Crystal Structure of AhpE from Mycobacterium tuberculosis, a 1-Cys Peroxiredoxin

doi:10.1016/j.jmb.2004.12.046 J. Mol. Biol. (2005) 346, 1035–1046

Crystal Structure of AhpE from Mycobacteriumtuberculosis, a 1-Cys Peroxiredoxin

Simon Li1,2, Neil A. Peterson1,2, Min-Young Kim3, Chang-Yub Kim3

Li-Wei Hung4, Minmin Yu5, Timothy Lekin6, Brent W. Segelke6

J. Shaun Lott1,2* and Edward N. Baker1,2*

1Centre of MolecularBiodiscovery, University ofAuckland, Auckland, NewZealand

2School of Biological SciencesUniversity of AucklandAuckland, New Zealand

3Bioscience Division, LosAlamos National LaboratoryLos Alamos, NM 87545, USA

4Physics Division, Los AlamosNational Laboratory, LosAlamos, NM 87545, USA

5Physical Biosciences DivisionLawrence Berkeley NationalLaboratory, Berkeley, CA 94720USA

6Biology and BiotechnologyProgram, Lawrence LivermoreNational Laboratory, LivermoreCA 94551, USA

0022-2836/$ - see front matter q 2004 E

Abbreviations used: Prx, peroxireoxygen species; ORFs, open readingselenomethionine; MAD, multiwavdiffraction; TryP, tryparedoxin peroE-mail addresses of the correspon

[email protected]; ted.baker@au

All living systems require protection against the damaging effects ofreactive oxygen species. The genome of Mycobacterium tuberculosis, thecause of TB, encodes a number of peroxidases that are thought to be activeagainst organic and inorganic peroxides, and are likely to play a key role inthe ability of this organism to survive within the phagosomes ofmacrophages. The open reading frame Rv2238c in M. tuberculosis encodesa 153-residue protein AhpE, which is a peroxidase of the 1-Cysperoxiredoxin (Prx) family. The crystal structure of AhpE, determined at1.87 A resolution (RcrystZ0.179, RfreeZ0.210), reveals a compact single-domain protein with a thioredoxin fold. AhpE forms both dimers andoctamers; a tightly-associated dimer and a ring-like octamer, generated bycrystallographic 4-fold symmetry. In this native structure, the active siteCys45 is in its oxidized, sulfenic acid (S–O–H) state. A second crystal formof AhpE, obtained after soaking in sodium bromide and refined at 1.90 Aresolution (RcrystZ0.242, RfreeZ0.286), reveals the reduced structure. In thisstructure, a conformational change in an external loop, in two of the fourmolecules in the asymmetric unit, allows Arg116 to stabilise the Cys45thiolate ion, and concomitantly closes a surface channel. This channel isidentified as the likely binding site for a physiological reductant, and theconformational change is inferred to be important for the reaction cycle ofAhpE.

q 2004 Elsevier Ltd. All rights reserved.

Keywords: peroxiredoxin; Mycobacterium tuberculosis; antioxidant; crystalstructure; redox mechanism
*Corresponding authors
Introduction

An inevitable consequence of aerobic life is thegeneration of potentially-damaging peroxides andother reactive oxygen species. Protection is given byboth small-molecule reducing agents, such asglutathione, and enzymes such as catalases, peroxi-dases and superoxide dismutases. Pathogenicorganisms face particular challenges, because sitesof infection are often associated with conditions ofhypoxia, where the reduced efficiency of respiratory

lsevier Ltd. All rights reserve

doxin; ROS, reactiveframes; SeMet,

elength anomalousxidase.ding authors:ckland.ac.nz

pathways means that the side reactions thatproduce reactive oxygen species become morepronounced.Mycobacterium tuberculosis is a devastating human

pathogen, responsible for around three milliondeaths worldwide every year.1 Its success as apathogen is attributable in part to its ability to entera dormant or persistent state inside activatedmacrophages in the lung.2,3 Engulfment of theorganism by macrophages is associated both withexposure to the toxic environment within themacrophage phagosome and with the onset ofconditions where essential nutrients, includingoxygen, are in very low supply. In these conditions,the enzymes and small-molecule reducing agentsthat protect M. tuberculosis against peroxides andother reactive oxygen species (ROS) become veryimportant to its survival.4

d.

† http://www.doe-mbi.ucla.edu/TB/

1036 Structure of AhpE from Mycobacterium tuberculosis

Among the defences of M. tuberculosis againstROS are the low molecular mass thiol compound,mycothiol, which substitutes in mycobacteria forglutathione,5 the catalase-peroxidase enzyme,KatG,4 and a variety of thioredoxin-relatedenzymes.6 Mutations of KatG provide resistance tothe frontline TB drug isoniazid, which requiresactivation by KatG. The suppression of KatGactivity leads, however, to the upregulation ofexpression of other components of the protectiveresponse, notably the alkylhydroperoxidase AhpC,7

a member of the thioredoxin superfamily.8 Theelucidation of the complete genome sequence ofM. tuberculosis strain H37Rv9 has led to the dis-covery of further genes whose gene products mayfunction in the detoxification of ROS. In addition tokatG and ahpC, open reading frames (ORFs) can befound encoding other proteins related to AhpC.These include Rv2238c, annotated as ahpE, Rv1932,a putative thiol peroxidase, Rv1608c (bcpB) andRv2521 (bcp), all with putative roles in theprotective response. Another ORF, Rv2429 (ahpD),is immediately adjacent to ahpC (Rv2428) andencodes a protein that has peroxidase activity butbelongs to a very different structural family.10

