Journal of Structural Biology 176 (2011) 24–31

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Journal of Structural Biology

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Crystal structure of the TL29 protein from Arabidopsis thaliana: An APXhomolog without peroxidase activity

Erik Lundberg 1, Patrik Storm, Wolfgang P. Schröder ⇑, Christiane FunkDepartment of Chemistry, Umeå University, SE-901 87 Umeå, Sweden

a r t i c l e i n f o a b s t r a c t

Article history:Received 3 March 2011Received in revised form 8 July 2011Accepted 12 July 2011Available online 21 July 2011

Keywords:APX4Thylakoid lumenAscorbate peroxidase

1047-8477/$ - see front matter � 2011 Elsevier Inc. Adoi:10.1016/j.jsb.2011.07.004

Abbreviations: APX, ascorbate peroxidase; PS, photsoybean APX; sbAPX, soybean APX; TL, thylakoid lum⇑ Corresponding author. Fax: +46 90786 7655.

E-mail address: wolfgang.schroder@chem.umu.se1 Present address: Department of Surgical and Pe

Umeå University, Umeå SE-901 85, Sweden.

TL29 is a plant-specific protein found in the thylakoid lumen of chloroplasts. Despite the putativerequirement in plants for a peroxidase close to the site of photosynthetic oxygen production, and thesequence homology of TL29 to ascorbate peroxidases, so far biochemical methods have not shown thisenzyme to possess peroxidase activity. Here we report the three-dimensional X-ray crystal structure ofrecombinant TL29 from Arabidopsis thaliana at a resolution of 2.5 Å. The overall structure of TL29 ismainly alpha helical with six longer and six shorter helical segments. The TL29 structure resembles thatof typical ascorbate peroxidases, however, crucial differences were found in regions that would be impor-tant for heme and ascorbate binding. Such differences suggest it to be highly unlikely that TL29 functionsas a peroxidase.

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1. Introduction

Oxygenic photosynthesis in plants occurs in specializedorganelles – the chloroplasts. Whereas the enzymes involved inCO2 fixation are located in the stroma, photosystems I and II, whichare responsible for electron transport coupled to ATP synthesis, aresituated in the thylakoid membrane. The thylakoid membrane en-closes a continuous aqueous space known as the thylakoid lumen(Albertsson, 2001), which was once considered to be of limitedfunctional significance in photosynthesis, serving mainly as a sinkfor protons in chemiosmosis. The protein composition of the thyla-koid lumen was formerly regarded as being simple and dominatedby the extrinsic photosystem (PS) II proteins and plastocyanin.However, since the first biochemical characterization of thelumen’s content (Kieselbach et al., 1998) interest in this cellularcompartment has increased. Systematic proteomic studies haveestimated that the lumen of Arabidopsis thaliana contains 80–200different proteins (Kieselbach et al., 2000; Schubert et al., 2002;Peltier et al., 2002; Kieselbach and Schröder, 2003). Moreover,biochemical studies have elucidated the metabolic processes ofdistinct protein families found in the thylakoid lumen (Speteaet al., 2004; Kapri-Pardes et al., 2007; Sun et al., 2007; Gupta

ll rights reserved.

osystem; rsAPX, recombinanten.

(W.P. Schröder).rioperative Sciences, Surgery,

et al., 2002; Edvardsson et al., 2003). However, the function ofseveral lumenal proteins remains to be clarified (Kieselbach andSchröder, 2003).

The thylakoid lumen protein TL29 was first identified in thelumenal fraction of spinach chloroplasts (Kieselbach et al., 1998);orthologous genes have since been detected in the genomes oftomato (Kieselbach et al., 2000), pea (Peltier et al., 2000) and A.thaliana (Kieselbach et al., 2000). Bioinformatic analysis combinedwith in vitro import experiments confirmed the location of TL29: atwin-arginine motif found in the pre-sequence directs the proteinto the thylakoid lumen via the Tat pathway, where the character-istic bipartite signal peptide of the lumenal proteins is cleavedoff, leaving a mature protein with molecular mass of 29 kDa(Kieselbach et al., 2000). Because it exhibits sequence homologyto ascorbate peroxidases (APXs), TL29 was putatively assigned asan APX (EC 1.11.1.11) and was consequently renamed APX4 byPanchuk et al. (2002, 2005) and Mittler et al. (2004). APX proteinsare found in various cellular compartments, where they scavengehydrogen peroxide using ascorbate as an electron donor. In eukary-otic photosynthetic organisms, APXs are members of the class Iheme peroxidase gene family (Welinder et al., 1992; Kitajima,2008). Chloroplasts of higher plants contain two APX isoforms fordefense against reactive oxygen species (ROS): one is localized inthe stroma (chs) and the other is bound to the thylakoid membrane(cht) (Jespersen et al., 1997; Kitajima, 2008). In many higher plants,both of these chloroplastic APXs are produced from a commongene by alternative splicing (Shigeoka et al., 2002). Clearly thereis an ostensible need for a peroxidase inside the thylakoid lumen,located near the site of photosynthetic oxygen evolution, where

E. Lundberg et al. / Journal of Structural Biology 176 (2011) 24–31 25

ROS production is likely to take place. The identification of TL29 inisolated PSII, its electrostatic interaction to the complex (NaCl-wash treatments released the protein), combined with its sequencehomology to ascorbate peroxidases therefore seemed compelling(Kieselbach et al., 2000; Granlund et al., 2009). However, in-depthbiochemical in vitro and in vivo analyses did neither indicate anyperoxidase activity, nor heme or ascorbate binding to TL29(Granlund et al., 2009). Furthermore, various reconstitution exper-iments and calorimetric investigations revealed no interaction ofTL29 with heme and ascorbate (Granlund et al., 2009).

