Báo cáo khoa học: Antioxidant Dps protein from the thermophilic cyanobacterium Thermosynechococcus elongatus An intrinsically stable cage-like structure endowed with enhanced stability potx

Antioxidant Dps protein from the thermophilic cyanobacterium Thermosynechococcus elongatus An intrinsically stable cage-like structure endowed with enhanced stability Stefano Franceschini*, Pierpaolo Ceci*, Flaminia Alaleona, Emilia Chiancone and Andrea Ilari C.N.R. Institute of Molecular Biology and Pathology, University of Rome ‘La Sapienza’, Italy The family of DNA-binding proteins from starved cells (Dps) is part of a complex bacterial defence system that protects DNA against oxidative damage [1–3]. Dps proteins use hydrogen peroxide to oxidize intracellular Fe(II) and thereby simultaneously remove the two molecules that produce highly toxic hydroxyl radicals via the Fenton reaction [4,5]. Subsequent sequestration of the ferric ions thus formed in the protein cavity as a micellar hydroxide core completes the detoxification process. Thus, the structural features central to the detoxifying activity of Dps are the characteristic cage- like dodecameric assembly endowed with 23-tetrahedral symmetry and the ferroxidase centre with its unique intersubunit location at the twofold symmetry axes [2]. Keywords Dps from Thermosynechococcus elongatus; hydrogen peroxide; iron oxidation; thermostability; X-ray structure Correspondence A. Ilari, Istituto di Biologia e Patologia Molecolari CNR Dipartimento di Scienze Biochimiche, Universita ` di Roma ‘La Sapienza’, P.le A. Moro, 5 00185 Rome, Italy Fax: +39 06 444 0062 Tel. +39 06 494 0543 ⁄ 499 10761 E-mail: andrea.ilari@uniroma1.it Database The atomic coordinates and structure fac- tors have been deposited in the Protein Data Bank, Research Laboratory for Struc- tural Bioinformatics, Rutgers University, New Brunswick (http://www.rcsb.org, PDB code 2C41) *These authors contributed equally to this work (Received 21 July 2006, revised 4 September 2006, accepted 5 September 2006) doi:10.1111/j.1742-4658.2006.05490.x DNA-binding proteins from starved cells (Dps proteins) protect bacteria primarily from oxidative damage. They are composed of 12 identical subunits assembled with 23-symmetry to form a compact cage-like struc- ture known to be stable at temperatures > 70 °C and over a wide pH range. Thermosynechococcus elongatus Dps thermostability is increased dramatically relative to mesophilic Dps proteins. Hydrophobic interac- tions at the dimeric and trimeric interfaces called Dps-like are replaced by salt bridges and hydrogen bonds, a common strategy in thermophiles. Moreover, the buried surface area at the least-extended Dps-like inter- face is significantly increased. A peculiarity of T. elongatus Dps is the presence of a chloride ion coordinated with threefold symmetry-related arginine residues lining the opening of the Dps-like pore toward the internal cavity. T. elongatus Dps conserves the unusual intersubunit ferr- oxidase centre that allows the Dps protein family to oxidize Fe(II) with hydrogen peroxide, thereby inhibiting free radical production via Fenton chemistry. This catalytic property is of special importance in T. elongatus (which lacks the catalase gene) in the protection of DNA and photosys- tems I and II from hydrogen peroxide-mediated oxidative damage. Abbreviations Dps, DNA-binding proteins from starved cells; Dps-Te, DNA-binding protein from starved cells of Thermosynechococcus elongatus. FEBS Journal 273 (2006) 4913–4928 ª 2006 The Authors Journal compilation ª 2006 FEBS 4913 The cage-like structure of the Dps dodecamer is expected to be resistant to dissociation into subunits because closed symmetric systems, in which intersub- unit interactions are maximized, tend to have lower energies than asymmetric assemblies. Detailed studies, performed as a function of pH, on Listeria innocua [6] and Mycobacterium smegmatis Dps [7] confirm this con- tention, but also highlight significant differences in the tendency of the dodecamer to dissociate into subunits. Thus, L. innocua Dps forms dimers only below pH 2.0 and monomers below pH 1.0, whereas dissociation of M. smegmatis Dps into dimers is evident at pH 5.0 and proceeds to the monomer stage at pH 4.0. No spe- cific information is available on the stability of the dodecameric assembly as a function of temperature, although this property is currently exploited during the purification of Dps proteins. To investigate Dps ther- mostability, a protein from the thermophilic cyano- bacterium Thermosynechococcus elongatus (Dps-Te) was chosen. Thermosynechococcus elongatus inhabits Japanese hot springs and grows optimally at around 55 °C [8]. It is a model