The ahpC gene is widespread in microorganisms,and its gene product AhpC belongs to the non-heme peroxiredoxin (Prx) family, whose membersare found in a wide range of biological systems,from bacteria to mammalian cells.11,12 The import-ance of the Prxs, throughout living systems, isunderlined by their extremely high abundance inboth prokaryotic and mammalian cells.12 In thelatter, Prxs have multiple roles including bothantioxidant activity and the regulation of H2O2-mediated signaling processes.12 In bacteria, Prxscan detoxify deleterious ROS, such as H2O2 andalkyl hydroperoxides, catalyzing their reduction towater and alcohols, respectively, as well as reactivenitrogen species such as peroxynitrite.13 Theactivity of M. tuberculosis AhpC against a broadrange of such substrates thus explains its ability tocompensate for the loss of catalase-peroxidaseactivity in M. tuberculosis katG mutants.7

All Prxs share a common reactive Cys residue intheir N-terminal region, which is oxidized byperoxides to a cysteine sulfenic acid intermediate.14

Depending on the presence or otherwise of otherconserved cysteine residues in the sequence, Prxproteins can then be divided into 2-Cys and 1-Cystypes.12 In 2-Cys Prxs, the oxidized cysteine sulfenicacid intermediate can be regenerated to the reducedstate by disulfide bond formation with the secondcysteine, followed by reduction by an externalsmall-molecule or protein reductant. The 2-CysPrxs have been further subdivided into typical oratypical types depending on whether inter-molecular or intramolecular disulfide bond for-mation occurs, respectively. The 1-Cys Prx proteinsare less well characterized, however, with three-dimensional structural information available foronly one family member, the human hORF6enzyme,15 to date. As in the 2-Cys Prxs, the active

cysteine undergoes oxidation to a sulfenic acid, butthe full catalytic mechanism of 1-Cys Prxs isunclear.

AhpE (Rv2238c) inM. tuberculosis is a homologueof AhpC with 34% sequence identity. The presenceof only one cysteine, Cys45, in the AhpE sequenceidentifies it as belonging to the 1-Cys class of Prxs,in contrast to AhpC which belongs to the 2-Cyssubgroup. Sequence alignment of AhpE with thehuman 1-Cys Prx, hORF6, showed that it ishomologous with this protein also, but withlower sequence identity (24%), and that it lacksthe C-terminal domain that mediates dimerizationof hORF6.15 Here, we describe the crystal structureof native AhpE from M. tuberculosis, at 1.87 Aresolution, together with a second AhpE structureobtained after soaking native crystals in sodiumbromide. The crystal structures reveal that theserepresent the oxidized and reduced forms of theenzyme, respectively, and identify a conformationalchange involving the closure of a surface channelwith the adoption of the reduced state. Thesestructural changes may be relevant to differentstages in the catalytic cycle.

Results

Structure determination and model quality

The ORF Rv2238c fromM. tuberculosiswas clonedby PCR from genomic DNA. Its gene product,AhpE, was then expressed, purified, and crystal-lized, both as the native protein and as itsselenomethionine (SeMet)-substituted derivative,using the high-throughput facilities of the Myco-bacterium tuberculosis Structural GenomicsConsortium†. The structure of AhpE was solvedby X-ray crystallography, using multiwavelengthanomalous diffraction (MAD) methods16 on theSeMet-substituted protein. The resulting structurewas then refined at 1.87 A resolution, to a finalcrystallographic R-factor of 0.179 and free R of 0.210(Table 1). Subsequent analysis of native crystals thathad been soaked in sodium bromide prior to datacollection gave a second structure in a different, butrelated, space-group, which was refined at 1.90 Aresolution (RcrystZ0.242, RfreeZ0.286; Table 1).

The final model for native oxidized AhpEcomprises 2473 protein atoms from the two inde-pendent molecules in the asymmetric unit of thecrystal, together with 185 water molecules (Table 1).The two molecules comprise the complete 153-residue polypeptide, together with six extraresidues from the linker to which the N-terminalpoly-histidine tag was attached. Both molecules arewell restrained to standard bond distances andangles, and their main chain torsion angles corre-spond well with expected values; 88.9% of non-glycine residues are in the most favored regions of

http://www.doe-mbi.ucla.edu/TB/

Table 1. Refinement statistics

Native Bromide-soaked

Resolution range (A) 50.0–1.87 40.0–1.90Number of reflections(working/test)

28,807/1828 56,673/2397

Rcryst/Rfree 0.179/0.210 0.242/0.286

Number of atoms (non-hydrogen)Protein atoms 2473 4760Water molecules 185 190

Average B (A2)Protein 23.7 22.3Water 47.4 50.7

rms deviations from idealityBonds (A) 0.012 0.012Angles (deg.) 1.58 1.61Residues in mostfavored region (%)

88.9 87.6

Structure of AhpE from Mycobacterium tuberculosis 1037

the Ramachandran plot, as defined inPROCHECK,17 with no outliers. The bromide-soaked reduced AhpE structure has four indepen-dent molecules in the crystal asymmetric unit, in adifferent space-group (P42, as opposed to I4).