In the work described in this paper, we determined the three-dimensional X-ray crystal structure of recombinant TL29 from A.thaliana and compared it to that of the ascorbate-binding peroxi-dase complex APX1 from soybean (sbAPX) (Sharp et al., 2003),which displays very high sequence similarity. In terms of its overallstructure TL29 was found to be similar to APX1. However, crucialstructural differences were discovered in the region of the activesite and the ascorbate binding site, presumably rendering TL29inactive as an ascorbate peroxidase. We therefore suggest thatTL29 has lost ascorbate peroxidase activity and instead has analternative function in the cell.

2. Materials and methods

2.1. Cloning, protein expression and purification

The sequence of mature TL29/APX4, i.e. the complete 268-ami-no acid sequence without the signal peptide, was amplified from acDNA clone U16014 (Arabidopsis Biological Resource centre, DNAstock centre, Ohio State University) by PCR, using the forward pri-mer 50-GACGACGACAAGATGGCTGACTTGAATCAAC-30 and the re-verse primer 50-GAGGAGAAGCCCGGTTTATAGCTTGAGTTTG-30.The amplified construct was cloned into a pET-46 Ek/LIC vectorusing the Ek/LIC cloning kit (Novagen), which introduced a His-tag followed by an enterokinase cleavage site upstream of theTL29 gene product. TL29 incorporating seleno-methionine (Se-Met) was expressed in the non-auxotrophic Escherichia coli(E. coli) strain Rosetta2 (Novagen), based on the method describedby Van Duyne et al. (1993) and Granlund et al. (2009).

The His-tag of the purified TL29 protein was removed by adding100 U enterokinase (New England Biolabs, InVitro Sweden AB,Stockholm, Sweden) and CaCl2 to a final concentration of 2 mM,and incubating for 2.5 h at 23 �C. Immobilized metal affinity chro-matography (IMAC) was performed as described above (Granlundet al., 2009) to remove the cut His-tag as well as uncut protein, fol-lowed by ion exchange chromatography with a 1 mL HighTrap SPFF column at pH 7 to remove enterokinase. The bound TL29 wasthen eluted, and further purified on size-exclusion chromatogra-phy as above, using buffer containing 16 mM sodium phosphate,pH 7.0, 0.1 M NaCl, and 10% glycerol. Fractions containing Se-Met-labelled TL29 were pooled and concentrated to 10 mg mL�1

using Vivaspin 500 centrifugal concentrators (Vivascience, Lincoln,UK), and stored at �20 �C.

2 For interpretation of color in Figs. 1, 3 and 4, the reader is referred to the webversion of this article.

2.2. Protein crystallization and data collection

Diffracting crystals of Se-Met-incorporated TL29 without His-tag were obtained at room temperature using the hanging-drop va-por-diffusion method. A 4 lL drop was used comprising 2 lL ofreservoir solution (16% PEG 4000, 200 mM potassium phosphatebuffer, (pH 4.8), 10% glycerol) and 2 lL of protein solution(16 mM sodium phosphate, pH 7.0, 0.1 M NaCl, 10% glycerol andprotein at 10 mg mL�1). Crystals grew in space group P41212 withcell dimensions a = b = 92.35 Å, c = 143.46 Å, and with two mole-cules in the asymmetric unit. The crystal was flash frozen in a

stream of liquid nitrogen at 100 K in the absence of cryo buffer,and a single-wavelength anomalous dispersion (SAD) dataset to2.5 Å resolution was collected at a fixed wavelength of 0.97926 Åon a MarMosaic 225 detector at beamline I911-3 at MAX-lab, Lund,Sweden. Diffraction images were processed using MOSFLM (Leslie,2006) and scaled using SCALA, a component of the CCP4 programsuite (Collaborative Computational Project No. 4, 1994). Data col-lection and processing statistics are given in Table 1.

2.3. Structure determination and refinement

The structure of TL29 was solved by SAD phasing using the pro-gram AutoSol from the PHENIX software suite (Adams et al., 2002).Automatic model building using PHENIX AutoBuild resulted in agood electron density map and starting model. Data to 2.5 Å reso-lution were used in the refinement, which was performed using95% of the data; the remaining 5% were removed and used forthe calculation of Rfree. The asymmetric unit was found to containtwo TL29 molecules. The final model was built into the electrondensity map using COOT (Emsley and Cowtan, 2004), and modelrefinement was performed using REFMAC (Collaborative Computa-tional Project No. 4, 1994). The translation–liberation–screw (TLS)method was used in the last rounds of refinement, with each of thetwo monomers being treated as an individual TLS group. Modelquality was analyzed using WHAT-CHECK (Hooft et al., 1996). De-tails of the refinement statistics are shown in Table 1. Figures weregenerated using CCP4MG (Potterton et al., 2004). The coordinatesand structure factors of the final model have been deposited inthe Protein DataBank (PDB ID 3RRW).

