Structural and functional characterization of Schistosoma mansoni Thioredoxin

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ARTICLE in PROTEIN SCIENCE · JUNE 2011

Impact Factor: 2.85 · DOI: 10.1002/pro.634

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Giovanna Boumis Sapienza University of Rome 38 PUBLICATIONS 503 CITATIONS

SEE PROFILE Francesco Angelucci Università degli Studi dell'Aquila 44 PUBLICATIONS 482 CITATIONS

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Adriana Erica Miele

Sapienza University of Rome

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Available from: Giovanna Boumis

Retrieved on: 02 November 2015

PROTEIN STRUCTURE REPORT

Structural and functional characterization of Schistosoma mansoni Thioredoxin

Giovanna Boumis,Francesco Angelucci,Andrea Bellelli,Maurizio Brunori, Daniela Dimastrogiovanni,and Adriana E.Miele*

Dipartimento di Scienze Biochimiche and Istituto Pasteur—Fondazione Cenci Bolognetti,‘‘Sapienza’’University of Rome,

P.le Aldo Moro5,00185Rome,Italy

Received1February2011;Revised18March2011;Accepted22March2011

DOI:10.1002/pro.634

Published https://www.wendangku.net/doc/f812796545.html,

Abstract:Schistosomiasis,the human parasitosis caused by various species of the blood-fluke Schistosoma,is a debilitating disease affecting200million people in tropical areas.The massive administration of the only effective drug,praziquantel,leads to the appearance of less sensitive parasite strains,thus,making urgent the search for new therapeutic approaches and new suitable targets.The thiol-mediated detoxification pathway has been identified as a promising target,being essential during all the parasite developmental stages and sufficiently different from the host counterpart.As a part of a project aimed at the structural characterization of all the proteins involved in this pathway,we describe hereby the high-resolution crystal structure of Schistosoma mansoni Thioredoxin(SmTrx)in three states,namely:the wild-type oxidized adult enzyme and the oxidized and reduced forms of a juvenile isoform,carrying an N-terminal extension.SmTrx shows

a typical thioredoxin fold,highly similar to the other components of the superfamily.Although probably unlikely to be a reasonable drug target given its high similarity with the human counterpart,SmTrx completes the characterization of the whole set of thiol-mediated

detoxification pathway components.Moreover,it can reduce oxidized glutathione and is one of the few defence proteins expressed in mature eggs and in the hatch fluid,thus confirming an

important role in the parasite.We believe its crystal structure may provide clues for the formation

of granulomas and the pathogenesis of the chronic disease.

Keywords:schistosomiasis;structural genomics;X-ray crystallography;thioredoxin;detoxification metabolism

Abbreviations:DNase,deoxyribonuclease;DTT,dithiothreitol;EcTrx,Escherichia coli thioredoxin;GSH,glutathione;GSSG,oxidized glutathione;HsTrx,Homo sapiens thioedoxin;IPTG,isopropyl b-D-1-thiogalactopyranoside;K3E-SmTrx,Lys3Glu-mutated isoform of juvenile thioredoxin from Schistosoma mansoni;NEM,N-ethylmaleimide;PEG,polyethylene glycol;PMSF,phenyl-methane-sulfonyl-fluoride;ROS,reactive oxygen species;TCEP,tris(2-carboxyethyl)phosphine;Trx,thioredoxin;WHO,World Health Organization;wt-SmTrx,wild-type thioredoxin from Schistosoma mansoni.;b-ME,b-MercaptoEthanol

Grant sponsor:‘‘Sapienza’’University of Rome;Grant sponsor:MIUR of Italy;Grant number:FIRB RBRN07BMCT_007;Grant sponsor:Fondazione Roma;Grant number:Rational approach to the specific inhibition of Plasmodium falciparum and Schistosoma mansoni;Grant sponsor:EU FP7;Grant number:226716.

