Exploring the catalytic mechanism of the first dimeric Bcp: Functional, structural and docking analyses of Bcp4 from Sulfolobus solfataricus

Biochimie 92 (2010) 1435e1444

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Research paper

Exploring the catalytic mechanism of the first dimeric Bcp: Functional, structural and docking analyses of Bcp4 from Sulfolobus solfataricus Danila Limauro a,1, Katia D’Ambrosio b,1, Emma Langella b, Giuseppina De Simone b, Ilaria Galdi a, Carlo Pedone b, Emilia Pedone b, **, Simonetta Bartolucci a, * a b

Dipartimento di Biologia Strutturale e Funzionale, Università degli Studi di Napoli ‘‘Federico II’’, Complesso Universitario Monte S. Angelo, Via Cinthia, 80126 Naples, Italy Istituto di Biostrutture e Bioimmagini-CNR, via Mezzocannone 16, 80134 Naples, Italy

a r t i c l e i n f o

a b s t r a c t

Article history: Received 8 February 2010 Accepted 6 July 2010 Available online 14 July 2010

The detoxification from peroxides in Sulfolobus solfataricus is performed by the Bacterioferritin comigratory proteins (Bcps), Bcp1 (Sso2071), Bcp2 (Sso2121), Bcp3 (Sso2255) and Bcp4 (Sso2613), antioxidant enzymes belonging to one of the subfamilies of the Peroxiredoxins. In this paper we report on the functional, structural and docking analyses of Bcp4, characterized by the CXXXXC motif in the active site. Bcp4 represents the first dimeric Bcp so far investigated. Biochemical studies showed that the protein has a non-covalent dimeric structure and adopts an atypical 2-Cys catalytic mechanism. The X-ray structure of the double mutant C45S/C50S, representative of the fully reduced enzyme state, described the protein dimeric arrangement. Finally, concurrent availability of the crystallographic structure of the monomeric Bcp1 allowed comparative analysis of the interaction with Protein Disulfide Oxidoreductase SsPDO (Sso0192), involved in the reduction of both Bcp1 and Bcp4, through a proteineprotein docking approach. Ó 2010 Elsevier Masson SAS. All rights reserved.

Keywords: Peroxiredoxin Bacterioferritin comigratory proteins Protein disulfide oxidoreductase Sulfolobus solfataricus Disulfide bond

1. Introduction Reactive oxygen species (ROS) are potent oxidants generated both during aerobic metabolism and in response to external factors capable of damaging the main biomolecules in cells. Aerobic organisms have developed various mechanisms to self-protect against the toxicity of ROS [1]. Among the enzymatic defenses, the peroxiredoxins (Prxs) are able to reduce a wide array of substrates, such as hydrogen peroxide, peroxynitrite and organic peroxides [2e5]. Unlike other peroxidases, they do not possess redox cofactors such as metal or prosthetic groups but are characterized by catalytic cysteine residues [6]. Several Prxs have been discovered in species from prokaryotes to eukaryotes, although their different physiological functions are still unclear [7e13]. According to the catalytic mechanism involved, Prxs can be divided into typical 2Cys, atypical 2-Cys, and 1-Cys [3]. All three categories contain in the N-terminal region one conserved cysteine residue called the peroxidatic cysteine (CPSH), which is oxidized to sulphenic acid (CPSOH) during the catalytic reaction. In the 1-Cys Prxs the CPSOH is

* Corresponding author. Tel.: þ39 081 679052; fax: þ39 081 679053. ** Corresponding author. Tel.: þ39 081 2534521; fax: þ39 081 2534574. E-mail addresses: [email protected] (E. Pedone), [email protected] (S. Bartolucci). 1 These authors contributed equally to the paper. 0300-9084/$ e see front matter Ó 2010 Elsevier Masson SAS. All rights reserved. doi:10.1016/j.biochi.2010.07.006

regenerated by an unidentified electron donor. In contrast, in typical and atypical 2-Cys Prxs a second cysteine residue, the socalled resolving cysteine (CRSH) is involved in the resolution of the CPSOH via a disulfide bond formation. In particular, typical 2-Cys Prxs function as homodimeric enzymes, containing two identical active sites. During resolution of the CPSOH, the CRSH from one subunit attacks the CPSOH intermediate of the other subunit, forming an intermolecular disulfide bridge. The atypical 2-Cys Prxs can be both monomeric and dimeric, even though the CRSH is always located on the same polypeptide chain as the CPSH. Thus the resolution of the intermediate CPSOH results in the formation of an intramolecular disulfide bond [14]. The recycling of 2-Cys Prxs involves different disulfide reduction systems, such as Thioredoxin (Trx)/Thioredoxin reductase (Tr) [15,16]. Due to the recent characterization of a huge amount of Prxs from different sources, these enzymes have been further classified into seven subfamilies based on sequence and structure analysis [17]. Among these subfamilies, Bcps have received particular attention. These enzymes, generally monomeric, were originally identified as homologues to Escherichia coli bacterioferritin comigratory protein [18], and are found in all kingdoms of the phylogenetic tree, from the most ancient forms of life, namely cyanobacteria and archaea, to eukaryotes, such as yeast and plants [2,17e21]. Analysis of the primary structure of different Bcps revealed that these proteins can be further divided into two groups: those that have the CXXXXC