3. Results and discussion

3.1. General description of the TL29 structure and its interaction withmetal ions

The mature TL29 protein comprises 268 amino acid residues.The structure was solved to a resolution of 2.5 Å using SAD on asingle seleno-methionine (Se-Met) substituted protein crystal.The final model, which includes residues A4-A258 and B4-B255,43 water molecules, two phosphate ions, two calcium ions and fiveglycerol molecules, is well ordered and has a crystallographic R-factor of 22.5% (Rfree = 27.6%). The asymmetric unit of the TL29crystal contains two monomers of TL29 (A and B) with a root–mean–square (rms) deviation of 0.6 Å in the position of all super-imposed Ca atoms in each monomer (Leu4-Thr255). All residueswere found to lie in the most favored or additionally allowed re-gions of the Ramachandran plot (Ramachandran and Sasisekharan,1968). According to the protein interfaces, surfaces and assembliesservice (PISA) at the European Bioinformatics Institute (Krissineland Henrick, 2007), the monomer–monomer interfaces betweenthe TL29 monomers within the asymmetric unit and also the sym-metry-related monomers, all have a complexation significancescore of 0, suggesting the interfaces lack any biological relevanceand are merely a result of crystal packing. The overall structureof TL29 is mainly alpha-helical (Fig. 1) with six long and six shorthelical segments.

Two metal atoms were identified in the asymmetric unit ofTL29, one in each monomer, positioned at the same site in bothmonomers (green2 sphere, Fig. 1). These metal ions are found inthe vicinity of the BC- and DE-loops in TL29, distant from the me-tal-ion-binding and active sites of sbAPX. The resolution of theTL29 structure does not allow certainties whether these metal ions

Table 1Data collection (merged data), refinement, and model quality statistics for TL29.

(a) Data collectionSpace group P41212Unit cell distances a, b, c (Å) 92.35, 92.35, 143.46Unit cell angles (a, b, c) (�) 90.00, 90.00, 90.00Wavelength (Å) 0.97926Resolution rangea (Å) 48.28–2.50 (2.64–2.50)Number of observationsa 159104 (23066)Number of unique observationsa 22173 (3160)Completenessa (%) 99.9 (100.0)Rmerge (%) 9.2 (34.6)Mean I/r(I)a 12.8 (4.3)Overall redundancya 7.2 (7.3)

(b) RefinementResolution rangea (Å) 46.17–2.5 (2.565–2.500)Reflections in the working seta 20985 (1524)Reflections in the test seta 1130 (84)R-factor working seta,b (%) 22.5 (32.3)R-free test seta,b (%) 27.6 (38.0)Average B-factor (Å2) 28.5Rms deviation from ideal valuesBonds (Å) 0.015Angles (�) 1.336Ramachandran plot:Most favored region (%) 92.0Additionally allowed region (%) 8.0Generously allowed region (%) 0.0Disallowed region (%) 0.0No. of protein atoms 3904No. of metal ions 2No. of water molecules 43No. of glycerol molecules 5No. of phosphate ions 2

a Values in parenthesis indicate statistics for the highest resolution shell.b R-factor =

Phkl||Fobs(hkl)| � |Fcalc(hkl)||/

Phkl|Fobs(hkl)|.

Fig.1. The structure of TL29. The TL29 monomer is composed of 12 alpha-helices ofvarious sizes organized in a similar way to the structure of the ascorbateperoxidases (APXs). Domain 1 of the APXs consists of the N- and C-termini andthe helices A, B, B0 , C and D; it is connected to domain II via helix E. Domain IIconsists of the helices F, F0 , G, H, I and J. The TL29 alpha-helices are designatedaccording to the nomenclature used for the APXs. Helices D2 and D20 do not exist inthe APXs, while helices G and H of the APXs are reduced to a single alpha-helix inTL29, which occupies a fairly similar position. The metal ion (presumed to be Ca) isshown as a light green sphere (For interpretation of the references to color in thisfigure legend, the reader is referred to the web version of this article.).

26 E. Lundberg et al. / Journal of Structural Biology 176 (2011) 24–31

are calcium or sodium ions (see Harding, 2002, 2004). We assumecalcium ions to be bound because (i) small chelate loops (donorpairs) are formed with sequence differences of two amino acids

between the donor atoms (Gly54-O, Asn56-OD1, and Ser58-OG),which is a common feature of calcium binding sites (Harding,2004); (ii) sodium ions typically interact with water moleculesand carbonyl oxygen, but the metal ions in TL29 interact withAsp42-O, Asp42-OD1, Gly54-O, Asn56-OD1, Ser58-OG, Asp158-OD2, and (iii) during refinement the temperature factors for themetal ions and interacting amino acid atoms differed markedly.In the A monomer, the temperature factors were 30 (for Ca) and7 (for Na); in comparison, the temperature factors of the aminoacid atoms interacting with either Ca or Na varied between 33and 46. However, one should note that the metal ion in the TL29monomer B did not exhibit full occupancy (0.5), even though thecalcium-binding amino acid residues are conserved in all TL29family members (Supplementary Fig. 1). The functional importanceof these metal ions is not yet clear.