*Correspondence to:Adriana E.Miele,PhD,Dept.Biochemical Sciences,‘‘Sapienza’’University of Rome,P.le Aldo Moro5,00185 Rome,Italy.E-mail:adriana.miele@uniroma1.it

Published by Wiley-Blackwell.V C2011The Protein Society PROTEIN SCIENCE2011VOL20:1069—10761069

Introduction

Neglected tropical diseases are a major threat to human health.Under WHO auspices some academic research groups and governmental agencies have undergone systematic biological and epidemiological studies of the causative agents of these diseases.The research group at the‘‘Sapienza’’University of Rome has chosen a focussed structural genomics approach to find suitable drug targets against Schistosoma mansoni,a blood fluke,which is endemic in75tropi-cal and subtropical countries.The pathway we decided to tackle is the thiol-mediated detoxification one,in which electrons flow from NADPH through several redox proteins to finally reduce hydrogen and organic peroxides.In schistosomes,this metabolic pathway is peculiar because the two parallel path-ways present in mammals,based on Thioredoxin (Trx)and on glutathione(GSH)are condensed in one single enzyme called thioredoxin glutathione reduc-tase(TGR).1This feature combined with the fact that all proteins belonging to this pathway are expressed throughout all life stages of schistosomes2makes them appealing structural targets that our group has been characterizing in the last years.3–8

Thioredoxins(Trx)are a family of small(about 12kDa)proteins sharing a conserved catalytic site (WCGPC),which undergoes reversible oxidation to cystine disulfide(Trx-S2)through the transfer of reducing equivalents from the two catalytic Cys usu-ally to a disulfide substrate(X-S2).In mammals,oxi-dized Trx is then reduced back to the Cys form[Trx-(SH)2]by the NADPH-dependent flavoprotein thiore-doxin reductase,9whereas in schistosomes,the same reaction is undertaken by the peculiar TGR enzyme.1 In addition to their antioxidant properties,Trxs are involved in a variety of cellular redox reactions, ranging from protein folding to transcription regula-tion,as well as being growth factors for a variety of cells(reviewed in Ref.9).

Here,we present the work carried out on two isoforms of SmTrx,which we have cloned in E.coli, expressed in high yield,purified,and functionally and structurally characterized.

Results

Cloning,expression,and purification of SmTrx According to its latest release,the complete sequence of S.mansoni genome contains one gene for Trx,whose construct is processed differently in the juvenile and in the adult stages.In the former, an extra N-terminal sequence of four amino acids, namely QLVI,is present before the first Met residue. These amino acids are absent in the adult form.

Herein,we shall refer to the adult isoform as wt-SmTrx,and to the juvenile one as K3E-SmTrx, because the gene originally fished out from a cDNA library contained the above-mentioned residues and a mutation leading to a substitution in positiont3 from Lys to Glu.Both proteins,expressed in good yield in E.coli(15mg/L culture),were purified to homogeneity(SDS-PAGE not shown),tested for in vitro activity and crystallized at high resolution.

Structural characterization

The structure of oxidized wt-SmTrx was solved at 1.6A?.It crystallized in space group P212121,with one molecule per asymmetric unit.Crystals of K3E-SmTrx were obtained both in the oxidized and reduced forms.K3E-SmTrx ox structure was solved at 1.56A?resolution;it crystallized in space group P212121,with one molecule per asymmetric unit. K3E-SmTrx red crystals diffracted at1.67A?in space group P32;this time,two molecules were found in the asymmetric unit.The statistics of the diffraction and refinement data for the three structures are summarized in Table I.The coordinates and the structure factors of wt-SmTrx,K3E-SmTrx ox,and K3E-SmTrx red have been deposited in the Protein Data Bank and assigned the following accession numbers:2xbi,2xc2,2xbq.

The structure of the oxidized wt-SmTrx is shown in Figure1.The map gave the possibility to fit residues fromà1to106.The two additional resi-dues before M1(G-1and S0)belong to the thrombin cleavage site of the expression vector.wt-SmTrx con-served the typical thioredoxin fold,made up of a five-stranded b sheet(b1-5)capped on each side by two a helices(a1and3and a2and4).The sequence identity between SmTrx and those from other organ-isms ranges between45%and65%.2In particular, the identity between SmTrx and human Trx(HsTrx) is47%[Fig.2(B)].The conserved active site amino acids W33–C34–G35–P36–C37link the strand b2to helix a2and are arranged in a b-turn shape.The re-dox site,formed by C34and C37,is oxidized,the dis-tance between the two sulphurs being2.1A?.This cystine pair is shielded from the solvent by the C-ter-minal portion of helix a2,by the loop between a3and b4,and by the active site loop itself.As a result of this architecture,C34acts as the nucleophilic Cys to-ward its substrates,being more exposed to the sol-vent,whereas C37is buried and could act as the resolving one.A conserved Asp residue(D28)with a H-bonded water molecule(D28(OD1)A O? 2.7A?) responsible for the deprotonation of the resolving Cys during the redox cycle10is present(Fig.1).