Monomer structure

The AhpE monomer (Figure 1) is folded into acompact, spherical, single domain based on acentral mixed b-sheet of five strands, in the orderb5-b4-b3-b8-b9. One face of this central b-sheet hastwo helices (a2 and a5) packed against it, with theother face being covered by helix a4 and a bba

Figure 1. The AhpE monomer, shown as a ribbondiagram. Secondary structural elements are labeled, andthe N and C termini are indicated.

structure from the N-terminal part of the protein,comprising a b-hairpin (b1-b2) and following helix(a1). Another helix a3, which joins strands b4 andb5, is on the periphery of the monomer where itparticipates in oligomerization (see below). Otherfeatures are a short b-ribbon (strands b6 and b7) thatextends from the main body of the molecule, and anextended N-terminal region that packs alongsidestrand b9, making several hydrogen bonds with it.The strands b4-b3-b8-b9 and helices a2, a4, a5

make up the core thioredoxin fold, with the othersecondary structural elements being embellish-ments that are typical of the Prx family; theextended N-terminal region, which is antiparallelwith b9, appears to be unique to AhpE, however.The active site cysteine, Cys45, is located at the Nterminus of helix a2 from the central bab motif ofthe thioredoxin fold. This helix has a pronouncedkink near its C-terminal end, as in many Prxs. InAhpE, this kink is associated with a proline residue,Pro57, which disrupts the a-helix hydrogen bond-ing, although this proline is not conserved in otherPrxs.A search of the currently available protein

structures in the Protein Data Bank, using theprogram SSM† shows that the closest structuralhomologues are the tryparedoxin peroxidase (TryP)from Crithidia fasciculata18 (PDB accession code1E2Y), AhpC from Salmonella typhimurium8

(1KYG) and a putative thiol peroxidase fromStreptococcus pneumoniae (PDB accession code1PSQ). These are all 2-Cys Prxs that share 25–30%sequence identity with AhpE (Figure 2). Super-positions of AhpE on to the other Prxs show that140–145 residues can be matched with an rmsdifference of 1.4–1.5 A in Ca positions. Comparisonof AhpE with hORF6,15 the only 1-Cys Prx whosestructure has previously been available, shows asomewhat lower level of structural correspondence(136 Ca atoms can be matched with an rmsdifference of 1.8 A), probably reflecting the lowerlevel of sequence identity (24%) with this eukaryoticprotein. The major structural differences betweenPrxs of different sub-classes (1-Cys, typical 2-Cysand atypical 2-Cys) appear to depend primarily onthe appearance of extra domains or extensionsoutside the basic fold in some members. Forexample, whereas AhpE comprises a compactsingle domain, both the 1-Cys hORF615 and the2-Cys PrxII (HPB23)19 have a small additionalC-terminal domain that contributes to dimerization.

Oligomerization

Several 2-Cys (but to date no 1-Cys) Prxs areknown to form decamers (pentamers of dimers) inresponse to a variety of factors, including varyingionic strength, pH and redox state.8 Analysis of thecrystal packing of AhpE suggests the existence ofboth dimer and octamer (tetramer of dimers)

† http://www.ebi.ac.uk/msd-srv/ssm/

http://www.ebi.ac.uk/msd-srv/ssm/

Figure 2. Structure-based sequence alignment of AhpE with other Prxs. Sequences are given for tryparedoxinperoxidase (TryP), rat PrxI (also known as HPB23), S. typhimurium AhpC, human PrxV (also known as PRDX5), theputative thiol peroxidase from S. pneumoniae (THIOL) and the human 1-Cys Prx hORF6. The secondary structure andresidue numbering for AhpE are shown above its sequence, and the secondary structure found in the hORF6 structure isgiven below its sequence. Fully conserved residues (white letters on red background) and conservatively-substitutedresidues (red letters on white background) across this group of Prxs are indicated.


species. The two independent molecules in thecrystal asymmetric unit pack across a non-crystal-lographic 2-fold axis, forming a dimer (Figure 3(a))that buries a total of 1427 A2 of solvent accessiblesurface (713.5 A2 or 9.5% of the surface of eachmonomer). The main contributors to this interfaceare residues 39–42 from the b3-a2 loop, 72–76, 79and 83 from helix a3, 94–97 from the b5-a4 loop, and110–113 from the small b6-b7 hairpin loop. Analysiswith the protein–protein interaction server† showsthat the interface is mostly (70%) hydrophobic, withprincipal contributors being the side-chains ofLeu39, Phe41, Pro75, Ile79, Phe94, Trp95 andHis97 (Figure 3(a)). Comparisons with other Prxsshow that the dimerization interface in AhpE is the