3.2. Structural comparison of TL29 and ascorbate peroxidases

A basic protein BLAST (blastp) search was performed with TL29sequences from various organisms against the Protein Data Bank(Supplementary Fig. 1) and a search using the full-length recombi-nant TL29 amino acid sequence was performed on the DALI server;the best match obtained was with the soybean ascorbate peroxi-dases (sbAPX, e.g. pdb code: 1OAF) (Sharp et al., 2003). Therefore,we have compared the structure of TL29 to the one of sbAPX usingthe nomenclature assigned to APX domains (Patterson and Poulos,1995) (see Fig. 2, Supplementary Fig. 1). In Fig. 2, the structures ofthe two proteins are compared; segments that differ significantlybetween the two are highlighted in color. In contrast to sbAPX,TL29 contains two additional helices and two very different loopregions (shown in red, Fig. 2A and B), of which one appears to blockthe region that in sbAPX contains the heme binding site. In addi-tion, sbAPX contains one structural element (marked in green,Fig. 2C and D) that is not present in the TL29 protein. In both struc-tures, TL29 and sbAPX, the N- and C-termini are in close proximityto each other, however, compared to sbAPX the N-terminus of TL29is shortened, and the C-terminus enlarged. Domain I in sbAPX(Fig. 2C and D) includes the N- and C-termini as well as the helicesA, B, B0, C and D. Helix E connects domain I to domain II, which con-tains the helices G, H, I and J. Transferring this nomenclature toTL29 we observed that the first helices A, B and B0, and the AB-,BB0-, and B0C-loop regions are similar in both proteins. Further,the C-helix and CD-loop are longer in TL29, but the D-helix startsat an equivalent position in both proteins. At the end of the D-helixthere are a number of interesting differences (Fig. 2E and F). The D-helix is much longer in TL29, and instead of the long loop found insbAPX, the D-helix in TL29 is followed by two novel peripheral al-pha-helices (one longer helix (D2) followed by a short one-turn al-pha-helix (D2

0) (Figs. 1 and 2A). The two novel helices are separatedby a short loop of amino acids with the sequence Gly137-SAGQW-Gly143, which is likely to be highly flexible; this supposition wasconfirmed by the higher temperature factors obtained for this areathan for the rest of the protein (excluding the N- and C-termini).The loop connecting the extended D-helix and the D2-helix con-tains a solvent-exposed cysteine residue (Cys122), and two glycineresidues (Gly123 and Gly124), which may indicate that this area isfunctionally important in the TL29 protein (Fig. 2A). Between the F-and G-helices in sbAPX there is a region containing two short beta-strands, and several loops that interact with a sodium ion. This re-gion is absent in TL29. Even though the sodium-binding loop ofsbAPX is similar in size and charge to the hydrophobic FG-loopof TL29, the glycine residue is missing in TL29 and steric hindrancecreated by the extended D-helix prevents the TL29-loop fromadopting a position similar to that observed in the correspondingloop of sbAPX. Instead, this loop prevents the binding of heme toTL29 (it is denoted the ‘‘heme-blocking loop’’ in Fig. 2A).

Fig.2. TL29 (A and B) and soybean APX1 (PDB ID: 1OAF) (C and D) are shown at two angles differing by 90�. Areas in TL29 that differ significantly from sbAPX are shown in red(A and B), i.e. the extended D-helix and the novel D2- and D20-helices, the ‘‘heme-blocking loop’’, and the extended IJ-loop (in the vicinity of the D- and D2-helices). The twocysteine residues are also shown. Regions that are missing in TL29 are highlighted in green (C and D). E is an overlay of A (TL29) and C (APX1), F an overlay of B (TL29) and D(APX1).

E. Lundberg et al. / Journal of Structural Biology 176 (2011) 24–31 27

Differences between sbAPX and TL29 in this area are discussed inmore detail below. The G-helix in sbAPX is followed by a loopand the short H-helix. TL29 has only one helix in this region; it issimilar in size to the sbAPX G-helix, but is positioned further fromthe main protein body (Figs. 1 and 2). The equivalent position insbAPX, would be between the G- and H-helices, further away fromthe sbAPX heme binding site. The I- and J-helices are similar in sizeand position in both proteins.