Unlike many other eukaryotic Trxs(such as human,Plasmodium falciparum and Drosophila melanogaster),SmTrx has no Cys residues other than those of the active site.2Side chains around the active site,including their rotamers and electro-static potential,are conserved between wt-SmTrx and HsTrx[highlighted in green in Fig.2(A,B)].A few differences can be located in the second sphere residues,including the additional C69and C73in

https://www.wendangku.net/doc/f812796545.html, Crystal Structure of Reduced and Oxidized Smtrx

HsTrx,replaced by Y70and A74in wt-SmTrx,and a few charged residues[highlighted in grey in Fig. 2(A,B)].We cannot assess whether these differences might be enough to account for any selectivity between the two proteins within their multiple func-tions.Nevertheless,we have proven that SmTrx is indeed a substrate of human Trx-Reductase(see later).

The two oxidized structures,wt-SmTrx and K3E-SmTrx ox,are completely superimposable[r.m.s.d. overall0.33A?calculated with SSM11;Fig.2(C)],the main differences being confined to the N-terminal, encompassing the extra residues and the mutation K3E.This region is floating into the solvent,on the opposite side of the molecule with respect to the active site.Moreover,K3E-SmTrx was found to have the same enzymatic properties as wt-SmTrx(see later). We have no obvious explanation for the presence of the acidic mutation in the cDNA prepared from worm extract,nor for its in vivo role,if any.

The Trx fold is very rigid and the only detecta-ble movements on reduction(overall rmsd?0.61A?between K3E-SmTrx ox and K3E-SmTrx red)are lim-ited to a rotamer change in the Cys couple and to a slight opening of the active site stretch W33-K38[Fig.2(D)],besides the differences in the C-terminal stretch due to crystallographic packing.

Functional characterization:Insulin reduction SmTrx can reduce insulin if maintained in the reduced state by the addition of DTT.The activity of wt-SmTrx and K3E-SmTrx was assessed by record-ing the start of insulin precipitation and its rate(see Methods).In the presence of SmTrx,the start time was100s and the rate(D A650minà1)was0.35.The activity of SmTrx is thus comparable with that of HsTrx(80s and0.41D A650minà1)and slightly higher than that of EcTrx(240sec and0.28D A650 minà1)assayed under the same conditions.

When GSH was used as reducing agent,insulin reduction activity was lower than in the presence of DTT.Also in this case,both schistosome enzymes are slightly more efficient than HsTrx and EcTrx(start-ing times1100,1080,1020,and1440s and rates 0.09,0.10,0.06,and0.07D A650minà1for wt-SmTrx, K3E-SmTrx,HsTrx,and EcTrx,respectively).

Functional characterization:GSSG reduction

In the presence of NADPH and human Trx Reduc-tase,wt-SmTrx,but not EcTrx,is able to reduce

Table I.Summary of Data Collection and Refinement Statistics

wt-SmTrx(2xbi)K3E-SmTrx ox(2xc2)K3E-SmTrx red(2xbq) Data Collection statistics

Space Group P212121P212121P32

Cell dimensions

a,b,c(A?)39.2,46.8,56.433.9,52.0,59.362.1,62.1,58.3 a,b,c( )90,90,9090,90,9090,90,120 Resolution in A?(last shell range)36.00–1.60(1.64–1.60)39.00–1.56(1.60–1.56)53.80–1.67(1.71–1.67) R merge(last shell value)0.11(0.49)0.06(0.18)0.10(0.54)

I/r I(last shell)14.6(4.7)26.9(5.1)15.2(2.2)

%Completeness(last shell)99.8(99.5)100(96.5)99.9(99.3) Redundancy(last shell) 6.9(6.9)10.1(9.2) 5.7(5.7) Refinement statistics

Resolution in A?20.00–1.6020.00–1.5625.0–1.67 Number of reflections134871428127704

R work0.190.170.18

R free0.240.220.21

No.molecules in asymmetric unit112

Total number of atoms9599971852

Protein853*******

Ions622

Water100107137

B factor analysis(A?2)

B Wilson9.418.619.9

Overall10.218.617.1

Protein8.917.215.4

Ions23.426.017.5

Water21.429.728.5

R.M.S.deviations

bond lengths(A?)0.0160.0120.009

bond angles( ) 1.41 1.46 1.32 Ramachandran plot%of residues in

Most favored regions96.797.996.3 Additionally allowed 3.3 2.1 3.7 Generously allowed000 Disallowed000 Boumis et al.PROTEIN SCIENCE VOL20:1069—10761071

oxidized glutathione.The steady state parameters for the reduction of GSSG were measured as:k cat ?0.085s à1and K m ?253.5l M for SmTrx.These val-ues are comparable with those obtained for HsTrx (k cat ?0.115s à1and K m ?202.1l M ).

