The Crystal Structure of Trypanosoma cruzi Glucokinase Reveals Features Determining Oligomerization and Anomer Specificity of Hexose-phosphorylating Enzymes

J. Mol. Biol. (2007) 372, 1215–1226

doi:10.1016/j.jmb.2007.07.021

The Crystal Structure of Trypanosoma cruzi Glucokinase Reveals Features Determining Oligomerization and Anomer Specificity of Hexose-phosphorylating Enzymes Artur T. Cordeiro 1 , Ana J. Cáceres 2 , Didier Vertommen 3,4 Juan Luis Concepción 2 , Paul A. M. Michels 1,4 ⁎ and Wim Versées 5,6 1

Research Unit for Tropical Diseases, Christian de Duve Institute of Cellular Pathology Avenue Hippocrate 74, B-1200 Brussels, Belgium 2

Unidad de Bioquímica de Parásitos, Centro de Ingeniería Genética, Faculdad de Ciencias Universidad de Los Andes Mérida 5101, Venezuela 3

Hormone and Metabolic Research Unit, Christian de Duve Institute of Cellular Pathology, Avenue Hippocrate 75, B-1200 Brussels, Belgium 4

Laboratory of Biochemistry Université catholique de Louvain, Avenue Hippocrate 75 B-1200 Brussels, Belgium 5

Department of Ultrastructure Vrije Universiteit Brussel Pleinlaan 2, B-1050 Brussels Belgium 6

Department of Molecular and Cellular Interactions, VIB Pleinlaan 2, B-1050 Brussels Belgium

Glucose is an essential substrate for Trypanosoma cruzi, the protozoan organism responsible for Chagas' disease. The glucose is intracellularly phosphorylated to glucose 6-phosphate. Previously, a hexokinase responsible for this phosphorylation has been characterized. Recently, we identified an ATP-dependent glucokinase in T. cruzi exhibiting a tenfold lower substrate affinity compared to the hexokinase. Both enzymes, which belong to very different groups of the same family, are located inside glycosomes, the peroxisome-like organelles of Kinetoplastida that are known to contain the first seven glycolytic steps as well as enzymes of the oxidative branch of the pentose phosphate pathway. Here, we present the crystallographic structure of T. cruzi glucokinase, in complex with glucose and ADP. The structure suggests a loose tetrameric assembly formed by the association of two tight dimers. TcGlcK was previously reported to exist in a concentration-dependent equilibrium of monomeric and dimeric states. Here, we used mass spectrometry analysis to confirm the existence of TcGlcK monomeric and dimeric states. The analysis of subunit interactions and comparison with the bacterial glucokinases give insights into the forces promoting the stability of the different oligomeric states. Each T. cruzi glucokinase monomer contains one glucose and one ADP molecule. In contrast to hexokinases, which show a moderate preference for the α anomer of glucose, the electron density clearly shows the D-glucose bound in the β configuration in the T.cruzi glucokinase. Kinetic assays with α and β-D-glucose further confirm a moderate preference of the T. cruzi glucokinase for the β anomer. Structural comparison of the glucokinase and hexokinases permits the identification of a possible mechanism for anomer selectivity in these hexose-phosphorylating enzymes. The preference for distinct anomers suggests that in T. cruzi hexokinase and glucokinase are not directly competing for the same substrate and are probably both present because they exert distinct physiological functions. © 2007 Elsevier Ltd. All rights reserved.

*Corresponding author

Keywords: glucokinase; hexokinase; anomer selectivity; glycolysis; ATPase

Introduction Abbreviations used: LDH, lactate dehydrogenase; GlcK, glucokinase; HK, hexokinase; PyK, pyruvate kinase; Se-Met, seleno-methionine; Tc, Trypanosoma cruzi; Ec, Escherichia coli; Ar, Arthrobacter sp.; MAD, multi-wavelength anomalous dispersion. E-mail address of the corresponding author: [email protected]

Trypanosoma cruzi is the causative agent of American trypanosomiasis or Chagas' disease. This parasite is transmitted between its mammalian hosts by hematophagous reduviid insects. It possesses a complex life-cycle, living essentially as a replicative intracellular parasite (amastigote) in the

0022-2836/$ - see front matter © 2007 Elsevier Ltd. All rights reserved.