† http://www.biochem.ucl.ac.uk/bsm/PP

same as is used in a hybrid PrxV from Haemophilusinfluenzae,20 and in the homologous humanPrxV.20–22 An equivalent interface is also used fordecamer formation of AhpC and other decamericPrxs (the reported oligomerization regions I–IV).8

Operation of the crystallographic 4-fold axis ofthe I4 space group generates an octamer, shown inFigure 3(b). Although the dimer–dimer interfacesare smaller (470 A2 per monomer), the cooperativityimplicit in the circular octamer suggests that thepacking in the crystal is a stable arrangement thatmodels the solution species. The same octamer isseen in the P42 space group of the bromide-soakedcrystals; in both cases the octameric ring has anouter diameter of about 115 A and its central hole adiameter of 55 A. This is very similar in itsdimensions to the decameric structures seen for

http://www.biochem.ucl.ac.uk/bsm/PP

Figure 3. Oligomerization of AhpE. (a) The AhpE dimer, characterized by mostly hydrophobic interactions across anon-crystallographic 2-fold axis. Prominent contributors to the interface are the two a3 helices and the two b6-b7 hairpinloops. Side-chains in the interface are shown in stick mode. (b) The AhpE octamer, formed by operation of thecrystallographic 4-fold axis, relating four dimers.


TryP, PrxI and AhpC, with outer diameter 115–120 A, inner diameter w60 A.12

Biophysical studies of AhpE confirmed thepresence of both dimers and octamers in solution,as implied by the crystal structure. Size exclusionchromatography gave two discrete peaks, a largepeak corresponding to an AhpE dimer preceded by

a small peak for the octamer (Figure 4). Bothfractions were monodisperse by dynamic lightscattering, with the octamer fraction having amolecular mass of 136 kDa and monodispersityindex Cp/RH of 24%, whereas the dimer fractioncorresponded to a molecular mass of 40 kDa andCp/RH of 17.2%.

Figure 4. Size exclusion chromatography elutionprofile, showing presence of octamer and dimer species.


Active site

The active site in AhpE is marked by the presenceof the single cysteine residue, Cys45, which islocated at the N terminus of helix a2 and identifiesthe protein as a 1-Cys Prx. The Cys45 side-chain sitsin a pocket whose inner surface is made up of theside-chains of Phe37, Pro38, Leu49 and Trp80.These hydrophobic residues limit the solventaccessibility and shield the reactive Cys45 fromfurther oxidation by peroxides. A shallow groove,about 12–15 A in length, and 7–8 A in width, leadsfrom the Cys45 pocket, and in the native structure isoccupied by a number of bound water molecules.The inner wall of this channel is formed by the side-chain of Pro38, the a4-b8 loop (residues 107–115)

Figure 5. AhpE surface in the vicinity of the active site. (asulfenic acid, Arg116 is directed away, and a large surface chamolecule B of the bromide-soaked structure, with Arg116 diredeep, positively charged cavity leading to Cys45.

and the side-chain of Met132, and the outer wall bythe side-chain of Ile44 and the solvent-exposed b9-a5 loop (residues 134–139). The shape of thischannel suggests that it could accommodate anextended substrate that passes between the side-chains of Met132 and Pro135 and enters the activesite cavity between Pro38 and Ile44.

Two arginine residues, Arg116 and Arg139,contribute to an overall positively charged environ-ment (Figure 5), as is characteristic of all Prxs. Theside-chain of Arg139 is directed towards Cys45such that the Cz atom of the guanidinium group is7.7 A away from Cys45 Sg, but the side-chain ofArg116, which forms the floor of the putativesubstrate channel, is directed away from Cys45,such that the distance between 116 Cz and 45 Sg is8.4 A. Both arginine residues make multiple hydro-gen bonds, notably with the side-chain of Glu48.

In the native AhpE structure, the electron densityshows that Cys45 has been oxidized to cysteine-sulfenic acid (Cys-SOH), as was also observed in the1-Cys Prx, hORF615. Electron density for the singleoxygen atomwas seen at a level ofO3s in an FoKFcdifference map, and refinement with a full-occupancy oxygen atom resulted in an oxygenB-factor that was very similar to those of the Cys45Cb and Sg atoms (in molecule A, B-factors are 23 A2,24 A2 and 28 A2 for Sg, Cb and Od, respectively, andin molecule B 24, 25 and 26 A2). The Cys45 side-chain also adopts a rotamer that directs Sg towardsthe hydrophobic back of the pocket, perhapsprotecting against further oxidation. Three watermolecules around the sulfenic acid oxygen, W43,W71,W105may further stabilize this oxidized form.

