The Crystal Structure of the C45S Mutant of Annelid Arenicola Marina

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The crystal structure of the C45S mutant of annelid Arenicola marina peroxiredoxin 6 supports its assignment to the mechanistically typical 2-Cys subfamily without any formation of toroid-shaped decamers

AUDE SMEETS,1 ELE´ ONORE LOUMAYE,2 ANDRE´ CLIPPE,2 JEAN-FRANCxOIS REES,2 2 1 BERNARD KNOOPS, AND JEAN-PAUL DECLERCQ 1Unit of Structural Chemistry (CSTR), Universite´ catholique de Louvain, B-1348 Louvain-la-Neuve, Belgium 2Laboratory of Cell Biology, Institut des Sciences de la Vie, Universite´ catholique de Louvain, B-1348 Louvain-la-Neuve, Belgium

(RECEIVED December 10, 2007; FINAL REVISION January 24, 2008; ACCEPTED January 28, 2008)

Abstract The peroxiredoxins (PRDXs) define a superfamily of thiol-dependent peroxidases able to reduce hydrogen peroxide, alkyl hydroperoxides, and peroxynitrite. Besides their cytoprotective antioxidant function, PRDXs have been implicated in redox signaling and chaperone activity, the latter depending on the formation of decameric high-molecular-weight structures. PRDXs have been mechanistically divided into three major subfamilies, namely typical 2-Cys, atypical 2-Cys, and 1-Cys PRDXs, based on the number and position of cysteines involved in the catalysis. We report the structure of the C45S mutant of annelid worm Arenicola marina PRDX6 in three different crystal forms determined at 1.6, 2.0, and 2.4 A˚ resolution. Although A. marina PRDX6 was cloned during the search of annelid homo- logs of mammalian 1-Cys PRDX6s, the crystal structures support its assignment to the mechanistically typical 2-Cys PRDX subfamily. The protein is composed of two distinct domains: a C-terminal domain and an N-terminal domain exhibiting a thioredoxin fold. The subunits are associated in dimers compatible with the formation of intersubunit disulfide bonds between the peroxidatic and the resolving cysteine residues in the wild-type enzyme. The packing of two crystal forms is very similar, with pairs of dimers associated as tetramers. The toroid-shaped decamers formed by dimer association and observed in most typical 2-Cys PRDXs is not present. Thus, A. marina PRDX6 presents structural features of typical 2-Cys PRDXs without any formation of toroid-shaped decamers, suggesting that it should function more like a cytoprotective antioxidant enzyme or a modulator of peroxide-dependent cell signaling rather than a molecular chaperone. Keywords: peroxiredoxin; crystal structure; Arenicola marina; toroid-shaped decamer; thioredoxin fold

Reprint requests to: Jean-Paul Declercq, Unit of Structural Chem- Besides classical cellular antioxidant enzymes involved istry (CSTR), Universite´ catholique de Louvain, 1 place Louis Pasteur, in the reduction of hydrogen peroxide and alkyl hydro- B-1348 Louvain-la-Neuve, Belgium; e-mail: [email protected]; peroxides in animal cells, such as glutathione peroxidases fax: 32-10-472707. Article and publication are at http://www.proteinscience.org/cgi/ (GPXs) and catalase (CAT), peroxiredoxins (PRDXs) doi/10.1110/ps.073399308. have emerged more recently as a major superfamily of

700 Protein Science (2008), 17:700–710. Published by Cold Spring Harbor Laboratory Press. Copyright Ó 2008 The Protein Society

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Crystal structure of A. marina peroxiredoxin 6

