an important role in the bacterial defense system toward oxidative stress.     

Redox switch of hsp33 has a novel zinc-binding motif

The chaperone activity of the heat shock protein Hsp33 is regulated by reversible disulfide bond formation. Oxidized Hsp33 is active, and reduced Hsp33 is inactive. We show that zinc binding is essential for the function of this redox switch. Our results reveal that Hps33 contains a new, high affinity (K(a) > 10(17) m(-)(1)), zinc-binding motif in the form Cys-X-Cys-X(27-32)-Cys-X-X-Cys. All four conserved cysteines within this motif act to coordinate a single zinc atom. Experiments where reduced wild type Hsp33 is reconstituted with cobalt or cadmium demonstrate that the metal-coordinating cysteines are present as highly reactive thiolate anions. This ionization may allow for the fast and successful activation of the chaperone function of Hsp33 upon incubation in oxidizing agents.     

Crystal structure of constitutively monomeric E. coli Hsp33 mutant with chaperone activity

Heat shock protein 33 (Hsp33) from Escherichia coli is a redox-regulated molecular chaperone that protects cells from oxidative stress. To understand the molecular basis for the monomer-dimer switch in the functional regulation of E. coli Hsp33, we generated a constitutively monomeric Hsp33 by introducing the Q151E mutation in the dimeric interface and determined its crystal structure. The overall scaffold of the monomeric Hsp33(1-235) (Q151E) mutant is virtually the same as that of the dimeric form, except that there is no domain swapping. The measurement of chaperone activity to thermally denatured luciferase showed that the constitutively monomeric Hsp33 mutant still retains chaperone activity similar to that of wild-type Hsp33(1-235), suggesting that a Hsp33 monomer is sufficient to interact with slowly unfolded substrate.     

The redox-switch domain of Hsp33 functions as dual stress sensor

The redox-regulated chaperone Hsp33 is specifically activated upon exposure of cells to peroxide stress at elevated temperatures. Here we show that Hsp33 harbors two interdependent stress-sensing regions located in the C-terminal redox-switch domain of Hsp33: a zinc center sensing peroxide stress conditions and an adjacent linker region responding to unfolding conditions. Neither of these sensors works sufficiently in the absence of the other, making the simultaneous presence of both stress conditions a necessary requirement for Hsp33's full activation. Upon activation, Hsp33's redox-switch domain adopts a natively unfolded conformation, thereby exposing hydrophobic surfaces in its N-terminal substrate-binding domain. The specific activation of Hsp33 by the oxidative unfolding of its redox-switch domain makes this chaperone optimally suited to quickly respond to oxidative stress conditions that lead to protein unfolding.     

Roles of the N- and C-terminal sequences in Hsp27 self-association and chaperone activity

The small heat shock protein 27 (Hsp27 or HSPB1) is an oligomeric molecular chaperone in vitro that is associated with several neuromuscular, neurological, and neoplastic diseases. Although aspects of Hsp27 biology are increasingly well known, understanding of the structural basis for these involvements or of the functional properties of the protein remains limited. As all 11 human small heat shock proteins (sHsps) possess an α-crystallin domain, their varied functional and physiological characteristics must arise from contributions of their nonconserved sequences. To evaluate the role of two such sequences in Hsp27, we have studied three Hsp27 truncation variants to assess the functional contributions of the nonconserved N- and C-terminal sequences. The N-terminal variants Δ1-14 and Δ1-24 exhibit little chaperone activity, somewhat slower but temperature-dependent subunit exchange kinetics, and temperature-independent self-association with formation of smaller oligomers than wild-type Hsp27. The C-terminal truncation variants exhibit chaperone activity at 40 °C but none at 20 °C, limited subunit exchange, and temperature-independent self-association with an oligomer distribution at 40 °C that is very similar to that of wild-type Hsp27. We conclude that more of the N-terminal sequence than simply the WPDF domain is essential in the formation of larger, native-like oligomers after binding of substrate and/or in binding of Hsp27 to unfolding peptides. On the other hand, the intrinsically flexible C-terminal region drives subunit exchange and thermally-induced unfolding, both of which are essential to chaperone activity at low temperature and are linked to the temperature dependence of Hsp27 self-association.     

