Crystal Structure of Constitutively Monomeric E. Coli Hsp33 Mutant with Chaperone Activity

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FEBS Letters 585 (2011) 664–670

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

Seung-Wook Chi a,1, Dae Gwin Jeong a,1, Joo Rang Woo a, Hye Seon Lee a, Byoung Cheol Park a, Bo Yeon Kim a, ⇑ ⇑ Raymond L. Erikson a,b, Seong Eon Ryu a,c, , Seung Jun Kim a,

a Medical Proteomics Research Center, Korea Research Institute of Bioscience and Biotechnology, 52 Eoeun-Dong, Yuseong-Gu, Daejeon 305-333, Republic of Korea b Department of Molecular and Cellular Biology, Harvard University, Cambridge, MA 02138, USA c Department of Bio Engineering, Hanyang University, Seongdong-Gu, Seoul 133-791, Republic of Korea

article info abstract

Article history: Heat shock protein 33 (Hsp33) from Escherichia coli is a redox-regulated molecular chaperone that Received 18 November 2010 protects cells from oxidative stress. To understand the molecular basis for the monomer–dimer Revised 18 January 2011 switch in the functional regulation of E. coli Hsp33, we generated a constitutively monomeric Accepted 18 January 2011 Hsp33 by introducing the Q151E mutation in the dimeric interface and determined its crystal struc- Available online 23 January 2011 ture. The overall scaffold of the monomeric Hsp331–235 (Q151E) mutant is virtually the same as that Edited by Kaspar Locher 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 , suggesting that a Hsp33 monomer Keywords: 1–235 Heat shock protein 33 (Hsp33) is sufﬁcient to interact with slowly unfolded substrate. Chaperone Ó 2011 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. Domain-swapping Redox-sensitive

1. Introduction exposure to heat stress, the linker region undergoes the unfolding transition that is required for the activation of Hsp33. Stress proteins such as molecular chaperones protect bacterial Under normal condition, Hsp33 remains inactive as a zinc- cells from oxidative stress [1,2]. Escherichia coli heat shock protein bound reduced monomer. It was proposed earlier that Hsp33 is 33 (Hsp33) is a redox-regulated molecular chaperone that is acti- activated by a sequential and stepwise manner [1,6,7]. (1) Upon vated in response to oxidative stress [1,3,4]. Activated Hsp33 pre- exposure to oxidative stress, the ﬁrst disulﬁde bond is formed be-

vents partially denatured proteins from aggregation during tween Cys265 and Cys268, which triggers the release of bound zinc oxidative stress. In the Hsp33, there are four absolutely conserved and the unfolding of the zinc-binding domain. (2) Upon sensing

cysteines (Cys232, Cys234, Cys265 and Cys268) that are capable of heat stress, the linker region is unfolded and the second disulﬁde coordinating zinc in the reduced state. Hsp33 consists of N-terminal bond (Cys232–Cys234) is then formed. (3) Eventually, the fully active substrate-binding domain (residues 1–178) and C-terminal Hsp33 chaperone is formed by dimerization and thus exerts its zinc-binding domain (residues 232–294) [5], which are connected function as an oxidized dimer. via a linker region (residues 179–231). The compactly folded Although the functional switch on/off of Hsp33 is well coupled substrate-binding domain of Hsp33 is thought to bind unfolded to the oxidation/reduction process, its correlation with the mono- substrates via its hydrophobic surface. Recently, Jakob et al. eluci- mer–dimer transition remains still unclear. Previous in vivo studies dated that both oxidative stress and heat stress are required for full showed that the dimerization of Hsp33 is required to protect cells activation of Hsp33 and its redox-switch domain (residues 179–294) against combined heat and oxidative stress [8]. However, it was serves as dual stress sensor [6]. The zinc-binding domain of Hsp33 previously reported that reduced Hsp33 dimers are still active senses oxidative stress by disulﬁde-bond formation and zinc release, and stabilized by substrate binding [9], and reduced Hsp33 mono- whereas the linker region functions as a themosensor. Upon mers are also partially active [8]. In our previous study, we presented the crystal structure of dimeric N-terminal fragment

(residues 1–235) of E. coli Hsp33 (Hsp331–235) with chaperone activity (1I7F) [10]. The N-terminal Hsp33 fragment containing ⇑ Corresponding authors. Fax: +82 42 860 4598. the substrate-binding domain and the linker region, by itself, can E-mail addresses: [email protected] (S.E. Ryu), [email protected] (S.J. Kim). dimerize and bind unfolded substrates as an active molecular 1 These authors contributed equally to this work.