system for studying the interplay of gen- etic, biochemical and physiological phenomena in pho- tosynthesis due to the availability of the complete genome sequence [9], but it is also the source of highly stable protein complexes that have been crystallized, e.g. those of photosystems I and II [10,11]. The occur- rence of oxygenic photosynthesis in T. elongatus adds to the reaction of free Fe(II) with hydrogen peroxide as an important source of reactive oxygen species. Thus, superoxide radicals, hydrogen peroxide and hydroxyl radicals are generated as a result of the pho- tosynthetic transport of electrons from water to plasto- quinone such that photosystems I and II are the main targets of photodamage [12–17]. The T. elongatus genome contains two putative Dps- encoding genes. The antioxidant activity provided by the corresponding proteins is likely to have particular importance in protecting DNA and photosystems I and II against oxidative damage. In fact, the organism does not appear suited to manage hydrogen peroxide given the absence of a catalase gene coupled to the presence of two superoxide dismutase genes [9]. Fol- lowing expression in Escherichia coli, Dps-Te has been characterized in terms of its X-ray crystal structure, thermostability and antioxidant activity at various pH values. Analysis of the Dps-Te structure showed an increased number of salt bridges at the subunit interfa- ces with respect to mesophilic members of the family. Such interactions, which are known to promote ther- mostability in a number of proteins from thermophiles and hyperthermophiles therefore play a crucial role in conferring additional stability to an intrinsically stable cage-like structure. In the structural comparison spe- cial attention has been paid to the two types of pore formed at the threefold interfaces. Pores at the so-called ferritin-like interface are all of similar size and are lined with negatively charged residues pointing to a common function in the iron-uptake process; those at the so-called Dps-like interface show marked variabil- ity in their dimensions and chemical nature. Their function may therefore differ in different organisms. Results Sequence analysis Alignment of the Dps-Te sequence with the sequences of six members of the Dps family was performed using multalin [18] and is presented in Fig. 1A. The Dps- Te sequence was compared with: (a) Dlp2 from Bacil- lus anthracis (35% sequence identity) [19], used as search model to solve the Dps-Te structure by molecu- lar replacement; (b) L. innocua and M. smegmatis Dps (sequence identity 30 and 24.2%, respectively) [20,21], whose stability has been studied previously [6,7] and E. coli Dps (22.7% sequence identity), the family pro- totype; (c) Dps from the cyanobacterium Trichodes- mium erythraeum (30% sequence identity) [22]; and (d) Dps from the halophile Halobacterium salinarum (32% sequence identity) [23]. Dps-Te contains all the distinc- tive residues of the Dps family despite the low degree of identity, namely the residues diagnostic of the inter- subunit ferroxidase centre (His33, Asp60, His45 and Glu64), the near-by Trp34 residue, and aspartates 125 and 130 lining the pore along the threefold symmetry axes. Alignment also shows that Dps-Te lacks the long, positively charged N-terminus involved in Dps–DNA complex formation in E. coli Dps [3,24]. Further analy- sis of the sequence using the Predict Protein server (http://www.predictprotein.org) shows the presence of three potential protein kinase C phosphorylation sites, namely TLK (residues 6–8 and 14–16) and TVK (residues 94–96). The first two are positioned on the N-terminal tail and the third is on the BC loop, located on the surface of the molecule in the assembled protein. Monomer fold and dodecameric assembly The Dps-Te monomer folds into the four-helix bundle typical of Dps proteins and ferritins (Fig. 1B). The four helices, A–D, are stabilized mainly by hydrophobic interactions, an additional short a helix (BC) is in the long loop connecting helices B and C. Superposition of The thermostable T. elongatus Dps S. Franceschini et al. 4914 FEBS Journal 273 (2006) 4913–4928 ª 2006 The Authors Journal compilation ª 2006 FEBS the Dps-Te monomer onto those of B. anthracis Dlp2, L. innocua Dps and M. smegmatis Dps yields very small RMSD values (0.893, 0.910 and 1.07 A ˚ , respect- ively). A higher RMSD value pertains to superposition of the Dps-Te monomer onto the E. coli Dps (1.54 A ˚ ). Figure 1B shows that the only significant differences occur in the N- and C-terminal regions. Twelve monomers assemble to form a hollow pro- tein cage with 23-tetrahedral symmetry (external and internal diameters 90 and 45 A ˚ , respectively). The