Three Dimensional Structure and Implications for the Catalytic Mechanism of...

doi:10.1016/j.jmb.2006.11.063 J. Mol. Biol. (2007) 366, 868–881

Three Dimensional Structure and Implications for theCatalytic Mechanism of 6-Phosphogluconolactonasefrom Trypanosoma brucei

Marc Delarue1⁎, Nathalie Duclert-Savatier2, Emeric Miclet3

Ahmed Haouz4, David Giganti2, Jamal Ouazzani5, Philippe Lopez5

Michael Nilges2 and Véronique Stoven6⁎
1Unité de DynamiqueStructurale des Macromolécules,CNRS URA 2185, InstitutPasteur, 25 rue du DocteurRoux, 75015 Paris, France2Unité de BioinformatiqueStructurale, CNRS URA 2185,Institut Pasteur, 25 rue duDocteur Roux, 75015 Paris,France3FR 2769, UMR 7613,Université Pierre et MarieCurie, 4 place Jussieu, 75252Paris cedex 5, France4Plateforme de cristallogénèse,Institut Pasteur, 25 rue duDocteur Roux, 75015 Paris,France5Institut de Chimie desSubstances Naturelles,Avenue de la Terrasse, 91198Gif-sur-Yvette cedex, France6Centre de Bioinformatique,Ecole des Mines de Paris, 33 rueSaint Honoré, 77305Fontainebleau cedex, France
Abbreviation used: PPP, pentoseE-mail addresses of the correspon

[email protected]; veronique

0022-2836/$ - see front matter © 2006 E

Enzymes from the pentose phosphate pathway (PPP) are potential drugtargets for the development of new drugs against Trypanosoma brucei, thecausative agent of African sleeping disease: for instance, the 6-phosphoglu-conate dehydrogenase is currently studied actively for such purposes.Structural and functional studies are necessary to better characterize theassociated enzymes and compare them to their human homologues, inorder to undertake structure-based drug design studies on such targets. Inthis context, the crystal structure of 6-phosphogluconolactonase (6PGL)from T. brucei, the second enzyme from PPP, was determined at 2.1 Åresolution. Comparison of its sequence and structure to other relatedproteins in the 6PGL family with a known structure (Thermotoga maritimaTm6GPL 1PBT and Vibrio cholerae Vc6PGL (1Y89), which have not beendiscussed in print), or in the glucosamine-6-phosphate-deaminase family(hexameric Escherichia coli 1DEA and monomeric Bacillus subtilis 2BKV),allowed the identification of the 6PGL active site. In addition to the analysisof the crystal structure, 3D NMR interaction studies and dockingexperiments are reported here. Key residues involved in substrate bindingor in catalysis were identified.

© 2006 Elsevier Ltd. All rights reserved.

Keywords: 6-phosphogluconolactonase; Trypanosoma brucei; pentose phos-phate pathway; X-ray structure; NMR, docking
*Corresponding authors
Introduction

Glucose metabolism in Trypanosoma brucei

Glucose is metabolised in the cell either byglycolysis or by the pentose phosphate pathway

phosphate pathway.ding authors:[email protected]

lsevier Ltd. All rights reserve

(PPP). Glycolysis provides pyruvate and energy bygeneration of ATP, while PPP provides reductivepower by generation of NADPH through three steps,converting glucose-6-phosphate (G-6-P, compound 1in Figure 1(a)) into ribulose-5-phosphate (Ru-5-P,compound 4 in Figure 1(a)). Ru-5-P is later used inthe non-oxidative branch of PPP for the synthesis ofother sugar derivatives, in particular for nucleotidebiosynthesis. Trypanosomes and Leishmania havedeveloped particular sub-cellular compartments,glycosomes, where the first seven steps of glycolysis,

d.

mailto:[email protected]

mailto:[email protected]

http://dx.doi.org/10.1016/j.jmb.2006.11.063

Figure 1. Enzymatic reactions. (a) The first three steps of the pentose phosphate pathway (PPP), corresponding to theoxidative branch of the PPP. The three enzymes are glucose-6-phosphate dehydrogenase (G6PDH: G-6-P → δ-6-P-G-L),6-phosphogluconolactonase (6PGL: δ-6-P-G-L→ 6-P-G-A) whose structure andmechanism is discussed in this study, and6-phosphogluconate dehydrogenase (6PGDH: 6-P-G-A→ Ru-5-P). (b) Deamination reaction of glucosamine-6-phosphate(compound 1′) into fructose-6-phosphate (compound 2′) catalyzed by NagB enzymes.

869Structure and Mechanism of 6PGL

and the first three steps of PPP take place.1 Such anunusual localization of these major pathways insidea specific organelle has endowed the parasiticenzymes with distinct properties compared to thoseof their human homologues located in the cytosol.Therefore, current research in the development ofnew drugs for this re-emerging disease includesevaluation of enzymes of the carbohydrate metabo-lism as potential drug targets. Indeed, rational drugdesign relies on the specific features of the parasiticenzymes in order to identify selective inhibitors withlimited effect on human homologues.2

Glycolysis has been investigated extensively in thatcontext, and it has been shown that several of itsassociated enzymes are good targets for drugdevelopment.3 The rationale of this strategy is thatthe bloodstream form of Trypanosoma brucei isabsolutely dependent upon glycolysis for ATPsynthesis.1 In contrast, a limited number of studiesare available on enzymes of the PPP, for parasites andfor mammalian homologues.4 Nevertheless, thispathway is of particular importance for these para-sites, since the NADPH generated serves as ahydrogen donor in various biosynthetic processes,and has an important role in the defense against theoxidative attack by the infected host. In this context,the crystal structure of 6-phosphogluconate dehydro-genase (6PGDH, the third enzyme of the PPP) hasbeen solved recently for T. brucei (PDB entry 1PGJ)and for sheep (PDB entry 2PGD). Interestingly, struc-tural differences between the parasitic and mamma-lian 6PGDH are currently exploited to designmolecules that would be specific inhibitors of the T.brucei enzyme.5