0014-5793/$36.00 Ó 2011 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.febslet.2011.01.029 S.-W. Chi et al. / FEBS Letters 585 (2011) 664–670 665 chaperone. Recently, it was shown that reduced dimeric Hsp33 Table 1 proteins from Bacillus subtilis (1VZY) [11] and Thermotoga maritima Data collection and reﬁnement statistics. (1VQ0) [12] have virtually the same structures as that of E. coli Data collection

Hsp331–235 dimer [10,13], albeit no domain swapping. However, Space group P41212 all the crystal structures of Hsp33 solved until now are confined Cell constant a = b = 65.98 Å, c = 145.95 Å, a = b = c =90° Resolution (Å) 2.9 to dimers. Total observation 33 240 To gain insight into a molecular basis for the monomer–dimer Unique reflections 7120 transition of Hsp33 and its correlates with the functional switch, Completeness (%) 91.5 (76.5)a b we constructed constitutively monomeric mutant of Hsp33 by intro- Rmerge (%) 6.6 (19.8) ducing Q151E mutation in the dimeric interface and determined its Average I/r(I) 11.8 (2.6) crystal structure. The chaperone activity of the constitutively mono- Refinement meric mutant Hsp33 was analyzed to provide its structure–function No. of reflections 6752 Total No. of atoms 1811 relationship. Rcryst/Rfree 22.7/28.1 rmsd bond lengths (Å) 0.018 2. Materials and methods rmsd bond angles (°) 2.89 rmsd improper angles (°) 3.95 rmsd dihedrals (°) 26.2 2.1. Cloning and purification of E. coli Hsp33 proteins Temperature factor (Å2) 43.3

a Values inP parenthesesP are for theP highestP resolution bin. Wild-type E. coli Hsp331–294 and Hsp331–235, and mutant Hsp331–294 b Rmerge ¼ hkl jjIhkl; j hIhklij= hkl j Ihkl; jj, where I is the intensity for the jth (Q151E) and Hsp331–235 (Q151E) and Hsp331–294 (Q151E) mutant measurement of an equivalent reflection with the indices h,k,l. were subcloned into the pET11a vector (Novagen) and expressed in E. coli BL21(DE3) in 1 mM ZnCl2. All the Hsp33 proteins contained the mutations in nonconserved cysteines (C141D and C239S) and aggregation of firefly luciferase (Boehringer Mannheim). Reaction they were purified as previously described [10]. The purified pro- buffer (40 mM Hepes-NaOH, pH 7.5) alone or with each Hsp33 proteins were dialyzed against 10 mM ADA (pH 6.5), 2 mM DTT, tein of 0.075 lM were pre-incubated at 43 °C and luciferase was 0.1 mM ZnCl2 for crystallization and concentrated to 23 mg/ml by then added to them under continuous stirring to a final concentra- centricon-10 units (Amicon). tion of 0.15 lM. Light scattering of the thermally aggregated luciferase was monitored in the absence or presence of the each Hsp33