Unique Unfoldase/Aggregase Activity of a Molecular Chaperone Hsp33 in its Holding-Inactive State

The various chaperone activities of heat shock proteins contribute to ensuring cellular proteostasis. Here, we demonstrate the non-canonical unfoldase activity as an inherent functionality of the prokaryotic molecular chaperone, Hsp33. Hsp33 was originally identified as a holding chaperone that is post-translationally activated by oxidation. However, in this study, we verified that the holding-inactive reduced form of Hsp33 (^RHsp33) strongly bound to the translational elongation factor, EF-Tu. This interaction was critically mediated by the redox-switch domain of ^RHsp33 and the guanine nucleotide-binding domain of EF-Tu. The bound ^RHsp33, without undergoing any conformational change, catalyzed the EF-Tu aggregation by evoking the aberrant folding of EF-Tu to expose hydrophobic surfaces. Consequently, the oligomers/aggregates of EF-Tu, but not its functional monomeric form, were highly susceptible to proteolytic degradation by Lon protease. These findings present a unique example of an ATP-independent molecular chaperone with distinctive dual functions—as an unfoldase/aggregase and as a holding chaperone—depending on the redox status. It is also suggested that the unusual unfoldase/aggregase activity of ^RHsp33 can contribute to cellular proteostasis by dysregulating EF-Tu under heat-stressed conditions.     

Hsp72 chaperone function is dispensable for protection against stress-induced apoptosis

In addition to its role as a molecular chaperone, heat shock protein 72 (Hsp72) protects cells against a wide range of apoptosis inducing stresses. However, it is unclear if these two roles are functionally related or whether Hsp72 inhibits apoptosis by a mechanism independent of chaperone activity. The N-terminal adenosine triphosphatase domain, substrate-binding domain and the C-terminal EEVD regulatory motif of Hsp72 are all essential for chaperone activity. In this study, we show that Hsp72 mutants with a functional substrate-binding domain but lacking chaperone activity retain their ability to protect cells against apoptosis induced by heat and tumor necrosis factor alpha. In contrast, a deletion mutant lacking a functional substrate-binding domain has no protective capacity. The ability of the Hsp72 substrate-binding domain to inhibit apoptosis independent of the regulatory effects of the adenosine triphosphate-binding domain indicates that the inhibition of apoptosis may involve a stable binding interaction with a regulatory substrate rather than Hsp72 chaperone activity.     

The lack of chaperonelike activity of Caenorhabditis elegans Hsp12.2 cannot be restored by domain swapping with human αB-crystallin

The small heat shock proteins Hsp12.2 and alphaB-crystallin differ in that the former occurs as tetramers, without chaperonelike activity, whereas the latter forms multimers and is a good chaperone. To investigate whether the lack of chaperone activity of Hsp12.2 is primarily due to its tetrameric structure or rather to intrinsic sequence features, we engineered chimeric proteins by swapping the N-terminal, C-terminal, and tail regions of Hsp12.2 and alphaB-crystallin, designated as n-c-t and N-C-T, respectively. Three of the chimeric sHsps, namely N-c-T, n-c-T, and N-C-t, showed nativelike secondary and quaternary structures as measured by circular dichroism and gel permeation chromatography. Combining the conserved alpha-crystallin domain of Hsp12.2 with the N-terminal and tail regions of alphaB-crystallin (N-c-T) resulted in multimeric complexes, but did not restore chaperonelike activity. Replacing the tail region of Hsp12.2 with that of alphaB-crystallin (n-c-T) did not alter the tetrameric structure and lack of chaperone activity. Similarly, providing alphaB-crystallin with the tail of Hsp12.2 (N-C-t) did not substantially influence the multimeric complex size, but it reduced the chaperoning ability, especially for small substrates. These results suggest that the conserved alpha-crystallin domain of Hsp12.2 is intrinsically unsuitable to confer chaperonelike activity and confirms that the tail region in alphaB-crystallin modulates chaperonelike capacity in a substrate-dependent manner.     

The zinc-dependent redox switch domain of the chaperone Hsp33 has a novel fold

The Escherichia coli chaperone Hsp33 contains a C-terminal zinc-binding domain that modulates activity by a so-called "redox switch". The oxidized form in the absence of zinc is active, while the reduced form in the presence of zinc is inactive. X-ray crystal structures of Hsp33 invariably omit details of the C-terminal domain, which is truncated in protein constructs that are capable of forming crystals. We report the solution structure of a recombinant 61-residue protein containing the zinc-binding domain (residues 227-287) of Hsp33, in the presence of stoichiometric amounts of Zn2+. The zinc-bound protein is well folded, and forms a novel structure unlike other published zinc-binding domains. The structure consists of two helices at right-angles to each other, a two-stranded B-hairpin and a third helix at the C terminus. The zinc site comprises the side-chains of the conserved cysteine residues 232, 234, 262 and 265, and connects a short sequence before the first helix with the tight turn in the middle of the B-hairpin. The structure of the C-terminal zinc-binding domain suggests a mechanism for the operation of the redox switch: loss of the bound zinc ion disrupts the folded structure, allowing the ligand cysteine residues to be oxidized, probably to disulfide bonds. The observation that the C-terminal domain is poorly structured in the active oxidized form suggests that the loss of zinc and unfolding of the domain precedes the oxidation of the thiolate groups of the cysteine residues, since the formation of disulfides between distant parts of the domain sequence would presumably promote the formation of stable three-dimensional structure in the oxidized form.Hsp33 provides an example of a redox signaling system that utilizes protein folding and unfolding together with chemical modification for transduction of external stimuli, in this case oxidative stress, to activate the machinery of the cell that is designed to deal with that stress.     