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motif in the active site constitute the a-group, while those that have not, belong to the b-group [21]. The a-group proteins adopt always a 2-Cys catalytic mechanism, while those belonging to the b-group can be both 2-Cys and 1-Cys Prxs, depending on the presence of the CRSH. When it is present, CRSH is located 35-residues downstream the CPSH. We recently reported a functional and structural analysis of a Bcp belonging to the a-group, namely Bcp1 (Sso2071) from the archaeon Sulfolobus solfataricus [17]. This enzyme utilizes the Protein Disulfide Oxidoreductase SsPDO (Sso0192)/SsTr (Sso2416) system for recycling, in place of the common Trx/Tr [10]. Functional studies have demonstrated that the enzyme carries out the catalytic reaction using an atypical 2-Cys mechanism. However, when the CRSH is mutated in serine, the enzyme can still work utilizing an alternative mechanism. Moreover, crystallographic studies provided for the first time a detailed description of the structural rearrangement necessary for a member of the Bcp family to perform the catalytic reaction [17]. In this paper we present a detailed study on Bcp4 (Sso2613), another Bcp from S. solfataricus belonging to the a-group. This protein presents two conserved cysteine residues (Cys45 and Cys50) and unlike Bcp1, was recently shown to be dimeric [10]. Two different dimeric interfaces were observed in Prxs so far: type A and type B. Type B is a b-sheet-based interface, while type A is a loopbased-interface [2]. Interestingly, it has been suggested that, due to the juxtaposition of type A interface with the peroxidatic active site, oligomerization using this interface may be associated to the catalytic activity [2]. In order to verify whether the quaternary structure could play a role also in the catalytic mechanism of Bcp4, we report on the characterization of the catalytic activity of this enzyme, and the crystal structure of its C45S/C50S double mutant. Moreover, interaction with SsPDO, the first component of the reducing cascade, was analyzed through a molecular modeling approach.

2. Material and methods 2.1. Strains, media and growth conditions E. coli Top F’10 was used as a general host for DNA manipulation, E. coli XL1-Blue (Invitrogen) was utilized to transform the mutagenesis products and E. coli BL21-CodonPlus(DE3)-RIL (Stratagene) was used for expression of the recombinant proteins [10]. These strains were cultivated in Luria-Bertani (LB) medium at 37  C. When necessary 100 mg/ml of ampicillin, or 50 mg/ml of kanamycin and 33 mg/ml chloramphenicol were added to the medium to maintain plasmids as needed.

2.2. Light scattering Purified proteins (oxidized or reduced with 10 mM DTT) were analyzed by size exclusion chromatography connected to a multiangle light scattering (MALS) equipped with a QELS module (quasi-elastic light scattering) for RH measurements. 100 ml samples (10 mg/ml) were loaded on an S75 10/30 column (GE Healthcare), equilibrated in 20 mM Tris/HCl (pH 8.0) and 0.15 M NaCl. A constant flow rate of 0.5 ml/min was applied. Elution profiles were detected by a Shodex interferometric refractometer and a mini Dawn TREOS light scattering system at 685 nm (Wyatt Technology Corp.). Data were analyzed by using Astra 5.3.4.14 software (Wyatt Technology). Determination of molecular masses and hydrodynamic radii are reported as mean values of triplicate experiments.

2.3. Chemical modification Chemical modification of Bcp4 with Iodoacetamide was performed. Bcp4 (100 mg) was incubated in the absence or presence of 2.5 mM DTT at 30  C for 30 min and then chemical modification was carried out in a 100 ml reaction mixture, containing 50 mM Tris/HCl (pH 8.0) and 20 mM Iodoacetamide for 30 min at room temperature. An aliquot of the mix was analyzed by LC/MS, as described in D’Ambrosio et al. [17]. 2.4. Site-directed mutagenesis and purifications of recombinant proteins To generate the single mutants C45S, C50S and the double mutant C45S/C50S the protocol outlined in the QuickChange II sitedirected mutagenesis kit (Stratagene) was followed, using primers complementary to the coding and non-coding template sequence (pET30Bcp4) containing a double mismatch. To generate mutants C45S, C50S and C45S/C50S the following forward primers were used: 50 -CTGCTGCGTTCACTTCAGTTAGTACTAAAGAGATGTGC-30 50 -GTACTAAAGAGATGAGCACTTTTAGGGACTCCATGGC-30 50 -CGTTCACTTCAGTTAGTACTAAAGAGATGAGCACTTTTAGGGAC-30 The base pair mismatches were underlined. Complementary reverse primers were used. The mixture (50 ml) contained 50 ng of template DNA (pET30Bcp4), 125 ng of each primer, 200 mM dNTP and 2.5 U of Pfu Ultra HF DNA polymerase. Twelve cycles of 95  C for 30 s, 55  C for 1 min and 68  C for 6 min were carried out in a Mini Cycler followed by a cycle at 4  C for 2 min. To digest methylated template, each reaction mixture was treated with 10 U of DnpI at 37  C for 1 h. The mutagenesis products were transformed into XL-1 Blue cells. Single colonies were selected on LB plates containing kanamycin, and isolated plasmid DNA were sequenced at Primm (DNA sequencing service Naples, Italy). The plasmids pETBcp4 (C45S), pETBcp4(C50S), and pETBcp4(C45S/C50S) containing the mutations were used to transform BL21-CodonPlus (DE3)-RIL competent cells. C45S, C50S and C45S/C50S were expressed and purified with the same procedure reported for Bcp4 [10]. E. coli cells containing the expressed recombinant proteins were harvested by centrifugation and pellets from 1000 ml cultures were suspended in 20 mM Tris/HCl (pH 8.0) and disrupted by sonication with 20 min pulses at 20 Hz (Sonicator Ultrasonic liquid processor; Heat System Ultrasonics Inc.). The suspensions were clarified by ultracentrifugation at 160 000 g for 30 min. The soluble fractions (140 mg) of the cell extracts were heated at 80  C for 15 min, and then centrifuged at 15 000 g at 4  C for 30 min, removing almost 70% of the mesophilic host proteins. Purification of the mutant proteins was carried out as already described for the wild-type enzyme [10]. The molecular mass of the recombinant proteins was confirmed by LC/MS, as described in D’Ambrosio et al. [17]. 2.5. Peroxidase assays Peroxidase activities of Bcp4 and its mutants were analyzed for their ability to reduce H2O2 in an in vitro assay using DTT (DTTassay) or NADPH/SsTr/SsPDO as regenerating enzyme systems. DTT-assay: the reaction was started by adding H2O2 at a final concentration of 0.2 mM to the reaction mixture containing 50 mM HEPES (pH 7.0), 10 mM DTT in the presence of different concentrations of the proteins (0e5 mM) in a final volume of 0.1 ml as reported previously [10]. The reaction was incubated at 80  C for 1 min and stopped by adding 0.9 ml of trichloroacetic acid solution (10% w/v). Peroxidase activity was determined from the amount of

