The Molecular Chaperone Mechanism of the C-Terminal Domain of Large-Size Subunit Catalases

Large-size subunit catalases (LSCs) have an additional C-terminal domain (CT) that is structurally similar to Hsp31 and DJ-1 proteins, which have molecular chaperone activity. The CT of LSCs derives from a bacterial Hsp31 protein. There are two CT dimers with inverted symmetry in LSCs, one dimer in each pole of the homotetrameric structure. We previously demonstrated the molecular chaperone activity of the CT of LSCs. Like other chaperones, LSCs are abundant proteins that are induced under stress conditions and during cell differentiation in bacteria and fungi. Here, we analyze the mechanism of the CT of LSCs as an unfolding enzyme. The dimeric form of catalase-3 (CAT-3) CT (TDC3) of Neurospora crassa presented the highest activity as compared to its monomeric form. A variant of the CAT-3 CT lacking the last 17 amino acid residues (TDC3Δ17aa), a loop containing hydrophobic and charged amino acid residues only, lost most of its unfolding activity. Substituting charged for hydrophobic residues or vice versa in this C-terminal loop diminished the molecular chaperone activity in all the mutant variants analyzed, indicating that these amino acid residues play a relevant role in its unfolding activity. These data suggest that the general unfolding mechanism of CAT-3 CT involves a dimer with an inverted symmetry, and hydrophobic and charged amino acid residues. Each tetramer has four sites of interaction with partially unfolded or misfolded proteins. LSCs preserve their catalase activity under different stress conditions and, at the same time, function as unfolding enzymes.


Introduction
Proteins can have several metastable states and many cellular proteins become partially denatured or acquire a non-functional conformation particularly under stressful conditions. Molecular chaperones are proteins that assist other proteins to gain their native functional conformation which usually is the system's state of least energy. Molecular chaperones are unfolding enzymes-this is probably the unifying function of these proteins, and not, or not mainly, preventing aggregation of partially unfolded or misfolded proteins, as has been emphasized in the scientific literature [1,2]. Some unfolding enzymes/molecular chaperones are disaggregases, indicating that the unfolding activity is also critical for protein disaggregation [3].
Unfolding enzymes are essential to all cells and are present in many if not all cellular compartments, the extracellular space, and the bacterial periplasm [4][5][6][7][8][9][10]. They are abundant proteins [11], nevertheless the expression of many of these proteins, but not all, increases under different stress conditions [12,13]. Expression of numerous chaperones rises during heat shock and thus many unfolding enzymes are called heat shock proteins (Hsp). Unfolding enzymes can have overlapping functions and usually low specificity in their interactions with partially unfolded or misfolded proteins and they often display

Plasmid Constructs and Expression in E. coli
The catalase domain C3 ∆TD (residues 1-518) and the TDC3 (residues 568-719) were cloned into the pCold TM I plasmid as described in [33]. The Hsp31 gene was obtained from the DNA of E. coli by amplification with specific oligonucleotides and also cloned into the multicloning site of pCold I as described in [33].
CAT-3 ∆17aa and TDC3 ∆17aa variants were amplified from the cat-3 gene without introns, using oligonucleotides designed to eliminate the carboxy-terminal region. The variants were introduced into a pCold I plasmid using the XbaI restriction site as was done for the complete variants. The single mutants TDC3 Q652C , TDC3 K662L , TDC3 L702E , TDC3 F705E , TDC3 R710W , TDC3 F711E , and TDC3 D714I were produced by site-directed mutagenesis using the QuickChange II kit (Agilent, Santa Clara, CA, USA, 200524) following the indications of the manufacturer.
From an isolated single colony, bacteria were grown overnight in LB Lennox medium containing 100 mg/mL ampicillin at 37 • C, agitating at 200 rpm. The preculture was diluted 1:10 into fresh LB medium and incubation was continued to an OD at 600 nm of 0.4. Expression of the protein was induced with 1 mM IPTG and incubated at 16 • C, with 200 rpm agitation for 48 h. Hemin, 30 mM, pH 9.6, was added to the medium when catalases were expressed.
The expressed protein, tagged with six histidine residues, was purified with a Niagarose (QIAGEN N.V., Germantown, MD, USA, 30250) affinity column following instructions of the dealer (5th edition). Fractions containing most of the protein were concentrated by centrifugation in an Amicon filter (either 30,000 or 10,000 Da cutting size) and verified by PAGE-SDS (either 8% or 15% acrylamide) and stained with Coomassie Brilliant Blue.