Both the N- and C-terminal chaperone sites of Hsp90 participate in protein refolding.

Hsp90 is able to bind partially unfolded firefly luciferase and maintain it in a refoldable state; the subsequent successive action of the 20S proteasome activator PA28, Hsc70 and Hsp40 enables its refolding. Hsp90 possesses two chaperone sites in the N- and C-terminal domains that prevent the aggregation of denatured proteins. Here we show that both chaperone sites of Hsp90 are effective not only in capturing thermally denatured luciferase, but also in holding it in a state prerequisite for the successful refolding process mediated by PA28, Hsc70 and Hsp40. In contrast, the heat-induced activity of Hsp90 to bind chemically denature dihydrofolate reductase efficiently and prevent its rapid spontaneous refolding was detected in the N-terminal site of Hsp90 only, while the C-terminal site was without effect. Thus it is most likely that both the N- and C-terminal chaperone sites may contribute to Hsp90 function as holder chaperones, however, in a significantly distinct manner.     

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.     

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.     

Modulation of chaperone function and cochaperone interaction by novobiocin in the C-terminal domain of Hsp90: evidence that coumarin antibiotics disrupt Hsp90 dimerization

The C-terminal domain of Hsp90 displays independent chaperone activity, mediates dimerization, and contains the MEEVD motif essential for interaction with tetratricopeptide repeat-containing immunophilin cochaperones assembled in mature steroid receptor complexes. An alpha-helical region, upstream of the MEEVD peptide, helps form the dimerization interface and includes a hydrophobic microdomain that contributes to the Hsp90 interaction with the immunophilin cochaperones and corresponds to the binding site for novobiocin, a coumarin-related Hsp90 inhibitor. Mutation of selected residues within the hydrophobic microdomain significantly impacted the chaperone function of a recombinant C-terminal Hsp90 fragment and novobiocin inhibited wild-type chaperone activity. Prior incubation of the Hsp90 fragment with novobiocin led to a direct blockade of immunophilin cochaperone binding. However, the drug had little influence on the pre-formed Hsp90-immunophilin complex, suggesting that bound cochaperones mask the novobiocin-binding site. We observed a differential effect of the drug on Hsp90-immunophilin interaction, suggesting that the immunophilins make distinct contacts within the C-terminal domain to specifically modulate Hsp90 function. Novobiocin also precluded the interaction of full-length Hsp90 with the p50(cdc37) cochaperone, which targets the N-terminal nucleotide-binding domain, and is prevalent in Hsp90 complexes with protein kinase substrates. Novobiocin therefore acts locally and allosterically to induce conformational changes within multiple regions of the Hsp90 protein. We provide evidence that coumermycin A1, a coumarin structurally related to novobiocin, interferes with dimerization of the Hsp90 C-terminal domain. Coumarin-based inhibitors then may antagonize Hsp90 function by inducing a conformation favoring separation of the C-terminal domains and release of substrate.     

Crystal structure of the E. coli Hsp100 ClpB N-terminal domain

E. coli Hsp100 ClpB can disaggregate denatured polypeptides by employing ATP hydrolysis. The ClpB N-terminal domain (ClpBN) has been proposed to play important roles in ClpB molecular chaperone activities. We have determined the crystal structure of ClpBN to 1.95 A resolution by MAD methods. The ClpBN monomer contains two subdomains that have similar folds. The crystal structure revealed a hydrophobic groove on the molecular surface. We have constructed ClpB mutants in which the hydrophobic residues within the putative peptide binding groove were replaced by glutamine. These ClpB mutants exhibited severe defects in molecular chaperone activity but retained the wild-type ATPase activity.     

Analysis of the alphaB-crystallin domain responsible for inhibiting tubulin aggregation