threefold symmetry-related subunits make two types of interaction. One defines the so-called ‘ferritin-like’ interface because the interactions resemble those of ferritin subunits along the threefold symmetry axes [20], the other defines the interface specific to this pro- tein family named ‘Dps-like’. In Dps-Te, residues 2–7 (visible only in subunit C) form a structured tail that protrudes from the dodeca- meric assembly towards the solvent. These residues have been refined without imposing non-crystallo- graphic symmetry (NCS) restraints, indicating that they assume different conformations. In the other known Dps crystal structures the N-terminus is either involved in interactions with the protein scaffold or is not visible because of its flexibility. Thus, in L. innocua Dps, the first six residues are not visible, whereas in Dlp2 the first three are anchored to the C helix of the same subunit via the Ser2 OH group, which is hydro- gen bonded to the main chain oxygen of Val115 (Fig. 1B). In the E. coli Dps X-ray structure, the first eight residues (containing two positively charged lysines) are not visible, residues 9–15 are oriented towards the solvent and residues 16–21 form a cove that is bent toward the ‘ferritin-like’ interface. In M. smegmatis Dps, the N-terminal tail formed by the first 14 amino acids is likewise bent towards the ferritin-like interfaces. Figure 1B also shows that Dps-Te is characterized by a relatively long C-terminus (residues 151–158) which is visible in all subunits and forms a hook bent towards the Dps-like interface. The longest C-terminal tail is found in M. smegmatis Dps [7,21]. The few residues of this long tail that are visible are likewise bent towards the Dps-like interface. Trimeric ‘ferritin-like’ interface The surface area buried at the ferritin-like interface is quite extended (966 A ˚ 2 per monomer) as it comprises A B Fig. 1. Primary structure alignment (A) and monomer fold (B) of Dps proteins. (A) Proteins from T. elongatus (Dps-Te), B. anthracis (Dlp2), L. innocua (Dps-Li), E. coli (Dps-Ec), M. smegmatis (Dps-Ms), H. salinarum Dps (DPS-HS) and T. erithraeum Dps (Dps-Er). The residues of the ferroxidase centre are depicted in red, those lining the two types of pore are shown in green. The a helices are indicated by upper case letters. (B) Structural overlay of the Ca trace of the Dps-Te monomer (Te, blue) with those of Dlp2 (Ba, red), Dps-Li (Li, green), Dps-Ec (Ec, azure) and Dps-Ms (Ms, salmon). The N- and C-terminal regions are indicated by (N) and (C), respectively. Pictures were generated using PYMOL (Delano Scientific LLC, San Carlos, LA; http://www.pymol.org). S. Franceschini et al. The thermostable T. elongatus Dps FEBS Journal 273 (2006) 4913–4928 ª 2006 The Authors Journal compilation ª 2006 FEBS 4915 the CD loop, the beginning of the D helix and the last part of the B helix. It is stabilized by hydrophobic and hydrophilic interactions and displays the features des- cribed for E. coli and M. smegmatis Dps and Dlp2 [2,7,19]. The most buried hydrophobic side chains belong to the highly conserved Trp144 residues Val136 (D helix), Ala117 (CD loop), Leu67 and Leu69 (B helix). The hydrophilic residues stabilizing the interface are the conserved Arg65 and Asp125 residues and Asp70, Arg143 and Gln140, which are not conserved in the other Dps proteins considered. In particular (Fig. 2A, panels 1 and 2), Arg143 forms two strong electrostatic bonds with Asp70 (distances: Asp O-d1– Arg N-g1 ¼ 2.8 A ˚ , Asp O-d2–Arg N-g2 ¼ 2.8 A ˚ ). As in the other Dps proteins considered, the conserved Arg65 residue contributes to stabilize the ferritin-like interface (Table 1). In Dps-Te it is hydrogen bonded to Gln140 (distance Gln140 O-e2–Arg65 N-g2 ¼ 2.84 A ˚ ) which also forms a weak electrostatic interac- tion with Asp125, another conserved residue (distance Asp125 O-d2–Arg65 N-e2 ¼ 4.8 A ˚ ). Trimeric ‘Dps-like’ interface In all the mesophilic Dps proteins whose structure has been solved to date the trimeric Dps-like interfaces are the least extended ones and are stabilized mostly by hydrophobic interactions. Thus, in L. innocua and E. coli Dps hydrophilic interactions are absent and in M. smegmatis Dps there is only a strong salt bridge between Arg99 and Glu157 and two hydrogen bonds (Table 1). At variance with these proteins, the Dps-like interface of Dps-Te is stabilized by a large number of hydrophilic interactions and is the most extended one (1711 A ˚ 2 per monomer), because it comprises the C-terminal tail, the last part of the D helix and the first B helix residues (Fig. 2B, panels 1 and 2). The hydrophobic residues buried most deeply at the interface are Val95, on the C helix and Trp144, Phe145, Phe149 