2.2. Crystallization and diffraction data collection proteins: wild-type Hsp331–294 and Hsp331–235, and mutant Hsp331–235 (Q151E) and Hsp331–294 (Q151E). The light scattering Crystals were grown from 0.9 M lithium sulfate and 0.4 M was time-kinetically measured on the spectrofluorophotometer ammonium sulfate in 0.1 M Na-citrate (pH 5.6) at 25 °C from hang- (RF-5301PC, Shimadzu) at 350 nm for both excitation and emission. ing drops by vapor diffusion method and crystals grew at their full Excitation and emission slit widths were set to 1.5 nm and 3 nm, lengths for 5 days. X-ray diffraction data were collected at 6B respectively. beamline at Pohang accelerator laboratory. The crystal in the drop- let was transferred to a cryo-solution containing mother liquor 2.5. Gel filtration experiment supplemented with 20% (v/v) glycerol and then fresh-frozen in a nitrogen gas stream at 93 K. The diffraction data were processed Gel filtration was carried out using a Superose 12 HR 10/300 and scaled using DENZO and Scalepack from HKL2000 package column (Pharmacia). The wild type and variant Hsp33 proteins [14]. The crystals belong to P41212 space group with cell dimen- were eluted in a buffer containing 20 mM Hepes-NaOH, pH 7.0, sions of a = b = 65.98 Å and c = 145.95 Å. 200 mM NaCl and 10% (v/v) glycerol at 25 °C. All Hsp33 proteins were concentrated to 45 lM. The sample volume was 100 ll

2.3. Structure determination and refinement and the protein was detected by measuring OD280. For the reduced form of wild type Hsp331–294, 2 mM DTT was supplemented to a The crystal structure of Hsp331–235 (Q151E) mutant was deter- buffer to avoid oxidation during the experiment. mined by molecular replacement using the program AMoRe [15]. For the structure determination of Hsp331–235 (Q151E) mutant, 3. Results the previously determined structure of wild-type Hsp331–235 (PDB code:1I7F) was used as a search model. The programs O 3.1. Generation of constitutively momomeric Hsp33 mutant [16] and Refmac [17] were used in the model building and refinement, respectively. During the refinement, the randomly selected In our previously study, we determined the dimeric structure of 5% of data were set aside for the Rfree calculation. The structure an active E. coli Hsp33 fragment (residues 1–235) (Hsp331–235) of 2.9 Å resolution was refined to an Rcryst of 22.8% and an Rfree of [10]. In the structure, most of the highly conserved residues of 28.0%. Crystallographic and refinement statistics are shown in Hsp33 are located in the dimeric interface (Fig. 1A), indicating Table 1. The Ramachandran plots drawn by the program the importance of the dimerization in the function of Hsp33. Re- PROCHECK [18] show that, except for Pro and Gly, 98% of all resi- cently, the dimerization of Hsp33 was shown to be required to pro- dues fall within the most favored and additional allowed regions. tect cells against a combination of heat and oxidative stress [8].To There are no residues in the disallowed region. Figures were drawn explore whether the monomer–dimer switch of Hsp33 is indeed by using the programs PyMOL (www.pymol.org). strongly correlated with its functional regulation, a Hsp33 mutant that is incapable of forming a dimer was designed. To disrupt the 2.4. Measurement of chaperone activity closest contacts between the two subunits of a dimer, the abso-

lutely conserved Gln151 (Fig. 1A) at the dimerization interface Chaperone activities of the wild-type Hsp331–294 and Hsp331–235, was replaced with glutamate. In the structure of Hsp331–235 dimer and mutant Hsp331–235 (Q151E) and Hsp331–294 (Q151E) were mea- [10] (Fig. 1B), Gln151 provides one of the most signiﬁcant contribu- sured by monitoring the effect of the Hsp33 proteins on the thermal tions to the dimerization of Hsp331–235 by forming the closest 666 S.-W. Chi et al. / FEBS Letters 585 (2011) 664–670

Fig. 1. Design of constitutively monomeric Hsp33 mutant. (A) Functional domains of Hsp33 and sequence conservation of Gln151 in bacterial Hsp33 homologues. The domain structures of the substrate-binding domain, the linker region and the zinc-binding domain are presented with each domain colored differently. The sequence alignment of bacterial Hsp33 homologues in dimeric interface is presented below the substrate-binding domain with the absolutely conserved residues and homologous residues colored 10 red and yellow, respectively. (B) A ribbon diagram of Hsp331–235 dimer . Two Hsp33 monomers are colored differently. Side chains of Gln151 are drawn in stick representation. (C) The hydrogen bonds of Gln151 between the subunits. The side-chains of the Hsp33 residues involved in the hydrogen bonds with Gln151 are drawn and labeled.