Here, we focus on the second enzyme of the PPP, 6-phosphogluconolactonase (6PGL), which catalyzeshydrolysis of δ-6 phosphogluconolactone (δ-6-P-G-L,compound 2 in Figure 1(a), called lactone in the

following) into 6-phosphogluconic acid (6-P-G-A,compound 3 in Figure 1(a)). We have already clonedand enzymatically characterized the human and T.brucei forms of 6PGL (entry O95336 in UniprotKB/Swiss-Prot and entry Q385D6 in the UniprotKB/TrEMBL databases, respectively), and found signifi-cant differences in the activities of these twohomologues,6,7 which urged us to characterize theseenzymes further. Structure determinationwas under-taken, and the X-ray structure ofTb6PGL is presentedhere. A comparisonwith other 6PGL structures in thePDB (Tm6PGL and Vc6PGL which have not beendiscussed in print) is presented. Analysis of thestructure, togetherwithNMR interaction studies anddocking approaches have allowed us to identify thecatalytic site of 6PGL enzymes.

The 6PGL family

Enzymes from the 6PGL family correspond to EC3.1.1.31 and are usually encoded by the pgl genes.However, it was shown recently that, in the case of afewprocaryotes that lack the pgl gene, likeEscherichiacoli, this function is borne by ybhE genes, encodingproteins of totally different sequence and structure.8A BLAST9 search shows that Tb6PGL presents

significant sequence similarity to bacterial DevBenzymes (devB gene products, 20–25% sequenceidentity with Tb6PGL), and Sol1-4 yeast enzymes(30–35% sequence identity). Indeed, the DevBprotein from Pseudomonas aeruginosa was shown todisplay a 6PGL activity,10 together with some mem-bers of the Sol family.11 Therefore, these proteinshave been referred to as the Sol/DevB/6PGL family,but wewill use 6PGL in the following, for the sake ofsimplicity.They also share similarity with N or C-terminal

regions of some predicted bifunctional dehydrogen-

870 Structure and Mechanism of 6PGL

ase/lactonase eucaryotic enzymes. For example,Tb6PGL has 25% sequence identity with the C-terminal part of the human enzyme, and 22% withthe N-terminal part of the malaria parasite's enzyme(entries O95479 andX74988 of theUniprotKB/Swiss-Prot database, respectively).12 It was confirmed thatthe isolated N and C-terminal regions of thesebifunctional enzymes display 6PGL activity.7,13

Finally, 6PGL enzymes present about 20%sequence identity with glucosamine-6-phosphatedeaminase enzymes, also known as the NagB geneproducts (EC 3.5.99.6). These enzymes were knownformerly as glucosamine-6-phosphate isomerase(EC 5.3.1.10). They interconvert glucosamine6-phosphate into fructose 6-phosphate (Figure1(b)), and it is striking how similar in structureNagB and 6PGL substrates are. However, it wasshown that human 6PGL has no deaminaseactivity.13 One major difference between the twofamilies of proteins is that NagB can be allostericenzymes (e.g. in E. coli) activated by N-acetylglucosamine 6-phosphate (GlcNac6P),14 or not (e.g.in Bacillus subtilis),15 whereas 6PLG enzymes are, toour knowledge, never allosteric. Since enzymes fromthe two homologous families 6PGL and NagBappear to catalyze different reactions on relatedsubstrates, we have combined the new structuralinformation with sequence comparisons to identifythe residues involved specifically in the distinctfunctions, or shared by both types of enzymes.

Results

Structure determination and overall description

TbPGL has 266 amino acids and a molecular massof 30 kDa. The refined structure consists of aminoacids 2-264 (the N-terminal methionine residue andtwo C-terminal residues were not observed in theelectron density map), and a zinc ion. Two prolineresidues (P37 and P196) out of 16 are observed in thecis conformation.The global fold of the enzyme belongs to the α/β

structural family (Figure 2(a) and (b)). The structurepresents eight α-helices (residues 13–31, 46–61, 87–93, 120–137, 174–177, 205–210, 224–231, and 257–259) and five 310 helices (33–35, 96–98, 101–103, 221–223, and 240–246). These helices surround oneseven-stranded parallel β-sheet (residues 6–10, 38–42, 69–73, 104–105, 155–158, 213–218, and 250–256)corresponding to 1-7-6-5-2-3-4 order with respect tothe protein sequence, one two-stranded antiparallelsheet (residues 140–142, and 149–151) and one three-stranded antiparallel β-sheet (residues 75–77, 185–188, and 200–203) corresponding to a 1-3-2 orderwith respect to the protein sequence. (Labeledsecondary structure elements are displayed alongthe protein sequence in the Supplementary DataFigure.) A few salt-bridges are observed involvingD122 and R125 (d=3.1 Å), D102 and R67 (d=2.65 Å),E222 and K223 (d=3.1 Å). In the hydrophobic core,