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peroxide remaining, which was detected by measuring the absorbance at 490 nm of the purple-colored ferrithiocyanate complex developed after addition of 0.2 ml of 10 mM Fe(NH4)2(SO4)2 and 0.1 ml of 2.5 M KSCN, using H2O2 as a standard [10]. The percentage of peroxides removed was calculated on the basis of the change in A490nm obtained with Bcp4 and its mutants relative to that obtained without these enzymes. Experiments were done in triplicate. Peroxidase activity was also measured indirectly by oxidation of NADPH using a disulfide reductase system formed of SsPDO/SsTr [22]. The complete reaction mixture (500 ml) contained 100 mM potassium phosphate pH 7.0, 2 mM EDTA, 0.2 mM SsTr, 0.05 mM FAD, 0e10 mM SsPDO, 5 mM Bcp4 and 0.25 mM NADPH. After preincubation at 60  C for 2 min, the reaction was started by adding H2O2 to the mixture at the final concentration of 0.2 mM, and the absorbance of NADPH was monitored spectrophotometrically at 340 nm. 2.6. Crystallization and X-ray data-collection Crystallization experiments were carried out using the hangingdrop vapor-diffusion method [23]. The search for initial crystallization conditions was performed using Hampton Research Crystal Screen kits I and II [24,25]. The wells contained 500 ml of precipitant solution and the drops were composed of 1 ml of reservoir solution and 1 ml of protein solution at a concentration of 8 mg/ml. Initial crystals of irregular shape were obtained using a reservoir solution consisting of 0.2 M ammonium acetate, 30% PEG 4000, 0.1 M sodium acetate pH 4.6. Several parameters such as buffer composition, pH and protein concentration were varied in order to improve crystal quality. Optimal conditions for crystallization were achieved at a protein concentration of 5 mg/ml, with reservoir solutions consisting of 0.15 M ammonium acetate, 25% PEG 4000, 0.1 M sodium acetate pH 3.6, at 298 K. The crystals grew to maximum dimensions of 0.25  0.25  0.15 mm3 within one week. X-ray diffraction data at 2.55 Å resolution were collected at a temperature of 100 K, at the Synchrotron source Elettra in Trieste, using a Mar CCD detector. Since the crystallization solution was not suitable to provide cryoprotection, the crystals were rapidly washed in the reservoir solution containing 20% (v/v) glycerol and immediately flash-frozen in a nitrogen-gas stream at 100 K. The data were processed using DENZO and scaled with SCALEPACK [26]. Table 1 summarizes the crystal parameters and data-collection statistics. 2.7. Structure determination and refinement The structure was solved by molecular replacement using the program CNS [27] and Bcp1 from S. solfataricus [17] (PDB code: 3DRN) as search model. The rotation and translation functions were calculated using data between 15.0 and 4.0 Å resolution. The asymmetric unit of Bcp4 contained four molecules. The first cycles of the refinement were carried out with 4-fold NCS-restraints with an energy barrier of 300 kcal/mol Å2. After Rfactor and Rfree reached 0.225 and 0.267, respectively, the NCS-restraint was removed. The ordered water molecules were added automatically and checked individually. Each peak contoured at 3s in the jFo  Fcj maps was identified as a water molecule, provided that hydrogen bonds would be allowed between this site and the model. Many cycles of manual rebuilding, using the program O [28] and positional and temperature refinement using the program CNS [27], were necessary to reduce the crystallographic Rfactor and Rfree values (in the 20.00e2.55 Å resolution range) to 0.204 and 0.263, respectively. Table 1 summarizes the refinement statistics. Coordinates and structure factors have been deposited with the Protein Data Bank (accession code 3HJP).