3.3. Structural consideration of a hypothetical APX function of the TL29protein

The peroxidase activity of APX depends upon the binding of aprosthetic heme group to the protein. The D20E-loop that connectsthe short D20-helix and the E-helix in TL29 is structurally similar tothe downstream end of the DE-loop in sbAPX. However, while thisDE-loop in sbAPX is part of the heme-binding site (Fig. 3A, gray

Fig.3. The active site of sbAPX (PDB ID: 1OAF) (A) in comparison to the equivalent area in TL29 (B). The Ca-trace is shown in dark gray for sbAPX and in light gray for TL29. In(A) the key residues of the sbAPX active site are shown, together with water molecules displayed as red spheres. Hydrogen bonds are shown as green dotted lines. In (B) thecorresponding residues in TL29 are shown (if present) and the Ca-trace of the ‘‘heme-blocking loop’’ is shown in red. C is an overleay of A (sbAPX) and B (TL29). The ‘‘heme-blocking loop’’ in TL29 is shown in red, regions that are missing in TL29 are highlighted in green. (For interpretation of the references to color in this figure legend, the readeris referred to the web version of this article.)

28 E. Lundberg et al. / Journal of Structural Biology 176 (2011) 24–31

loop in front of the heme molecule), the corresponding D20E-loopof TL29 is positioned closer to the main protein body. Therefore,in TL29 heme binding is precluded by steric restrictions (Fig. 3Band C). Also the E-helix in TL29 is positioned slightly closer tothe area corresponding to the heme-binding site in sbAPX (Figs.1 and 2). Major differences between TL29 and sbAPX are visiblein the region of the F-helix and FG-loop, which is involved in hemebinding in APXs (Fig. 3C). Hydrophobic residues in TL29 give rise toa bend in the FG-loop. As a result the site in TL29 that would beanalogous to the heme-binding site of sbAPX is totally blocked(Fig. 3B).

Since ascorbate peroxidases (APX) catalyze the decompositionof H2O2 to water using ascorbate as an electron donor, both hydro-gen peroxide and ascorbate must bind to the protein. The bindingsite of ascorbate in APX has been shown to be close to the c-hemeedge (Sharp et al., 2003). Comparing the structural data of sbAPXand TL29, distinct differences were found at both the heme- andsubstrate-binding sites (Figs. 3 and 4). It has been reported thatin the active site of sbAPX, nine specific amino acid residues areimportant in the coordination of heme and the functioning of theactive site, namely Arg38, Trp41, His42, Ser160, His163, His169,Arg172, Trp179, and Asp208 (Patterson and Poulos, 1995; Sharpet al., 2003). In TL29, five of these residues are missing (His163,His169, Arg172, Trp179, and Asp208), and three (Arg38, Trp41,

and His42 in sbAPX) are different (Lys37, Leu40, and Asn41 inTL29). Only the Ser192 residue (Ser160 in sbAPX) is conserved inTL29, however, its side chain points in the opposite direction tothat in sbAPX. This Ser192 is the first amino acid of a novel loopconsisting of the hydrophobic residues Ala193, Phe194, Leu195,and Pro197, which occupies the space that is analogous to thesbAPX heme binding site (Figs. 2–4). Thus, instead of this site thisarea partly constitutes the hydrophobic core in TL29. Further, asmentioned previously, the entire region of loops and two shortbeta-strands between the F- and G- helix in sbAPX, containing,e.g. His169 and Arg172, is missing in TL29 (Fig. 4A–C). In sbAPXthe side-chains of His169 and Arg172, together with some of themain-chain atoms of this region, are involved in both the coordina-tion of heme and ascorbate molecules. While the side-chain ofsbAPX-Lys30, situated in the AB-loop opposite His169 andArg172 and also interacting with ascorbate in APXs (Sharp et al.,2003) is conserved in TL29 (Lys28), it is followed by Pro29, whichforces the main-chain to adopt a different orientation. Also cyto-chrome c peroxidase (CCP, Poulos, 2010) contains a long surfaceloop preventing APX activity. This loop had little effect on struc-ture, stability or the ability to form well-ordered crystals. Interest-ingly, removal of seven-residues of this loop and replacing it byresidues 27–32 of APX led to formation of an ascorbate site(Meharenna et al., 2008).

Fig.4. Substrate interaction in sbAPX (A and D) compared with the equivalent areas in TL29 (B and E). C is an overlay of A and B, F an overlay of D and E. The ‘‘heme-blocking loop’’in TL29 is shown in red, regions that are missing in TL29 are highlighted in green. (A) Ascorbate (ASC) binding in sbAPX (PDB ID: 1OAF, Sharp et al., 2003). Key residues involvedin ascorbate binding are shown as sticks, and an interacting water molecule as a red sphere. Hydrogen bonds are displayed as green dotted lines. (B) The area in TL29corresponding to (A). None of the residues interacting with ascorbate are conserved except Lys28 (Lys30 in sbAPX), which has a different orientation. The entire regioncontaining His169 and Arg172 is missing. The Ca-trace of the ‘‘heme-blocking loop’’ is shown in red. (C) Overlay between sbAPX (A) and TL29 (B), this time as helices, strands andloops to more clearly show the proteins and how the loop in TL29 reaches towards the ascorbate (ASC) site. Colors are chosen as in Fig. 3. (D) Salicylhydroxamic acid (SHA)-binding in sbAPX (PDB ID: 1V0H, Sharp et al., 2004). The carbon atoms in SHA are shown in purple to more clearly display this molecule. (E) The area in TL29 corresponding to (D).Several structural differences are found between TL29 and sbAPX, both, in the region where SHA and heme bind in sbAPX. Together they hinder binding of SHA in TL29. TheCa-trace of the ‘‘heme-blocking loop’’ is shown in red. Binding of similar aromatic molecules cannot be excluded, but heme-dependant ascorbate peroxidase activity is highlyunlikely. (F) Overlay between sbAPX (D) and TL29 (E). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