F107 and Y130 aromatic side-chains present stackinginteractions.A strong peak in the electron density is observed

at the protein surface for each monomer, whichcorresponds to a co-crystallized ion with octahedralgeometry with B-factors refined to about 29 Å2 and36 Å2. This density was assigned to a zinc divalention, because a fluorescence spectrum on the K-absorption edge of zinc showed a peak at theexpected characteristic wavelength (1.2837 Å). Nofluorescence was detected on the K-absorption edgeof Ni or Fe. A peak was observed at the wavelengthcharacteristic of Co with intensity one-third of thatof Zn, which is presumably due to a partialexchange between Zn and Co that occurred on theCo-NTA column during purification. In addition,two separate mass spectrometry measurements(one on the selenomethionylated protein and oneon the wild-type protein) indicated the presence of astrong complex with a species of 65(±10) Da, whichis compatible with a bound zinc atom; moreover,isothermal microcalorimetry experiments showedthat the untagged protein was able to bind Zn2+ invitro with a Kd of 8(±1) μM (data not shown). Thedivalent ion lies at the center of an octahedron,involving side-chains of H56, H97 and D98 andwater molecules. H56 and H97 occupy two cornersof a square containing the ion at its center (thedistances between the N atom of these histidineresidues and Zn being 2.1 Å and 2.3 Å, respec-tively), while two water molecules occupy the twoother corners at 2.2 Å and 2.3 Å from the Zn atom(Figure 3(a)); the oxygen atom of the carboxylicside-chain of D98 is an axial ligand in a mono-dentate manner (d=2.1 Å). The sixth axial ligand(d=2.1 Å) was also assigned to a water molecule,although there is clearly continuous density joiningthe zinc atom of the protein monomer A to the zincatom of an equivalent of monomer B (see Figure3(b)), generated by the following crystallographicoperator (−x+1/2, −y, z−1/2). Therefore, this elec-tron density forms a bridge between the two zincatoms of the two monomers building the crystal.This density was interpreted in the current model asindividualwatermolecules. Attempts to identify thisligand by collecting data at higher resolution areunder way.The structure displays one main internal crevice,

as shown in Figure 2(c). Interestingly, the analysis ofthe 3D structure of Tb6PGL shows that the 11conserved residues are close to each other, and arelocated on the edge and inside this pocket surface,constituting the active site of the enzyme (seebelow). The zinc ion is far from this region, and istherefore not expected to play a catalytic role, butmore probably a structural role. We have furtherconfirmed this last statement by doing kineticexperiments as described7 on both the nativeenzyme and the enzyme in the presence of an excessof orthophenanthroline, a well-known strong ligand(and scavenger) of zinc ions. There was no differencein the activity of the enzyme in the two experiments(data not shown).

Figure 2. Overall views of Tb6PGL structure. (a) and (b) Two orthogonal views of Tb6PGL structure, in a ribbonrepresentation highlighting secondary structures. (c) Surface representation of Tb6PGL, showing the active site pocketand the side-chains of the 11 conserved residues (in white).


Description of the dimeric interface

There are two molecules in the asymmetric unit,related by a pure translation. The twomonomers arevery similar, with an rmsd value of 0.85 Å over allCα atoms. Residues displaying significant higherrmsd between the two monomers are located inloops at the surface of the protein, and exhibit higherB-factors.To assess whether the non-crystallographic dimer

is biologically relevant, we first generated all thesymmetry-related mates of monomer B, andselected the first three with the smallest distancebetween the centers of mass of monomers A and B.Then the amount of accessible surface area buriedupon dimerisation was calculated as described.16

The largest value was found to be 470 Å2, indicatingthat the protein is probably not a dimer in solu-

tion.17 This is in line with a τc correlation time of14.7 ns measured by NMR at 25 °C under conditionswhere the enzyme was fully active,7 which isconsistent with a monomer of 30 kDa.18

Sequence alignments

To identify residues specifically involved in the6PGL function, we first performed CLUSTALW19

multiple sequence alignment including all known6PGL proteins. This showed that ten residues arestrictly conserved in this family, corresponding toG44, G45, D75, R77, H165, S168, F170, R200, G220,and K223 in the T. brucei enzyme (see the Supple-mentary Data Figure). We add D163 to this list, sincea carboxylate side-chain is always found at thisposition, the aspartic acid residue being replaced bya glutamic acid residue in only two cases, and

Figure 3. Zinc-binding site in an octahedral geometry.(a) Four ligands (H97 and H56, two water molecules)occupy the four corners of a plane, with a zinc ion in themiddle. The electron density (2Fobs–Fcalc) is in blue andcontoured at 1 σ. (b) The two axial ligands are (i) anoxygen atom of the carboxylate group of D98, and (ii) anunknown ligand with continuous density that links thetwo zinc atoms (CPK in pink) of the two monomers of theasymmetric unit. This unknown ligand has been inter-preted as individual water molecules in the current model.The electron density (2Fobs–Fcalc) is in blue and contouredat 1 σ.


because this carboxylate side-chain appeared to berequired for function (see below).Similarly, a multiple sequence alignment was

obtained between all known sequences in theNagB family (data not shown) and a structuralalignment (Figure 4) was performed between allknown 6PGL structures involving Tb6PGL fromT. brucei (2J0E, this study), Tm6PGL from Thermotogamaritima (1PBT) and Vc6PGL from Vibrio cholerae(1Y89), and two structures of the NagB family (E. coli1DEA, Bacillus subtilis 2BKV), in order to obtain areliable multiple alignment between proteins of

these two families despite their relatively lowsequence similarity. This last alignment showedthat six of the 11 conserved residues in the 6PGLfamily (G45, D75, D163, H165, G220, and K223 inTb6PGL) are also conserved through the wholeNagB family (Figure 4). The five other residues (G44,R77, S168, F170, and R200) appear to be specific ofthe 6PGL family, and correspond to conservedresidues of another type in the NagB family, asshown in Figure 4. This is in agreement withprevious studies suggesting that R77, F170, andR200 could constitute the 6PGL signature.13 Thecorrespondence between the conserved residueswithin these two families is listed in Table 1.