evolutionarily conserved peroxidases (Hofmann et al. typical 2-Cys subfamily without any formation of toroid- 2002; Wood et al. 2002; Rhee et al. 2005a). shaped decamers. A similar situation was already ob- PRDXs are selenium- and heme-free peroxidases that served for the archaeal PRDX from Aeropyrum pernix depend on cysteines for their catalytic activity (for K1, but with the formation of toroid-shaped decamers review,seeWoodetal.2003).Mechanistically,PRDXs (Mizohata et al. 2005). have been classified into three subfamilies depending on the number and the position of cysteines implicated in Results and Discussion the catalysis. All PRDXs contain a conserved peroxidatic cysteine residue in the N-terminal domain of the protein. Quality of the models During the catalytic cycle, the peroxidatic cysteine is first oxidized by peroxide or peroxynitrite to sulfenic acid. We report here the crystal structure of the C45S mutant of Then, this intermediate is transformed either by forming a Arenicola marina PRDX6 in three different crystal forms. disulfide bond with a resolving C-terminal cysteine of Thefirsttwoformsaremonoclinic with four monomers another subunit (typical 2-Cys subfamily) or the same (A–D) in the asymmetric unit, and the last one is ortho- subunit (atypical 2-Cys subfamily). In the 1-Cys sub- rhombic with eight monomers (A–H) in the asymmetric family, only an N-terminal peroxidatic cysteine is present unit. In all cases, pairs of monomers are associated to in the enzyme, and the sulfenic acid of the peroxidatic form dimers. cysteine is reduced by a heterologous thiol-containing In the monoclinic structures, the electron density is reductant such as glutathione. usually well defined along the protein chain except for Functionally, PRDXs are important cytoprotective anti- the first residue at the N-terminal part and a few residues oxidant enzymes, although their reactivity with peroxides (including the 63His tag) at the C-terminal part of the hasbeenpreviouslyquestionedcomparedwithGPXs polypeptides. The situation is similar for the first four and CAT (Hofmann et al. 2002). However, reassessments chains (A–D) of the orthorhombic structure, but in the of the kinetic values have shown that PRDXs may exhibit four remaining chains (E–H) the electron density is poorly high catalytic efficiencies compatible with their role as defined in some loop (residues 188–201), and in this region cytoprotective antioxidant enzymes (Parsonage et al. 2005; the structural model was built according to the well-defined Oguscu et al. 2007; Peskin et al. 2007; Trujillo et al. 2007). chains. The Ramachandran diagrams computed by the pro- Mammalian typical 2-Cys PRDXs have been also implicated gram Molprobity (Lovell et al. 2003) indicate that about as regulators of redox signaling due to their ability to be 98% of the residues lie in favored regions and that all of reversibly inactivated by peroxides during the catalytic them adopt an allowed conformation. process that accommodates the intracellular messenger All the monomers are very similar, as well as their function of hydrogen peroxide (Rhee et al. 2005b). Finally, association to form dimers. For this reason, one monomer more recently, bacteria, yeast, and human 2-Cys PRDXs and one dimer from the first monoclinic form were have been shown to act alternatively as peroxidases and selected for a detailed description since the resolution molecular chaperones, the functional switch necessitating of 1.6 A˚ achieved for this structure is substantially higher the formation of toroid-shaped homodecamers and high- than the two other ones (2.0 A˚ and 2.4 A˚ ). The avail- molecular-weight complexes (Jang et al. 2004; Chuang et al. ability of three different crystal structures remains impor- 2006; Lee et al. 2007). tant, however, because they will present differences in the In search of a homolog of mammalian 1-Cys PRDX6 packing of the dimers. Indeed, it has been shown that in the marine annelid Arenicola marina,wehavecloned in the case of typical 2-Cys PRDXs, the oligomerization and initiated the biochemical characterization of a PRDX of the dimers may play an important role in the redox with high sequence homology with mammalian PRDX6s mechanism and that enzyme activity could be linked to (E.Loumaye,A.Andersen,A.Clippe,H.Degand,M. the oligomeric state (Alphey et al. 2000; Schro¨der et al. Dubuisson, F. Zal, P. Morsomme, J.F. Rees, and B. 2000; Wood et al. 2002; Parsonage et al. 2005). Knoops, in prep.). Interestingly, although A. marina PRDX6 presents 63% identity and 85% similarity with Structure of the monomer human 1-Cys PRDX6, the protein possesses five cysteines, among which two cysteines function as peroxi- In the first monoclinic crystal form, the average RMS de- datic and resolving cysteines of typical 2-Cys PRDXs viation between the Ca atoms of the four chains is 0.184 (E.Loumaye,A.Andersen,A.Clippe,H.Degand,M. A˚ , and chain B can be considered as the ‘‘central subunit’’ Dubuisson, F. Zal, P. Morsomme, J.F. Rees, and B. with an average RMS deviation of 0.165 A˚ between this Knoops, in prep.). The crystal structures of the C45S chain and the three other ones. Figure 1 shows the fold mutant of annelid worm Arenicola marina PRDX6 pre- of this monomer and a topological diagram. The structure sented here support its assignment to the mechanistically is composed of two domains. The N-terminal domain 1