Enzyme Purification and Analysis
CAT-1 was purified directly from N. crassa conidia following the method described in [32]. The purified CAT-1 was treated with subtilisin at a 7:1 protease/protein ratio at 37 • C for 1 h to give the active C63 catalase [33].

TDC Analysis and TDC3 Dimer and Monomer Formation
The molecular weight of the purified TDC3 was determined with a Superdex 75 HR 10/300 column coupled to a FPLC system (GE Healthcare Life Sciences, San Diego, CA, USA) according to the specifications by the company. TDC3 (1 mg/mL) was injected to the column with a flux of 0.75 mL/min. The molecular mass was determined with a mix of gel filtration standards (BioRad, Hercules, CA, USA, 1511901).
To form the cysteine disulfide in TDC3 Q652C the protocol described in reference [34] was followed: TDC3 Q652C (6 µM) in 100 mM Tris, pH 8.5, was mixed with 10 volumes of 9 M urea (HPLC grade), treated subsequently with DTT (0.5 M), incubated overnight at 4 • C and thereafter dialyzed by passing through a Sephadex G25 column in 0.1 M Tris-HCl buffer, pH 8.0. Then, 160 µM of H 2 O 2 was added to the reduced TDC3 Q652C , incubated at 25 • C for one hour, and thereafter dialyzed by passing through a Sephadex G25 column.
Monomer and dimer formation was verified by SDS-PAGE using 15% of acrylamide ( Figure S1). Folding of the TDC3 monomer, dimer, and the TDC3 mutant variants was confirmed by near-UV CD spectra ( Figure S2A). Spectra were run between 200 and 260 nm, at 25 • C, with a protein concentration of 0.2 mg/mL in phosphate buffer 50 mM, pH 7.8, in a Chirascan TM spectropolarimeter (Applied Photophysics ® , Leatherhead, UK).

Chaperone Assay
ADH (6.2 µM calculated as tetramers) in 50 mM phosphate buffer, pH 7.0, was heatdenatured at 45 • C for 150 min and light scattering at 360 nm was followed in a Beckman Coulter DU-650 spectrophotometer. The control sample contained 6.2 µM BSA instead of a chaperone. CAT-1 and CAT-3 was added at either 1.5 or 3 µM, calculated as tetramers; C63 and C3 ∆TD at 6 µM, calculated as tetramers; and TDC3 and Hsp31 at 6 µM, calculated as dimers. Mixtures were done at RT in a 500 µL quartz cuvette with a final volume of 400 µL.

Detection of Hydrophobic Regions
A concentration of 0.8 µM of each protein in 50 mM phosphate buffer, pH 7.4, was incubated with bis-ANS (1.2 µM), either at RT or 45 • C, for 30 min. Fluorescence was measured in a BioTek Synergy Mx Microplate Reader, excited at 370 nm, and emission determined between 400 and 600 nm at RT.
CAT-1, CAT-3 (each at 3 µM), Hsp31, and TDC3 (each at 6 µM) in 50 mM phosphate buffer, pH 7.4, were incubated with bis-ANS and thereafter exposed to UV light 360-370 nm for 10 min at RT. Proteins were dialyzed by passing them through a Sephadex G25 resin and used for the chaperone assay.  [37] and tested on the heat denaturation assay of ADH, as described.
CAT-1, CAT-3, and Hsp31 at 3 and 6 µM were tested in the ADH chaperon assay using either 50 mM sodium phosphate at pH 6.0, 50 mM Na/K phosphate at pH 7.8, or 50 mM sodium borate at pH 9.0.

