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• W2084665652 abstract "The molecular chaperone Hsp33 in Escherichia coli responds to oxidative stress conditions with the rapid activation of its chaperone function. On its activation pathway, Hsp33 progresses through three major conformations, starting as a reduced, zinc-bound inactive monomer, proceeding through an oxidized zinc-free monomer, and ending as a fully active oxidized dimer. While it is known that Hsp33 senses oxidative stress through its C-terminal four-cysteine zinc center, the nature of the conformational changes in Hsp33 that must take place to accommodate this activation process is largely unknown. To investigate these conformational rearrangements, we constructed constitutively monomeric Hsp33 variants as well as fragments consisting of the redox regulatory C-terminal domain of Hsp33. These proteins were studied by a combination of biochemical and NMR spectroscopic techniques. We found that in the reduced, monomeric conformation, zinc binding stabilizes the C terminus of Hsp33 in a highly compact, α-helical structure. This appears to conceal both the substrate-binding site as well as the dimerization interface. Zinc release without formation of the two native disulfide bonds causes the partial unfolding of the C terminus of Hsp33. This is sufficient to unmask the substrate-binding site, but not the dimerization interface, rendering reduced zinc-free Hsp33 partially active yet monomeric. Critical for the dimerization is disulfide bond formation, which causes the further unfolding of the C terminus of Hsp3 and allows the association of two oxidized Hsp33 monomers. This then leads to the formation of active Hsp33 dimers, which are capable of protecting cells against the severe consequences of oxidative heat stress. The molecular chaperone Hsp33 in Escherichia coli responds to oxidative stress conditions with the rapid activation of its chaperone function. On its activation pathway, Hsp33 progresses through three major conformations, starting as a reduced, zinc-bound inactive monomer, proceeding through an oxidized zinc-free monomer, and ending as a fully active oxidized dimer. While it is known that Hsp33 senses oxidative stress through its C-terminal four-cysteine zinc center, the nature of the conformational changes in Hsp33 that must take place to accommodate this activation process is largely unknown. To investigate these conformational rearrangements, we constructed constitutively monomeric Hsp33 variants as well as fragments consisting of the redox regulatory C-terminal domain of Hsp33. These proteins were studied by a combination of biochemical and NMR spectroscopic techniques. We found that in the reduced, monomeric conformation, zinc binding stabilizes the C terminus of Hsp33 in a highly compact, α-helical structure. This appears to conceal both the substrate-binding site as well as the dimerization interface. Zinc release without formation of the two native disulfide bonds causes the partial unfolding of the C terminus of Hsp33. This is sufficient to unmask the substrate-binding site, but not the dimerization interface, rendering reduced zinc-free Hsp33 partially active yet monomeric. Critical for the dimerization is disulfide bond formation, which causes the further unfolding of the C terminus of Hsp3 and allows the association of two oxidized Hsp33 monomers. This then leads to the formation of active Hsp33 dimers, which are capable of protecting cells against the severe consequences of oxidative heat stress. The molecular chaperone Hsp33 belongs to a novel class of redox-regulated proteins, whose activity is regulated by their redox state (1Paget M.S. Buttner M.J. Annu. Rev. Genet. 