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b-sheet (b10, b11, b13, b12) and one a-helix (a6) located between strands b11 and b12. There are only a very few intrasubunit contacts between the two domains. They involve residues located in helix a5indomain1and in the loop between b10 and b11 in domain 2. Hydrogen bonds are formed between Ser162 Og andThr173Og1 and between Arg158 Nh1 and the carbonyl oxygen of Trp177. A. marina PRDX6 presents 63% identity with human PRDX6 (hORF6), whose crystal structure has been determined and corresponds to PDB code 1prx (Choi et al. 1998). The folds of these two monomers of PRDX6 are very similar: 195 residues can be aligned with an RMS deviation of 0.97 A˚ between the Ca atoms. Large differences occur mainly in domain 1, in two regions that do not belong to the thioredoxin fold: the loop between a3 and b5, and the short two-stranded b-sheet (b6, b7), which is a unique feature of A. marina PRDX6 and is replaced by a large loop without secondary structure in human PRDX6. Human PRDX6 belongs to the 1-Cys subfamily of PRDXs. According to the sequence homology, it was initially supposed that A. marina PRDX6 would also belong to this subfamily. However, it has been shown that from a mechanistic point of view this enzyme must be classified into typical 2-Cys PRDXs (E.Loumaye,A.Andersen,A.Clippe,H.Degand,M. Dubuisson, F. Zal, P. Morsomme, J.F. Rees, and B. Knoops, in prep.). The crystal structure of another PRDX showing the same particularity is already known: The primary sequence of the archaeal PRDX from Aeropyrum pernix K1 is similar to those of the 1-Cys PRDXs, while its catalytic properties resemble those of the typical 2-Cys PRDXs (Mizohata et al. 2005). The wild-type A. marina PRDX6 contains five Cys residues in positions 45, 71, Figure 1. (A) Topological diagram of a monomer of A. marina PRDX6. 86, 127, and 183. It was proposed that Cys45 could be the Secondary structure elements belonging to the thioredoxin fold in domain 1: (green) a-helices, (red) b-strands. The remaining elements of domain peroxidatic cysteine and Cys183 the resolving cysteine 1 are yellow and domain 2 is colored blue. (B) Ribbon diagram of a (E.Loumaye,A.Andersen,A.Clippe,H.Degand,M. monomer colored as in A. The side chains of the Cys residues and of Dubuisson, F. Zal, P. Morsomme, J.F. Rees, and B. Ser145, as well as the benzoate ion, are represented as balls and sticks. Knoops, in prep.). It seemed interesting to examine how these Cys residues are positioned in comparison with the structures of PRDXs belonging to different subfamilies. (residues 1–169) is characterized by the presence of a A multiple structural alignment was thus worked out thioredoxin fold. It is composed of a central four-stranded using the program Mustang (Konagurthu et al. 2006) and b-sheet (b4, b3, b8, b9)flankedbythreea-helices (a2, applied to the present molecule and to PRDXs belonging a4, a5). Domain 1 is completed at its N terminus by a to different subfamilies. The results are given in Table 1. short b-sheet (b1, b2) and by the short helix a1. Between It can be seen that all peroxidatic cysteine residues are b4anda4 of the thioredoxin fold there is an insertion of well aligned in the structures.Furthermore,threeofthe helix a3 and of strand b5thatrunsparalleltotheb4 four additional cysteine residues of A. marina PRDX6 strand of the thioredoxin fold and thus constitutes a fifth are aligned with cysteine residues of Plasmodium yoelii strand associated with the thioredoxin fold motif. There is PRDX. However, except for the latter, none of the addi- an additional insertion of a short two-stranded b-sheet tional cysteine residues of A. marina PRDX6, including (b6, b7) between helix a4andstrandb8. The C-terminal the resolving residue Cys183, structurally corresponds to domain 2 (residues 170–220) is connected at the a cysteine residue in the other PRDXs structures. Based C-terminal end of the a5 helix belonging to the thiore- on SDS-PAGE analysis and on mass spectrometry results, doxin fold. It is composed of a four-stranded antiparallel it was proposed that in A. marina PRDX6, an intrasubunit

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Table 1. Structural alignment of various PRDX sequences which correspond to the wild-type enzymes

a A. marina PRDX6 (2v2g, this work); b 1-Cys PRDX, human PRDX6 hORF6 (1prx, Choi et al. 1998); c typical 2-Cys PRDX, human PRDX2 (1qmv, Schro¨der et al. 2000); d archaeal PRDX from Aeropyrum pernix K1 (2cv4, Mizohata et al. 2005); e Plasmodium yoelii PRDX (1xcc, Vedadi et al. 2007). The Cys residues are bold-faced and highlighted gray. Residues taking part to the active site, as described in the Discussion section, are bold-faced and underlined. a-Helices denoted by double underline; b-strands denoted by dotted underline.

disulfide bond could be formed between Cys71 and One can thus expect a considerable rearrangement at the Cys127, but only when Cys45 is present in the enzyme level of the thioredoxin fold if this intrasubunit disulfide (E.Loumaye,A.Andersen,A.Clippe,H.Degand,M. bond is formed. Dubuisson, F. Zal, P. Morsomme, J.F. Rees, and B. Knoops, in prep.). In the present structure of the C45S The active site mutant, we confirm the absence of this disulfide bond in all the monomers. Crystals were grown in the presence of In the present study, the conserved peroxidatic residue DTT, thus favoring the absence of a disulfide bond, but Cys45 is mutated into a Ser. As is the case in all the the crystal of the monoclinic form 2 was soaked in H2O2 reduced forms of PRDX structures, this residue is located before data collection. Even in this case, a disulfide bond in the N-terminal part of the kinked a2 helix, in a posi- between Cys71 and Cys127 is missing. The formation of tively charged pocket. The Og oxygen atom of Ser45 is this hypothetical disulfide bond would require important in direct contact with the side chains of Arg128, Thr42, structural rearrangements. As can be seen in Figure 1, the His37, and the carboxylate group of a benzoate ion that two residues are not very close to each other, with a covers the entrance of the pocket. The side chain of distance of 10.2 A˚ between the two sulfur atoms, and the Arg151 is not in direct contact with Ser45 Og but also lies side chain of Cys127 is not oriented in the direction of in the vicinity. In the wild-type enzyme, the surroundings Cys71. The two residues are located at the extremity of of Cys45 would thus be very similar to what is observed b-strands b4andb8, respectively. These two strands in other PRDXs, with the exception of the benzoate ion, belong to the thioredoxin fold. They are not direct neigh- which was observed only in the crystal structure of human bors in the b-sheet since they are separated by strand b3. PRDX5 (Declercq and Evrard 2001; Declercq et al. 2001;