Structural differences in the bromide-soakedcrystals

In the bromide soaked AhpE crystal, the changein space group from I4 to P42 gives an asymmetric

) The oxidized AhpE structure, in which Cys45 forms annel approaches the active site. (b) Reduced AhpE, seen incted towards Cys45 Sg, and no surface channel except for a

Figure 6. Conformational change in AhpE. Stereo view showing the oxidized structure (pink) superimposed on thereduced structure (blue), represented by molecule B of the bromide-soaked structure. The movement of Arg116 and theb9-a5 loop in towards Cys45, closing the channel that exists in the oxidized form, together with the rotamer change inCys45, are apparent in the reduced form.


unit comprising four molecules (molecule A tomolecule D). The monomer structure is essentiallythe same as for the native crystals, with thedifference in space group appearing to originatefrom local conformational differences in two of thefour molecules (B and C). Molecules A and D can besuperimposed on to the native structure withan rms difference of 0.43 A for all Ca atoms(residues 1–153), but for molecules B and C therms difference is 0.78 A. The asymmetric unit thuscontains two dimers (AB and CD) in which the twomolecules are slightly different. In none of the fourmolecules, however, is there any evidence ofoxidation of the Cys45 side-chain, indicating thatthis crystal structure represents the reduced form ofAhpE.

The conformational change that occurs inmolecules B and C on bromide soaking, shown inFigure 6, involves an external loop, residues 133–139 (the b9-a5 loop), which moves w4 A inwardstowards the a4-b8 loop, completely closing theputative substrate channel seen in the nativestructure, and partly covering the side-chain ofArg116. Associated with this loop closure is a largeconformational change of the side-chain of Arg116,which moves inwards to interact with Cys45 Sg.These and other changes in the bromide-soakedcrystals relative to the oxidized native structurehave important implications for the active sitestructure and function, as described below.

Themovement of the side-chain of Arg116, linkedwith the movement of the b9-a5 loop in molecules Band C, dramatically changes the environment of the

catalytic cysteine, Cys45. In the native, oxidized,structure, and in molecules A and D, the guani-dinium group of Arg116 is oriented away fromCys45, with its Cz atom 8.5 A from Cys45 Sg, its Nh1

and Nh2 atoms hydrogen bonded to the peptideoxygen of Glu137 and Nh1 additionally hydrogenbonded to Glu48 O31. In molecules B and C,however, Arg116 flips inwards such that Nh1 andNh2 form a bifurcated hydrogen bonding inter-action (w3.5 A) with Cys45 Sg. This position isfurther stabilized by hydrogen bonds betweenArg116 Nh1 and Glu48 O32, Nh2 and Pro135 O,and N3 and Pro135 O. This position for Arg116parallels that of the equivalent Arg128 in reducedTryP18 and Arg127 in reduced PrxV (PRDX5).21 Theinteraction of this conserved arginine side-chain isbelieved to play a key role in lowering the pKa ofthe thiol group of the reactive cysteine, by stabiliz-ing its ionized state.21

Several other conformational changes are foundin the active site, in some or all of the molecules ofthe bromide-soaked structure. In three of the fourmolecules (B, C and D), the rotamer of the Cys45side-chain changes from c1w608, as in the native(oxidized) structure, to a value of wK608. Thismoves the sulfur atom further out of the active sitecleft, where it can interact with Arg116. In contrast,in molecule A, Cys45 has a c1 value of 588, like thatin the native, and the sulfur atom lies closer to thehydrophobic interior. In this position it hydrogenbonds with Thr42 Og1 (3.0 A) and the peptideoxygen of Leu39 (3.2 A); the latter interaction wouldbe consistent with a sulfur atom in its protonated


(reduced) state. The side-chain of Thr42 also under-goes a rotamer change to hydrogen bond to theCys45 sulfur.

Discussion

Both the sequence and structure of AhpE fromM. tuberculosis establish it as a typical member of theperoxiredoxin (Prx) family.12 All members of thisfamily have in common an active Cys residue (theperoxidatic cysteine CP) that attacks peroxidesubstrates and is itself oxidized to a sulfenic acidderivative, S–O–H. This residue, Cys45 in AhpE, isalways in the N-terminal portion of the protein, atthe N terminus of an a-helix which serves to lowerits pKa. A conserved arginine residue, Arg116 inAhpE, further lowers the pKa by its ability tostabilize the cysteine thiolate ion. The activecysteine can be regenerated by subsequentreduction of the sulfenic acid species; for the2-Cys Prxs this involves reaction with another thiolto form a disulfide intermediate, followed byreaction with one of a number of cell-specificdisulfide reductases (such as thioredoxin, AhpF,tryparedoxin or AhpD) to produce the thiol form ofthe Prx12. In the 2-Cys Prxs, the disulfide is formedwith a second cysteine (the resolving cysteine, CR)which is either provided by a C-terminal arm froman adjacent subunit (in the oligomeric typical 2-CysPrxs) or from within the same polypeptide (inatypical 2-Cys Prxs).12 Although AhpE is closelyrelated in sequence and structure to 2-Cys Prxs suchas AhpC and TryP, its single cysteine residuedefines it as a 1-Cys Prx, a class for which onlyone other representative, the eukaryotic hORF6, hasbeen structurally characterized.