2003; 37: 91-121Crossref PubMed Scopus (254) Google Scholar). In prokaryotes, Hsp33 is located in the highly reducing environment of the cytosol, where it is monomeric and largely devoid of chaperone function. Upon exposure to oxidative stress in vitro or in vivo, Hsp33 is quickly activated as a potent molecular chaperone (2Jakob U. Muse W. Eser M. Bardwell J.C.A. Cell. 1999; 96: 341-352Abstract Full Text Full Text PDF PubMed Scopus (429) Google Scholar). The switch that regulates the activity of Hsp33 is a novel, very high affinity zinc-binding motif (CXCX27-32CXXC) that is located in the C terminus of the protein (3Jakob U. Eser M. Bardwell J.C.A. J. Biol. Chem. 2000; 275: 38302-38310Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar). The four absolutely conserved cysteines that constitute this redox switch are kept in the reduced deprotonated thiolate anion state and together coordinate one zinc(II) ion (KD ∼ 10-18 m) (3Jakob U. Eser M. Bardwell J.C.A. J. Biol. Chem. 2000; 275: 38302-38310Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar). Under oxidative stress conditions, these four cysteines release zinc and rapidly form two intramolecular disulfide bonds, connecting the two pairs of neighboring cysteines, Cys232 with Cys234 and Cys265 with Cys268 (4Barbirz S. Jakob U. Glocker M.O. J. Biol. Chem. 2000; 275: 18759-18766Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar). Disulfide bond formation and concomitant zinc release then induces the dimerization of two oxidized Hsp33 monomers (5Graumann J. Lilie H. Tang X. Tucker K.A. Hoffmann J.H. Vijayalakshmi J. Saper M. Bardwell J.C.A. Jakob U. Structure (Camb.). 2001; 9: 377-387Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar). Once activated, Hsp33 is a highly efficient chaperone holdase, which is able to prevent the aggregation of a variety of unfolded proteins. To prime Hsp33 for substrate protein release, reducing conditions have to be restored. This turns Hsp33 into a reduced dimer, which is still active but now able to transfer the substrate proteins to the DnaK/DnaJ/GrpE foldase system for refolding once this system becomes available (6Hoffmann J.H. Linke K. Graf P.C.F. Lilie H. Jakob U. EMBO J. 2004; 23: 160-168Crossref PubMed Scopus (117) Google Scholar). Biochemical studies showed that Hsp33 senses reactive oxygen species through its C-terminal four-cysteine zinc center, with zinc playing an important role for the formation of the correct disulfide bonds and the rapid activation of the chaperone function of Hsp33 (3Jakob U. Eser M. Bardwell J.C.A. J. Biol. Chem. 2000; 275: 38302-38310Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar). The precise structural changes that accompany disulfide bond formation and permit the dimerization of Hsp33 are, however, largely unknown. This is in part because no structural information exists for the redox-active C terminus of Hsp33. Both of the crystal structures that were solved were of N-terminal fragments, which ended just after the first pair of redox-active cysteines (7Vijayalakshmi J. Mukhergee M.K. Graumann J. Jakob U. Saper M.A. Structure (Camb.). 2001; 9: 367-375Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar, 8Kim S.J. Jeong D.G. Chi S.W. Lee J.S. Ryu S.E. Nat. Struct. Biol. 2001; 8: 459-466Crossref PubMed Scopus (39) Google Scholar). Previously, Raman et al. (9Raman B. Siva Kumar L.V. Ramakrishna T. Mohan Rao C. FEBS Lett. 2001; 489: 19-24Crossref PubMed Scopus (30) Google Scholar) used circular dichroism to demonstrate that Hsp33 undergoes dramatic conformational changes upon oxidation. They also showed that Hsp33 exposes hydrophobic surfaces in the oxidized conformation, but not in the reduced, inactive state. Because most molecular chaperones interact with their substrate proteins through hydrophobic interactions, these exposed hydrophobic surfaces are likely to represent the substrate-binding site of Hsp33. Thus, it is conceivable that the substrate-binding site is masked in the reduced, inactive form but exposed in the oxidized, active form. At the time of these studies, however, neither the activation mechanism nor the fact that oxidized Hsp33 can form both monomers and dimers were known. We show here that the C-terminal zinc-binding domain is fully folded in the reduced zinc-coordinated Hsp33 conformation. This appears to mask both the dimerization interface and the substrate-binding site. Upon zinc release and disulfide bond formation, the C-terminal domain of Hsp33 dramatically unfolds. This exposes the substrate-binding site and unmasks the dimerization interface. NMR and CD studies of the isolated C-terminal domain and the full-length Hsp33 wild type protein suggested that only the C-terminal redox switch domain unfolds, while the structure of the N-terminal chaperone domain appears largely unaltered by the oxidation and dimerization process. Proteins—Wild type Hsp33 and the cysteine-free Hsp33 mutant protein were purified in the absence of reducing agents as described previously (2Jakob U. Muse W. Eser M. Bardwell J.C.A. Cell. 1999; 96: 341-352Abstract Full Text Full Text PDF PubMed Scopus (429) Google Scholar, 5Graumann J. Lilie H. Tang X. Tucker K.A. Hoffmann J.H. Vijayalakshmi J. Saper M. Bardwell J.C.A. Jakob U. Structure (Camb.). 