contacts between the subunits. The NE2 and N atoms in Gln151 of the Hsp33 mutant is characterized by a four-layered abab struc- from one subunit form hydrogen bonds with the O atom in Thr60 ture, which contains two b sheets and six a-helices. For clarity in and the OE1 atom in Glu150 from the other subunit, respectively structural comparison, the same designation for domain (domain (Fig. 1C). The Q151E mutation could impair the dimerization of I and II) and secondary structure (H1–H7 and S1A–S10E) was used

Hsp33 by disrupting one crucial hydrogen bond between the sub- as previously described in wild-type Hsp331–235 [10]. The structure units. Therefore, Hsp331–235 (Q151E) was generated as a candidate of Hsp331–235 (Q151E) mutant consists of domain I that runs from for a constitutively monomeric mutant and used for crystal struc- residue 1 to 179 and domain II, a smaller domain that runs from ture determination and chaperone activity assay. the residue 184–235 (Fig. 3A). The domain I is composed of two discrete b-sheets (S1 and S2) with a total of nine b-strands and 3.2. Oligomeric status of Hsp33 (Q151E) mutant in solution three a-helices (H1, H2 and H4), whereas the domain II is of three a-helices (H5–H7), followed by a b-strand (S10E). The two promi-

To characterize oligomeric status of Hsp331–235 (Q151E) mutant nent domains are connected by a folded loop (residues 180–183). in solution, we performed gel ﬁltration chromatography experi- In the domain I, the three a-helices (H1, H2 and H4) are sand- ment with wild-type and mutant Hsp33 proteins (Fig. 2). The re- wiched between b-sheet S1 on one side and b-sheet S2 on the other duced wild-type Hsp331–294 was present as a monomer in side. Compared to wild-type E. coli Hsp331–235, both a-helix H3 and 0 solution, while oxidized Hsp331–294 eluted as a major peak corre- b-strand S2 F are absent in the structure of Hsp331–235 (Q151E) sponding to monomer with signiﬁcant fractions of dimer peak indi- mutant. cating an equilibrium between monomeric and dimeric species. The interactions between the domain I and the domain II are

However, Hsp331–235 (Q151E) exclusively eluted as a monomer very extensive. First, three consecutive a-helices (H5–H7) in the while peaks corresponding to Hsp331–294 (Q151E) contain substan- domain II and their interconnecting loops cover the hydrophobic tially smaller fraction of dimer compared to the oxidized wild-type patches of b-sheet S1 in the domain I, which would be otherwise 0 Hsp331–294, conﬁrming that Q151E mutation renders Hsp33 to be a solvent-exposed. Second, the short b strand (S1 E) in the domain monomer irrespective of oxidation status. II interacts with the domain I by forming the edge of b-sheet S1.

Cys232 and Cys234 are located at the C-terminal loop, which is away 3.3. Overall structure of constitutively monomeric Hsp33 mutant from the body of molecule and goes perpendicular to b-sheet S2. Fig. 3B shows the electrostatic potential distribution of the pro-

The crystal structure of Hsp331–235 (Q151E) mutant was deter- posed substrate-binding surface of Hsp331–235 (Q151E) mutant. mined by molecular replacement (MR) method and reﬁned to 2.9 Å The proposed substrate-binding surface is composed of b-sheets resolution to a crystallographic R factor of 22.8% and an Rfree of S2A–S2E in domain I and its predominantly hydrophobic patch is 28.0% (Table 1). As expected, Hsp331–235 (Q151E) mutant is folded solvent-exposed. This electrostatic potential distribution is quite as a monomer with no domain swapping. No indications of oligo- similar to that of dimeric Hsp33 [10] although the size of binding meric interactions were found in the crystal. The overall global fold surface is signiﬁcantly reduced. S.-W. Chi et al. / FEBS Letters 585 (2011) 664–670 667

Fig. 2. Oligomeric status of Hsp33 proteins in solution. Hsp33 proteins were loaded onto Superose 12 (Pharmacia) column. The eluted volumes of marker proteins are indicated as vertical arrows. The position of void volume (Vo) is determined by blue dextran 2000 (2000 kDa).