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Table 1 Crystal parameters, data collection and refinement statistics. Crystal parameters Space group a (Å) b (Å) c (Å) b ( ) Number of independent molecules

P 21 74.2 64.1 76.7 114.8 4

Data collection Resolution range (Å) Outermost resolution shell (Å) Wavelength (Å) Temperature (K) Rsym (%)a Mean I/s(I) Completeness (%)

50.00e2.55 2.64e2.55 1.10000 100 5.5 (33.3) 20.0 (2.2) 96.1 (77.4)

Refinement Resolution range (Å) Rfactor (%) Rfree (%) rmsd from ideal geometry: Bond lengths (Å) Bond angles ( ) Average B factor (Å2)

20.00e2.55 20.4 26.3 0.007 1.3 47.5

a Rsym ¼ SjIi  j/SIi; over all reflections. Values under brackets refer to the outermost resolution shell.

2.8. Homology modelling The SsPDO 3-D homology model was built based on the 3-D structure of Aeropyrum pernix PDO (ApPDO) (PDB code: 2HLS) [29] by means of the SWISS-MODEL server [30]. The structure of ApPDO was identified as the best template for modelling SsPDO by a PSI-BLAST [31] search against the non-redundant database with an E-value of 1e  60 (sequence identity 50%, positives 70%). The model built (SsPDOred) represents the reduced form of SsPDO (only the structural disulfide bond between C173 and C178 is present). The model of the oxidized form of Bcp4, i.e., with C45 and C50 bridged by a disulfide bond (called Bcp4ox), was built on the basis of the reduced X-ray structure reported herein by modifying the region 39e61 encompassing the two active cysteines, since in Bcps this has been shown [17] to be the only region undergoing a significant reorganization in the transition from the reduced to the oxidized form. In particular, the 3-D structure of the region 39e61 was built using the SWISS-MODEL server on the basis of the corresponding region in the only X-ray structure of a Bcp in its oxidized state determined so far (Bcp from A. pernix: PDB code 2CX3). Similarly the 3-D model of the oxidized form of Bcp1 (Bcp1ox) was built by modifying only region 39e61. All the models obtained were energy-minimized by 10 000 steps of conjugate gradient minimization using the DISCOVER module of the INSIGHT package (Accelrys). In the case of Bcp4ox as well as Bcp1ox, Ca atom positions in the region 41e51 were kept fixed during energy minimization. 2.9. Proteineprotein dockings The ZDOCK [32] program was used to predict the rigid-body docking complexes of SsPDOred with Bcp4ox and Bcp1ox, respectively. ZDOCK performs rigid-body dockings using a scoring function which combines pair-wise shape complementarity (PSC), desolvation and electrostatics in its calculation. ZDOCK version 2.3 was used with a 6 Euler angle increment. For each system up to 2000 rigid-body docking complexes were obtained and we selected the top-scoring ones that satisfied the biological requirement of

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having Cys155 of SsPDOred in close proximity to the disulfide bond of the partner (Bcp4ox or Bcp1ox). Since the docking protocol utilized allows only 2-body docking studies to be performed, in the case of Bcp4ox, which could bind two SsPDOred molecules (i.e., the 3-body problem), one for each active site, docking analysis was carried out in two steps. In the first step ZDOCK was used to obtain a 1:1 complex between the Bcp4 dimer and one SsPDO molecule, thus involving only one of the two Bcp4 catalytic sites. In the second step, a second SsPDO molecule was manually docked to the other Bcp4 active site, in agreement with the binding mode observed in the first active site, thus obtaining the 1:2 complex. Obtained complexes were subsequently energy-minimized by 10 000 steps of conjugate gradient minimization using the DISCOVER module of the INSIGHT package. The MOLMOL program was employed for molecular visualization and analysis [33]. The proteineprotein interfaces of the docking complexes were further analyzed by the PROTORP proteineprotein interface analysis server [34].

increment in molecular weight after alkylation, indicating the presence of an intramolecular disulfide bond between Cys45 and Cys50 and suggesting an atypical 2-Cys catalytic mechanism. The functional role of Cys45 and Cys50 in the catalysis was investigated by site-directed mutagenesis. In particular, two single mutants C45S and C50S were obtained as described in Material and methods. The mutant proteins were overexpressed in soluble forms in E. coli. The cell extracts were treated at 80  C for 15 min and, using a combination of affinity chromatography on His trap HP and anion exchange chromatography, the proteins were purified to homogeneity. The overall yield ranges from 14 to 20 mg/ml of E. coli cultures. The peroxidase activities of the single mutants were tested using H2O2 as substrate and DTT or the NADPH/SsTr/SsPDO system as reductants. In the first case, H2O2 removal was detected spectrophotometrically as showed in Material and methods, in the second case the ability to remove H2O2 was determined indirectly following NADPH oxidation according to the scheme here reported:

3. Results

In the DTT-assay the mutant C45S has no residual activity while the mutant C50S is still active. In the NADPH/SsTr/SsPDO-assay inactivity of both C45S and C50S mutants was observed (Fig. 2). All together these results indicate that Cys45 is the essential CPSH while Cys50 is the CRSH. To examine the specificity of the interaction between Bcp4 and SsPDO, the activity of Bcp4 was measured at different concentrations of SsPDO and the KM value (0.75 mM) was determined (Fig. 3). To gain insight into the catalytic behaviour of the C50S mutant, its quaternary structure in both oxidized and fully reduced conditions was analyzed by a combination of size exclusion chromatography, multi-angle light scattering, and quasi-elastic light scattering. The light scattering analysis in non-reducing conditions