on the D helix. The residues engaged in electrostatic interactions are Tyr37 and Gly38 on the AB loop, Asp43, Arg42 and Glu50 on the first part of the B helix, Lys96 on the last part of the CD loop, Glu148 on the last part of the D helix, and Gly153 and Asp154 on the C-terminus. Arg42 provides the strongest electrostatic interactions at this interface because it forms a salt bridge via N-g1 with O-e2 of Glu50 (distance N-g1–O-e2 ¼ 2.8 A ˚ ). Moreover, three hydrogen bonds are formed between Lys96 and Asp154, Glu148 and Gly38 and Tyr37 and Gly153 (Table 1). In the halophilic H. salinarum Dps, the Dps–like interface is less extended (1008 A ˚ 2 ⁄ monomer) than in Dps-Te but is likewise stabil- ized by a great number of hydrophilic interactions (Table 1). In the Dps protein from the N 2 -fixing cyanobacteri- um T. erythraeum the interface lacks many of the resi- dues involved in electrostatic interactions in Dps-Te, i.e. Arg42 is Asn62 and Asp43 is Gln63. Dimeric interface and ferroxidase centre The dimeric interface is formed by helices A and B and by the short BC helix placed at the centre of the long loop connecting helices B and C (Fig. 2C). It contains the two symmetry-related characteristic inter- subunit ferroxidase centres. The absence of peaks with values > 4 r in the F obs ) F calc difference Fourier map calculated before the introduction of water molecules indicates that the ferroxidase centres are iron free. However, two water molecules (A and B), placed at a distance of  3A ˚ , are present at the iron-binding sites (Fig. 3A). The A water molecule is coordinated by N-e2 of His33 (distance N-e2–O ¼ 2.7 A ˚ ) and by the carboxylic oxygen atoms O-d1 and O-d2 of Asp60 (dis- tances O-d1–O ¼ 2.53 A ˚ and O-d1–O ¼ 3.25 A ˚ ). Water molecule B is placed at 3.16 and 2.57 A ˚ , respectively, from the Glu64 carboxylic oxygen O-e2 and O-e1 and at 2.86 A ˚ from N-e2 of His45. The surface area buried upon dimerization (1180 A ˚ 2 per monomer) is similar to those calculated for the other members of the family [7]. In Dps-Te it is stabil- ized mostly by hydrophilic interactions, whereas in the mesophilic Dps proteins the dimeric interface is mainly hydrophobic (Table 1, Fig. 2C, panel 2). In particular, Lys30 is salt bridged to Asp60 (distance O-d2–N-f ¼ 2.78 A ˚ ), the Asp76 carboxylic oxygen forms a salt bridge with the Lys31 nitrogen atom (distance O-d2– N-f ¼ 2.83 A ˚ ) and the O-d1 carboxylic oxygen of Asp76 is hydrogen bonded to the Gly91 nitrogen atom. Interestingly, also in the Dps protein from the halophilic archaebacterium H. salinarum the dimeric interface is stabilized mostly by hydrophilic interac- tions and by two salt bridges between Arg8 and Glu56 (N-g2–O-e2 ¼ 2.8 A ˚ ) of the twofold symmetry-related subunits (Table 1). The ‘ferritin-like’ and ‘Dps-like’ pores Residues at the ferritin-like interface of Dps-Te form a pore that connects the oligomer cavity to solvent and is lined by negatively charged residues. In particular, the opening on the protein surface ( 13.5 A ˚ diameter) contains Glu118 and Glu122, respectively, whereas that facing the protein cavity ( 7.5 A ˚ diameter) con- tains the highly conserved Asp130 residues. The thermostable T. elongatus Dps S. Franceschini et al. 4916 FEBS Journal 273 (2006) 4913–4928 ª 2006 The Authors Journal compilation ª 2006 FEBS A B C Fig. 2. Trimeric ferritin-like (A), trimeric Dps-like (B) and dimeric (C) interfaces of T. elongatus Dps. Panel 1, view along the interfaces; panel 2, blow-up indicating relevant interactions as detailed in the text. Pictures were generated using PYMOL. S. Franceschini et al. The thermostable T. elongatus Dps FEBS Journal 273 (2006) 4913–4928 ª 2006 The Authors Journal compilation ª 2006 FEBS 4917 Comparison of the Dps-Te ‘ferritin-like’ pore with those of other family members, such as L. innocua, E. coli, H. salinarum and M. smegmatis Dps, shows that its structural features are largely conserved. Thus, the length is  10 A ˚ with the exception of H. salinarum Dps in which it is  18 A ˚ , the diameter of outer open- ing ranges between 9.0 and 13.5 A ˚ , and the opening on the protein cavity between 7.0 and 8.0 A ˚ (Table 2). These values pertain to distances between Ca atoms. In particular, the negatively charged residues lining both openings are conserved in all Dps proteins. At the ‘Dps-like’ interface the subunits form another pore with threefold symmetry. This pore shows great variability among the Dps proteins considered (Table 2). Thus, the length of the pore ranges from 7 to 21 A ˚ , whereas the size of the openings on the pro- tein cavity and surface vary between 5 and 9.0 A ˚ , respectively. Because these values refer to distances between Ca