Comparison to known structures of homologousproteins

Two structures of other 6PGL enzymes areavailable at the PDB databank: 6PGL from T.maritima (PDB entries 1PBT and 1VL1, which differonly by the nature of co-crystallized anions), andfrom V. cholerae (PDB entry 1Y89). These structureswere solved as part of structural genomic programs,and have not been analyzed in a publication.Tb6PGL shares 32% and 30% sequence identitywith Tm6PGL (1PBT and 1VL1) and Vc6PGL (1Y89),respectively. The three structures are similar, andpresent the same secondary structure elements,except for the two-stranded antiparallel sheet (β6and β7), which is absent from 1PBT (and 1VL1)because the corresponding loop is too short to allowformation of this sheet (see the sequence alignmentin the Supplementary Data Figure). The structure ofTb6PGL has an rmsd of 1.8 Å with 1PBT on 760equivalent backbone atoms, and of 2.1 Å with 1Y89on 824 equivalent backbone atoms. Figure 5(a)shows backbone superposition of the structures ofTb6PGL (2J0E) and Tm6PGL (1PBT). It is noteworthythat the 11 conserved residues are almost super-imposed, as shown in Figure 5(c). The rmsd valueson all backbone atoms of these residues are 0.8 Åand 1.05 Å, for T. maritima and V. cholerae,respectively. Interestingly, A167 of Tb6PGL, whichis the only residue in the generously allowed regionof the Ramachandran diagram (lower left quarter) isequivalent to A140 in 1VL1, and to A146 in 1Y89,which are also situated in the same lower left regionof the Ramachandran diagram in the structures ofthese two homologues. Therefore, this unusualbackbone geometry is likely to be a characteristicstructural feature shared by this family of enzymes.As for the Tb6PGL structure, two molecules arefound in the asymmetric unit of Vc6PGL (1Y89), butthe Tm6PGL homologue is a monomer both in 1PBTand 1VL1. In 1Y89, the two monomers are main-tained by an inter-monomer antiparallel associationof their first N-terminal extended strand (I2 to F7),which is far from the active site pocket; this buildsup a remarkable cross-subunit continuous β-sheet of14 strands with a large twist.The structures of 6PGL from T. maritima and V.

cholerae do not contain a zinc ion. The presence of

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Figure 4. Sequence alignment of all known 3D structures in the 6PGL family, and of two NagB known 3D structures.Alignment was obtained by multiple sequence and structure alignment, using all available sequences and structures inthe two families of proteins. However, for the sake of clarity, only sequences of the proteins discussed in the text areshown: Tb6PGL (this work), Tm6PGL (1PBT), Vc6PGL (1Y89), and NagB enzymes from E. coli (1DEA) and B. subtilis(2BKV). Residues printed in bold red are strictly conserved and common to the two families of enzymes. Residues printedin bold cyan and black are specific to the 6PGL and NagB families, respectively.


this additional ion seems to be specific to T. brucei.This can be explained by the fact that none of theresidues binding the zinc ion in Tb6PGL (H56, H97,and D98), is conserved in Tm6PGL and Vc6PGL.This is consistent with the absence of a catalytic rolefor the zinc ion in Tb6PGL.Several free and bound structures are available in

the NagB family, for human (PDB entry 1NE7), B.subtilis (2BKV and 2BKX entries), and E. coli (PDBentries 1DEA, 1FS6, 1FSF, 1FS5, 1HOR, 1HOT),respectively, sharing 24%, 23%, and 22% sequenceidentity with Tb6PGL. Superposition of 1DEA withTb6PGL shows that the overall folds of NagBproteins are highly similar, with an rmsd of 2.1 Åon 208 backbone atoms, and of 1.0 Å for the sixconserved residues, as shown in Figure 5(b) and (d).

Interaction studies by NMR

Since no inhibitor has been identified in the 6PGLfamily, we tried to obtain the structure of Tb6PGL incomplex with 6-P-G-A (compound 3 in Figure 1), theproduct of the reaction, using soaking experiments.The analysis of structures of such complexes is oftenused to gain insights into the enzyme mechanism.Unfortunately, soaked crystals for which we couldcollect X-ray diffraction data revealed no extradensity in the vicinity of the active site. However,we had previously undertaken an NMR study ofTb6PGL, and achieved heteronuclear backbone

NMR resonance assignment for backbone 1Hamide protons, 15N amide nitrogen and 13C for Cα,Cβ and carbonyl carbon atoms.20 The spectralassignment is deposited in the BMRB databank†under accession number 5468. These NMR dataallowed us to undertake interaction studies byNMR.The identification of residues in contact with 6-P-

G-A relies on the fact that the NMR resonance linesof these residues are affected by the presence of 6-P-G-A, in frequency and/or line width. Because thesize of the protein (30 kDa) would lead to extensivespectral overlap on 2D spectra, we followed theinteraction using 3D NMR experiments on a 13C and15N double-labeled protein sample. The 3D HNCOspectra21 were recorded in the absence and in thepresence of increasing concentrations of 6-P-G-A. Inthe HNCO spectra, each residue is represented byone peak defined by three frequencies: its amideproton 1H chemical shift, its amide nitrogen 15Nchemical shift, and the carbonyl 13C chemical shift ofits preceding residue in the sequence. A 2Dprojection of the HNCO spectrum in the (HN)plane, including assignment, is shown in Figure 6(a).For each residue, the chemical shift perturbationupon 6-P-G-A binding was measured in the 1Hand 15N dimensions (the 13C dimension being usedto avoid spectral overlap), as shown in Figure 6(b).Besides the variations observed near the N and

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Table 1. Sequence numbers of equivalent residues between amino acid residues from Tb6PGL (2J0E), its homologues in T.maritima (1PBT) and V. cholerae (1Y89), and E. coli NagB protein (1DEA)

2J0E (Tb6PGL) G44 G45 D75 R77 D163 H165 S168 F170 R200 G220 K2231PBT (Tm6PGL) G41 G42 D68 R70 D136 H138 S141 F143 R167 G187 K1901Y89 (Vc6PGL) G37 G38 D68 R70 D142 H144 S147 F149 R173 G193 K1961DEA (E.coli NagB) T41 G42 D72 Y74 D141 H143 F146 E148 A209 G205 K208

Residues in bold are strictly conserved in all known 6PGL sequences and those in italics are strictly conserved in all known NagBsequences.