E. Lundberg et al. / Journal of Structural Biology 176 (2011) 24–31 29

30 E. Lundberg et al. / Journal of Structural Biology 176 (2011) 24–31

Ascorbate peroxidase is a bifunctional enzyme that can also usevarious aromatic substances as substrates. These compounds bindat a different site than ascorbate, close to the so called d-heme edge(Sharp et al., 2004). It has been proposed that the binding of thesearomatic substances occurs at an aromatic-substrate binding pocket(Sharp et al., 2004). Salicylhydroxamic acid (SHA) is an aromaticmolecule that has been shown to bind to the aromatic pocket (pdbcode: 1V0H) via hydrogen bonds with Arg38-NE, Trp41-NE1, andthe carbonyl oxygen of Pro132 (Sharp et al., 2004). As shown inFig. 4D–F, this pocket was found to be altered in TL29. SbAPX-Pro132 is conserved in TL29 as Pro164, but Arg38 and Trp41 ofsbAPX correspond to Lys37 and Leu40, respectively, in TL29. Further,in TL29 Pro164 is positioned differently: the downstream part of theDE-loop in sbAPX is situated almost parallel to the E-helix, whereasin TL29 it forms a bend destroying the protein environment neces-sary for heme and SHA binding. The flexibility of TL29-Gly167 allowsTrp166 to move into this region and to form a hydrogen bond be-tween the side-chain NE1 atom and Asn41-OD1.

While the overall structure of TL29 is similar to APXs, there aresignificant differences, which preclude TL29 from functioning as anascorbate peroxidase. The structural data presented here may ex-plain why TL29 neither binds heme, ascorbate, nor aromatic sub-strates (at least not in the same manner as APXs) and stronglysupport the biochemical analyses performed by Granlund et al.(2009).

3.4. Membrane interaction and regulation of the TL29 protein

TL29 is located in the thylakoid lumen of higher plant chloro-plasts. This narrow compartment has been estimated to have aprotein concentration of roughly 20 mg mL�1 (Kieselbach et al.,1998) and TL29 has been identified as one of the most abundantof these proteins (Schubert et al., 2002). Therefore, even thoughTL29 is a highly globular and hydrophilic protein, it is likely to con-tact the thylakoid membrane and/or other proteins. Hydrophobicinteractions are possible due to a small hydrophobic patch formedby Leu131, Leu132, Ala135, Tyr136, and Val226. The IJ-loop forms abulge where Val226 extends towards the novel D2-helix, interact-ing hydrophobically with Leu132. These amino acid residues aresituated on one side of the exposed cysteine Cys122 residue (whichis positioned at the end of the D-helix). On the other side of Cys122are the charged residues Glu127 and Lys128. Recently, Hall et al.(2010) showed that TL29 is one of 19 potential thioredoxin targetproteins found in the thylakoid lumen. Interestingly, the cysteinesuggested to be involved in the thioredoxin interaction was of iso-lated or recombinant TL29 Cys244 (Cys325, as the signal peptidewas included in the numbering of Hall et al., 2010) which wasfound to be buried within the protein structure, whereas the sec-ond cysteine found in TL29, Cys122, is exposed (Fig. 2A). Therefore,we propose that it is Cys122 that is important for interaction withand/or regulation of TL29 rather than Cys244. An alignment of theknown TL29 protein sequences from different species supports thishypothesis: Cys122 is conserved in all species investigated, butCys244 is conserved only in Arabidopsis and Vitis vinifera (Supple-mentary Fig. 1). A possible explanation for the results different tothe one of Hall et al. (2010) could be that these authors usedIAM in combination with trypsin digestion, followed by MALDI-TOF analysis. The fragment generated with this method that con-tained Cys122, would be quite small and therefore may have beendifficult to detect. Further work is required to determine whetherCys122 is involved in the redox-regulated activity of TL29.

3.5. The function of the TL29 protein in the chloroplast lumen

The TL29 structure at 2.5 Å resolution presented in this papersupports earlier findings showing that this protein does not func-

tion as an ascorbate peroxidase, despite its high homology withAPX proteins (Granlund et al., 2009). TL29 neither binds hemenor ascorbate, the corresponding heme- and ascorbate-binding do-mains of sbAPX are absent in its structure. Why then does TL29have a structure resembling ascorbate peroxidases, but no peroxi-dase activity? To address this question an InterPro search was per-formed for proteins containing peroxidase domains (hemeperoxidases (InterPro domain IPR002016 and IPR010255) andplant ascorbate peroxidases (IPR002207)); a summary of thissearch is provided in Supplemental Table 1. Interestingly, whilethe majority of protein hits belonged to the family of catalasesand peroxidases, several proteins with other hypothetical orknown functions were identified that contain a peroxidase domainnear the N- or C-termini including tyrosyl-tRNA synthetase, li-pases, and adenylate cyclase. The peroxidase domain thereforemay confer an evolutionary advantage to an enzyme structurewithout being functionally active. Alternatively, TL29 may oncehave functioned as a peroxidase, but evolved to take on other rolesin the cell. These so far unknown functions required structuralchanges, which then precluded the binding of heme or ascorbate.