2001; 9: 377-387Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar). The Glu150 to Arg150 point mutation was introduced using the QuikChange site-directed mutagenesis kit (Stratagene) using matching primers. The forward primer that was used read 5′-GATTACTTTATGCGTTCTAGACAGCTGCCGACGCGCC-3′. A pET11a vector containing wild type Hsp33 (pUJ30) was used as template (2Jakob U. Muse W. Eser M. Bardwell J.C.A. Cell. 1999; 96: 341-352Abstract Full Text Full Text PDF PubMed Scopus (429) Google Scholar). The coding sequence of the region was sequenced. BL21 strains containing an insertion mutation in hslO, the gene encoding Hsp33 (hslO::Km) (JH13) were transformed with this plasmid (5Graumann J. Lilie H. Tang X. Tucker K.A. Hoffmann J.H. Vijayalakshmi J. Saper M. Bardwell J.C.A. Jakob U. Structure (Camb.). 2001; 9: 377-387Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar), and the mutant protein (Hsp33-E150R) was overexpressed and purified according to the purification protocol of wild type Hsp33 (2Jakob U. Muse W. Eser M. Bardwell J.C.A. Cell. 1999; 96: 341-352Abstract Full Text Full Text PDF PubMed Scopus (429) Google Scholar). The coding sequences for each of the C-terminal domain fragments of Hsp33 were obtained using standard PCR methods and pUJ30 as template DNA. The sequences were re-cloned into a pET21a expression vector, and DNA sequencing was used to confirm the identity of the inserts. The plasmids were transformed into JH13 strains, and the Hsp33 fragments were purified under native conditions, using an anion exchange column (HiTrap Q-Sepharose) followed by a gel filtration column (Superdex 75 or Sephacryl S100 HR). Isotopically labeled NMR samples of the C-terminal constructs were prepared by growing the cells at 15 °C in M9 minimal medium (0.67% Na2HPO4, 0.3% KH2PO4, 0.05% NaCl, 1 mm MgCl2, 0.1 mm CaCl2) containing 100 μg/ml carbenicillin and supplemental basal Eagle's medium vitamins. Protein expression was induced with isopropyl-β-d-thiogalactopyranoside, and the medium was supplemented with 15NH4Cl and [13C]glucose. At the time of induction, ZnSO4 was added to a final concentration of 150 μm. The labeled protein fragments were purified using the same protocol that was employed for the unlabeled proteins. The purity of the protein fragments was checked by SDS-PAGE and matrix-assisted laser desorption ionization mass spectrometry. Firefly luciferase was obtained from Sigma. Pig heart mitochondrial citrate synthase (CS) 1The abbreviations used are: CS, citrate synthase; bis-ANS, 4,4′-dianilino-1,1′-binaphthyl-5,5′-disulfonic acid; DTT, dithiothreitol; TPEN, N,N,N′,N′-tetrakis(2-pyridylmethyl)ethylenediamine; aa, amino acid. was from Roche Applied Science and prepared as described (10Buchner J. Grallert H. Jakob U. Methods Enzymol. 1998; 290: 323-338Crossref PubMed Scopus (197) Google Scholar). All protein concentrations are listed in the monomer concentration. Analytical Ultracentrifugation—The oxidized species of wild type Hsp33 and its variants were analyzed at initial protein concentrations of 0.02-0.6 mg/ml in 40 mm HEPES-KOH, pH 7.4, with a Beckman Optima XL-A centrifuge equipped with an An50Ti rotor. Sedimentation equilibrium measurements (absorption at 230 and 280 nm) were carried out in double sector cells at 10,000 rpm, and 20 °C sedimentation velocity was analyzed at 40,000 rpm with scans taken every 10 min. Data were analyzed with the software provided by Beckman Instruments (Palo Alto, CA). Bis-ANS (4,4′-Dianilino-1,1′-binaphthyl-5,5′-disulfonic acid) Fluorescence—To probe for the presence of hydrophobic surfaces in Hsp33, the fluorescent probe bis-ANS (Molecular Probes) was used. Hsp33 (3 μm) and bis-ANS (10 μm) were mixed in 40 mm HEPES-KOH, pH 7.5, and fluorescence emission spectra (Hitachi F4500) from 400-600 nm were recorded at 25 °C. The excitation wavelength was set to 370 nm, and the slit widths were set to 2.5 and 5 nm, respectively. The spectrum of bis-ANS in the absence of protein was used as buffer control. Chaperone Assays—The influence of the different Hsp33 preparations on the aggregation of chemically unfolded luciferase was determined as described previously with some minor modifications (5Graumann J. Lilie H. Tang X. Tucker K.A. Hoffmann J.H. Vijayalakshmi J. Saper M. Bardwell J.C.A. Jakob U. Structure (Camb.). 