C-terminal ends of the protein, we can distinguishmainly five regions of the protein sequence in whichNMR resonances are significantly affected by thepresence of 6-P-G-A. Interestingly, these regions arecentered on the conserved residues G44, D75, D163,R200, and K223, and include the 11 conservedresidues discussed above. These results are consis-tent with a role of the conserved residues insubstrate binding and/or activation, and confirmour identification of the active site pocket.

Figure 5. Comparison of Tb6PGL structure with other kTb6PGL (red) and Tm6PGL (yellow, 1PBT). (b) Overall backbdeaminase (green). (c) Superposition of conserved residues inindicate the numbers of these 11 conserved residues in the casecrystallized in 1PBT are represented in yellow and red in the acTb6PGL (red), and of the shared common conserved residues iThe two phosphate ions that are co-crystallized in 1DEA are rethe active site.

Docking of substrate and product in a 6PGLstructure

A more detailed picture of the protein/ligandinteractions in the 6PGL family can be proposed bydocking the lactone and 6-P-G-A, substrate andproduct of the reaction (compounds 2 and 3 inFigure 1(a)), respectively, into the 6PGL structure.We used the FlexX software,22 allowing for flexibledocking of the ligand, and generating several

nown structures. (a) Overall backbone superposition ofone superposition of Tb6PGL (red) and the E. coli NagBTb6PGL in red, and in Tm6PGL (1PBT) in yellow. Labelsof the Tb6PGL sequence. The two sulfate ions that are co-tive site pocket. (d) Superposition of conserved residues inn the NagB deaminase family, in the case of 1DEA (green).presented in bronze and red, one of them being situated in

Figure 6. NMR interaction studies. (a) 1H-15N projection of the Tb6PGL HNCO spectrum, recorded at 600 MHz,25 °C. Assignment has been reported for each 2D cross-peak.19 NMR peaks corresponding to strictly conserved residuesare colored red. (b) Chemical shift changes of Tb6PGLNMR peaks upon 6-P-G-A binding (1.5 mM). Bars corresponding tostrictly conserved residues are colored red.


energetically favored ligand conformations in theactive site.For the lactone, two clusters of conformations are

observed among those of best scores, in a total of 525calculated conformations. In one cluster (Figure7(c)), the phosphate group is close to K223 ofTb6PGL, and the Nε atom of H165 is close to cyclicoxygen O5. In the other cluster (Figure 7(a)), thephosphate group is close to R200, the cyclic oxygenO5 is close to R77, and the C1 carboxyl group is closeto D75.Similarly, for 6-P-G-A, two similar global clusters

of orientations are found in the docking solutions ofbest scores, among 421 calculated docking solutions.In the first cluster (Figure 7(d)), the terminalcarboxyl group interacts with R77 and R200 of

Tb6PGL, and the phosphate group interacts withK223. In the second cluster (Figure 7(b)), the 6-P-G-Amolecule is rotated by 180° with respect to the firstorientation so that the phosphate group interactswith R200 and R77, and the terminal carboxyl groupinteracts with K223.These two main orientations are consistent with

otherwise known crystal complexes in the 6PGLfamily. For 6-P-G-A, the two negatively chargedregions of the molecule (i.e. the phosphate groupand the carboxylate group) in both orientations ofthe docked structures (Figure 7(a) and (c)) aresituated close to the two sulfate ions in the 1PBT ofTm6PGL, to the two terminal carboxyl groups of thecitrate ligand in 1VL1 of Tm6PGL (Figure 8(a)), andto two of the three phosphate groups in 1Y89 of

Figure 7. Docking of the substrate and product of 6PGL enzymes. (a) A view of one conformation of the δ-6-phosphogluconolactone in Tb6PGL, belonging to the first cluster of solutions. Only the residues that are conserved in thewhole 6PGL family are represented in red and in the same orientation as in Figure 5, and labeled according to thissequence. The docked lactone substrate is in CPK representation. (b) A view of one conformation of 6-P-G-A in Tb6PGL,belonging to the first cluster of solutions, as in (a). The docked 6PGA is represented in CPK representation. (c) A view ofone conformation of the δ-6-phosphogluconolactone in Tb6PGL, belonging to the second cluster of solutions. (d) Aview ofone conformation of 6-P-G-A in Tb6PGL, belonging to the second cluster of solutions, as in (c).


Vc6PGL. The same situation holds for the phosphategroup and the carbonyl group in the lactonesubstrate of 6PGL.Taken together, the multiple alignments, the X-ray

structure, the docking results, the NMR resultsallow us to discuss possible features for themechanism of the 6PGL enzymes.

Discussion

Local conformational change upon ligandbinding in the 6PGL family

All other known structures in the 6PGL families(1PBT and 1VL1 of Tm6PGL and 1Y89 of Vc6PGL)contain co-crystallized negatively charged ions inthe active site: two sulfate ions in 1PBT, a citratemolecule in 1VL1 of T. maritima, and three phos-phate ions in 1Y89 of V. cholerae. When the structuresare superimposed, two anion groups occupy similarpositions in each structure. The first group includesSO4 303 in 1PBT and a carboxylate group of citratein 1VL1 from Tm6PGL, and PO4 202 in 1Y89 fromVc6PGL. The second group includes SO4 302 in1PBT and a carboxylate group of citrate in 1VL1from Tm6PGL, and PO4 201 in 1Y89 from Vc6PGL.