Since TL29 is only present in plants, it is likely to have a plant-specific role. It has been suggested to be regulated via interactionswith thioredoxin, which is a common mechanism for coupling en-zyme activity to light-mediated photosynthesis in plants (Lindahlet al., 2011). The fact that TL29 is one of the most abundant pro-teins in the thylakoid lumen (Schubert et al., 2002) may indicateit has an important function. However, the precise nature of thisfunction remains obscure despite biochemical, molecular biologi-cal and crystallographic analyses. On the basis of the results pre-sented in this paper we propose that TL29 may have lost itsperoxidase activity and its ascorbate-binding capacity in the courseof evolving to fulfill a new and as-yet unknown, function.

Acknowledgments

The authors would like to thank Prof. Sauer-Eriksson, UmeåUniversity, for valuable comments on the manuscript, and Dr. Tho-mas Kieselbach for helpful discussions and for MS analysis. Thework was supported by the Swedish Research Council (to WPS;VR, 621-2008-3207), the Swedish Energy Agency (to CF), the RoyalSwedish Academy of Sciences (to CF) and Umeå University (to CF).We are very grateful to the Kempe foundation for granting Post-doctoral fellowships to E.L. and P.S.

Appendix A. Supplementary data

Supplementary data associated with this article can be found, inthe online version, at doi:10.1016/j.jsb.2011.07.004.

References

Adams, P.D., Grosse-Kunstleve, R.W., Hung, L.W., Ioerger, T.R., McCoy, A.J., et al.,2002. PHENIX: building new software for automated crystallographic structuredetermination. Acta Crystallogr. D Biol. Crystallogr. 58, 1948–1954.

Albertsson, P., 2001. A quantitative model of the domain structure of thephotosynthetic membrane. Trends Plant Sci. 6, 349–358.

Collaborative Computational Project No. 4, 1994. The CCP4 suite: programs forprotein crystallography. Acta Crystallogr. D Biol. Crystallogr. 50, 760–763.

Edvardsson, A., Eshaghi, S., Vener, A.V., Andersson, B., 2003. The major peptidyl-prolyl isomerase activity in thylakoid lumen of plant chloroplasts belongs to anovel cyclophilin TLP20. FEBS Lett. 542, 137–141.

Emsley, P., Cowtan, S., 2004. Coot: model-building tools for molecular graphics. ActaCrystallogr. D Biol. Crystallogr. 60, 2126–2132.

Granlund, I., Storm, P., Schubert, M., Garcìa-Cerdàn, J.G., Funk, C., et al., 2009. TheTL29 Protein is lumen located, associated with PSII and not an ascorbateperoxidase. Plant Cell Physiol. 50, 1–13.

Gupta, R., Mould, R.M., Zengyong, H., Luan, S., 2002. A chloroplast FKBP interactswith and affects the accumulation of Rieske subunit of cytochrome bf complex.Proc. Natl. Acad. Sci. USA 99, 15806–15811.

E. Lundberg et al. / Journal of Structural Biology 176 (2011) 24–31 31

Hall, M., Mata-Cabana, A., Åkerlund, H.E., Francisco, J., Florencio, F.J., Schröder, W.P.,Lindahl, M., Kieselbach, Thomas., 2010. Thioredoxin targets of the plantchloroplast lumen and their implications for plastid function. Proteomics 10,987–1001.

Harding, M.M., 2002. Metal-ligand geometry relevant to proteins and in proteins:sodium and potassium. Acta Crystallogr. D Biol. Crystallogr. 58, 872–874.

Harding, M.M., 2004. The architecture of metal coordination groups in proteins.Acta Crystallogr. D Biol. Crystallogr. 60, 849–859.

Hooft, R.W., Vriend, G., Sander, C., Abola, E.E., 1996. Errors in protein structures.Nature 381, 272.

Jespersen, H.M., Kjærsgård, I.V.H., Østergaard, L., Welinder, K.G., 1997. Fromsequence analysis of three novel ascorbate peroxidases from Arabidopsisthaliana to structure, function and evolution of seven types of ascorbateperoxidase. Biochem. J. 326, 305–310.

Kapri-Pardes, E., Naveh, L., Adam, Z., 2007. The thylakoid lumen protease Deg1 isinvolved in the repair of photosystem II from photoinhibition in Arabidopsis.Plant Cell 19, 1039–1047.

Kieselbach, T., Hagman, A., Andersson, B., Schröder, W.P., 1998. The thylakoid lumenof chloroplasts. Isolation and characterization. J. Biol. Chem. 273, 6710–6716.