2001; 9: 377-387Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar). Briefly, luciferase was denatured in 4.5 m guanidine hydrochloride, 40 mm HEPES-KOH, pH 7.5, at room temperature. To initiate refolding, denatured luciferase was diluted 1:160 (final concentration: 48 nm) into 40 mm HEPES-KOH, pH 7.0, at 30 °C with or without 192 nm Hsp33. To analyze the influence of Hsp33 on the aggregation of thermally unfolding luciferase, native luciferase (final concentration: 0.1 μm) was incubated in 40 mm HEPES-KOH, pH 7.5, at 43 °C in the absence or presence of 0.1 μm Hsp33. In both experiments, light scattering was measured using a Hitachi F4500 fluorescence spectrophotometer equipped with a thermostated cell holder and stirrer. The excitation and emission wavelengths were set to 350 nm, and the slit widths were set to 2.5 nm. To analyze the influence of Hsp33 on thermally unfolding CS, native CS (final concentration: 0.15 μm) was diluted into 40 mm HEPES-KOH, pH 7.5, at 43 °C in the absence or presence of 0.3 μm Hsp33. Light scattering was monitored at excitation and emission wavelengths set to 500 nm. The slit widths were set again to 2.5 nm. Oxidation-induced Zinc Release and Activation of Hsp33—The oxidation-induced release of zinc from Hsp33 by H2O2 incubation was performed as described previously (5Graumann J. Lilie H. Tang X. Tucker K.A. Hoffmann J.H. Vijayalakshmi J. Saper M. Bardwell J.C.A. Jakob U. Structure (Camb.). 2001; 9: 377-387Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar). 50 μm reduced, zinc-replete wild type Hsp33 or Hsp33-E150R mutant protein was incubated with 2 mm H2O2 in 40 mm HEPES-KOH, pH 7.5, at 43 °C. At various time points, aliquots were taken to determine the extent of zinc release as well as the extent of chaperone activity using thermally unfolding citrate synthase as a substrate protein. Circular Dichroism and NMR Spectroscopy—To prepare reduced, zinc-reconstituted wild type Hsp33, Hsp33-E150R and Hsp33-218-287 aa fragments for CD measurements, 200 μm Hsp33 were incubated in 5 mm DTT and equimolar concentration of ZnCl2 for 30 min at 37 °C. To prepare reduced, metal-free Hsp33-218-287 aa fragment, 200 μm Hsp33-218-287 aa fragment was incubated in 5 mm DTT and 5 mm TPEN (N,N,N′,N′-tetrakis(2-pyridyl-methyl)ethylenediamine) at 43 °C for 30 min. Then, NAP-5 columns (Amersham Biosciences) equilibrated in 20 mm potassium phosphate, pH 7.5, 0.2 mm DTT were used for desalting and buffer exchange according to the protocol of the manufacturer. Oxidation of the zinc-reconstituted fragment (36 μm) was achieved with 2 mm H2O2 at 37 °C for 30 min. Oxidized wild type Hsp33 and Hsp33-E150R were prepared by incubating 200 or 5 μm reduced, zinc-replete Hsp33 with 2 mm H2O2 at 43 °C for 4 or 1 h, respectively. The oxidized samples were then dialyzed against 20 mm potassium phosphate, pH 7.5. Far-UV CD spectra were measured on a Jasco J-810 spectropolarimeter in a quartz cell with 1-mm path length at 25 °C. All spectra were first buffer-corrected and then normalized for slight variations in Hsp33 concentration. For NMR, the Hsp33 proteins were oxidized by using a mixed redox buffer of reduced and oxidized glutathione (11Mo H. Moore R.C. Cohen F.E. Westaway D. Prusiner S.B. Wright P.E. Dyson H.J. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 2352-2357Crossref PubMed Scopus (141) Google Scholar). The buffer contained 20 mm Tris-Cl, pH 8.0, 100 mm NaCl, 1 mm GSSG, 0.5 mm GSH, and 100 mm EDTA, with a protein concentration of ∼0.1 mg/ml. NMR spectra were obtained on Bruker spectrometers operating at 500 and 600 MHz. The Four Hsp33 Conformations—The activation mechanism of Hsp33 is at least a two-step process (5Graumann J. Lilie H. Tang X. Tucker K.A. Hoffmann J.H. Vijayalakshmi J. Saper M. Bardwell J.C.A. Jakob U. Structure (Camb.). 