The cytoskeleton has a unique property such that changes of conformation result in polymerization into a filamentous form. alphaB-Crystallin, a small heat shock protein (sHsp), has chaperone activities for various substrates, including proteins constituting the cytoskeleton, such as actin; intermediate filament; and tubulin. However, it is not clear whether the "alpha-crystallin domain" common to sHsps also has chaperone activity for the protein cytoskeleton. To investigate the possibility that the C-terminal alpha-crystallin domain of alpha-crystallin has the aggregation-preventing ability for tubulin, we constructed an N-terminal domain deletion mutant of alphaB-crystallin. We characterized its structural properties and chaperone activities. Far-ultraviolet (UV) circular dichroism measurements showed that secondary structure in the alpha-crystallin domain of the deletion mutant is maintained. Ultracentrifuge analysis of molecular masses indicated that the deletion mutant formed smaller oligomers than did the full-length protein. Chaperone activity assays demonstrated that the N-terminal domain deletion mutant suppressed heat-induced aggregation of tubulin well. Comparison of chaperone activities for 2 other substrates (citrate synthase and alcohol dehydrogenase) showed that it was less effective in the suppression of their aggregation. These results show that alphaB-crystallin recognizes a variety of substrates and especially that alpha-crystallin domain binds free cytoskeletal proteins. We suggest that this feature would be advantageous in its functional role of holding or folding multiple proteins denatured simultaneously under stress conditions.     

Structure, stability, and chaperone function of alphaA-crystallin: role of N-terminal region

Small heat shock protein alphaA-crystallin, the major protein of the eye lens, is a molecular chaperone. It consists of a highly conserved central domain flanked by the N-terminal and C-terminal regions. In this article we studied the role of the N-terminal domain in the structure and chaperone function of alphaA-crystallin. Using site directed truncation we raised several deletion mutants of alphaA-crystallin and their protein products were expressed in Escherichia coli. Size exclusion chromatography of these purified proteins showed that deletion from the N-terminal beyond the first 20 residues drastically reduced the oligomeric association of alphaA-crystallin and its complete removal resulted in a tetramer. Chaperone activity of alphaA-crystallin, determined by thermal and nonthermal aggregation and refolding assay, decreased with increasing length of deletion and little activity was observed for the tetramer. However it was revealed that N-terminal regions were not responsible for specific recognition of natural substrates and that low affinity substrate binding sites existed in other part of the molecule. The number of exposed hydrophobic sites and the affinity of binding hydrophobic probe bis-ANS as well as protein substrates decreased with N-terminal deletion. The stability of the mutant proteins decreased with increase in the length of deletion. The role of thermodynamic stability, oligomeric size, and surface hydrophobicity in chaperone function is discussed. Detailed analysis showed that the most important role of N-terminal region is to control the oligomerization, which is crucial for the stability and in vivo survival of this protein molecule.     

Beyond transcription--new mechanisms for the regulation of molecular chaperones

Molecular chaperones are an essential part of the universal heat shock response that allows organisms to survive stress conditions that cause intracellular protein unfolding. During the past few years, two new mechanisms have been found to control the activity of several chaperones under stress conditions-the regulation of chaperone activity by the redox state and by the temperature of the environment. Hsp33, for example, is redox-regulated. Hsp33 is specifically activated by disulfide bond formation during oxidative stress, where it becomes a highly efficient chaperone holdase that binds tightly to unfolding proteins. Certain small heat shock proteins, such as Hsp26 and Hsp16.9, on the other hand, are temperature regulated. Exposure to heat shock temperatures causes these oligomeric proteins to disassemble, thereby changing them into highly efficient chaperones. The ATP-dependent chaperone folding system DnaK/DnaJ/GrpE also appears to be temperature regulated, switching from a folding to a holding mode during heat stress. Both of these novel post-translational regulatory strategies appear to have one ultimate goal: to significantly increase the substrate binding affinity of the affected chaperones under exactly those stress conditions that require their highest chaperone activity. This ensures that protein folding intermediates remain bound to the chaperones under stress conditions and are released only after the cells return to non-stress conditions.     

Mammalian Hsp70 and Hsp110 proteins bind to RNA motifs involved in mRNA stability.

In this study, in vitro RNA binding by members of the mammalian 70-kDa heat shock protein (Hsp) family was examined. We show that Hsp/Hsc70 and Hsp110 proteins preferentially bound AU-rich RNA in vitro. Inhibition of RNA binding by ATP suggested the involvement of the N-terminal ATP-binding domain. By using deletion mutants of Hsp110 protein, a diverged Hsp70 family member, RNA binding was localized to the N-terminal ATP-binding domain of the molecule. The C-terminal peptide-binding domain did not bind RNA, but its engagement by a peptide substrate abrogated RNA binding by the N terminus of the protein. Interestingly, removal of the C-terminal alpha-helical structure or the alpha-loop domain unique to Hsp110 immediately downstream of the peptide-binding domain, but not both, resulted in considerably increased RNA binding as compared with the wild type protein. Finally, a 70-kDa activity was immunoprecipitated from RNA-protein complexes formed in vitro between cytoplasmic proteins of human lymphocytes and AU-rich RNA. These findings support the idea that certain heat shock proteins may act as RNA-binding entities in vivo to guide the appropriate folding of RNA substrates for subsequent regulatory processes such as mRNA degradation and/or translation.