Functional characterization:Interaction with SmTGR

By the effective reduction of insulin in the presence of NADPH and SmTGR,we were able to assess the functional interaction between the two natural part-ners.Both wt-SmTrx and K3E-SmTrx proved to be fully functional.

Functional characterization:p K a determination of the active site Cysteines

The p K a values of the nucleophilic C34and the bur-ied C37of wt-SmTrx were determined by pH titra-tion at 240nm.Generally,the p K a of Cys is about 8.7.The pH titration yielded a first p K a ? 6.5,which was attributed to the solvent-exposed C34,by analogy with other known Trx.A second,less evi-dent,p K a %9was attributed to C37,which is buried in the structure.

Discussion

In the quest for novel drugs to combat schistosomia-sis as well as other parasitic diseases,the biochemi-cal approach is to identify a suitable macromolecular target.This implies a deep knowledge of the biology of the schistosome parasite,which is able of digene-sis,because it enters the human body as monomor-

phic nonfeeding cercaria and then develops into a sex-differentiated blood-feeding worm.To be effective against the juvenile and the adult forms,a drug needs to target proteins,which are expressed by all life cycles and are crucial to the survival in the host blood stream.In this respect,undertaking a system-atic study of the thiol-mediated detoxifying meta-bolic pathway makes sense,because all the proteins involved are expressed in high yield from cercaria to the adult stage.This is a necessary but not sufficient condition to succeed,because the parasite target(s)ought to be significantly different from the human ortholog(s),to avoid cross-inhibition leading to severe side effects.

The thiol-mediated detoxifying system under study has been validated as a good target,but in refining the search to a few candidates,we had to exclude SmTrx,because its physical–chemical prop-erties do not significantly differ from HsTrx.This result is not surprising given that this small protein is multitasking,acting as a potent antioxidant,as redox regulator in signal transduction,and as elec-tron donor during DNA synthesis.Moreover,it is present and conserved in all species from Archebac-teria to mammals.Not only the structural features are conserved but also the structure and stereo-chemistry around the active site,which is designed to fit different proteins,do not change during the redox cycle.

Despite its limited usefulness in drug targeting,SmTrx structural and functional studies are impor-tant to complete the knowledge of Schistosoma

redox

Figure 1.Stereo ribbon representation of the overall fold of oxidized wt-SmTrx.Residues of the active site (Asp28,Trp33,Cys34,and Cys37)are in stick representation,together with the water molecule putatively involved in catalysis (see text).The electron density 2F o –F c contoured at 1.5r is also shown for the same residues.An interactive view is available in the electronic version of the article.

https://www.wendangku.net/doc/f812796545.html,

Crystal Structure of Reduced and Oxidized Smtrx

metabolism.We have also performed in vitro experi-ments of SmTrx reduction by the endogenous reduc-tase SmTGR,confirming the full functionality of the recombinant enzyme not only toward small molecules (DTT,insulin,and GSSG)but also toward macromo-lecular ligands.We used two SmTGR isoforms,a truncated one lacking the two C-terminal Sec-Gly residues,6and a full-length Sec597Cys mutant.7The electron transfer from NADPH to insulin through TGR and Trx was efficient with the full-length reduc-tase but not with the truncated one,thus demonstrat-ing the role of TGR C-terminus in Trx reduction.

The observed ability of SmTrx to reduce GSSG may have a great importance in the worm survival under high oxidative stress,also in view of the rela-tively low efficiency versus glutathione of the worm’s TGR.This additional way to lower the GSSG/GSH ratio is active in other parasitic platyhelminths 12and protozoa such as Plasmodium falciparum 13that have to fight the host immune system attack

but

Figure 2.Structure comparison.Panel A:Electrostatic surface of wt-SmTrx (left)and HsTrx (1ERT )(right).The view is rotated 90 on x -axis from Fig.1,looking at the molecule through the active site.Side chains of the active site are shown as green sticks,residues of the second sphere are in grey.Panel B:Sequence alignment between wt-SmTrx (SCHMA)and HsTrx (HUMAN).The residues shown in Panel A are highlighted with the same colour scheme.Stars represent identity,colons

represent high similarity;dots represent low similarity.Panel C:Superposition of oxidized wt-SmTrx (blue)and oxidized K3E-SmTrx (cyan).Panel D:Enlarged view of the active site of superposed oxidized (cyan)and reduced (magenta)K3E-SmTrx.An interactive view is available in the electronic version of the article.