Does the TDC3 Function as a Dimer or as a Monomer?
The CT origin from a bacterial Hsp31 [25] and the requirement of the dimeric form for chaperone activity of Hsp31 [17,38], led us to question whether the TDC3 also functions as a dimer. Size-exclusion chromatography showed that TDC3 in solution had monomers and dimers in similar proportions, and a small fraction of aggregates of high molecular weight. The molecular mass of the monomer obtained was 18.24 kDa, very close to the theoretical value 18.02 kDa (calculated by Expasy); while for the dimer, it was 44.61 kDa, approximately 5 kDa higher than the theoretical value of 39.04 kDa ( Figure S3).
To evaluate if the TDC3 dimer is required for its activity, the Gln652 was substituted by a cysteine (TDC3 Q652C ) to form a disulfide bond between TDC3 monomers allowing, in this way, the stabilization of the TDC3 dimer. The Q652 is found in the alpha helix located at the center of the interface of the inverted symmetric dimers in the CAT-3 tetrameric structure [39] (PDB 3EJ6). No cysteines are present in the native TDC3. To obtain TDC3 monomers that do not form dimers in solution, the cysteine residue of the Q652C variant was derivatized with NEM ( Figure S3A). The unfolding activity of the TDC3 Q652C dimer was similar to the TDC3 without modification. The TDC3 Q652C monomer derivatized with NEM also showed chaperone activity but it increasingly failed to preserve the ADH in its active conformation through the incubation time ( Figure 1A,B). When the concentration of the NEM-derivatized TDC3 Q652C monomer was increased two times, a complete ADH protection effect was observed ( Figure 1A,B). The monomer having the Cys derivatized with glutathione gave similar results ( Figure 1A,B).  Figure S3A). The unfolding activity of the TDC3 Q652C dimer was similar to the TDC3 without modification. The TDC3 Q652C monomer derivatized with NEM also showed chaperone activity but it increasingly failed to preserve the ADH in its active conformation through the incubation time ( Figure 1A,B). When the concentration of the NEM-derivatized TDC3 Q652C monomer was increased two times, a complete ADH protection effect was observed ( Figure 1A,B). The monomer having the Cys derivatized with glutathione gave similar results ( Figure 1A,B).

Hydrophobic Regions Are Involved in the Unfolding Activity of the CT
Exposure of hydrophobic patches at the interacting protein surface of the molecular chaperone is a general mechanism by which unfolding enzymes recognize partially unfolded or misfolded polypeptides. We therefore assayed the bis-ANS probe that fluoresces when it interacts with hydrophobic regions at the protein surface. CAT-1, CAT-3, and TDC3 had a high fluorescence that increased further when incubated at 45 °C; in contrast, the SSCs CAT-A or BSA showed a low fluorescence and a small increase at 45 °C ( Figure  2A-C). This indicates that CAT-1, CAT-3, and TDC3 exposed more hydrophobic regions when incubated at 45 °C, similar to Hsp31 (

Hydrophobic Regions Are Involved in the Unfolding Activity of the CT
Exposure of hydrophobic patches at the interacting protein surface of the molecular chaperone is a general mechanism by which unfolding enzymes recognize partially unfolded or misfolded polypeptides. We therefore assayed the bis-ANS probe that fluoresces when it interacts with hydrophobic regions at the protein surface. CAT-1, CAT-3, and TDC3 had a high fluorescence that increased further when incubated at 45 • C; in contrast, the SSCs CAT-A or BSA showed a low fluorescence and a small increase at 45 • C (Figure 2A-C). This indicates that CAT-1, CAT-3, and TDC3 exposed more hydrophobic regions when incubated at 45 • C, similar to Hsp31 (Figure 2A,C) [40].
To assure that the exposed hydrophobic regions that interact with bis-ANS are involved in the unfolding activity, the bis-ANS, by UV light treatment, was covalently bond to its binding residues. Proteins treated with bis-ANS plus UV light lost the ability to inhibit heat denaturation of ADH ( Figure 3A,B). The mere presence of bis-ANS, added 10 min after initiation of the reaction, also hampered the unfolding activity ( Figure 3C). To assure that the exposed hydrophobic regions that interact with bis-ANS are involved in the unfolding activity, the bis-ANS, by UV light treatment, was covalently bond to its binding residues. Proteins treated with bis-ANS plus UV light lost the ability to inhibit heat denaturation of ADH ( Figure 3A,B). The mere presence of bis-ANS, added 10 min after initiation of the reaction, also hampered the unfolding activity ( Figure 3C).