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Evrard et al. 2004a) and in the C47S mutant of the same gen atoms of the benzoate are hydrogen bound with enzyme (Evrard et al. 2004b). As can be seen on the residues belonging to the active site: O1 to Val44 N structural alignment presented in Table 1, Arg128 and (2.91 A˚ ), O2 to Thr42 Og1 (2.88 A˚ ), Ser45 N (2.94 A˚ ), Thr42areconservedinallthePRDXspresentedhere, Ser45 Og (3.09 A˚ ), and Arg128 Nh2 (2.86 A˚ ). These while the equivalent of His37 is missing only in human bonds are exactly equivalent to those described in human PRDX2. Arg151 is also well conserved. Figure 2 shows a PRDX5 (Evrard et al. 2004b). A very similar situation view of the accessible surface of a monomer, colored was also described in the crystal structure of chloroper- according to the electrostatic potential. The presence of oxidase from Streptomyces aureofaciens Tu¨24, in which arginine and histidine residues is thus responsible for an organic acid is required for the haloperoxidase activity the positively charged pocket and for lowering the pKa of (Hofmann et al. 1998). In spite of the crystallization pH the peroxydatic cysteine Cys45 in the wild type. The (5.5), it seems that the O2 atom of the benzoate is presence of a benzoate ion was completely unexpected protonated because Thr42 Og1 takes part in three hydro- since benzoate was never used during the production, the gen bonds: It is the donor in a bond with the carbonyl purification, or the crystallization. In the case of human oxygen atom of Arg39 (2.70 A˚ ) and thus an acceptor in PRDX5, the presence of the benzoate in the protein the bonds with Ser45 Og (3.14 A˚ ) and the benzoate O2 sample before crystallization was confirmed by mass (2.88 A˚ ). It is thus likely that the neighborhood of the spectrometry. The presence of the benzoate in the C45S benzoate has increased its pKa value (4.2). In the crystal mutant of A. marina PRDX6 thus suggests that it should structure of human PRDX5, it was noticed that the probably also be present in the wild-type enzyme. As aromatic ring of the benzoate ion was in contact with shown in Figure 2B, the benzoate is positioned at the hydrophobic residues belonging to helix a5, which is a entrance of the pocket containing the active site and at characteristic of PRDX5, since it is not observed in other least partially obstructs its access. The carboxylate oxy- PRDX structures. Interestingly, in A. marina PRDX6, this

Figure 2. (A) Accessible surface of a monomer colored according to the electrostatic potential: blue for positive, and red for negative. (B) Similar to A with the benzoate ion covering the active site pocket. (C) Details of the active site. Important residues discussed in the text are represented as balls and sticks. (D) Electron density in the region of the active site. The sigma map is contoured at a level of 1.0 s.

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region is occupied by a short two-stranded b-sheet (b6-b7) that is not frequently observed in PRDXs but is present in the typical 2-Cys PRDX AhpC (Wood et al. 2002). The shortest contact of the aromatic ring is with Glu117 Cd (3.30 A˚ ), which belongs to the b6 strand. The exact role of this benzoate has not been elucidated up to now, but it was speculated (Karplus and Hall 2007) that it could bind as a substrate analog, with the two carboxylate oxygens mim- icking the two oxygen positions of a peroxide substrate.

