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

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.. Pores a

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 temperastruc-tures > 70C 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.

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 55C [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 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 oxyhydro-gen 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).

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-g1¼ 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-g2¼

2.84 A˚) which also forms a weak electrostatic

interac-tion with Asp125, another conserved residue (distance

Asp125 O-d2–Arg65 N-e2¼ 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-e2¼ 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 N2-fixing

cyanobacteri-um T erythraecyanobacteri-um 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 Fobs) Fcalcdifference 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  3 A˚, are present at the iron-binding sites (Fig 3A) The A water molecule is coordinated by N-e2 of His33 (distance N-e2–O¼ 2.7 A˚) and by the carboxylic oxygen atoms O-d1 and O-d2 of Asp60 (dis-tances O-d1–O¼ 2.53 A˚ and O-d1–O¼ 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-f¼ 2.78 A˚), the Asp76 carboxylic oxygen forms a salt bridge with the Lys31 nitrogen atom (distance O-d2– N-f¼ 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-e2¼ 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

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

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 openopen-ing 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–Cl¼ 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.

T elongatus Dps

H salinarum Dps

E coli Dpsa

a Taken from Ceci et al [3]

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 1Lb vibronic transitions of the Trp34 and Trp144 residues, and a negative peak at 297 nm due to 1La vibronic transitions In the other members

of the Dps family the tryptophan 1Lb vibronic trans-itions produce negative peaks [6] Spectra measured at acid pH values show that the signal corresponding

to the Trp 1La and 1Lb 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 100C 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

Arg42

H salinarum Gln178

Leu181 (surface)

Glu59, Arg61 Asp62 (cavity)

E coli Ala57 (surface)

Ala61 (cavity)

L innocua His37

Asn38 (surface) 6.80 6.26  13 Thr41 (cavity)

Glu44

M smegmatis Pro45 (surface)

Gly49 (cavity)

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 Tm 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 H2O2 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

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

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 55C, 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 55C, 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

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 m M hydrogen per-oxide, 50 l M Fe(II) and 3 l M 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)1were 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 (Æ Æ Æ), 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

1 mgÆmL)1; pH 3.3.

Dps proteins [4] At pH 7.0 and 25C 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 H2O2 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 55C 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

> 55C, 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 temperastruc-ture At this pH, the melting temperature of Dps-Te is 10 or 30C 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 m M 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 m M NaCl

buffer at pH 7.0 were mixed with 50 l M H2O2in the same buffer in

a stopped flow apparatus (Applied Photophysics).