3.1. Biochemical studies Recombinant Bcp4 was expressed in E. coli and purified to homogeneity as previously described [10]. The nature of Bcp4 dimeric form was investigated by using a combination of size exclusion chromatography, multi-angle light scattering, and quasielastic light scattering on the oxidized and reduced forms of the enzyme. These experiments demonstrated that Bcp4 has a noncovalently linked dimeric structure. Indeed, it was observed that molecular mass (about 38 kDa) and RH values (4.4) of the oxidized (Fig. 1) and reduced Bcp4 were the same. To gain insight into the Bcp4 catalytic mechanism, chemical modification of the two cysteine residues, Cys45 and Cys50, was performed. Iodoacetamide, a thiol-specific alkylating agent, was used in the presence and absence of DTT. The obtained products were analyzed by LC/MS. The reduced and alkylated enzyme showed an increase of 114 Da in molecular mass, corresponding to the alkylation of both cysteines. By contrast, the oxidized enzyme did not show an

Fig. 1. The hydrodynamic radius versus the elution volume plot for the oxidized Bcp4 obtained from SEC coupled to on-line MALSeQELS detection.

Fig. 2. SsTr/SsPDO dependent peroxidase activity of Bcp4 (C), and mutants C45S (:) and C50S (>). H2O2 reduction in the presence of SsTr/SsPDO coupled system was followed measuring NADPH oxidation at 340 nm. The assay mixture in 100 mM potassium phosphate pH 7.0, 2 mM EDTA, contained 0.2 mM SsTr, 0.05 mM FAD, 5 mM SsPDO, and 0.25 mM NADPH. Negative control without Bcp4 (-).

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Fig. 3. MichaeliseMenten plot of Bcp4 using SsPDO as substrate. The activity was monitored by consumption of NADPH via a decrease in absorbance at 340 nm.

shows the presence of both a dimeric (about 40 kDa) and a tetrameric form (about 100 kDa) (Fig. 4). On the other hand, a shift to a dimeric form is observed in reducing conditions, indicating that the tetrameric form is originated by a covalent bond between two non-covalent dimers. 3.2. Crystallographic studies 3.2.1. Overall structure and active site C45S/C50S mutant was obtained through site-directed mutagenesis of bcp4 as described in Materials and Methods and expressed and purified to homogeneity as reported above. The structure of this enzyme was solved by X-ray diffraction studies. Representing the fully reduced enzyme state, this mutant was used to avoid the potential crystallization problems caused by a mixture of structurally different reduced and oxidized forms. C45S/C50S was crystallized in the space group P21 with four molecules per asymmetric unit (called A, B, C and D). The structure was solved by molecular replacement, using Bcp1 from S. solfataricus (PDB code: 3DRN) [17] as starting model and refined with the CNS program [27] to a crystallographic Rfactor 20.4% and an Rfree of 26.3% in the 20.0e2.55 Å resolution range. The final model consisted of 4988 non-hydrogen atoms, 206 water molecules and 4 chloride ions. All protein residues were well defined in the electron

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density maps. The refined structure presented a good geometry with rmsd from ideal bond lengths and angles of 0.007 Å and 1.3 , respectively. The average temperature factor (B) for all atoms was 47.5 Å2. The stereochemical quality of the model was assessed by Procheck [35]. The most favored and additionally allowed regions of the Ramachandran plot contained 85.4% and 14.6%, of the nonglycine residues, respectively. Refinement statistics are summarized in Table 1. The four molecules in the asymmetric unit revealed only minor differences. Indeed, selecting A as the reference, B, C and D showed a rmsd for the superposition of all Ca atoms of 0.44 Å, 0.38 Å and 0.44 Å, respectively. For this reason only monomer A is arbitrarily chosen for the following discussion and comparisons unless otherwise stated. C45S/C50S appears as a compact globular protein, whose roughly ellipsoidal shape is approximately 28  28  44 Å3 in size. As already observed for other Prxs with known three-dimensional structures [3], C45S/C50S presents a typical thioredoxin fold, which comprises a central four-stranded b-sheet (b3,b4,b8 and b9) and three a-helices (a1,a3 and a4), flanking the sheet (Fig. 5). The structure includes an extra a-helix (a2) and five additional b-strands (b1, b2, b5, b6 and b7). b1, b2, b5 associate with the Trx fold, forming a central seven-stranded mixed b-sheet, while the two remaining strands, b6 and b7, form a b-hairpin which is located on the protein surface (Fig. 5). Finally, a 310 helix (G1), located between strand b2 and b3, is also present. The four a-helices are distributed asymmetrically on either side of the central b-sheet, with the helix a3 located on one side and helices a1, a2 and a4 on the other (Fig. 5). As observed in many other Prxs, helix a1 has a marked kink at the level of Ser55 [3,36e39]. Ser45, which replaces the CPSH is located in the N-terminal part of helix a1 and is well defined in the electron density maps. It is situated within a small cavity delimited by residues Pro38-Val44, Pro139, Thr140. The Ser45OG atom is hydrogen bonded to the Arg119NH1 atom (Ser45OG—Arg119NH1 ¼ 2.7 Å) and to a chloride ion (Ser45OG—Cl ¼ 3.0 Å), derived from the crystallization solution, which in turn interacts with residues Thr42 and Arg119 (Thr42OG1— Cl ¼ 2.7 Å; Arg119NH2—Cl ¼ 3.1 Å). A weak polar interaction with Thr42OG1 atom (Ser45OG—Thr42OG1 ¼ 3.6 Å) is also observed (Fig. 5). Ser50, which replaces the CRSH is also well defined in the maps and is located in the middle of helix a1. Its side chain is hydrogen bonded to the carbonyl oxygen of Thr46 (Ser50OG—Thr46O ¼ 2.7 Å) and to a water molecule (Ser50OG—HOH185 ¼ 2.9 Å) (Fig. 5).