Boumis et al.

PROTEIN SCIENCE VOL 20:1069—10761073

has also a fundamental role in insects,such as Dro-sophila melanogaster,Anopheles gambiae,and Apis mellifera,13–15that lack glutathione reductase and have a tracheal respiratory system highly exposed to ROS.Interestingly,free living,not parasitic platy-helminths maintain the two typical separate path-ways for thioredoxin and glutathione reductions.12

A further interesting aspect of SmTrx lies in its presence in the mature egg secretory products.2It is well known that eggs are mainly responsible for the pathology correlated to schistosomiasis,because they elicit the formation of granulomas in liver and blad-der.Moreover,anti-Trx antibodies are able to elicit circumoval precipitin reaction,2which is still consid-ered a useful diagnostic test for schistosomiasis, being highly specific and sensitive.In summary, even though SmTrx cannot be exploited as a drug target,its functional and structural characteriza-tions may help to design more specific antibodies to develop new diagnostic tests and to better under-stand the interplay of the enzymes belonging to the peculiar redox pathway of the parasite.

Materials and Methods

Cloning,expression,and purification of SmTrx SmTrx gene(Accession number AF473536)was ini-tially amplified from S.mansoni cDNA prepared from a30RACE(by courtesy of Dr Cristiana Valle, CNR-IBC Monterotondo).The gene was then cloned into pGEX-4T-1(GE Healthcare)expression vector via EcoRI and XhoI restriction sites.After sequenc-ing,we found that the amplified protein contained four extra amino acids at the N-terminus(QLVI), which belong to the deposited preprotein,typical of schistosomula and cercaria larval stages(EST from GenBank AM043494).Moreover,a single-base muta-tion occurred in the DNA,and Lys3was replaced by Glu.Hence,the N-terminal sequence of this prepro-tein isoform of SmTrx is QLVIMSE.In addition,the expressed protein has seven extra residues,due to the restriction sites used and the thrombin cleavage site.The resulting N-terminal arm(fromà10tot3) is the following:GSPEFTSQLVIMSE,and we shall refer to this long construct as K3E-SmTrx.The pro-tein was expressed fused to GST-tag in BL21(DE3) bacterial cells on induction with0.3m M IPTG,incu-bating overnight at20 C.Successful expression of soluble K3E-SmTrx was confirmed by SDS-PAGE, highlighting a band at$40kDa.Cells were lysed by sonication in20m M Tris/HCl pH7.4,0.2M NaCl buffer,on addition of10m M beta-mercaptoethanol, 0.1%Triton-X100,3U DNase,1m M PMSF,and1 m M EDTA.Protein was purified from the soluble fraction by affinity chromatography on glutathione sepharose(GE Healthcare);the GST tag was cleaved by thrombin(Sigma-Aldrich),which was finally removed by elution on a1mL benzamidine FF(HS)column(GE Healthcare).K3E-SmTrx was exchanged into crystallization buffer[20m M Tris/HCl(pH7.4), 5m M b-ME,and50m M NaCl],concentrated to15 mg/mL by ultrafiltration(Amicon,Millipore),ali-quoted,and stored atà20 C.

To avoid the extra amino acids due to the plas-mid and to the preprotein and to eliminate the mutation,we decided to clone wt-SmTrx between BamHI and XhoI sites of pGEX-4T-1,using proper PCR primers.The protein was successfully expressed and purified using the same protocol as for the mutant,and it was used for functional and structural experiments.

Crystallization of SmTrx

Crystals of K3E-SmTrx and wt-SmTrx were grown by vapour diffusion according to standard hanging drop methods.Crystals of reduced K3E preprotein (K3E-Trx red)grew over3days in a drop composed by1l L protein(15mg/mL)and1l L well solution (24%(w/v)PEG8000,5m M zinc acetate,5mM TCEP,and0.1M sodium cacodylate,pH 6.5).The oxidized form(K3E-Trx ox)was obtained in the same manner,with the addition of10m M zinc acetate to the well solution in the absence of TCEP.

wt-SmTrx crystals grew in0.1M BisTris,pH6.5, 45%polypropylene glycol P400.We were unable to obtain crystals of wt-SmTrx more that20%reduced, despite the use of TCEP,b-ME,and N-ethymalei-mide(NEM).