A H5 H5

H7 S1B H7 S1B S1A S1C S1A S1C S1’E S1’E S1D S1D N H6 N H6

H4 H4 H2 H2 S2D S2E S2D S2E

H1 S2C H1 S2C

S2B S2B S2A S2A

C C B Potential substrate- binding region

Fig. 3. Crystal structure of constitutively monomeric Hsp33 mutant. (A) A stereo ribbon diagram of the Hsp331–235 (Q151E) mutant is drawn. Domains I (residues 1–179) and a folded loop (residues 180–183) are colored blue and domain II (residues 184–235) is colored red. (B) Electrostatic potential distribution of the proposed substrate-binding 1 surface of Hsp331–235 (Q151E) mutant. Electrostatic potentials of Hsp331–235 (Q151E) mutant were calculated with contours from 20 (red) to +20 (blue) kTe (k, Boltzmann’s constant; T, temperature; e, electron) using the program GRASP [19]. The point of view is consistently maintained between left and right panels. 668 S.-W. Chi et al. / FEBS Letters 585 (2011) 664–670

3.4. Structural comparison between dimeric wild-type and monomeric Another structural difference between dimeric Hsp331–235 and mutant Hsp33 monomeric Hsp331–235 (Q151E) mutant is observed at the dimeric interface of the domain I. In the Hsp331–235 dimer, the interactions A search of the EMBL database using the program DALI [20] between the two domain Is around the two-fold axis occur in the shows that E. coli Hsp331–235 (Q151E) mutant shares the highest cleft of the domain I, which is composed of central helix (H2) structural homology with oxidized E. coli Hsp331–235 dimers (PDB and two b-sheets (S1 and S2). The residues 145–153 in the neigh- code 1I7F and 1HW7) [10,13] with root mean square deviations boring molecule are buried deepest in the cavity (Fig. 1C). Through (RMSDs) of 1.2 Å and 1.6 Å (over 175 and 176 residues), respec- these interactions, reduced Hsp33 from B. subtilis can form a dimer tively (Fig. 4A). The second and the third highest structural similar- even without domain swapping (Fig. 4B). In contrast, these ity were found in the reduced dimeric structures of B. subtilis interactions are unlikely to take place between the monomers of

Hsp33 (1VZY) [11] and T. maritima Hsp33 (1VQ0) [12] with RMSDs Hsp331–235 (Q151E) mutant because the crucial intersubunit of 2.7 Å and 2.8 Å (over 223 and 226 residues), respectively (Fig. 4B hydrogen bond described above is disrupted by the substitution and C). of Gln151 by glutamate. Such dimeric interface is absent in the

Structural comparison between dimeric Hsp331–235 and mono- structure of reduced T. maritima Hsp33 dimer (Fig. 4C). meric Hsp331–235 (Q151E) mutant shows that the structure of the monomeric mutant is virtually the same as that of the dimeric 3.5. Chaperone activity of constitutively monomeric Hsp33 mutant form, with the except that there is no domain swapping with a cross-over (Fig. 4A). The dimerization of Hsp331–235 forms a large To investigate the effect of Q151E mutation of Hsp33 on chap- intermolecular b-sheet (S2 + S20) composed of 10 antiparallel erone activity, we tested its activity using thermally unfolded lucif- b-strands. In the dimeric structure of Hsp331–235 (Fig. 1B), the res- erase and compared with those of wild-type Hsp33 proteins idues 179–235 in the domain II and the interconnecting loop (Fig. 5). The molar ratio of all Hsp33 proteins to luciferase was crosses between monomers to form an intersubunit b-sheet. In set to 0.5:1. Light scattering increased in an exponential manner short, the domain II serves as a cross-over arm, making extensive due to the aggregation of thermally unfolded luciferase at 43 °C contacts with the domain I of the other subunit to stabilize the when no additive proteins was present (Fig. 5, trace a). In the pres- dimerization. On the other hand, the domain I of Hsp331–235 ence of oxidized wild-type Hsp331–294 (trace e) or wild-type (Q151E) mutant folds back onto the domain II of its parent mole- Hsp331–235 (trace c), the light scattering did not increase upon cule as observed in Zn2+-bound reduced Hsp33 dimers from B. sub- the addition of luciferase because these proteins prevented ther- tilis and T. maritima Hsp33 (Fig. 4B and C). Even in the cross-over mally unfolded luciferase from aggregation. In contrast, the re- region of the domain II, high structural similarity with a RMSD of duced wild-type Hsp331–294 (trace f) showed no inﬂuence on the 2.7 Å was observed between wild-type Hsp331–235 and aggregation of thermally unfolded luciferase. Constitutively mono- Hsp331–235 (Q151E) mutant. Since Hsp331–235 (Q151E) mutant is meric Hsp331–235 (Q151E) mutant (trace c) also has the chaperone stable as a monomer, a fairly large conformational change appears activity similar to that of wild-type Hsp33, indicating that there to be necessary for domain-swapped dimerization to occur. This was negligible effect of the Q151E mutation on the chaperone conformational change can be caused by disulﬁde-bond formation activity of Hsp331–235. It is noticeable that Hsp331–235 (Q151E) mu- and high temperature-induced unfolding. tant incapable of forming a dimer is still active. We performed the