Kieselbach, T., Bystedt, M., Hynds, P., Robinson, V., Schröder, W.P., 2000. Aperoxidase homologue and novel plastocyanin located by proteomics to theArabidopsis chloroplast thylakoid lumen. FEBS Lett. 480, 271–276.

Kieselbach, T., Schröder, W.P., 2003. The proteome of the chloroplast lumen ofhigher plants. Photosynth. Res. 78, 249–264.

Kitajima, S., 2008. Hydrogen peroxide-mediated inactivation of two chloroplasticperoxidases, ascorbate peroxidase and 2-cys peroxiredoxin. Photochem.Photobiol. 84, 1404–1409.

Krissinel, E., Henrick, K., 2007. Inference of macromolecular assemblies fromcrystalline state. J. Mol. Biol. 372, 774–797.

Leslie, A.G., 2006. The integration of macromolecular diffraction data. ActaCrystallogr. D Biol. Crystallogr. 62, 48–57.

Lindahl, M., Mata-Cabana, A., Kieselbach, T., 2011. The disulfide proteome and otherreactive cysteine proteomes: analysis and functional significance. Antioxid.Redox Signal. PubMed PMID: 21275844.

Meharenna, Y.T., Oertel, P., Bhaskar, B., Poulos, T.L., 2008. Engineering ascorbateperoxidase activity into into cytochrome c peroxidase. Biochemistry 47, 10324–10332.

Mittler, R., Vanderauwera, S., Gollery, M., Van Breusegem, F., 2004. Reactive oxygengene network of plants. Trends Plant Sci. 9, 490–498.

Panchuk, I.I., Volkov, R.A., Schöffl, F., 2002. Heat stress and heat shock transcriptionfactor-dependent expression and activity of ascorbate peroxidase in Arabidopsis.Plant Physiol. 129, 838–853.

Panchuk, I.I., Zentgraf, U., Volkov, R.A., 2005. Expression of the Apx gene familyduring leaf senescence of Arabidopsis thaliana. Planta 222, 926–932.

Patterson, W.R., Poulos, T.L., 1995. Crystal structure of recombinant pea cytosolicascorbate peroxidase. Biochemistry 34, 4331–4341.

Peltier, J.B., Friso, G., Kalume, D.E., Roepstroff, P., Nilsson, F., et al., 2000. Proteomicsof the chloroplast: systematic identification and targeting analysis of lumenaland peripheral thylakoid proteins. Plant Cell 12, 319–342.

Peltier, J.B., Emanuelsson, O., Kalume, D.E., Ytterberg, J., Friso, G., et al., 2002. Centralfunctions of the lumenal and peripheral thylakoid proteome of Arabidopsisdetermined by experimentation and genome-wide prediction. Plant Cell 14,211–236.

Potterton, L., McNicholas, S., Krissinel, E., Gruber, J., Cowtan, K., et al., 2004.Developments in the CCP4 molecular-graphics project. Acta Crystallogr. D Biol.Crystallogr. 60, 2288–2294.

Poulos, T., 2010. Thirty years of heme peroxidase structural biology. Arch. Biochem.Biophys. 500, 2–12.

Ramachandran, G.N., Sasisekharan, V., 1968. Conformation of polypeptides andproteins. Adv. Protein Chem. 23, 283–438.

Schubert, M., Petersson, U.A., Haas, B.J., Funk, C., Schröder, W.P., et al., 2002.Proteome map of the chloroplast lumen of Arabidopsis thaliana. J. Biol. Chem.277, 8354–8365.

Sharp, K.H., Mewies, M., Moody, P.C.E., Raven, E.L., 2003. Crystal structure of theascorbate peroxidase–ascorbate complex. Nat. Struct. Biol. 10, 303–307.

Sharp, K.H., Moody, P.C.E., Katherine, B., Raven, E.L., 2004. Crystal structure of theascorbate peroxidase–salicylhydroxamic acid complex. Biochemistry 43, 8644–8651.

Shigeoka, S., Ishikawa, T., Tamoi, M., Miyagawa, Y., Takeda, T., et al., 2002.Regulation and function of ascorbate peroxidase isoenzymes. J. Exp. Bot. 53,1305–1319.

Spetea, C., Hundal, T., Lundin, B., Heddad, M., Adamska, I., et al., 2004. Multipleevidence for nucleotide metabolism in the chloroplast thylakoid lumen. Proc.Natl. Acad. Sci. USA 101, 1409–1414.

Sun, X., Peng, L., Guo, J., Chi, W., Ma, J., et al., 2007. Formation of DEG5 and DEG8complexes and their involvement in the degradation of photodamagedphotosystem II reaction center D1 protein in Arabidopsis. Plant Cell 19, 1347–1361.

Van Duyne, G.D., Standaert, R.F., Karplus, P.A., Schreiber, S.L., Clardy, J., 1993. Atomicstructures of the human immunophilin FKBP-12 complexes with FK506 andrapamycin. J. Mol. Biol. 229, 105–124.

Welinder, K.G., Mauro, J.M., Nørskov-Lauritsen, L., 1992. Structure of plant andfungal peroxidases. Biochem. Soc. Trans. 20, 337–340.