The first group of ions interacts with a conservedlysine side-chain equivalent to K223 of Tb6PGL(K190 for 1PBT or 1VL1, and K196 for 1Y89), asshown in Figure 8(a), and the second group withtwo arginine side-chains and one histidine equiva-lent to R77, R200, and H165 in Tb6PGL (R70, R167,and H138 for 1PBT or 1VL1 from Tm6PGL, and R70,R173 and H144 for 1Y89 from Vc6PGL). Closerinspection of the active sites reveals that someresidues are almost superimposed, corresponding toresidues G44, G45, S168, R200, D75, and R77 inTb6PGL, while the positions of some other residues,corresponding to H165, G220, K223 in Tb6PGL,diverge more significantly (see Figure 8(a)). Inparticular, the N terminus of helix α8 G220-K223in Tb6PGLmoves away from its equivalent region inthe other structures (G187-K190 in 1PBT or 1VL1,and G193-K196 in 1Y89), although this shortsegment contains two strictly conserved residues(see Figure 8(c)). These local rearrangements may bedue to anion binding in 1VL1, 1PBT and 1Y89, andare not observed in the Tb6PGL structure, which isdevoid of any anion bound to the active site.

Implications for the enzymatic mechanism

As presented in Results, the docking studies leadto two possible orientations of the lactone and the

Figure 8. Active site superposition. (a) Superposition of Tb6PGL residues that are conserved in the whole 6PGL family(red) with the corresponding residues in 1PBT from Tm6PGL (yellow), together with the two co-crystallized sulfate ions inCPK (red and yellow). The co-crystallized citrate ion in 1VL1 from Tm6PGL is superimposed in CPK representation (redand blue). The labels correspond to the sequence numbers of conserved residues in Tb6PGL. (b) Superposition of Tb6PGLresidues that are conserved in the whole 6PGL family (red) with the corresponding residues from E. coliNagB (green) andthe co-crystallized phosphate ion in CPK (bronze and red) in 1DEA, and the co-crystallized inhibitor 2-deoxy-2aminoglucitol-6-phosphate (AGP), in 1HOR. (c) Cα trace representation of superimposed Tb6PGL (red) with Tm6PGL (1PBT,yellow and 1VL1, green).


6-P-G-A molecules in the active site. It is importantto note that both orientations found in the dockingresults, leading to different catalytic mechanisms,are compatible with other known complexes in the6PGL family. Sulfate ions are close to K190, R70 andR167 in 1PBT of Tm6PGL (Figure 8(a)). Carboxylategroups of the citrate ligand are at similar positions in1VL1 of Tm6PGL (Figure 8(a)). Similarly, phosphateions are close to K196, R70 and R173 in Vc6PGL.Interestingly, a more detailed analysis of these two

orientations is possible in the light of previousworks in the NagB and the 6PGL families ofproteins.In the first type of orientations (Figure 7(c) and

(d)), the phosphate group of 6-P-G-A and of thelactone is close to K223 in Tb6PGL, a residuecommon to 6PGL and NagB enzymes. For the latter,a mechanism has been proposed in which K208 ofE. coli NagB binds a free phosphate group in 1DEA(PO4 268), and the phosphate group of the compe-titive inhibitor 2-deoxy-2-amino glucitol-6-phos-phate (A-G-P) in 1HOR (Figure 8(b)). In the 2BKXstructure of the NagB enzyme from B. subtilis, thephosphate group of fructose 6-phosphate is also

close to K202, the residue equivalent to K223 fromTb6PGL. Therefore, this lysine residue could beexpected to bind to the phosphate group ofglucosamine 6-phosphate, its natural substrate.23

However, in the NagB family, the phosphate groupof the substrate is maintained by the guanidiniumgroup of an arginine side-chain (R172 in EcoNagBand R167 in BsuNagB) belonging to the lid regionwhich is conserved in the NagB family, but not in the6PGL family. In the case of Tm6PGL, R43 could be itsfunctionally equivalent,15 but this arginine residue isnot conserved in the 6PGL family. In Tb6PGL, thereis no other basic side-chain that could bind aphosphate group in a similar position.However, under the hypothesis of a global

orientation shown in Figure 7(c) and (d), K223 inTb6PGL could still bind the phosphate group of itsnatural substrate, δ-6-phosphogluconolactone, simi-larly to the equivalent residue in the NagB family. Asimilar activation mechanism would then be sharedby 6PGL and NagB enzymes, involving the proto-nated Nε2 atom of a histidine residue common toboth families, and in close contact with the O5oxygen atom of the pyranose ring of the substrate.


This histidine residue is H143 in EcoNagB, H138 inBsuNagB, and H165 in Tb6PGL. In the case ofEcoNagB, H143 is activated by D141 and E148,23

while in BsuNagB H138 is activated by E143,15

equivalent to E148 in EcoNagB. In the case ofTb6PGL, the Nδ1 atom of H165 is in contact withOδ1 of D163 (2.5 Å), equivalent to D141 in EcoNagB,in an arrangement that resembles the proton-relaysystem present in many enzymes.23 Furthermore,under the hypothesis of the currently discussedorientation (Figure 7(c)), the protonated Nε2 atom ofH165 is close to the O5 oxygen atom of the lactonering, as observed in the deaminase family. Since thisfirst step of the deamination reaction is very similarto the lactone ring opening in the case of 6PGL(Figure 1(b)), H165 of Tb6PGL activated by D163would provide a generalized acid catalysis of thering. Ring opening would be facilitated by protontransfer from H165 to the lactone O5 oxygen atom.The resulting carboxylate group in the 6-P-G-Areaction product would be stabilized by R200 andR77, as observed in the corresponding dockedstructure (Figure 7(d)). Note that this mode ofactivation via protonation of the ring oxygen atomis observed also in ribose-5-phosphate isomerase.24