Fig. 4. Structural comparison of constitutively monomeric Hsp33 mutant with other Hsp33 dimers. The structure of E. coli Hsp331–235 (Q151E) mutant (blue) was superimposed with E. coli Hsp331–235 dimer [10] (red) (A), reduced B. subtilis Hsp33 dimer (orange) [11] (B) and reduced T. maritima Hsp33 dimer (gray) [12] (C). S.-W. Chi et al. / FEBS Letters 585 (2011) 664–670 669

30 substrate-binding hydrophobic patch. Subsequently, the second a disulfide bond is formed, triggering the fully opening of the linker 25 region (to fully open state) and dimerization, eventually leading to f full functional activation of Hsp33. Our chaperone activity assay showed that monomeric Hsp33 (Q151E) mutant retains the chap- 20 erone activity similar to those of dimeric wild-type Hsp33. These results indicated that the basal level of Hsp33’s chaperone activity 15 can be gained only by disulfide-bond formation and linker unfolding, even without dimerization. This is consistent with the step- 10 wise activation process of Hsp33 where the disulfide-bond formation, the linker unfolding and the dimerization then occur d in a sequential manner [1,6]. The stepwise process of Hsp33 activa- Light scattering (arb. units) scatteringLight (arb. 5 e tion involves at least three different species of Hsp33; inactive re- c duced monomer, active oxidized monomer and active oxidized b 0 dimer. As suggested earlier [8], a dimerization-defective mutant 0 100 200 300 400 500 600 could correspond to the intermediate of active oxidized mono- Time (s) meric Hsp33, which seems to be stably present during the activa-

tion pathway of Hsp33. Hence, the present structure of Hsp331–235 Fig. 5. Chaperone activity of constitutively monomeric Hsp33 mutant. Light scattering by the aggregation of thermally unfolded luciferase was measured at (Q151E) mutant may represent the structure of the oxidized mono- 43 °C in the absence of any added protein (a) or in the presence of mutant meric intermediate that is transiently present in the sequential