Although the Prxs share the same monomer corestructure, they differ both in the presence orotherwise of N or C-terminal extensions, and intheir oligomerization behavior. Thus AhpE lacksthe C-terminal domain that contributes to thedimerization of the other 1-Cys Prx, hORF6,15 andit lacks the C-terminal arm that carries the resolvingcysteine in 2-Cys Prxs such as AhpC8 and PrxI19

and creates arm-swapped dimers. In contrast, AhpEhas a short N-terminal extension that runs anti-parallel with strand b9, hydrogen bonding to it, andthus preventing the use of this strand in dimeriza-tion, as occurs in hORF6, AhpC, PrxI and TryP. Bothsize-exclusion chromatography and dynamic lightscattering show, however, that AhpE forms a stabledimer. The crystal structure reveals that this isformed through a different interface from thatemployed by most Prx dimers. This interface is,however, emerging as a common oligomerizationsurface in Prxs, being used for dimerization ofPrxV20–22 as well as AhpE, and for dimer–dimerinteractions in AhpC and TryP decamers.8

A striking feature of the Prx family is the ability ofsome members to form large oligomeric assemblies,typically doughnut-shaped decamers with 52symmetry, such as are formed by AhpC8 and

TryP,18 the closest structural homologues of AhpE.Because AhpE forms a different dimer it cannotform an equivalent decamer. Nevertheless, both itssolution behavior and the crystal structure showthat AhpE forms an octamer, which in the crystalforms long tubes of stacked rings. Similar cylindersof stacked rings have been observed for PrxII, a2-Cys Prx,12,23 although the biological significanceof such assemblies, if any, is unknown.Many factorshave been suggested as stabilizing higher-order Prxoligomers, with emerging evidence favoring adependence on redox state. Biophysical and crystal-lographic studies of AhpC suggest that the reducedstate favors decamer formation with a key“molecular switch” being provided by the so-calledCP-loop, which in the reduced state provides thefirst turn of helix a2, carrying the peroxidaticcysteine, but which unwinds in the oxidized stateand destabilizes the decamer.8 Although AhpEforms a different oligomer, residues 39–42,immediately prior to the CP-loop, are part of thedimer interface and thus provide a mechanismthrough which changes in the active site couldinfluence the oligomer structure.

The differences seen between the native andbromide-soaked structures for AhpE provide someinteresting and unexpected insights into possiblestructure-activity relationships. In the native AhpEcrystals the peroxidatic cysteine, Cys45, is found tobe in its oxidized, sulfenic acid (S–O–H) form; this isshown convincingly by the electron density and byrefinement of the structure. There is no evidence offurther oxidation. A similar sulfenic acid form hasbeen found for hORF6,15 the only other 1-Cys Prx tohave been characterized structurally, and bio-chemical studies of a bovine 1-Cys Prx show thatin this class of Prx the sulfenic acid intermediateappears to be extremely stable.24 In this oxidized,sulfenic acid, form of AhpE, the channel that leadsto Cys45, between the b8-a5 and a4-b7 loops, is inan open state, about 7–8 A wide. This suggests thatthis channel could be the binding site for thereducing agent that regenerates the active cysteine.

In contrast, the bromide-soaked crystals appearto contain the reduced form of AhpE. There is noevidence of oxidation of Cys45 Sg in the electrondensity, and the hydrogen bonding interactionsmade by Sg imply the reduced state. In particular,the hydrogen bonding of the guanidinium group ofArg116 to the cysteine Sg in molecules B and C isconsistent with the latter being a thiolate ion.Concomitant with this, the channel describedabove is closed and Arg116 is buried. It seemsunlikely that the altered redox state of the bromide-soaked crystals is a result of increased ionicstrength. Instead, it probably derives from differenttreatment of the two batches of protein. Thebromide-soaked crystals were from protein thatwas flash-frozen immediately after purification,with only ten days elapsed between thawing theprotein and collecting the data; accordingly theseare still in the reduced state. On the other hand, theoxidized crystals were from protein that was kept


for two months, without flash-freezing, beforecrystallization. Presumably this gave time forcomplete oxidation of the sample.

What is the trigger for the conformational changearound the active site, and what is its significancefor the reaction cycle of AhpE? It seems probablethat it is a consequence of the reduced state of theprotein, with Arg116 moving in to interact with theactive site Cys45 thiolate ion, as is seen forequivalent arginine residues in the reduced formsof TryP18 and PrxV.21 The observation of thisconformational change for only two of the fourmolecules in the asymmetric unit, even though allfour appear to be reduced, results in two asym-metric dimers. In the AB dimer, only molecule Bshows the conformational change, and in the CDdimer it is molecule C that undergoes the sametransition. Whether this asymmetry results fromcrystal packing constraints or hints at somecooperativity we cannot say at this point. What isclear, however, is that the structural transition islikely to be important for AhpE function.