Structure of the dimers In the crystal structure of C91S-hORF6 (Choi et al. 1998), the association of monomers to form homodimers was reported. In this structure, each monomer is composed of two domains, a large N-terminal domain (domain 1) that contains a thioredoxin fold, and a smaller C-terminal domain (domain 2). In the dimer association, domain 2 of Figure 3. (A) Ribbon diagram of a dimer of the C45S mutant of A. marina one monomer folds over domain 1 of the other to form an PRDX6. One subunit is red and the other green. The central 10-stranded b-sheet is visible. Ser45 and Cys183 are represented as balls and sticks. arm-exchanged dimer (Choi et al. 1998). The dimer forma- (B) Simulation of the oxidized form of the wild-type enzyme showing the tion implies a hydrogen-bonding network between two unfolding of the N-terminal part of helix a2 to bring Cys45 in the vicinity b-strands, one from each monomer, to form a central of Cys183 of the other subunit and allow the formation of the disulfide 10-stranded b-sheet. This big b-sheet is stabilized by bridges. In both cases, the upper part shows a close-up of the region intersubunit interactions between hydrophobic residues of containing residue 45 and Cys183. the two involved b-strands (Choi et al. 1998). Similar dimers are present in every crystal form of A. marina sification proposed by Copley et al. (2004), the presence of PRDX6. The structure of the crystal of monoclinic form 1 this dimer would place A. marina PRDX6 and human is used to describe the dimer (subunits A and B have been PRDX6 in class 4, to which also belong typical 2-Cys chosen) (Fig. 3A). As in the crystal structure of C91S- PRDXs represented in Table 1 by human PRDX2. Indeed, hORF6 (Choi et al. 1998), both monomers of a dimer are the definition of class 4 includes the active site motif related by a non-crystallographic twofold symmetry axis. TxxCxxE, the formation of the 10-stranded b-sheet, and the The estimation of the accessible surface by the solvent in presence of a C-terminal extension (Copley et al. 2004). the dimer was calculated by AREAIMOL (Lee and Richards 1971). The dimer formation decreases the acces- Oligomerization sible surface by an average value of 2323 A˚ 2 per monomer. That represents up to 20.5% of the total surface of the It has recently been shown that, from a mechanistic point monomer. This value is somewhat bigger than the one of view, A. marina PRDX6 must be classified into typical reported in the crystal structure of C91S-hORF6 (Choi et al. 2-CysPRDXs(E.Loumaye,A.Andersen,A.Clippe,H. 1998). Phe 41, Thr 42, Pro 43, Val 44, Thr 46, Thr 47, Glu Degand, M. Dubuisson, F. Zal, P. Morsomme, J.F. Rees, 48, Asp 83, Cys 86, and Leu 87 of domain 1 of one subunit and B. Knoops, in prep.). In this subfamily of PRDXs, a interact with Thr 148, Asn 152, Glu 155, and Thr 166 of redox-sensitive oligomeric state exists (Wood et al. 2002, domain 1 and with Ala 175, Met 184, Pro 187, Lys 204, Pro 2003). Toroid-shaped complexes consisting of a pentameric 207, and Tyr 215 of domain 2 of the other subunit. These arrangement of dimers ([a2]5 decamer) are observed when interactions are very similar to those found in the C91S- the intersubunit disulfide bond between the peroxydatic hORF6 dimer (Choi et al. 1998). At the time of the Cys residue of one monomer and the resolving Cys residue formation of the arm-exchanged dimer, the two b-strands of the other monomer of the dimer does not exist. This (b9) of each monomer are associated by the hydrogen- arrangement is thus present when the peroxidatic Cys bonding network. These interactions lead to the formation residue is reduced (–SH) or overoxidized to sulfinic of the central 10-stranded b-sheet (b3, b4, b5, b8, b9of (SO2H) or sulfonic acid (–SO3H). When the disulfide each monomer) (Fig. 3A). The dimer is also stabilized by bridge is present, the N-terminal part of helix a2 containing intersubunit interactions between hydrophobic residues the peroxidatic Cys residue is unwound and the comple- belonging to the two b9 strands (Leu 141, Ile 143, Leu mentary contact between regions I and II of two adjacent 144, and Tyr 145) as reported in the crystal structure of a2 dimers is lost. The consequence is a tendency toward C91S-hORF6 (Choi et al. 1998). According to the new clas- dissociation of the pentamer of dimers (Wood et al. 2002).

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Region I is part of the conserved loop helix active site exist with the adjacent monomer. The complementarity motif, and region II approximately corresponds to helix a3. between the surfaces of regions I and II of two adjacent Complementary contacts also exist between region III (the monomers is also lost. For example, in our superposition, loop between b5 and a4) and region IV (part of the loop the side chain of Phe41 belonging to region I is positioned following a4, which in A. marina PRDX6 is characterized at a short distance (2.51 A˚ ) from the side chain of Glu79 by the presence of short two-stranded b-sheets composed belonging to region II in the adjacent monomer. The of b6 and b7). In the present study of the C45S mutant of association observed in typical 2-Cys PRDXs is thus not A. marina PRDX6, the mutation of the peroxidatic Cys45 possible in A. marina PRDX6. into Ser45 prevents the possibility of oxidation, and the N- InthecaseofA. marina PRDX6, the packing of the terminal part of helix a2 remains folded. One would thus dimers is completely different in the monoclinic forms expect that the contacts between regions I and II and compared with the orthorhombic form. In both mono- between regions III and IV can occur and generate the clinic forms, two dimers are associated to form a formation of a toroid-shaped decamer. This expectation is tetramer. The accessible surface buried in this association further reinforced by the fact that these contacts are also is 460 A˚ 2 per monomer (4.1% of the total surface) for observed in dimers of human PRDX5 belonging to the chains A and C and 175 A˚ 2 (1.5%) for chains B and D. atypical 2-Cys subgroup. In that case, they do not generate The domain 2 of the monomers is very much implicated decamers because the dimers associating the two central in the formation of the tetramers. There is a strong salt b-sheets do not exist in PRDX5 (Declercq et al. 2001). bridge between Arg182 Nh2 of chain A and Asp181 Od1 In spite of this, we must notice that the toroid-shaped of chain D (2.89 A˚ ) and between Arg182 Nh2 of chain C decamers do not exist in any of the crystal forms of A. and Asp181 Od1 of chain B (2.95 A˚ ), and a weaker marina PRDX6 described here. On the other hand, the interaction between Arg182 Ne of chain D and Glu202 archaeal PRDX from Aeropyrum pernix K1 is also a PRDX Oe1 of chain A (3.32 A˚ ) and between Arg182 Ne of chain whose primary sequence is similar to those of 1-Cys B and Glu202 Oe1 of chain C (3.45 A˚ ). The distances PRDXs. Archaeal PRDX catalytic properties resemble were collected in monoclinic form I. those of the typical 2-Cys PRDXs, and the toroid-shaped There are only a few contacts between the tetramers. In decamer is observed in the crystal structure (Mizohata et al. both monoclinic forms, tetramers are aligned along a line 2005). The absence of the toroid-shaped decamer in parallel to the a-axis of the cell. The only difference A. marina PRDX6 seems to be due to the overcrowding between the two forms is the relative disposition of two of the zone corresponding to the region IV defined by adjacent lines of tetramers. It appears that the line close to Wood et al. (2002). This region corresponds to the loop the origin is exactly the same in both forms, but the between the two b-strands b6 and b7. In Figure 4, we have adjacent line has slid along the a-axis. simulated the association of two monomers belonging to In the orthorhombic form, the tetramers do not appear, two different dimers. We have superposed them on the and the contacts necessary to generate the toroid-shaped equivalent fragments of the reduced human PRDX2 (1qmv) decamer are also nonexistent. No particular arrangement in which the decamer is formed. It can be seen that this is observed, and each dimer is surrounded by a variable association is not allowed because region IV of A. marina number of other chains (between five and eight). PRDX6 is very bulky, and several close contacts would Simulation of the oxidized form From a mechanistic point of view, A. marina PRDX6 has been described as a typical 2-Cys PRDX, in which, upon oxidation, an intersubunit disulfide bond is formed between the peroxidatic and the resolving cysteine residues(E.Loumaye,A.Andersen,A.Clippe,H.Degand, M. Dubuisson, F. Zal, P. Morsomme, J.F. Rees, and B. Knoops, in prep.). In the wild-type A. marina PRDX6, these residues are Cys45 and Cys183, respectively. Up to Figure 4. Ca traces of two monomers belonging to two different dimers in now, all the trials to grow crystals of wild-type A. marina reduced human PRDX2 are represented by green and cyan, respectively. PRDX6 were unsuccessful. It is thus not possible to This association gives rise to the formation of the toroid-shaped decamer. obtain an experimental structure showing this disulfide Two monomers of A. marina PRDX6 superposed on the two monomers of bond. In the present structure of the C45S mutant, the PRDX2 are shown in red and blue, respectively. The region containing the distance between Ser45 Og of one chain of a dimer and short two-stranded b-sheet (b6, b7) in A. marina PRDX6 is much bulkier g than the equivalent region in PRDX2, and this kind of association is not Cys183 S of the other chain is ;14.9 A˚ .Ofcourse,this allowed. distance is much too large to allow the formation of a