Hsp331–294 (Q151E) (b), mutant Hsp331–235 (Q151E) (c), wild-type Hsp331–235 (d), activation process of Hsp33. oxidized wild-type Hsp331–294 (e) and reduced wild-type Hsp331–294 (f) at a 0.5:1 We previously hypothesized that a concave hydrophobic molar ratio of Hsp33 to luciferase. surface in Hsp33 dimer is a potential site for binding partially denatured substrates [10]. In the fold-back conformation of Hsp33 (Q151E) mutant (Fig. 3B), the exposure of substrate- same assay so as to investigate to what extent the intact 1–235 binding hydrophobic patch to solvent explains how it still retain Hsp33 (Q151E) mutant possesses the chaperone activity com- 1–294 chaperone activity. Dimerization results in doubling b-sheet pared to the truncated Hsp33 (Q151E) mutant. As shown in 1–235 (S2 + S20) of each monomer, thereby contributing to high-affinity trace d, 81% of chaperone activities were retained with binding to large substrates for effective chaperone function. This Hsp33 (Q151E) mutant. Overall, these data suggest that 1–235 is similar to what was observed in the oligomerization of dimerization is not a prerequisite for converting Hsp33 into a func- the molecular chaperone GroEL [21]. Our finding that Hsp33 tional chaperone that can bind heat inactivated aggregates. 1–235 (Q151E) monomer as well as intact Hsp331–294 (Q151E) was as active as wild-type Hsp33 dimer suggests that the single substrate- 4. Discussion binding site of Hsp33 monomer, without the formation of ex- panded intersubunit b-sheets (S2 + S20), is sufficient for the basal Oligomerization is known to be biologically important for the level of chaperone activity such as that to thermally inactivated function of molecular chaperones. It is an efficient mechanism to luciferase. increase the size and affinity of substrate-binding site although It is noteworthy to find that the constitutively monomeric form their oligomeric status depends on chaperone. Several molecular of Hsp33 without the zinc-binding domain is still as active as chaperones such as GroEL [21] and small heat shock proteins Hsp331–235 dimer. This is reminiscent of the previous reported re- (e.g., Hsp25, Hsp26) function as oligomers [22], whereas others sults of a constitutively monomeric variant of full-length Hsp33 such as DnaK function as a monomer [23]. In addition, all the mem- [8]. Graf et al. generated the redox-regulated Hsp33-E150R, where bers of Hsp90 family exist as homodimeric species [24]. Previous highly conserved Glu150 at the dimeric interface was replaced with in vivo studies showed that the dimerization of Hsp33 is necessary arginine. The oxidized monomers of full-length Hsp33-E150R to protect bacterial cells against combined heat and oxidative exhibited partial chaperone activity to thermally denatured lucif- stress [8]. While the oxidation/reduction process of Hsp33 is erase. Taken together with our results, we suggest that dimeriza- known to be strongly coupled to its functional switch, how the tion is not a prerequisite for converting Hsp33 into a functional dimerization is related to the functional switch is not still clear. chaperone that can bind slowly unfolded substrates such as ther- Although monomers, dimers and oligomers of Hsp33 exhibited mally inactivated luciferase. It seems, therefore, that domain- chaperone activity [8,10,25], all the structures of Hsp33 solved to swapped dimerization is not absolutely required for slow kinetics date were dimers. In this study, we presented the first structure aggregation such as thermal denaturation. In contrast, it was pre- of constitutively monomeric Hsp33 species by generating the viously observed that oxidized Hsp33-E150R has negligibly little