The physiologically relevant oxidants andreductants for AhpE are not known. We havedemonstrated that AhpE is readily oxidized byH2O2 (data not shown), but many oxidized lipidsand other small molecules are potential substrates.Molecules B and C of the bromide soaked structureprovide the best model for the reduced AhpEstructure, since Arg116 hydrogen bonds to thethiolate sulfur of Cys45, in an analogous interactionto that seen in the reduced states of TryP18 andPrxV.21 In this reduced structure, however, theabsence of any obvious surface cleft, other thanthe cavity in which Cys45 resides, means that thereis no strong indication as to the nature of thephysiological oxidant, or to its binding site. Incontrast, in the oxidized (sulfenic acid) form ofAhpE, the long surface channel that leads to Cys45is the probable binding site for the physiologicalreductant that forms a resolving disulfide bondwith Cys45. The cysteine-disaccharide conjugatemycothiol5 is one possibility as a small-moleculereductant, but a more likely candidate may comefrom among a number of thioredoxins (Trxs) thatare produced byM. tuberculosis; recent studies haveshown that two other Prxs, AhpC and the thiolperoxidase Rv1932, are reduced by Trxs.6

The reaction cycle for typical 2-Cys Prxs such asAhpC is believed to include a conformationalchange in the CP-loop that follows the formationof the sulfenic acid form, and allows the resolvingCys to form a disulfide bond.8,12 In AhpE, a 1-CysPrx, we also find a conformational change followingthe formation of this species, albeit of a differentnature; the movement of Arg116 away from Cys45Sg, and the associated loop movement, opens up achannel leading to the active site. We conclude thatthis is most likely related to the different reductantthat must be bound by AhpE. Finally, we do notknow the significance of the octamer found forAhpE but it parallels the decamers found for otherPrxs, and the involvement of the active site loop 39–

42 in the dimer interface suggests a mechanismthrough which the functional state could be linkedto oligomerization.

Materials and Methods

Protein expression and purification

The gene coding for Rv2238c was amplified by PCRfrom genomic DNA, and cloned into the expressionvector pProEX HTa (Life Technologies). This vector addsan N-terminal His6 tag and linker peptide to theexpressed protein. The plasmid was transformed intothe E. coli BL21(DE3)pRI expression strain, which con-tained the co-plasmid pRI952 to supplement the ileXisoleucyl and argU arginyl tRNAs.25 Expression of nativeRv2238c was induced at A600Z0.6–0.8 with 0.5 mMisopropyl-b-D-thiogalactopyranoside and incubation wascontinued overnight at 291 K. Cells were harvested bycentrifugation (SLC-4000 rotor, 6000g). For expression ofthe selenomethionine (SeMet)-substituted protein, theplasmid was transformed into the methionine-auxo-trophic E. coli strain BL41(DE3), cells were grown in 2 lof LeMaster medium supplemented with 0.5 mMSeMet,26 and the same expression protocol as above wasfollowed.Both native and SeMet-substituted proteins were

isolated with the same protocol, as follows. The cellswere resuspended in ice-cold lysis buffer (20 mM Hepes(pH 8.0), 150 mM NaCl, 10 mM imidazole). After lysiswith a cell disruptor, the cell debris was removed bycentrifugation (SS-34 rotor, 20,000g), and the supernatantwas filtered and applied to a Ni2C-resin column(Pharmacia Hi-Trap) pre-equilibrated with lysis buffer.The protein was eluted using a linear imidazole gradientfrom 0.01 M to 0.5 M. Fractions containing Rv2238c werepooled and dialyzed overnight against 20 mM Hepes(pH 8.0) containing 150 mM NaCl. The protein was thenconcentrated and loaded onto to a Superdex 75 gel-filtration column (Pharmacia) and was eluted as twodiscrete peaks. The fractions for each peak were pooledand concentrated to 10 mg mlK1. Both samples weremonitored by dynamic light scattering (Protein SolutionsDynapro) before use for crystallization experiments.

Crystallization

Crystals were grown in Intelli-Plate sitting drop vapordiffusion plates at 291 K by mixing 0.5 ml of the proteinsolution (w10 mg mlK1) with 0.5 ml of a well solutioncomposed of 1.8 M sodiummalonate (pH 5.0) mixed with0.1 M sodium acetate (pH 4.5). Both native and SeMetcrystals proved to be tetragonal, space group I4, with unitcell dimensions aZbZ148.0 A, cZ33.7 A (native) and aZbZ147.9 A, cZ33.6 A (SeMet). Assuming the presence oftwo AhpE molecules of w20 kDa (including the His-tag)in the asymmetric unit, the Matthews coefficient VM is2.3 A3 DaK1, corresponding to a solvent content of 47%.