2001; 9: 377-387Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar). In the first step, reduced zinc-coordinated Hsp33 undergoes oxidation-induced disulfide bond formation and releases its bound zinc. This causes the formation of zinc-free oxidized monomeric Hsp33 intermediates. In a second step, two oxidized Hsp33 monomers dimerize to form the fully active molecular chaperone. To dissect the roles of zinc binding and disulfide bond formation in the activation process of Hsp33, and to analyze and compare the functional and conformational changes that accompany this process, we required at least four different stable conformations of Hsp33: 1) reduced, zinc-coordinated monomeric Hsp33, 2) reduced, zinc-free monomeric Hsp33, 3) oxidized monomeric Hsp33, and 4) oxidized dimeric Hsp33. Both reduced, zinc-coordinated Hsp33 monomers as well as oxidized Hsp33 dimers are stable, easy to prepare, and have been characterized in detail before (2Jakob U. Muse W. Eser M. Bardwell J.C.A. Cell. 1999; 96: 341-352Abstract Full Text Full Text PDF PubMed Scopus (429) Google Scholar, 5Graumann J. Lilie H. Tang X. Tucker K.A. Hoffmann J.H. Vijayalakshmi J. Saper M. Bardwell J.C.A. Jakob U. Structure (Camb.). 2001; 9: 377-387Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar). To create a stably reduced, metal-free Hsp33 variant, we decided to use a mutant of Hsp33, in which all cysteine residues had been replaced (Cys-free Hsp33) (5Graumann J. Lilie H. Tang X. Tucker K.A. Hoffmann J.H. Vijayalakshmi J. Saper M. Bardwell J.C.A. Jakob U. Structure (Camb.). 2001; 9: 377-387Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar). This mutant protein should allow us to specifically analyze the role of zinc coordination on the activity and conformation of monomeric Hsp33 without the potential interference of disulfide bond formation. To generate oxidized monomeric Hsp33, which is presumably an intermediate in the activation pathway of Hsp33, and therefore only transiently present, we decided to introduce mutations into the highly conserved dimerization interface in an attempt to disrupt intersubunit contacts. Three residues, Ser149, Glu150, and Gln151 are located precisely at the dimerization interface and are highly conserved (7Vijayalakshmi J. Mukhergee M.K. Graumann J. Jakob U. Saper M.A. Structure (Camb.). 2001; 9: 367-375Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar). Ser149 and Gln151 are absolutely conserved residues found in every Hsp33 homologue identified so far, while Glu150 has been found in all but two Hsp33 homo-logues, where it is substituted with a glutamine residue. Both Glu150 and Gln151 make numerous stabilizing contacts between the two subunits. For instance, hydrogen bonds exist between the Glu150 carboxylate oxygen of one subunit and the backbone amide of Gln151 as well as the carboxylate group of Glu150 of the other subunit (7Vijayalakshmi J. Mukhergee M.K. Graumann J. Jakob U. Saper M.A. Structure (Camb.). 2001; 9: 367-375Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar). Our rationale was now that the mutation of these central residues should specifically impair dimerization without changing the overall fold or redox activity of Hsp33. This should allow us then to create Hsp33 variants that can form disulfide bonds but remain monomeric both in vitro and in vivo. In one mutant protein, we substituted the central Glu150 with the bulky, positively charged arginine residue, creating the Hsp33-E150R mutant protein. In another Hsp33 mutant, we substituted both central amino acids Glu150 and Gln151 with alanine residues, creating the Hsp33-E150A-Q151A mutant protein. Because both mutant proteins behaved nearly identically both in vitro and in vivo, we will focus here only on the results obtained with the Hsp33-E150R mutant protein. Generation of Redox-active, Constitutively Monomeric Hsp33 Species—The Hsp33-E150R mutant was first overexpressed and purified to homogeneity. The purification protocol of wild type Hsp33 was applied to purify this mutant protein, and Hsp33-E150R was found to behave identically to the wild type Hsp33 protein. The purified Hsp33-E150R mutant protein was then completely reduced and incubated with excess zinc. As is the case with wild type protein, once unbound zinc and excess DTT were separated from the protein, a 1:1 molar ratio of zinc to protein was determined (Fig. 1). In wild type Hsp33, disulfide bond formation induces the release of bound zinc (5Graumann J. Lilie H. Tang X. Tucker K.A. Hoffmann J.H. Vijayalakshmi J. Saper M. Bardwell J.C.A. Jakob U. Structure (Camb.). 