Charged Amino Acid Residues Are Involved in the Unfolding Activity of the CT
It has been shown that the charged amino acid residues at the interacting protein surfaces participate in the general mechanism by which molecular chaperones recognize partially unfolded or misfolded polypeptides [37]. To analyze the effect of charged amino acid residues in TDC3, the inhibition of ADH heat denaturation by CAT-3 and TDC3 was done in the presence of different concentrations of NaCl. The increase of the ionic strength led to a reduction in the unfolding activity of CAT-3 and TDC3 (Figure 4).

Charged Amino Acid Residues Are Involved in the Unfolding Activity of the CT
It has been shown that the charged amino acid residues at the interacting protein surfaces participate in the general mechanism by which molecular chaperones recognize partially unfolded or misfolded polypeptides [37]. To analyze the effect of charged amino acid residues in TDC3, the inhibition of ADH heat denaturation by CAT-3 and TDC3 was done in the presence of different concentrations of NaCl. The increase of the ionic strength led to a reduction in the unfolding activity of CAT-3 and TDC3 (Figure 4). circles), CAT-3 (3 µM) with incorporated bis-ANS (closed triangles); TDC3 (6 µM) without bis-ANS (open squares), TDC3 (6 µM) with incorporated bis-ANS (closed rhomboids); Hsp31 (6 µM) without bis-ANS (open triangles), and Hsp31 (6 µM) with incorporated bis-ANS (closed squares). (B) Average of three independent assays. (C) Light scattering of ADH (6 µM) when denatured at 45 °C for 150 min in the presence of BSA (6 µM) (rhomboids), TDC3 (6 µM) (triangles), and TDC3 (6 µM) but adding bis-ANS (1.2 µM) 10 min after starting the reaction (indicated by arrow) (squares). To avoid photo-incorporation of the bis-ANS probe, light scattering was determined at 500 nm in this assay.

Charged Amino Acid Residues Are Involved in the Unfolding Activity of the CT
It has been shown that the charged amino acid residues at the interacting protein surfaces participate in the general mechanism by which molecular chaperones recognize partially unfolded or misfolded polypeptides [37]. To analyze the effect of charged amino acid residues in TDC3, the inhibition of ADH heat denaturation by CAT-3 and TDC3 was done in the presence of different concentrations of NaCl. The increase of the ionic strength led to a reduction in the unfolding activity of CAT-3 and TDC3 (Figure 4).  This result indicates that charged amino acid residues are involved in the protein-protein recognition between the ADH and the unfolding enzymes. The chaperone activity of Hsp31 was more affected than the activity of CAT-3 and TDC3. Remarkably, even in the presence of 1 M NaCl, the CAT-3 and TDC3 preserved two thirds of its unfolding activity. We also assayed the participation of charged amino acid residues by changing the pH of the buffer. The pH had a general low effect on the unfolding activity; the effect was different for each of the enzymes tested ( Figure S4).