Fig. 4. The hydrodynamic radius versus molecular mass plots of C50S in non-reducing conditions.

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each other, while only one hydrogen bond is established between monomer B and monomer C (Leu18O—Lys85NZ ¼ 2.8 Å).Within each dimer the two monomers are related by a non-crystallographic 2fold symmetry axis and present the two active sites accessible to the substrate. They interact via two hydrogen bonds (Fig. 6) and several hydrophobic interactions, generating a large interface area that extends over 1669 Å2. A key role in the dimerization is played by the secondary structural elements a2 and b7. Moreover, some residues belonging to b1-b2, b3-a1, b5-a3 and b6-b7 loops, as well as to the b1 and a1 secondary structural elements, also contribute to the intermolecular packing stability. According to the classification of the Prx dimeric interfaces (see Introduction), Bcp4 dimers present an A-type interface [2,40,41].

Fig. 5. Overall fold of C45S/C50S. b-strands and a-helices belonging to the Trx fold are shown in blue, while all other secondary structural elements are shown in green. Residues involved in hydrogen bond interactions with catalytic cysteines are shown in ball and stick. Chloride ion and water molecule 185 are also reported.

The distance between the OG atoms of Ser45 and Ser50 is about 10 Å, suggesting that, as already described for Bcp1 [17], the enzyme would need a significant conformational change to allow the transition from the reduced to the oxidized state. 3.2.2. Dimeric arrangement The four independent molecules in the C45S/C50S asymmetric unit are arranged in two dimers consisting of the pairs AeB and CeD. The two dimers have a relatively small interface area (669 Å) and interact through van der Waals contacts, involving strand b7 and loops b6-b7 and b9-a4 of molecule A and helix a1 and loop a2-b5 of molecule C. Monomer B and monomer D do not interact between

3.2.3. Comparison with related proteins Bcp4 and Bcp1 [17], ApBcp (PDB code 2CX4) and the fungal nuclear protein Dot5p (PDB code 2A4V) [20] are the only examples of Bcps, all belonging to the a-group, to be structurally characterized so far. Among these proteins Bcp4 and ApBcp are dimeric, while Bcp1 and Dot5p are monomeric enzymes. Fig. 7 shows the structure-based sequence alignment of Bcp4 with these three proteins. As expected on the basis of high sequence identity (33.5% for Bcp4/Bcp1, 45.6% for Bcp4/ApBcp and 25.9% for Bcp4/Dot5p), the four proteins show a substantial degree of three-dimensional similarity, particularly for the elements taking part in the Trx fold. Most of the remaining secondary structure elements are also conserved in all the structures, with the exception of the two regions corresponding in Bcp4 to residues 24e28 and 109e115. The first region is a 310 helix in Bcp4 and Bcp1, while it is an a-helix in all the other proteins. The second region forms a b-hairpin, composed of strands b6 and b7, in Bcp4 and in ApBcp, while it is deleted in Bcp1 and Dot5p. This region is involved in the dimerization both in Bcp4 and ApBcp, and its deletion in Bcp1 and Dot5p is in agreement with their monomeric state. It is worth noting that several hydrophobic residues involved in the dimerization (Fig. 7) are conserved in ApBcp but not in Bcp1 and Dot5p. Most of the strictly conserved residues are located in the structural element a1, where the active site is located and are involved in catalytic mechanism [3,29,39e41]. 3.3. Proteineprotein docking studies To gain insights into Bcp4eSsPDO recognition, a proteineprotein docking study was carried out by means of the ZDOCK program [32]. Moreover, to shed light upon analogies and/or differences between

Fig. 6. Dimeric interface of C45S/C50S, also known as A-type interface, with one monomer in red, the other in green. Residues involved in polar interactions at the dimer interface are also shown.

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Fig. 7. Structure-based sequence alignment of Bcp4 with Bcp1, ApBcp, Dot5p. The b-strands are coloured in yellow, the a-helices in cyan and the 310 helices in red. The secondary structure refers to all the proteins in the reduced form. Strictly conserved residues are boxed. Hydrophobic residues of Bcp4 involved in dimeric interface are starred.