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Crystal structure of A. marina peroxiredoxin 6

disulfide bridge. It is, however, well known that upon two domains and the N-terminal domain is characterized oxidation, the N-terminal part of helix a2 that contains by a thioredoxin fold. Two monomers are associated to the peroxidatic Cys residue is unwound to allow the form dimers. In spite of these resemblances, it has been formation of the bridge. Since the resolving cysteine shown that from a mechanistic point of view, A. marina Cys183 of A. marina PRDX6 is not structurally aligned PRDX6 behaves as a typical 2-Cys PRDX (E. Loumaye, with the resolving cysteine of typical 2-Cys PRDXs (see A.Andersen,A.Clippe,H.Degand,M.Dubuisson,F. Table 1), it thus seemed interesting to see if unfolding the Zal, P. Morsomme, J.F. Rees, and B. Knoops, in prep.). loop helix active site motif in a way similar to what is On this basis, and taking into account that in the C45S observed in typical 2-Cys PRDXs would bring the perox- mutant studied here the disulfide bridge between the idatic Cys residue close enoughtotheresolvingoneand peroxidatic and the resolving cysteine residues cannot allow the formation of the bridge. For this simulation, we be formed, one would expect the formation of toroid- have used as a model the crystal structure of rat HBP23/ shaped decamers by the association of five dimers. Such PRDX1 (pdb 1qq2), a typical 2-Cys PRDX in which toroids do not exist in any of the three crystal forms dimers contain two disulfide bonds between the perox- presented here, but in two forms, pairs of dimers are idatic Cys52 residue of one subunit and the resolving associated as tetramers. This suggests that A. marina residue Cys173 of the other subunit (Hirotsu et al. 1999). PRDX6 should function more like a peroxidase and thus Using O (Jones et al. 1991), we have superposed the two as a cytoprotective antioxidant enzyme or a modulator of residues of HBP23/PRDX1 preceding the unfolding and peroxide-dependent cell signaling rather than a molecular the two residues following the unfolding on equivalent chaperone. Indeed, toroid-shaped oligomer formation residues of A. marina PRDX6. More precisely, residues appeared to be necessary, although not sufficient, for 44, 45 and 57, 58 of one subunit of HBP23/PRDX1 are su- 2-Cys PRDX molecular chaperone activities (Jang et al. perposed on residues 37, 38 and 50, 51, respectively, of one 2004; Chuang et al. 2006; Lee et al. 2007). Also, a subunit of A. marina PRDX6. After this superposition, resi- simulation of the structure of the oxidized form shows dues 46–56 of HBP23 are renumbered 39–49, are mutated that the formation of an intersubunit disulfide bond is to the corresponding sequence of A. marina PRDX6, and quite possible and that Cys183 is very well positioned to these residues are used to replace the original equivalent act as a resolving cysteine. residues in PRDX6. The same operation is performed with the two subunits of one dimer. After this operation and Materials and Methods without any modification of the region containing the resolving Cys183, the peroxidatic Cys45 residue comes Crystallization in the vicinity of the resolving Cys183 residue of the other subunit. This would allow the formation of the The cloning, expression, and purification of C-terminal hexa- disulfide bridge with a minimum of reorganization. Indeed, histidine (63His)-tagged recombinant A. marina PRDX6 and its the structure of this dimer was easily regularized using mutant C45S have been described (E. Loumaye, A. Andersen, A. Clippe, H. Degand, M. Dubuisson, F. Zal, P. Morsomme, REFMAC5 (Murshudov et al. 1997) after specification of J.F. Rees, and B. Knoops, in prep.). The C45S mutation was the two disulfide bridges using the SSBOND command in necessary for the crystallization. Three crystal forms were the PDB file. The result is presented in Figure 3B. In this obtained: two monoclinic forms and an orthorhombic one. The simulation, the disulfide bridge runs between strands b10 orthorhombic crystal was grown in 0.1 M Bis-tris pH 5.5, 25% and b11 of the terminal b-sheet. The two strands are thus (w/v) PEG3350, 0.001 M DTT, and 0.02% (w/v) sodium azide. Ammonium sulfate (0.1 M) was added to those conditions to drawn aside so that b10 no longer belongs to the sheet. This obtain the two monoclinic forms. The crystallization method simulation thus confirms that it is quite possible that, upon was hanging-drop vapor diffusion at 18°C, using a protein oxidation, A. marina PRDX6 uses the mechanism of a concentrationof10mgmL1. The volume of the crystallization typical 2-Cys PRDX, as previously suggested (E. Loumaye, solution was 500 mL, and the crystallization drop was formed by A. Andersen, A. Clippe, H. Degand, M. Dubuisson, F. Zal, mixing equal amounts (1 mL) of the protein solution and the crystallization solution. Crystals appeared after 2 d for all forms. P. Morsomme, J.F. Rees, and B. Knoops, in prep.). Crystals of the two monoclinic forms seemed very similar and grew to a size of 0.3 3 0.3 3 0.05 mm3. The size of the orthorhombic one was 0.2 3 0.5 3 0.1 mm3. The crystal of the Conclusions second monoclinic form was soaked in 1 mM H2O2. The second We report here the crystal structure of A. marina PRDX6. monoclinic form is not a consequence of the soaking because other crystals, not soaked, have the same unit cell parameters. The sequence of this enzyme is very similar to that of mammalian 1-Cys PRDXs, but there are several additional cysteine residues. We show here that the three- Data collection dimensional structure also resemblesverymuchthatof Before data collection, the crystals were soaked for a few mammalian 1-Cys PRDX6. A monomer is composed of seconds in a cryosolution similar to the mother liquor but