Data collection,processing,and refinement Diffraction data of SmTrx were collected at ESRF (Grenoble,France)and ELETTRA(Trieste,Italy) synchrotrons.Data were indexed with Mosflm and processed with programs of the CCP4Suite.16Crys-tals of K3E-SmTrx ox diffracted to 1.56A?,those of K3E-SmTrx red diffracted to1.67A?,and those of oxi-dized wt-SmTrx to 1.6A?.All the structures were solved by molecular replacement.In the case of K3E-SmTrx ox,the structure of D.melanogaster Trx(52% identity—PDB entry:1XWA10)was taken as a model. In the other two cases,we used K3E-SmTrx ox(with-out the extra N-terminal residues and the mutation) as a starting model.The structures were refined using REFMAC517and fitted to generated electron density maps by Coot.18The quality of the model was assessed with MolProbity19and ProCheck.20Data col-lection and refinement statistics are summarized in Table I.Figures were prepared with PyMOL(http:// https://www.wendangku.net/doc/f812796545.html,)and CCP4MG.21The alignment in the figure was made with ClustalW2.22

Insulin reduction assay

A standard turbidimetric assay was performed.Insu-lin solution was prepared according to Holmgren23 by dissolution of insulin in Tris/HCl buffer pH8, acidification to pH2–3and rapid titration of the

https://www.wendangku.net/doc/f812796545.html, Crystal Structure of Reduced and Oxidized Smtrx

solution back to pH8.This procedure leads to the cleavage of the polypeptide into two chains,linked by a disulfide.The Trx-catalyzed reduction of the S A S bond causes the separation of the two insulin chains and their precipitation,with consequent increasing of turbidity that can be followed spectro-photometrically at650nm.The assay mixture con-tained0.1M potassium phosphate buffer pH7.0, 1m M EDTA,130l M insulin,500l M dithiothreitol (DTT)and varying amounts of each protein:wt-SmTrx,K3E-SmTrx,EcTrx(SIGMA-Aldrich)and HsTrx(SIGMA-Aldrich).The baseline control reac-tion contained insulin and DTT but lacked Trx.The assay was performed also with GSH instead of DTT as reducing agent.

GSSG reduction assay

Rate constants were determined using an enzymatic Trx ox reducing system and subsequently transforming it to a GSSG reducing system.13In a cuvette contain-ing2m M EDTA,100l M NADPH,and100n M human Trx Reductase(SIGMA-Aldrich),GSSG was added at various concentrations(100l M to1m M). Reaction was started by the addition of6l M SmTrx ox, and NADPH consumption was followed at340nm. The rate represents a reducing flux from NADPH to Trx reductase to Trx,leading to GSSG reduction.

Functional interaction with SmTGR

The functional interaction of SmTrx with its endoge-nous reductase,SmTGR,was assessed by a coupled reaction between NADPH,TGR,Trx,and insulin. The mixture contained0.1M potassium phosphate pH 7.0,1m M EDTA,130m M insulin,300l M NADPH, and8l M SmTrx ox.The reaction was started by the addition of1.6l M SmTGR and was followed both at 340and650nm,to monitor NADPH consumption and insulin turbidity,respectively.

Determination of active site Cys p K a

The p K a values of the nucleophilic Cys34and the buried Cys37were determined by pH titration at 240nm,because the thiolate ion has a higher absorption at this wavelength than the thiol group.24 Spectra of oxidized and reduced Trx were recorded between200and400nm at25 C in1.3mL of1m M citrate,1m M borate,and1m M phosphate,0.2M KCl,pH 5.1,purged with nitrogen.The pH was adjusted from pH5.1to10.5by adding known vol-umes of either0.2M or0.1M KOH.The spectra were measured against air in a sealed quartz cuvette in a spectrophotometer.The spectrum of the buffer recorded in the same cuvette was subtracted from the spectrum of the respective protein solution.The absorbance was converted into molar extinction coef-ficients(e?5200Mà1cmà1)and corrected for the dilution due to pH adjustment.The protein concen-tration was calculated from absorbance at280nm.Acknowledgment

The authors thank Dr.Cristiana Valle(IBC CNR, Monterotondo,Italy)for supplying the cDNA and Dr. Louise J.Gourlay for cloning K3ESmTrx;ESRF, ELETTRA,BESSYII synchrotron facilities for having granted beam time to the project.FA is a fellow of Fon-dazione Roma.

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