2001; 9: 377-387Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar). Therefore, kinetic analysis of the oxidation-induced zinc release provides a convenient method to monitor the kinetics of disulfide bond formation in response to H2O2 (5Graumann J. Lilie H. Tang X. Tucker K.A. Hoffmann J.H. Vijayalakshmi J. Saper M. Bardwell J.C.A. Jakob U. Structure (Camb.). 2001; 9: 377-387Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar). As shown in Fig. 1, the apparent t1/2 of oxidation-induced zinc release is about 16 min for both wild type Hsp33 and the Hsp33-E150R mutant (Fig. 1, closed and open circles). Ellman's assays to determine the extent of disulfide bond formation in both wild type Hsp33 and the mutant confirmed the formation of two disulfide bonds in both proteins (data not shown). This showed that the introduction of a large, positively charged amino acid into a highly conserved region of Hsp33 did not alter the H2O2 sensitivity of Hsp33. To determine the oligomerization status of the oxidized Hsp33-E150R mutant protein, oxidized Hsp33-E150R was subjected to analytical ultracentrifugation. As shown previously, oxidized Hsp33 dimers sedimented at 2.8 S, while reduced Hsp33 monomers and the constitutively monomeric Cys-free Hsp33 mutant sedimented at 2.1 and 1.9 S, respectively (5Graumann J. Lilie H. Tang X. Tucker K.A. Hoffmann J.H. Vijayalakshmi J. Saper M. Bardwell J.C.A. Jakob U. Structure (Camb.). 2001; 9: 377-387Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar) (Fig. 1, inset). In excellent agreement with our structural predictions, the oxidized Hsp33-E150R mutant protein behaved very similarly to our monomeric Hsp33 preparations and sedimented with 2.1 S (Fig. 1, inset). To determine the KD for dimerization of the Hsp33-E150R mutant protein, sedimentation analysis was performed using various concentrations of the oxidized Hsp33-E150R mutant protein. At the highest protein concentrations (∼1 mg/ml) that can be used in these measurements, we were unable to detect any significant dimerization. We concluded that the KD of the Hsp33-E150R in the absence of substrate proteins must be at least 2 orders of magnitude greater than the KD of oxidized wild type Hsp33, which was determined to be 0.59 μm at 20 °C (5Graumann J. Lilie H. Tang X. Tucker K.A. Hoffmann J.H. Vijayalakshmi J. Saper M. Bardwell J.C.A. Jakob U. Structure (Camb.). 2001; 9: 377-387Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar). The monomeric state was also confirmed by sedimentation equilibrium experiments, in which a molecular mass of 33,160 ± 2,060 Da was observed independent of the protein concentration. These results showed that the substitution of the highly conserved Glu150 to Arg resulted indeed in a redox-regulated, constitutively monomeric Hsp33 variant. This mutant protein, therefore, should represent the stable oxidized monomeric Hsp33 species, which is presumably only transiently present during the activation process of Hsp33 in vitro. The Oxidized Monomer Has Partial Chaperone Activity—To analyze the activity of the oxidized monomeric variant of Hsp33, we used the same activity assays that have been previously employed to characterize the chaperone activity of wild type Hsp33 (5Graumann J. Lilie H. Tang X. Tucker K.A. Hoffmann J.H. Vijayalakshmi J. Saper M. Bardwell J.C.A. Jakob U. Structure (Camb.). 2001; 9: 377-387Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar). We first analyzed the influence of the oxidized, constitutively monomeric Hsp33-E150R mutant protein on the aggregation of chemically unfolded luciferase. Luciferase, once completely unfolded by incubation in high concentrations of guanidinium HCl, is unable to refold back to its native state upon dilution into refolding buffer. Instead, luciferase rapidly forms large, insoluble aggregates, which can be detected by light scattering (Fig. 2A, trace a). As previously shown, a 2-fold molar excess of oxidized Hsp33 dimer to luciferase was able to almost completely suppress the aggregation of luciferase (trace b). In contrast, the same monomer concentration of oxidized monomeric Hsp33-E150R mutant protein exhibited only a slight influence