Hsp331–235 (Q151E) mutant where the dimerization is impaired chaperone activity against chemically denatured luciferase [8]. by the Q151E mutation at the dimeric interface. The monomeric Similarly, we observed that oxidized Hsp331–294 (Q151E) mutant Hsp33 mutant exhibited stable ‘‘fold-back’’ conformation of which has no noticeable chaperone activity toward chemically denatured domain II is neither domain-swapped nor unfolded. substrates (data not shown), suggesting that the dimerization is re- What is physiological relevance for the structure of active quired for preventing substrates from the fast kinetics aggregation monomeric Hsp33 species? Recently, Jakob et al. proposed a model such as chemical denaturation. Thus, bacterial cells may deploy of Hsp33’s activation [1,6]. Under non-stress condition, Hsp33 two differential levels of Hsp33 activation to counter stress remains inactive as a reduced monomer where the linker region depending on nature or strength of stress. is tightly folded (closed-state) and buried in the proposed sub- Hsp33 is reported to exist mainly as an oligomer under the strate-binding hydrophobic surface. Upon exposure to oxidative cytosol mimic condition and oligomeric Hsp33 has enhanced chap- stress, the first disulfide bond is formed in the zinc-binding erone activity compared with the dimer [25]. It is likely that a di- domain, inducing the zinc release and unfolding the zinc-binding mer is another intermediate in a process of high degree of domain. Upon sensing heat stress, the linker region undergoes oligomerization, which can be easily achieved in the same way of the unfolding transition to open state, exposing a proposed dimerization through domain swapping without altering the rest 670 S.-W. Chi et al. / FEBS Letters 585 (2011) 664–670 of structure. It appeared that high order oligomer of Hsp33 would [7] Graumann, J., Lilie, H., Tang, X., Tucker, K.A., Hoffmann, J.H., Vijayalakshmi, J., generate large hydrophobic surface as shown in dimeric Hsp33, Saper, M., Bardwell, J.C. and Jakob, U. (2001) Activation of the redox-regulated molecular chaperone Hsp33 – a two-step mechanism. Structure 9, facilitating holding larger protein aggregates. To date, there is little 377–387. information that Hsp33 has various forms of oligomer. However it [8] Graf, P.C., Martinez-Yamout, M., VanHaerents, S., Lilie, H., Dyson, H.J. and is definitely worth investigating whether Hsp33 can be regulated Jakob, U. (2004) Activation of the redox-regulated chaperone Hsp33 by domain unfolding. J. Biol. Chem. 279, 20529–20538. on oligomeric-status-dependent manner to respond to the stress [9] Hoffmann, J.H., Linke, K., Graf, P.C., Lilie, H. and Jakob, U. (2004) Identification in terms of the varying strength or nature. of a redox-regulated chaperone network. EMBO J. 23, 160–168. In the present study, we report the first structure of monomeric [10] Kim, S.J., Jeong, D.G., Chi, S.W., Lee, J.S. and Ryu, S.E. (2001) Crystal structure of proteolytic fragments of the redox-sensitive Hsp33 with constitutive Hsp33 species by generating dimerization-defective Hsp331–235 chaperone activity. Nat. Struct. Biol. 8, 459–466. (Q151E) mutant. Its chaperone activity assay results suggest that [11] Janda, I., Devedjiev, Y., Derewenda, U., Dauter, Z., Bielnicki, J., Cooper, D.R., Hsp33 monomer is sufficient for substrate binding and thus the Graf, P.C., Joachimiak, A., Jakob, U. and Derewenda, Z.S. (2004) The crystal structure of the reduced, Zn2+-bound form of the B. subtilis Hsp33 chaperone dimerization is not a prerequisite for its chaperone activity, at and its implications for the activation mechanism. Structure 12, 1901–1907. least, against mild denaturing conditions such as elevated temper- [12] Jaroszewski, L., Schwarzenbacher, R., McMullan, D., Abdubek, P., Agarwalla, S., ature. This is consistent with the stepwise activation model of Ambing, E., Axelrod, H., Biorac, T., Canaves, J.M., Chiu, H.J., Deacon, A.M., Hsp33 where the disulfide-bond formation, linker unfolding, and DiDonato, M., Elsliger, M.A., Godzik, A., Grittini, C., Grzechnik, S.K., Hale, J., Hampton, E., Han, G.W., Haugen, J., Hornsby, M., Klock, H.E., Koesema, E., dimerization then occur in a sequential manner. Here, the present Kreusch, A., Kuhn, P., Lesley, S.A., Miller, M.D., Moy, K., Nigoghossian, E., structure of Hsp331–235 (Q151E) mutant may represent the struc- Paulsen, J., Quijano, K., Reyes, R., Rife, C., Spraggon, G., Stevens, R.C., van den ture of mimicking a stable intermediate of active monomeric Bedem, H., Velasquez, J., Vincent, J., White, A., Wolf, G., Xu, Q., Hodgson, K.O., Wooley, J. and Wilson, I.A. (2005) Crystal structure of Hsp33 chaperone Hsp33 during its activation process. (TM1394) from Thermotoga maritima at 2.20 Å resolution. Proteins 61, 669– 673. [13] Vijayalakshmi, J., Mukhergee, M.K., Graumann, J., Jakob, U. and Saper, M.A. PDB accession numbers (2001) The 2.2 Å crystal structure of Hsp33: a heat shock protein with redox- regulated chaperone activity. Structure 9, 367–375. [14] Otwinowski, Z. and Minor, W. 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