Identification of the Region Responsible for the Unfolding Activity
To identify those regions involved in the CT unfolding activity, we determined the interacting regions in the dimer formed by the CT at both poles of the tetrameric CAT-3 structure. For this, we obtained the surface electrostatic potential of the TDC3 dimer and the amino acid residues that are exposed to the solvent and that could interact with other proteins (Figure 5A,B). Many of the predicted amino acid residues are localized in the C-terminal loop and the cleft formed in the interface of the two CTs with inverted symmetry. The C-terminal loop has hydrophobic and charged amino acid residues only. We therefore eliminated the last 17 amino acid residues of CAT-3 and TDC3, consisting of seven hydrophobic and nine charged amino acid residues and one glycine (low hydrophobicity). Of these residues, V704 and F705 have a high probability, and K706, F707, R710, and F711 have intermediate probability of interaction with other proteins. CAT-3 and TDC3 without the C-terminal loop, CAT-3 ∆17aa and TDC3 ∆17aa , lost 85% of their unfolding activity ( Figure 5C,D).
To determine which of these amino acid residues participate in the unfolding activity of the TDC3 dimer, we substituted some residues-hydrophobic for charged and charged for hydrophobic. To select substitutions that had a less disturbing effect on the conformation of the domain a theoretical analysis was done using the server Duet [42]. All substitutions showed a clear effect on the TDC3 unfolding activity, between 43 and 50% of the control activity ( Figure 6A,B). However, it was possible to distinguish differences in their denaturation rates; rate was relatively fast for the K662L variant (maximal activity in 20 min), intermediate for R710W and F711E (maximal activity at 30 min), and slow for L702E, F705E, and D714I variants (maximal activity at 35-40 min) ( Figure 6A,C). Near-UV spectra showed similar amounts of alpha helices in all TDC3 variants with the exception of TDC3 D714 which had more beta strands and fewer unstructured regions ( Figure S2A). Many of the predicted amino acid residues are localized in the C-terminal loop and the cleft formed in the interface of the two CTs with inverted symmetry. The C-terminal loop has hydrophobic and charged amino acid residues only. We therefore eliminated the last 17 amino acid residues of CAT-3 and TDC3, consisting of seven hydrophobic and nine charged amino acid residues and one glycine (low hydrophobicity). Of these residues, V704 and F705 have a high probability, and K706, F707, R710, and F711 have intermediate probability of interaction with other proteins. CAT-3 and TDC3 without the C-terminal loop, CAT-3 Δ17aa and TDC3 Δ17aa , lost 85% of their unfolding activity ( Figure 5C,D).
To determine which of these amino acid residues participate in the unfolding activity of the TDC3 dimer, we substituted some residues-hydrophobic for charged and charged for hydrophobic. To select substitutions that had a less disturbing effect on the conformation of the domain a theoretical analysis was done using the server Duet [42]. All substitutions showed a clear effect on the TDC3 unfolding activity, between 43 and 50% of the control activity ( Figure 6A,B). However, it was possible to distinguish differences in their denaturation rates; rate was relatively fast for the K662L variant (maximal activity in 20 min), intermediate for R710W and F711E (maximal activity at 30 min), and slow for L702E, F705E, and D714I variants (maximal activity at 35-40 min) ( Figure 6A,C). Near-UV spectra showed similar amounts of alpha helices in all TDC3 variants with the exception of TDC3 D714 which had more beta strands and fewer unstructured regions ( Figure S2A).

Discussion
Unfolding enzymes are abundant proteins that assist other proteins to attain or maintain their native conformation. They do this essential task by interacting with hydrophobic regions of proteins partially denatured or misfolded in a non-functional conformation. Operating by hydrophobic and electrostatic interaction, unfolding enzymes drive their substrate proteins out of equilibrium allowing the exposed hydrophobic regions of these proteins to acquire their normal internal location and, in this way, recover the native functional conformation.