atoms, in solution the pore is likely to assume different conformations, e.g. ‘opened’ or ‘closed’, depending on the rotational conformations of the residues lining the pore. The nature of the residues along the Dps-like pores is likewise variable. Interestingly, in the two extremo- philic Dps proteins the opening on the protein surface is lined with a hydrophobic residue (Val157 in Dps-Te and Leu181 in H. salinarum Dps), whereas that on the protein cavity contains the positive charges of the sym- metry-related arginine residues (Arg42 in Dps-Te and Arg62 in H. salinarum Dps). In Dps-Te, these Arg42 residues bind a chloride ion which occludes the pore opening (distances: N-g1–Cl ¼ 3.2 A ˚ and N-g2–Cl ¼ 3.4 A ˚ ) (Fig. 3B). Figure 4 shows that in the proteins analysed with the exception of M. smegmatis Dps the Dps-like pores have a constriction. In E. coli Dps, as described by Grant et al. [2], the constriction is located near the protein cavity and is lined by hydrophobic residues (Ala61). In Dps-Te and L. innocua Dps the location is similar, whereas in H. salinarum Dps the constriction is in the middle of the pore. In the proteins, the con- striction is lined by charged residues (Arg42, Asp43 in Dps-Te and Arg61 Asp62 in H. salinarum Dps) or by hydrophilic ones (Thr41 in L. innocua Dps). Table 1. Electrostatic interactions stabilizing the interfaces in T. elongatus, H. salinarum, and E. coli Dps. Dimeric interfaces (A ˚ ) Ferritin-like interfaces (A ˚ ) Dps-like interfaces (A ˚ ) T. elongatus Dps Trp34 N-e1–O-d2 Asp60 2.7 Asp70 O-d1–N-g1 Arg143 2.8 Asp43 O-d1–N-e Arg42 2.4 Lys30 N-f –O-d2 Asp60 3.0 Asp70 O-d2–N-g2 Arg143 2.7 Asp43 O-d2 – H2O1206 2.7 Lys31 N-f –O-d2 Asp76 2.9 Arg65 N-d1–O-g1 Gln140 2.8 H2O1206–O Pro39 2.4 Gly91 N–O-d1 Asp76 2.8 Arg65 N-d2–O-g1 Gln140 2.9 Arg42 N-g1–O-e2 Glu50 2.8 Tyr37 O-g – O Gly153 2.7 Gly38 N–O Gln148 2.7 Lys96 N–O Asp154 3.0 Val95 N–O Asp154 2.4 H. salinarum Dps Arg8 N-g2–O-e2 Glu56 2.8 Asp128 O-d1–N-g1 Arg21 2.9 Asp62 O-d2–N-g1 Arg61 2.8 Arg8 N–O Val112 2.6 Glu131 O-e1–N-g1 Arg21 2.6 His168 N-d1–O Val55 3.0 Ala9 O–N Val112 2.9 Arg134 N-g1–O Leu22 3.0 His168 O–N Gly57 2.8 Ala11 N–O-d1 Asp111 2.9 Arg134 N-g2–O Leu22 2.8 Asp178 O- d2–N Ile115 2.8 Tyr42 O-g –O-g Tyr45 2.7 Glu138 O-e1–N-e2 His146 2.7 Thr174 O-c1–O-e1 Glu59 2.6 Tyr45 O-g –O-e2 Glu75 2.5 Glu160 O-e1–N-g1 Arg84 2.8 Leu175 N–O-e2 Glu59 2.8 His46 N-e2–N-e2 His46 2.7 Glu160 O-e1–N-g2 Arg84 2.6 Leu175 O–N-g1 Arg116 2.8 His52 N- e2–O- e1 Glu83 2.9 Glu167 O- e1–O-c Ser17 2.8 Trp53 N- e1–O-d1 Asp79 2.8 Asp172 O–N-g1 Arg8 3.0 Ala94 N–O- e2 Glu110 2.9 Asp172 O-d1–N-g2 Arg8 2.9 Ser95 N–O- e1 Glu110 2.9 Glu97 N–O- e1 Gln100 2.8 E. coli Dps a Lys48 N-f–O-d2 Asp78 2.8 Arg18 N-e –O-d1 Asp123 2.7 Arg70 N-g2–O-d2 Asp78 2.9 Arg83 N-g1–O-d1 Asp156 2.8 Arg133 N-g1–O-d1 Asp20 2.9 Lys134 N-f –O-d2 Asp20 3.0 a Taken from Ceci et al. [3] The thermostable T. elongatus Dps S. Franceschini et al. 4918 FEBS Journal 273 (2006) 4913–4928 ª 2006 The Authors Journal compilation ª 2006 FEBS State of association as a function of pH and temperature The state of association was studied over the pH range 1.0–7.0 by HPLC-gel filtration. Representative elution profiles presented in Fig. 5A show that the dodecamer- ic architecture of Dps-Te is stable between pH 7.5 and 3.0. At pH 2.5, the chromatogram shows two addi- tional small peaks, corresponding to a dimeric and a high molecular mass species. However, the decrease in the area of the peaks clearly points to marked precipi- tation of the protein on the column. At pH 2.0, the dimer peak disappears and the amount of precipitated material increases. At pH 1.0, the elution pattern dis- plays only one peak corresponding to a monomeric species whose area is indicative of almost complete precipitation of Dps-Te. It is worth noting that preci- pitation does not take place in the case of L. innocua and M. smegmatis Dps [6,7]. HPLC-gel filtration was supplemented by CD experi- ments in the near-UV region (Fig. 5B). The CD spectra at pH 7.0 showed two positive peaks at 289 and 282 nm due to 1 L b vibronic transitions of the Trp34 and Trp144 residues, and a negative peak at 297 nm due to 1 L a vibronic transitions. In the other members of the Dps family the tryptophan 1 L b vibronic trans- itions produce negative peaks [6]. Spectra measured at acid pH values show that the signal corresponding to the Trp 1 L a and 1 L b vibronic transition decreases dramatically at pH 2.0 and is lost completely at pH 1.0. Protein stability as a function of temperature was monitored in the far-UV CD region. The transition from the native to the denatured state could not be followed between pH 7.0 and 4.0 because of the high stability of Dps-Te even at 100 °C. Thus, heat- induced unfolding of Dps-Te was monitored at pH 3.0 where the quaternary structure is conserved at room temperature (Fig. 6). The denaturation process of the mesophilic L. innocua and E. coli proteins was followed under the same experimental conditions. Whereas Dps-Te and L. innocua Dps undergo full Fig. 3. Ferroxidase centre (A) and chloride-binding site (B) in T. elongatus Dps. (B) The view is along the threefold axis with the opening towards the protein cavity on the bottom. Water mole- cules are depicted in red. Pictures were generated using PYMOL. Table 2. Characteristics of the ‘Dps-like’ pores in T. elongatus, H. salinarum, E. coli, L. innocua and M. smegmatis Dps. The diam- eter and length of the pores refer to the Ca-Ca distances between the relevant symmetry-related residues. Proteins Residues lining the pore Diameter (A ˚ ) Length (A ˚ )surface cavity T. elongatus Val157 (surface) Leu40 Asp43 (cavity) 5.50 5.40  14 Arg42 H. salinarum Gln178 Leu181 (surface) Val176 8.95 6.10  19 Glu59, Arg61 Asp62 (cavity) E. coli Ala57 (surface) Asn58 7.02 5.37  9.5 Ala61 (cavity) L. innocua His37 Asn38 (surface) 6.80 6.26  13 Thr41 (cavity) Glu44 M. smegmatis Pro45 (surface) Asn46, Ile48 7.50 6.12  8.5 Gly49 (cavity) S. Franceschini et al. The thermostable T. elongatus Dps FEBS Journal 273 (2006) 4913–4928 ª 2006 The Authors Journal compilation ª 2006 FEBS 4919 denaturation, E. coli Dps still contains secondary structure at 320 K, the apparent melting temperature, an indication that protein aggregation takes place before completion of the thermal melting process. The T m values  353 for Dps-Te and  343 for L. innocua Dps (calculated over a range of three different experiments) can be taken as a measure of thermostability, because the irreversibility of the transitions depicted in Fig. 6 does not warrant the calculation of thermodynamic parameters. DNA-binding ability and DNA protection against hydroxyl radicals Binding of Dps-Te to DNA was analysed in vitro by means of agarose gel electrophoresis experiments under conditions where E. coli Dps is known to form large complexes with DNA that do not enter the gel [7]. Dps– DNA complexes were not detected when purified Dps- Te (3 lm) was added to 20 nm supercoiled pET-11a DNA in 30 mm Tris⁄ HCl containing 50 mm NaCl at pH 6.5, 7.0 or 8.0 (Fig. 7A). Thus, in accordance with the absence of the N-terminal extension used by E. coli Dps in the interaction with DNA [3,24], Dps-Te is unable to bind DNA. No complex formation was observed when the protein concentration was increased 10-fold while keeping DNA constant (data not shown). In order to establish whether T. elongatus Dps is able to prevent hydroxyl radical-mediated DNA clea- vage, an in vitro DNA damage assay was employed [25]. The combined effect of 50 lm Fe(II) and 10 mm H 2 O 2 on the integrity of plasmid pET-11a (5600 bp) was assessed in the presence and absence of Dps-Te in 30 mm Tris ⁄ HCl, 50 mm NaCl, pH 7.5. Under these conditions the hydroxyl radicals pro- duced by the Fenton reaction degrade plasmid pET- 11a completely (Fig. 7B, lane 2). By inhibiting hydroxyl radical formation the presence of Dps-Te (Fig. 7B, lane 3) confers full protection to the DNA plasmid. AC D E B Fig. 4. Pores at the Dps-like interface of T. elongatus Dps (A), H. salinarum Dps (B), E. coli Dps (C), L. innocua Dps (D) and M. smegmatis Dps (E). The residues forming the pores and their Van der Waal’s surfaces are indicated with a specific mention to the residues at the pore constric- tions. (Left) View down the threefold axis. (Right) View from the opening on the protein surface. Pictures were generated using PYMOL. The thermostable T. elongatus Dps S. Franceschini et al. 4920 FEBS Journal 273 (2006) 4913–4928 ª 2006 The Authors Journal compilation ª 2006 FEBS Iron incorporation kinetics Dps-Te is able to oxidize and incorporate ferrous iron in the presence of molecular oxygen at neutral pH at 25 and at 55 °C, the physiological temperature of the bacterium (Fig. 8A). Progress curves measured after the addition of 48 Fe(II) ⁄ dodecamer show that the half-times of the iron-uptake reaction correspond to 600 and 200 s, at 25 and 55 °C, respectively. In the absence of protein, the iron auto-oxidation process leads to the precipitation of iron hydroxide at both temperatures. Dps-Te ferroxidation is more efficient with hydro- gen peroxide as an oxidant, as described for other AB Fig. 7. DNA binding (A) and protection (B) by T. elongatus Dps. (A) Lane 1, plasmid DNA; lane 2, plasmid DNA with Dps-Te. (B) Lane 1, plasmid DNA; lane 2, plasmid DNA with 50 m M hydrogen per- oxide, 50 l M Fe(II); lane 3, plasmid DNA with 50 mM hydrogen per- oxide, 50 l M Fe(II) and 3 