In the second cluster of orientations (Figure 7(a)and (b)), the phosphate groups of the lactone and of6-P-G-A are found deeper in the active site pocket,close to R200, R77 and H165 in Tb6PGL. Thisorientation would differ from that observed for thesubstrates of NagB proteins, and from that antici-pated for the 6PGL substrates on the basis ofsequence analysis alone.13 It has been suggested byother authors on the basis of the BsuNagB struc-ture.15 Under this hypothesis, as observed in Figure7(a), cyclic oxygen O5 of the lactone would be closeto the guanidinium group of R77, allowing activa-tion for ring opening. This cluster of conformations isnot compatiblewith close interactions between cyclicoxygen O5 of the lactone and H165, which excludesin that case a catalytic role for this residue, althoughit could still have a role in substrate binding.Furthermore, the carboxylate group of the D75side-chain would be close to the C1 carboxylgroup, thus enhancing the electrophilic character ofC1 for subsequent attack by a water molecule.Finally, K223 would stabilize the resulting carbo-xylate group in 6-P-G-A, as observed in the dockedstructure (Figure 7(b)). Interestingly, the equivalentresidue in EcoNagB, D72, also plays an importantactivation role in the deamination reaction.At this point, it is interesting to discuss the above

results of the NMR interaction studies, in the light ofthe twodifferent possible orientations for the 6-P-G-Aproduct (Figure 7(b) and (d)). The buffer used forthe NMR sample is a phosphate buffer, because itturned out that the 15N, 13C, 2H triple-labeled proteinused for spectral assignment is very stable in aphosphate buffer,which is a critical feature in order torecord the complete set of required 3D spectra (phos-phate buffer is a classical buffer for NMR studies,avoiding strong 1H peaks arising from the buffer). Asa consequence, the interaction study performed here

is, in fact, a competitive experiment between thebuffer phosphate ions and the 6-P-G-A product.Therefore, one would anticipate that the displace-ment of a free phosphate ion by the phosphate groupof 6-P-G-A would lead to smaller chemical shiftvariations than the displacement of a free phosphateby a carboxylate group. The residues that exhibit thestrongest chemical shift variations of their backboneNH group are situated in the G44-G45-S46 loop. Thedocked 6-P-G-A structure presented in Figure 7(b)would be compatible with this observation, since itdisplays the shortest distance between this loop andthe 6-P-G-A carboxylate group.Future studies will require the structure determi-

nation of the human 6PGL homologue, in order tocompare its structure to that of Tb6PGL from thiswork, as well as the structure of the complex witheither an inhibitor or the product of the reaction. Theultimate goal will be to use this structural informa-tion to develop specific inhibitors against Tb6PGLand test their activity in vivo against the parasite.

Materials and Methods

Crystallization and data collection

The recombinant protein Tb6PGL was purified to 95%homogeneity in a single step with a Co-NTA columnthat makes use of the N-terminal His-tag, as described,7

and then concentrated to 10 mg/ml in 50 mM Tris–HCl(pH 8.0), 150 mM NaCl using Centricons (Amicon). Themonodispersity of the protein solution was checked withdynamic light-scattering. Crystallization was achievedwith the hanging-drop method, equilibrated against areservoir solution of 100 mM Hepes (pH 7.5), 30% (v/v)PEG MME 2000, 50 mM sodium acetate.Crystals grew within a week as thin plates and were

frozen in liquid nitrogen with 25% (v/v) glycerol ascryoprotectant. An initial data set at 2.8 Å was collectedwith a native orthorhombic crystal (Nat1 inTable 1) at ID14-EH2 (ESRF, Grenoble). All diffraction data were processedusing XDS.25 Given the cell parameters, the asymmetricunitwas likely to contain twomolecules. A native Pattersonmap revealed a purely translational non-crystallographicsymmetry with a peak at 23.5 σ at 3.5 Å resolution fortx=0.000 ty=0.176 and tz=0.500. Molecular replacementtechniques using AMoRe35 and either 1Y89 or 1PBT asmodels failed with this data set and the isomorphousreplacement method was used to obtain the phases.

Structure determination

Subsequently, two other data sets were collected forphasing at FIP (ESRF, Grenoble). One crystal was soakedfor 3 h in 1 mMEtHgCl and diffracted to 2.1 Å. A full singleanomalous dispersion data set was collected at a wave-length of 1.008014 Å. Four sites were found by inspection ofthe difference Patterson and confirmed by SOLVE.26 TheRESOLVE program30 was then used in the single anom-alous dispersion mode to obtain phases at 2.2 Å, usinga solvent content of 50% (v/v). This lead to an interpretablemap, with a figure-of-merit of 0.743. Another data set forSeMet derivative protein crystals (checked bymass spectro-metry) was collected at a wavelength of 0.980670 Å at FIP

Table 3. Refinement statistics of the 6PGL structure usingEtHgCl data (Bijvoet mates were merged)

Resolution (Å) 18–2.1Total reflections 30,438Reflections used for refinement 28,899Reflections used for Rfree calculation (5%) 1539Number of atoms per monomer

Protein 1971Ions 1 Zn2++2 HgWater molecules 465

Rfactor (%) 20.6Rfree (%) 24.6rmsd from idealBond lengths (Å) 0.006Bond angles (deg.) 1.3Ramachandran plot34

Residues in most favored regions (%) 91.9Residues in additionally allowed regions (%) 7.7Residues in generously allowed regions (%) 0.4G-factor (overall) 0.26


(ESRF, Grenoble) at 2.8 Å. However, its medium qualityprevented its use for phasing. Once a poly(Ala) model wasconstructed, calculated phases were, however, able to pullout seven of the eight expected selenium atoms using aFourier difference map at 3.0 Å resolution, thereby greatlyhelping the sequence assignment process during modelbuilding. In addition, the spatial proximity of eachHg atomto cysteine residues in each monomer was used to checksequence assignment and register.