Bcp4 and Bcp1, the Bcp1eSsPDO interaction was also analysed. To perform proteineprotein docking, 3-D models of Bcp4ox, Bcp1ox and SsPDOred were built as described in the Material and methods section. Docking studies on the Bcp4-SsPDO complex indicate that the contemporaneous binding of two SsPDOred molecules, one for each Bcp4ox active site, is allowed, since no steric clashes are observed (Fig. 8). Due to the identity of the two Bcp4ox active sites, the details of the binding to SsPDOred are discussed only for one active site [Fig. 9(a)]. Data analysis suggests that both monomers of Bcp4ox are involved in the binding with each SsPDOred molecule. The proteineprotein interface is mainly characterized by three contact regions [Fig. 9(a)]: one showing a hydrophobic character (region 1) and two characterized by the presence of charged residues (region 2 and region 3). Details of protein segments at the interface are shown in Fig. 9(b). Inspection of region 1 reveals that the protruding b-hairpin (41e52) of one Bcp4ox monomer, comprising the disulfide bridged

Fig. 8. 1:2 model complex between Bcp4ox dimer and SsPDOred. Bcp4ox is displayed as trace (one monomer in red, the other in green); SsPDOred molecules are shown as cartoon. The two Bcp4ox catalytic sites are indicated by arrows. Catalytic cysteines are displayed in ball-and-sticks.

catalytic cysteines, is involved in van der Waals interactions with the hydrophobic surface patch in proximity of the SsPDOred reactive cysteine. These hydrophobic interactions are strengthened by a pair of hydrogen bonds. In detail, Glu48 and Phe52 of Bcp4ox are H-bonded to Ser154 and Val199 of SsPDOred, respectively (Table 2). Analysis of regions 2 and 3 reveals that protein segments 75e87 and 17e30 of the other Bcp4ox monomer interact with residues 190e198 and 74e90 of SsPDOred, respectively [Fig. 9(aeb)]. These regions are characterized by the presence of charged residues which are involved in hydrogen bonds and salt bridges (Table 2). Findings obtained for the Bcp4oxeSsPDOred complex were compared with those for Bcp1oxeSsPDOred, revealing some interesting information on the molecular determinants of proteine protein recognition. On the whole, the main features of the binding are similar in both complexes, although non-negligible differences, which can be easily justified on the ground of the different quaternary structures, can be detected. More specifically, the hydrophobic interface (region 1), involving the Bcp1ox b-hairpin region and the hydrophobic patch surrounding SsPDOred Cys155 [Fig. 9(ced)], is still conserved in the Bcp1oxeSsPDOred complex. However, an appreciable roto-translation of the two corresponding hydrophobic patches with respect to Bcp4ox-SsPDOred was observed. This rototranslation is likely due to the fact that in the binding to Bcp1ox, SsPDOred is no longer subjected to structural constraints due to the presence of the second monomer as in Bcp4ox. This new orientation allows new van der Waals contacts to be established. Thus the b-hairpin region seems to play a key role in the recognition of the physiological partner in both complexes, as also supported by the highly conserved sequence of this region in all Bcps (Fig. 7) [19]. On the other hand, the charged recognition interface is preserved even if it is reduced in the Bcp1ox-SsPDOred complex due to the lack of the binding region 3 [Fig. 9(ced)]. In particular, the interaction with the SsPDOred charged surface patch (residues 190e198), which in Bcp4ox was mediated by residues belonging to the second monomer (K78, Q83 and K85), is now supplied by K39 and K83, which interestingly are conserved between the monomeric Bcps (Fig. 7) (Table 2) [19]. Moreover, in both complexes the negatively charged N-terminal tail of SsPDOred makes hydrogen bonds as well as salt bridges, which strengthen the binding to the redox partner (Table 2). However, the N-terminal tail is an intrinsically flexible region that could suffer from

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Fig. 9. Bcp4ox-SsPDOred (a) and Bcp1ox-SsPDOred (c) complex models. Bcp4ox and Bcp1ox are displayed as trace; SsPDOred is shown as cartoon. Catalytic cysteines are displayed in ball-and sticks. Protein fragments belonging to the complexes interface regions (regions 1e3) are shown in colours. (b, d) Bcp4ox, Bcp1ox and SsPDOred primary sequences; amino acids involved into proteineprotein recognition are highlighted in colour according to the interface region (regions 1e3) they belong to. SsPDOred N-terminal residues are included in the interface region 2 for shortness.

a rigid-body docking approach which does not take flexibility into account. Finally, it is worth noting that the disulfide bond seems to be more easily accessible to the redox partner in Bcp1 than in Bcp4 due to its monomeric form. Indeed, in the case of Bcp4 the second

Table 2 Interface hydrogen bonds and salt Bcp1eSsPDOred complexes. Bcp4oxeSsPDOred complex Bcp4ox

bridges in the Bcp4eSsPDOred and

Bcp1oxeSsPDOred complex

SsPDOred

Distance Bcp1ox (Å)

SsPDOred

Distance (Å)

Region 1 Lys47N Glu48O Glu48O Phe52NZ Lys141NZ

Glu4Oa Thr152OG1 Ser154N Val199O Glu4OE1a

2.7 2.5 3.3 3.0 2.7

Arg47N Glu48OE2 Phe52N Gly106N Phe107N Asn128O Ser129OG Met131N

Ser154OG Tyr157OH Val199O Glu2OE1a Tyr5OHa Gln3NE2a Glu4OE2a Glu4OE2a

3.0 2.6 3.0 2.9 2.9 3.0 2.5 2.9

Region 2 Lys78NZ Lys78NZ Gln83NE2 Lys85NZ

Glu195OE2 Glu191O Gln198OE1 Lys196O

2.9 3.0 3.0 3.1

Lys39NZ Lys83NZ

Glu195OE1 2.7 Glu195OE2 2.6

Region 3 Lys22NZ Asp26OD1 Asp26OD1 Lys30NZ

Asp38OD2 Arg74NH1 Arg80NH2 Glu77OE1

2.6 2.7 2.8 2.8

a SsPDOred N-terminal residues have been included into interface region 2 for shortness.