Discussion
Unfolding enzymes are abundant proteins that assist other proteins to attain or maintain their native conformation. They do this essential task by interacting with hydrophobic regions of proteins partially denatured or misfolded in a non-functional conformation. Operating by hydrophobic and electrostatic interaction, unfolding enzymes drive their substrate proteins out of equilibrium allowing the exposed hydrophobic regions of these proteins to acquire their normal internal location and, in this way, recover the native functional conformation.
LSCs are abundant proteins that are essential to reversing increasing H 2 O 2 concentrations during cellular oxidative stress. The CT of LSCs have an unfolding activity that assists other proteins to acquire or maintain their active conformation and increases the survival of E. coli under heat shock or oxidative stress [33]. Using heat denaturation of ADH as a molecular chaperone assay, we investigated the mechanism by which the CT of LSCs functions as an unfolding enzyme.
The CTs are structured as a dimers, one dimer in each pole of the LSC tetrameric structure. The dimer conformation is different from the Hsp31 and DJ-1 dimers, although the monomers are structurally very similar to the CT of LSCs. In the DJ-1/PfpI superfamily, four dimerization modes were described [17]; the CT of LSCs constitutes a fifth mode of dimerization into symmetric dimer [25].
Many low molecular chaperones, such as Spy, alpha-crystallin B, Hsp20, Hsp33, Hsp31, and DJ-1 are active as dimers [17,38,43]. The TDC3 spontaneously forms dimers in solution ( Figure S3). Docking experiments also indicated dimer formation of CTs, and some of the dimers obtained by docking had the inverted symmetry found in LSCs ( Figure S5). To have homogeneous populations of dimers and monomers, the Q652C variant of TDC3 was obtained to maintain the dimeric structure by forming a disulfide bridge. We showed that the dimer was more active than the monomer, whose formation was induced by derivatizing the cysteine of the Q652C variant with NEM or GSH.
The two CTs with inverted symmetry form a cleft between them with the two Cterminal loop positioned in the extremes of the cleft ( Figure 5A,B). In the C-terminal loop and along the cleft, several amino acid residues have a high probability of interaction with other proteins ( Figure 5B). The C-terminal loop contains 17 amino acid residues that have a surface potential of −10 kcal/mole (red in Figure 5A). This loop is composed of charged (9) and hydrophobic residues (8) only; six of its residues are predicted to interact with other proteins. We observed a reduction of 85% in the molecular chaperone activity by deleting the 17 C-terminal loop, indicating that this loop, probably together with other amino acid residues in the cleft, is required for the unfolding activity. Substitution of hydrophobic for charged amino acid residues and vice versa in the C-terminal loop of TDC3 resulted in a decrease in the unfolding activity of 43-50% of the control activity. This further indicates that hydrophobic and charged amino acid residues of this loop are important for the unfolding activity of the TDC3. Near-UV CD spectra indicate that the TDC3 D714I variant had an increased numbers of beta stands and less unstructured regions, suggesting a beta strand in the loose C-terminal loop ( Figure S2B), which could account for its slow rate of its chaperone activity.
Increased exposure of hydrophobic residues to temperature in CAT-1, CAT-3, and TDC3 was shown by an increase in the fluorescence of the bis-ANS probe, which involves the hydrophobic amino acid residues. Charged residues are also part of the general mechanism of unfolding enzymes for the recognition of unfolded or misfolded proteins. Increasing the ionic strength with NaCl reduced the ionic interactions and the unfolding activity of CAT-3 and TDC3. Together with the results of substitution of charged residues in the C-terminal loop this indicated that charged residues are important for the unfolding activity of CAT-3 and TDC3.
Interestingly, the SSCs of Arabidopsis thaliana [44] and Oryza sativa (rice) [45] are stabilized by catalase-specific chaperones, which are essential for the three catalase activities; furthermore, the Arabidopsis thaliana SSCs are also protected by a peroxisomal sHsp [46].
We observed general overlapping properties among of antioxidant and unfolding enzymes: both enzymes are abundant proteins, and they are induced under stress conditions and during cell differentiation [23,47]. Upon oxidation, peroxiredoxins are inactivated as peroxidases and acquire chaperone activity with oligomerization [48]. Thioredoxin, glutaredoxins, and disulfide isomerases also function as molecular chaperones [49,50]. In this work, we present further data supporting that the LSCs evolved to have antioxidant and unfolding activities in two separated domains of the protein.

Conclusions
LSCs are also unfolding enzymes. Molecular chaperone activity is due to an additional CT which is derived from a bacterial Hsp31. In the LSCs structure the CTs are structured as dimers with inverted symmetry in each pole of the tetrameric structure. The CT dimers of CAT-3 have a higher chaperone activity than the monomers. The unfolding enzyme activity of the CT is localized mainly in the C-terminal loop. Deletion of this loop resulted in major loss of TDC3 chaperone activity. Hydrophobic and charged amino acid residues of the C-terminal loop are critical for the chaperone activity. Substitution of hydrophobic for charged amino acid residues and vice versa in this loop considerably decreased the unfolding activity.