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containing 20% glycerol as a cryoprotectant, and were flash- [Choi et al. 2003], 1tp9 [Echalier et al. 2005], 1we0 [Kitano cooled at 100K. Some statistics of data collection and process- et al. 2005], 1x0r [Nakamura et al. 2006], 1xcc [Vedadi et al. ing of the three crystals are presented in Table 2. 2007], 1zye [Cao et al. 2005], and 2cv4 [Mizohata et al. 2005]). Electron density of an average map was interpreted using O (Jones et al. 1991) and COOT (Emsley and Cowtan 2004). After Data processing, structure solution, and refinement applying the NCS-phased refinement procedure available in the CCP4 suite (Collaborative Computational Project, Number 4 The diffraction images of the different crystals were processed 1994), the refinement was pursued with REFMAC5 (Murshudov and merged with the XDS program package (Kabsch 1993). et al. 1997). The second monoclinic and the orthorhombic There are eight subunits in the asymmetric unit of the ortho- structures were solved using the monoclinic form 1 structure rhombic form and four in the one of the two monoclinic forms. as a model. For the second monoclinic form, a rigid body at 4 A˚ The crystal of monoclinic form 1 was used to solve the structure. resolution was refined using REFMAC5 (Murshudov et al. In this form, a self-rotation function allowed us to identify the 1997). After applying the NCS-phased refinement procedure presence of five coplanar non-crystallographic twofold axes available in the CCP4 suite, only some manual adjustments with and another one perpendicular to this plane. The structure was COOT (Emsley and Cowtan 2004) were necessary and were solved by molecular replacement using the software PHASER followed by refinement using REFMAC5 (Murshudov et al. (Storoni et al. 2004) and the superposition of pdb files of 1997). The orthorhombic form was solved by molecular replace- different peroxiredoxins as models (pdb entries: 1hd2 [Declercq ment using PHASER (Storoni et al. 2004). After applying the et al. 2001], 1nm3 [Kim et al. 2003], 1prx [Choi et al. 1998], NCS-phased refinement procedure available in the CCP4 suite, 1qmv [Schro¨der et al. 2000], 1qq2 [Hirotsu et al. 1999], 1qxh the refinement was pursued with REFMAC5 (Murshudov et al.