monomer creates a cleft close to the active site which requires that SsPDOred approaches to the disulfide bond in a particular orientation. On the other hand, partner access to the active site seems to be not so challenging in the case of Bcp1, where the disulfide bond overlooks an essentially “open-free” surface [Fig. 9(aec)]. To the best of our knowledge, experimental structural studies on complexes between Bcp proteins and their redox partners with which to compare our results are still lacking. However, a recently proposed computer model of the complex between a plant Glutathione peroxidase (GPX) homologue and its in vitro recycling partner Trx h1 [42], shares some features with our interaction model. In particular, two regions of Trx h1 involved into the recognition of GPX were identified, one with a hydrophobic character and comprising the catalytic cysteine, and the other made of charged residues. Interestingly, the structure superposition between SsPDOred and Trx h1 (data not shown) reveals that the above-mentioned Trx interface regions accurately match the corresponding interaction regions (belonging to interface region 1 and region 2) of SsPDO. These similarities further support the proposed model of proteineprotein interaction. 4. Discussion Prxs are present in every branch of living organisms, underlying their key role in detoxification from peroxides. In particular, the Bcp subfamily found in the most ancient microorganisms, namely archaea and cyanobacteria, could have major evolutionary interest. The archaeon S. solfataricus is a good model system for these enzymes

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because it has an array of Bcps, namely Bcp1, Bcp2 (Sso2121), Bcp3 (Sso2255) and Bcp4, to detoxify the cell from peroxides. Bcp2 and Bcp3 can be induced by oxidative stress conditions, while Bcp1 and Bcp4 are expressed constitutively [10]. Bcp1, whose crystal structure was recently solved [17], and Bcp4 show many common features: the same CXXXXC motif of the catalytic site, similar peroxidase activity and the same SsPDO/SsTr reducing system to recycle the enzymes [10]. Moreover, Bcp1 and Bcp4 are capable to reduce both H2O2 and alkylhydroperoxide, although with different efficiencies, and to defend DNA from oxidative damage [10]. The main difference between Bcp1 and Bcp4 lies in the quaternary structure: Bcp1 has a monomeric structure while Bcp4 is dimeric. Since this structural difference could have important implications for the catalytic mechanism, we decided to investigate the function and structure of Bcp4 in detail. Dynamic light scattering analysis performed on Bcp4 in oxidizing and reducing conditions showed a non-covalent dimeric structure of the enzyme. Chemical modification experiments suggested that the enzyme adopts an atypical 2-Cys catalytic mechanism, while site-directed mutagenesis and activity assays helped to define the role played by the catalytic cysteines Cys45 and Cys50. In particular, Cys45 is the CPSH, as shown by the inactivity of C45S in all conditions tested and Cys50 is the CRSH, since C50S completely loses the SsPDO-dependent peroxidase activity. The latter result differs from those previously reported for Bcp1 [17] and E. coli Bcp (EcBcp) [14,18], whose corresponding C50S mutants showed a residual peroxidase activity. These data were explained by invoking an alternative mechanism, where the intermediate Cys45SOH was resolved by reaction with the Cys45 of a second molecule forming a dimer, which was then the substrate for reduction by the physiological partner [14,17]. In the case of the C50S Bcp4 mutant, the intermediate is a tetramer, as demonstrated by light scattering experiments, probably too cumbersome for the attack of the physiological partner SsPDO. Accordingly, the alternative mechanism cannot be adopted. To clarify the molecular basis responsible for the Bcp4 catalytic mechanism, structural studies were also performed. In particular, the X-ray structure of the double mutant C45S/C50S was solved, providing a detailed description of the protein dimeric arrangement and clear identification of residues responsible for dimerization. Interestingly, in the dimer both active sites were accessible to the substrate. Structural comparison with Bcp1 revealed that the two proteins present a high degree of structural similarity, with major differences observed in the region involved in dimerization. Availability of the crystallographic structures of Bcp4 and Bcp1 prompted us to use proteineprotein docking simulations to obtain insights into the Bcp4eSsPDO and Bcp1eSsPDO recognition. Overall, the main features of the binding are similar in both complexes which are stabilized by hydrophobic as well as charge interactions. More specifically, the complexes share an analogous hydrophobic interface region which comprises the b-hairpin containing the catalytic cysteines, whereas the charged interface regions slightly differ. Indeed, in the case of the Bcp4eSsPDO complex the charged interface is wider with respect to Bcp1eSsPDO, due to the presence of the second monomer. The obtained models also indicate that the contemporaneous binding of two SsPDO molecules, one for each Bcp4 active site, is allowed. However, in Bcp4 the disulfide bonds are more crowded with respect to Bcp1, due to its dimeric form. These results are in agreement with kinetic experiments which show that Bcp4 has a higher catalytic efficiency (kcat/KM ¼ 2.4 105 M1 s1), albeit not twice as high as that of Bcp1 (kcat/KM ¼ 1.7 105 M1 s1) [17]. In conclusion, Bcps can exist both in dimeric and monomeric form. The dimeric forms are present only in Archaea, while monomeric forms have also been found in Bacteria [18,21], and Eukarya [19,20]. The kinetic, structural and docking analyses

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