on the aggregation behavior of chemically denatured luciferase (trace d). This was very similar to the light scattering signal in the presence of reduced wild type Hsp33 monomers (trace c) or reduced Hsp33-E150R monomers (data not shown), which are considered to be largely inactive Hsp33 preparations. Very similar results were obtained when chemically denatured citrate synthase was used as a substrate protein instead of luciferase, suggesting that oxidized Hsp33 monomers are either unable to interact with highly unfolded proteins at all or that they do not exert sufficiently high affinity for these extremely aggregation-sensitive folding intermediates to effectively compete with their fast aggregation process. To distinguish between these two possibilities, we tested the activity of oxidized Hsp33-E150R monomers on the aggregation of thermally unfolding substrate proteins. Proteins that are incubated under slightly denaturing temperature conditions only slowly expose hydrophobic surfaces, and aggregate, therefore, with considerably slower kinetics (compare trace a in Fig. 2, A and B). To effectively compete with this slow aggregation process, lower chaperone concentrations, and in general, lower relative affinities of the chaperones for the substrate proteins, are required. This is shown in Fig. 2B, where a 0.5:1 molar ratio of oxidized Hsp33 dimers to luciferase was fully capable of preventing the heat-induced aggregation of luciferase (trace b). Here, the oxidized Hsp33-E150R monomer was active as a chaperone in that a 1:1 molar ratio of Hsp33-E150R monomers to heat-inactivated luciferase was as effective as a 0.5:1 molar ratio of oxidized wild type Hsp33 dimers to luciferase (compare Fig. 2B, traces b and d). In contrast, the reduced Hsp33 monomer showed again only a slight influence on the aggregation behavior of heat inactivated luciferase, which is most likely due to the oxidation of a subset of Hsp33 molecules during the time course of the experiment (trace c). Very similar results were obtained when thermally unfolding citrate synthase was used as a substrate protein instead (time point 140 min of Fig. 2C) or when the influence of Hsp33 on oxidatively damaged RrmJ was tested (data not shown). These results indicated that the influence of Hsp33 on slowly unfolding substrate proteins such as thermally unfolding luciferase or thermally unfolding citrate synthase was mainly determined by the absolute concentration of oxidized Hsp33 monomers and not by the concentration of Hsp33 dimers. This suggested that Hsp33, once oxidized, exposes a substrate-binding site, which has sufficiently high affinity for slowly unfolding proteins. To effectively compete against the fast aggregation that occurs during the refolding of completely unfolded proteins such as chemically denatured luciferase or citrate synthase, however, the substrate affinity of the chaperone must be significantly higher. This appears to be accomplished by the dimerization of two oxidized monomers, which might bring the two substrate-binding sites into close proximity and, therefore, enlarge the substrate-binding site. Oligomerization as a mechanism to increase the size and affinity of substrate binding sites is a common theme in chaperone biology and has been well studied with the 14-mer GroEL (12Farr G.W. Furtak K. Rowland M.B. Ranson N.A. Saibil H.R. Kirchhausen T. Horwich A.L. Cell. 2000; 100: 561-573Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar). This hypothesis is also supported by the structural analysis of active Hsp33 dimers, which identified two potential substrate-binding sites, both of which involve both Hsp33 subunits (7Vijayalakshmi J. Mukhergee M.K. Graumann J. Jakob U. Saper M.A. Structure (Camb.). 2001; 9: 367-375Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar, 8Kim S.J. Jeong D.G. Chi" @default.
• W2084665652 created "2016-06-24" @default.
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• W2084665652 date "2004-05-01" @default.
• W2084665652 modified "2023-10-11" @default.
• W2084665652 title "Activation of the Redox-regulated Chaperone Hsp33 by Domain Unfolding" @default.
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• W2084665652 doi "https://doi.org/10.1074/jbc.m401764200" @default.
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