lM Dps-Te. Fig. 5. Effect of pH on the state of association (A) and near-UV CD spectra (B) of T. elongatus Dps-Te. At any given pH, protein solu- tions at 1 mgÆmL )1 were incubated at 25 °C for 24 h. (A) Elution profiles upon HPLC-gel filtration after incubation at pH 1.0 (ÆÆÆ), 2.0 (—), 2.5 (ÆÆÆ), 3.0 (- - -), 7.0 (- Æ -). (B) Spectra recorded after incuba- tion at pH 7.0 (- Æ -), 3.3 ( ), 2.0 (—), 1.0 (ÆÆÆ). Fig. 6. Thermal denaturation of T. elongatus Dps-Te, L. innocua Dps and E. coli Dps. Spectra were recorded at 222 nm in 0.1 cm quartz cuvettes; protein concentration 1mgÆmL )1 ; pH 3.3. S. Franceschini et al. The thermostable T. elongatus Dps FEBS Journal 273 (2006) 4913–4928 ª 2006 The Authors Journal compilation ª 2006 FEBS 4921 Dps proteins [4]. At pH 7.0 and 25 °C the half-time of the reaction is 0.3 s upon addition of 100 Fe(II) ⁄ dodecamer (Fig. 8B), whereas at 55 °C the half-time decreases to  0.035 s. Thus, oxidation of Fe(II) by H 2 O 2 at room temperature is  2000- fold faster than by molecular oxygen at room temperature. As expected, when using both oxidants an increase in temperature from 25 to 55 °C results in an increase in the initial rates of the reaction. The increase in rate was approximately eightfold in the case of hydrogen peroxide, and only threefold in the case of molecular oxygen. Discussion The background to the present characterization of T. elongatus Dps is provided by the recent, numerous studies aimed at identifying the factors responsible for the increased stability of proteins from thermophiles. We were interested to establish which set of structural devices is utilized by the Dps family to further stabilize its characteristic shell-like assembly which is endowed with an intrinsically high stability. Comparison of the crystal structure of thermophilic Dps-Te with those of mesophilic homologues indicates that the strategy employed by T. elongatus is not only to increase the number of intersubunit ion pairs and hydrogen bond- ing interactions, a general strategy of thermophiles and hyperthermophiles, but also to increase the amount of buried surface of the least-extended Dps-like subunit interface. Prior to this study, the only reports that addressed the stability of the Dps dodecamer regarded its ten- dency to dissociate into subunits at acid pH and room temperature. The protein systems revealed significant differences in HPLC gel-filtration experiments. Thus, the L. innocua dodecamer preserves its quaternary structure at pH 2.0, whereas E. coli Dps starts dissoci- ating at pH 2.5 and M. smegmatis Dps at pH 5.0 [6,7]. In these systems, dissociation gives rise to stable dimers which in turn dissociate into stable monomers when the pH is lowered further. Quite unexpectedly, the thermophilic Dps-Te protein is less stable than the L. innocua protein at room temperature. Thus, disrup- tion of the Dps-Te assembly takes place at pH 2.5 as shown by the disappearance of the dodecamer peak in the HPLC patterns (Fig. 5A) and by the decrease in rotational strength in the near-UV CD spectra (Fig. 5B). Furthermore, the Dps-Te dimers and mono- mers tend to aggregate and ⁄ or precipitate at variance with those formed by L. innocua and M. smegmatis Dps. The instability of the Dps-Te subunits in turn implies that the subunit-dissociation process is irrevers- ible, again at variance with that of L. innocua Dps [6]. The increased stability of Dps-Te relative to L. inno- cua and E. coli Dps manifests itself at temperatures >55 °C, the optimal growth temperature for the bac- terium. At pH 7.0 and 80 °C, which corresponds to the melting temperature of E. coli Dps, there is no change in the secondary structure of Dps-Te (data not shown). Thus, given the extremely high stability of Dps-Te at neutral pH, thermal denaturation was stud- ied at pH 3.0, a condition where the quaternary struc- ture is conserved at room temperature. At this pH, the melting temperature of Dps-Te is 10 or 30 °C higher than those measured for the mesophilic L. innocua and A B Fig. 8. Kinetics of iron oxidation ⁄ incorporation by T. elongatus Dps using molecular oxygen (A) or hydrogen peroxide (B) as the oxidant. Traces were measured at 310 nm wavelength, which monitors for- mation of the ferric core because it corresponds to a d-d Fe(III) elec- tronic transition at 25 and 55 °C. (A) Solutions of 17.5 l M Fe(II) were added to solutions of 0.25 l M apoDps-Te [molar ratio 48 Fe(II) ⁄ Dps dodecamer] in 50 m M Mops, 150 mM NaCl buffer at pH 7.0. Tem- peratures: 25 °C (—) and 55 °C (- - -). Fe(II) auto-oxidation: 25 °C(ÆÆÆÆÆ) and 55 °C() Æ )). (B) Degassed solutions containing 1.0 l M apoDps- Te and 100 Fe(II) ⁄ Dps dodecamer in 50 m M Mops, 150 mM NaCl buffer at pH 7.0 were mixed with 50 l M H 2 O 2 in the same buffer in a stopped flow apparatus (Applied Photophysics). The thermostable T. elongatus Dps S. Franceschini et al. 4922 FEBS Journal 273 (2006) 4913–4928 ª 2006 The Authors Journal compilation ª 2006 FEBS [...]