Table 2. Statistics of data collection and refinement

Monoclinic form 1 Monoclinic form 2 Orthorhombic form

Data collection

Space group C2 C2 P212121 Cell dimension a(A˚ ) 127.246 128.261 77.110 b(A˚ ) 83.226 83.034 111.156 c(A˚ ) 98.349 107.444 229.800 a (°) 90.00 90.00 90.00 b (°) 100.71 116.72 90.00 g (°) 90.00 90.00 90.00 Number of subunits in cell 16 16 32 Synchrotron source ESRF-Grenoble DESY-EMBL,Hamburg ESRF-Grenoble Beamline BM30A BW7B BM30A Detector type MARCCD165 MAR345 MARCCD165 Wavelengh (A˚ ) 0.9798 0.8423 0.9798 Resolution (A˚ ) Overall (ov) 14–1.6 20–2.0 15–2.4 Highest shell (hs) 1.7–1.6 2.1–2.0 2.5–2.4 Measured reflections 481,364 203,635 282,787 Number of unique reflections 131,708 65,352 77,362 Completeness (%) ov/hs 99.2/99.2 95.8/85.8 99.0/96.9

RSym 0.064 0.112 0.061 I/s(I) ov/hs 13.1/2.2 8.9/3.0 15.2/3.3 Refinement a b Rcryst (Rfree ) Overall 0.160 (0.197) 0.192 (0.260) 0.177 (0.239) Highest shell 0.258 (0.324) 0.389 (0.530) 0.232 (0.343) Estimated overall coordinate error (A˚ ) based on maximum likelihood (Murshudov et al. 1997) 0.058 0.142 0.199 RMS deviations from ideality Bonds (A˚ ) 0.019 0.020 0.019 Angles (°) 1.773 1.864 1.716 Number of solvent molecules 1051 804 294 Mean B value (A2) Main chain 20.3 14.8 45.6 Side chain 22.7 16.1 45.9 Solvent 38.1 27.7 39.8

a Rcryst ¼ (S kFojjFck)/S jFoj, with jFoj as the observed structure factor amplitude and jFcj as the calculated structure factor amplitude. b Rfree is calculated in the same way as Rcryst, with 5% of the data that are not included in the refinement.

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Crystal structure of A. marina peroxiredoxin 6

1997). In all cases, during the final steps the hydrogen atoms type of mammalian peroxiredoxin at 1.5 A˚ resolution. J. Mol. Biol. 311: were incorporated in riding positions. In monoclinic form 1 and 751–759. in the orthorhombic structures, the mean-square displacements Echalier, A., Trivelli, X., Corbier, C., Rouhier, N., Walker, O., Tsan, P., Jacquot, J.P., Aubry, A., Krimm, I., and Lancelin, J.M. 2005. Crystal of rigid bodies were refined, two domains of each polypeptide structure and solution NMR dynamics of a D (type II) peroxiredoxin chain being defined as a different TLS group. Some refinement glutaredoxin and thioredoxin dependent: A new insight into the perox- statistics are given in Table 2. iredoxin oligomerism. Biochemistry 44: 1755–1767. Emsley, P. and Cowtan, K. 2004. Coot: Model-building tools for molecular graphics. Acta Crystallogr. D Biol. Crystallogr. 60: 2126–2132. Coordinates and structure factors Evrard, C., Capron, A., Marchand, C., Clippe, A., Wattiez, R., Soumillion, P., Knoops, B., and Declercq, J.P. 2004a. Crystal structure of a dimeric Atomic coordinates and structure factors have been deposited oxidized form of human peroxiredoxin 5. J. Mol. Biol. 337: 1079–1090. in the Protein Data Bank. Accession codes: 2v2g (monoclinic Evrard, C., Smeets, A., Knoops, B., and Declercq, J.P. 2004b. Crystal structure of the C47S mutant of human peroxiredoxin 5. J. Chem. Crystallogr. 34: form 1 of A. marina PRDX6), 2v32 (monoclinic form 2 of 553–558. A. marina PRDX6), and 2v41 (orthorhombic form). Hirotsu, S., Abe, Y., Okada, K., Nagahara, N., Hori, H., Nishino, T., and Hakoshima, T. 1999. Crystal structure of a multifunctional 2-Cys peroxiredoxin heme-binding protein 23 kDa/proliferation-associated gene prod- uct. Proc. Natl. Acad. Sci. 96: 12333–12338. Graphics Hofmann, B., To¨lzer, S., Pelletier, I., Altenbuchner, J., van Pe´e, K.H., and Hecht, H.J. 1998. Structural investigation of the cofactor-free chloroper- Figure 1A was drawn with TOPDRAW (Bond 2003). Figures oxidases. J. Mol. Biol. 279: 889–900. 1B, 2C, 3A,B, and 4 were drawn using MOLSCRIPT (Kraulis Hofmann, B., Hecht, H.J., and Flohe´, L. 2002. Peroxiredoxins. Biol. Chem. 1991) and RASTER3D (Merritt and Bacon 1997). Figure 2A,B 383: 347–364. was drawn with CCP4MG (Potterton et al. 2004). Figure 2D Jang, H.H., Lee, K.O., Chi, Y.H., Jung, B.G., Park, S.K., Park, J.H., Lee, J.R., was traced using COOT (Emsley and Cowtan 2004). Lee, S.S., Moon, J.C., Yun, J.W., et al. 2004. Two enzymes in one; two yeast peroxiredoxins display oxidative stress-dependent switching from a peroxidase to a molecular chaperone function. Cell 117: 625–635. Jones, T.A., Zou, J.Y., Cowan, S.W., and Kjeldgaard, M. 1991. 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