Data collection

All X-ray data were collected at 113 K on beamline 8.2of the Advanced Light Source (Lawrence BerkeleyLaboratory, CA). Data were first collected for a nativecrystal and for a second native crystal that had beensoaked in 0.5 M sodium bromide. In the latter case, thecrystal was soaked in mother liquor supplemented with

Table 2. Data collection and processing and phasing statistics

Data set NativeNativeCNaBrsoak SeMet peak SeMet inflection SeMet remote

Wavelength (A) 1.00000 0.91983 0.97918 0.97937 0.95370Space group I4 P42 I4

Cell parametersaZb (A) 147.99 148.20 147.89c (A) 33.71 33.86 33.57

Resolution range (outer shells) (A) 50.0–1.87(1.94–1.87)

40.0–1.90(1.97–1.90)

50.0–1.97(2.05–1.97)

50.0–2.00(2.07–2.00)

50.0–2.18(2.26–2.18)

Total observations 140,994 243,533 202,461 178,074 148,829Unique reflection 33,679 59,088 26,275 25,039 21,627Completeness (%) 97.6 (97.8) 99.8 (99.5) 99.9 (99.7) 99.9 (99.4) 99.7 (99.9)I/s 17.9 (2.5) 16.2 (3.2) 14.9 (2.5) 17.5 (2.2) 14.4 (2.5)Rsym (%) 7.4 (48.2) 8.2 (43.1) 11.5 (62.5) 10.8 (69.0) 12.3 (67.1)

MAD phasingNo. of Se sites 3

Overall figure of meritSolve 0.38Resolve 0.86

† http://www.pymol.org


20% (v/v) glycerol, 0.5 M sodium bromide for twominutes, followed by five seconds back-soaking. Afterflash-freezing the crystal, data were collected at threewavelengths around the bromide edge. The bromide-soaked crystal proved to be tetragonal, with very similarunit cell dimensions to the native, but with an alteredspace group, P42, and four molecules in the asymmetricunit. The anomalous signal proved to be insufficient forphasing, however, and when the SeMet crystals becameavailable, multiwavelength anomalous X-ray diffractiondata were collected from these at three wavelengths(peak, inflection, remote) around the selenium edge. Allthe measurements were indexed, integrated and scaledwith the HKL package.27 Data collection and processingstatistics are given in Table 2.The native structure was solved by multiwavelength

anomalous diffraction (MAD) methods16 using three-wavelength data from the SeMet crystal. Three seleniumsites (from the four expected for two molecules) werefound using SOLVE,28 and were refined with SHARP.29

These gave an initial figure of merit of 0.38 and Z score of14.5. The SHARP map was subsequently input toRESOLVE30 for density modification and automaticmodel building. RESOLVE gave nearly a completemodel (148 and 99 residues modeled from a total 153for each of the twomolecules in the asymmetric unit). Thestarting model for refinement was then completed bymanual model building in O.31

Crystal structure refinement was performed usingCNS,32 with a randomly chosen subset of 5% of reflectionsfor the calculation of the free R-factor,33 as a monitor ofrefinement. Rigid-body refinement of the startingmodel, followed by an initial round of grouped,unrestrained B-factor refinement reduced the R-factor to0.416 (Rfree 0.393). The model was then refined usingiterative cycles of CNS conjugate gradient minimizationrefinement, restrained individual B-factor refinement,and simulated annealing refinement. The model qualitywas assessed at intervals with PROCHECK,17 and 2FoKFcand FoKFc maps provided the basis for further manualrebuilding using TURBO-FRODO.34 A number of resi-dues from the N-terminal His-tag linker could bemodeled for both molecules. A single large peak ofpositive density adjacent to Cys45 Sg was modeled as asulfenic acid oxygen attached with a S–O bond length of1.66 A. Ordered water molecules were added at locations

where FoKFc density exceeded C3.0s and potentialhydrogen-bonding contacts could be made with theneighboring structure. This model resulted in an R-factorof 0.179 (RfreeZ0.210).The bromide-soaked crystal structure was solved by

molecular replacement of the refined native structure intothe bromide-soaked crystal unit cell using CNS, andgave a starting model for the bromide-soaked structure.Initial rigid-body refinement, followed by grouped,unrestrained B-factor refinement reduced the R-factor to0.364 (Rfree 0.367). The bromide-soaked crystal structurerefinement followed the same course as the nativestructure refinement, except that the first round ofrefinement started with simulated annealing refinement,to reduce bias, followed by conjugate gradient minimiz-ation refinement and restrained individual B-factorrefinement. Full details are in Table 1.Figures were prepared with RIBBONS35 and PYMOL

(Delano, W. L. (2002)).†, with the electrostatic surfacescalculated with APBS.36 The sequence alignment(Figure 2) was prepared with ESPRIPT.37

Atomic coordinates

The crystallographic coordinates and X-ray structureamplitudes have been deposited in the RCSB Protein DataBank, with accession codes 1XVW and 1XXU for thenative and bromide-soaked AhpE structures,respectively.

Acknowledgements

This work was supported by the Foundation forResearch, Science and Technology of New Zealand(NERF grant UOAX0301), the Health ResearchCouncil of New Zealand and the U.S. NationalInstitutes of Health (grant P50-GM062410) underthe Protein Structure Initiative.

http://www.pymol.org


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Edited by I. Wilson

(Received 12 November 2004; received in revised form 15 December 2004; accepted 20 December 2004)

Documents

Crystal Structure of AhpE from Mycobacterium tuberculosis, a 1-Cys Peroxiredoxin