... importance because it contains the unusual intersubunit bimetallic ferroxidase centre characteristic of the Dps family The two iron-binding sites, A and B have different affinities The thermostable T elongatus Dps for the metal In the available crystal structures, only the A site is occupied by the metal It is either fully occupied as in L innocua Dps, HP-NAP from H pylori and Dlp1 and Dlp2 from B anthracis... survival of the micro-organism Finally, the pores formed at the ferritin-like and Dps- like interfaces deserve a comment for their functional implications They connect the protein cavity with the outside and provide a passage for ions and FEBS Journal 273 (2006) 4913–4928 ª 2006 The Authors Journal compilation ª 2006 FEBS 4923 The thermostable T elongatus Dps S Franceschini et al small molecules The features... polypeptide chain, like the C-terminus, the C-terminal part of the D helix and the N-terminal part of the B helix It may be envisaged that these structural elements, although ‘frozen’ in a single conformation in the crystal structure, may move in solution and thereby enlarge the channel openings Intriguingly, in Dps- Te and H salinarum Dps, the two proteins from extremophiles examined, the pores are in a... to the proteins of E coli [34] and M smegmatis [7], where the A sites are empty and partially occupied, respectively In both proteins, a lysine residue in the vicinity of the A site is engaged in electrostatic interactions with an iron ligand (Lys48–Asp78 and Lys36–Asp66, respectively) The Dps- Te ferroxidase centre confers to the protein the ability to protect DNA from hydroxyl radicals even in the. .. involved in the iron reduction and exit processes and hence may serve as an entrance for ironreducing agents In turn, given their structural variability, this common role would require the nature of the endogenous reducing agents to differ widely in the different organisms If so, in Dps- Te the endogenous reducing agent should be anionic in nature In conclusion, the thermostability of the intrinsically stable. .. using as the search probe a polyalanine-truncated model built from the B anthracis Dlp2 tetramer (Protein Data Bank entry 1JIG) The rotational and translational searches, performed ˚ with molrep [41] in the resolution range 10–3.0 A, produced a clear solution Refinement of the atomic coordinates and displacement parameters were carried out applying the NCS restraints to the 12 subunits of Dps- Te from residue... Vijayan M (2004) X-ray analysis of Mycobacterium smegmatis Dps and a comparative study involving other Dps and Dps- like molecules J Mol Biol 339, 1103–1113 22 Castruita M, Saito M, Shottel PC, Elmegreen LA, Mineny S, Stiefel EI & Morel FM (2006) Overexpres- The thermostable T elongatus Dps 23 24 25 26 27 28 29 30 31 32 33 sion and characterization of an iron storage and DNA-binding Dps protein from. .. physiologically relevant macromolecular targets other than DNA, like photosystems I and II Recently, the Dps protein from the N2-fixing marine cyanobacterium T erythraeum has been characterized It shows  30% sequence similarity to Dps- Te, binds DNA, albeit with low affinity, but protects it from oxidative damage T erythraeum carries out photosynthesis in the presence of intense sunlight Thus, the Dps enzymatic... 943–954 9 Nakamura Y, Kaneko T, Sato S, Ikeuchi M, Katoh H, Sasamoto S, Watanabe A, Iriguchi M, Kawashima K, Kimura T et al (2002) Complete genome structure of the thermophilic cyanobacterium Thermosynechococcus elongatus BP-1 DNA Res 9, 123–130 10 Loll B, Kern J, Zouni A, Saenger W, Biesiadka J & Irrgang KD (2005) The antenna system of photosystem II ˚ from Thermosynechococcus elongatus at 3.2 A resolution... of Dps proteins is enhanced in T elongatus by means of two distinct structural features that do not affect the stability at room temperature, i.e an increased density of intersubunit salt bridges, a common strategy used by 4924 thermophiles and hyperthermophiles, and extension of the buried surface area of the smallest subunit interface These distinctive structural features do not increase the stability . Antioxidant Dps protein from the thermophilic cyanobacterium Thermosynechococcus elongatus An intrinsically stable cage-like structure endowed with enhanced stability Stefano. during the purification of Dps proteins. To investigate Dps ther- mostability, a protein from the thermophilic cyano- bacterium Thermosynechococcus elongatus