Model building and refinement

Model building proceeded with several rounds ofrefinement using CNS,27 and manual rebuilding usingO28 and then Coot.29 A zinc atom was spotted early on inthe refinement, at the surface of the protein. The refinedstructure showed six ligands in octahedral geometry: twohistidine residues, one aspartate residue, two watermolecules, and one unknown ligand. The final R-factoris 20.6% and Rfree is 24.6%, using all data between 18.0 Åand 2.10 Å. A bulk-solvent correction was applied in CNS,as well as constrained atomic isotropic B-factors. Themean B-factor was 25.8 Å2 for the protein atoms and29.1 Å2 for water molecules. It was 29 Å2and 36 Å2 for thetwo zinc ions. There are two cis-proline residues permonomer, and no outlier residue in the Ramachandrandiagram (see Table 2). Residues 2–264 were constructedfor each monomer (the N-terminal Met is missing fromthe electron density map, as well as the two last resi-dues), with all side-chain atoms, one zinc ion, twomercury atoms per monomer and 465 water molecules.A quick round of refinement of the model without the Hgatoms against the 2.8 Å native data set showed that thestructure was not perturbed in the two Hg-binding re-gions. The structure was analyzed using PROCHECK,30

and a G-factor of 0.26 was obtained.

Sequence alignment

The multiple sequence alignment of 6PGL enzymes(Figure 2) was performed with CLUSTALW,15 using allknown sequences in this family. Structural alignmentbetween Tb6PGL, 1PBT, 1Y89, 1DEA and 2BKV wasperformed using the STAMP algorithm.31

NMR interaction studies

Double-labeled 13C,15N Tb6PGL was prepared asdescribed.19 NMR samples contained 1.0 mM protein,

Table 2. X-ray diffraction data collection statistics

Nat1 EtHgCl SeMet

Concentration (mM) 1Soak. time (h) 3X-ray source (ESRF) ID14-EH2 FIP FIPWavelength (Å) 0.934 1.008014 0.980670Unit cell parameters

a (Å) 70.31 70.06 69.68b (Å) 80.85 80.75 80.57c (Å) 90.31 90.15 89.83

Resolution (Å) 2.8 2.1 2.8Total reflections 45,542 219,162 57,227Unique reflections 12,891 57,076 20,786Completeness (%) 96.6 (91.0) 98.9 (99.3) 86.4 (87.5)Rmerge (%) 7.7 (24.7) 5.9 (23.9) 11.9 (44.7)I/σ(I) 14.5 (5.6) 15.9 (5.9) 8.0 (2.6)

50 mM sodium phosphate buffer (pH 6.0), 200 mM NaCland 1 mM β-mercaptoethanol. NMR spectra wereacquired at 25 °C on a Varian INOVA 600 MHz spectro-meter, equipped with triple-resonance (1H, 13C, 15N)gradient probe. The 3D-HNCO spectra were processedon a Silicon Graphics workstation using the Gifasoftware.32 Processing included a 90°-shifted sinebellwindow function, appropriate phase correction and zerofilling, as well as linear prediction in the 15N dimension.The 1H chemical shifts were referenced to external 3,3,3-trimethylsilylpropionate (TSP) at 0 ppm. The 15N and13C shifts were referenced indirectly using the 1H/Xfrequency ratios of the zero point as described.33 Protonand heteronuclear resonances from backbone atomswere assigned previously,19 and have been depositedwith the BMRB databank‡ under accession number5468. The HNCO spectra were recorded in the absenceof 6-P-G-A, and in the presence of increasing concentra-tions of 6-P-G-A. Chemical shift changes were measuredfor each backbone amide group by quantifying displace-ment the of 15N/1H cross-peak using the relationshipΔδ1H+Δδ15N/7 (Δδ1H+Δδ15N/5 for glycine residues).34

Maximum chemical shift variations were reached at aconcentration of 1.5 mM in 6-P-G-A (higher concentrationsof 6-P-G-A did not lead to any more variation in chemicalshifts) (Table 3).

Docking

The FlexX software21 was used in the docking experi-ments of δ-6-phosphogluconolactone and 6-P-G-A into thestructure of Tb6PGL. In order to define the Tb6PGL activesite for the FlexX program, we have superimposed thestructure of Tb6PGL on the 1VL1 structure of Tm6PGL.The coordinates of the citrate molecule that occupies theactive site in 1VL1 were then transferred into the Tb6PGLstructure, and the active site of the latter was defined by asphere of 6.5 Å around the transferred citrate molecule.A total of 525 and 421 docking solutions (conformationsof the ligand in the active site) were generated andtheir energy evaluated for the lactone and 6-P-G-A com-plexes, respectively. The FlexX score was used for rankingthe solutions.35 The δ-6-phosphogluconolactone and

‡http://www.bmrb.wisc.edu

http://www.bmrb.wisc.edu


6-P-G-A molecules were generated in a mol2 format usingCORINA.36

Protein Data Bank accession number

The coordinates and the structure factors have beendeposited at the PDB under the code 2J0E.

Acknowledgements

We thank the staff at beamline FIP (ESRF,Grenoble) for help in data collection, and the proteinproduction and purification platform (PT5) from theInstitut Pasteur for preparation of the SeMet protein.We thank Felix Romain for numerous controlexperiments and sample preparation of theuntagged protein, and Sylviane Hoos for help withthe ITC experiments. Thanks are due to DidierNurizzo (ESRF, Grenoble, ID29-1), StéphaneDuquerroy and J. Cockburn (Institut Pasteur) forthe fluorescence X-ray data at ESRF and SLS,respectively. In its initial phase, this work wassupported by a postdoctoral fellowship from theEuropean Commission to Francis Duffieux, whoinitiated the work in the group of Paul Michels andFred Opperdoes (Brussels).

Supplementary Data

Supplementary data associated with this articlecan be found, in the online version, at doi:10.1016/j.jmb.2006.11.063

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Edited by M. Guss

(Received 2 August 2006; received in revised form 17 November 2006; accepted 18 November 2006)
Available online 22 November 2006

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