1216 mammalian host and extracellularly (replicative epimastigote or non-replicative trypomastigote) in the insect vector.1 It has been shown that T. cruzi depends on glucose, both for its differentiation from epimastigotes to trypomastigotes, and for its growth as amastigotes in axenic cultures.1,2 Indeed, functional glucose-consuming glycolytic and pentose phosphate pathways are present in the different life-cycle stages and consequently the enzymes of these pathways have been proposed to be good targets for anti-parasite drugs to be designed.3–5 The first seven steps of the glycolytic pathway are compartmentalized in peroxisome-like organelles known as glycosomes, and the enzymes of the pentose phosphate pathway (PPP) are partially present in these organelles.4,6,7 Previously, we have shown that in T. cruzi and the related parasites Trypanosoma brucei and Leishmania mexicana, the first shared reaction of these pathways involves the ATP-dependent phosphorylation of D-glucose or other hexoses catalyzed by hexokinase.8–11 According to Kawai et al.,12 two distinct nonhomologous families of enzymes responsible for the ATP (or ADP or inorganic polyphosphate) dependent phosphorylation of glucose and other hexoses are found in nature: the hexokinase (HK) and the ribokinase (RK) families. The RK family comprises glucokinases, i.e. glucose-specific kinases, from Euryarchaeota and a special glucokinase found in mammals. The HK family can be subdivided into three groups comprising respectively the eukaryotic hexokinases, i.e. kinases with a broad specificity for various hexoses (group HK), the glucokinases found in Gram-negative bacteria and amitochondriate protists (group A) and a third group of Crenarchaeota hexokinases and Gram-positive glucokinases (group B). The members of the three groups in this family have very different primary structures, except for a few conserved short motifs. Vertebrates contain four hexokinase isoenzymes, I–IV (or A–D). Isoenzymes I–III are dimeric enzymes with 100 kDa subunits, whereas isoenzyme IV is a 50 kDa monomeric enzyme.13 The 100 kDa subunits are the result of a gene duplication and fusion that must have occurred in an ancestral vertebrate.13,14 Unfortunately, and confusingly, the 50 kDa isoenzyme IV is often also called a glucokinase, because physiologically it acts almost exclusively on glucose, but actually on the basis of substrate specificity it should be considered as a hexokinase. Different from the mammalian hexokinases, but also from its T. brucei and L. mexicana homologues,3,11,15 the T. cruzi hexokinase is highly selective for D-glucose: it does not catalyze the phosphorylation of D-fructose, mannose or galactose, and is not inhibited by the product of the reaction, glucose 6-phosphate.10 Crystallographic structures are available for representatives of each of the three groups within the HK family. For the HK group, the human isoenzymes I (HsHKI) 16 and IV (HsHKIV or HsGlcK), 17 the Schistosoma mansoni hexokinase (SmHK)18 and the Saccharomyces cerevisiae hexokinases PI19 and PII20 have been solved. For groups A

Crystal Structure of Trypanosoma cruzi Glucokinase

and B, the representatives are Escherichia coli glucokinase (EcGlcK)21 and Arthrobacter sp. glucomannokinase (ArGMK),22 respectively. Although the trypanosomatids are not amitochondriated organisms, a second glucose-phosphorylating enzyme belonging to group A was discovered in the genome of T. cruzi and Leishmania species (Leishmania major, Leishmania brasiliensis and Leishmania infantum), but not in T. brucei. Subsequently, it was confirmed experimentally for T. cruzi and L. major that these enzymes are true glucokinases that specifically phosphorylate D-glucose in an ATP-dependent manner (A.J.C. et al., unpublished results). Trypanosomatids are the only organisms known to possess both hexokinases and group A glucokinase enzymes. The T. cruzi glucokinase (TcGlcK) has 41–44% sequence identity with the Leishmania glucokinases, whereas its sequence is difficult to align with that of EcGlcK (identity value below 16%). The T. cruzi 42-kDa GlcK and 50 kDa hexokinase (TcHK) differ considerably in their substrate affinity; their Km values for glucose are 0.7 mM and 0.06 mM, respectively. We present the crystallographic structure of TcGlcK as determined by seleno-methionine (Se-Met) multiwavelength anomalous dispersion (MAD) to 2.1 Å resolution. The structure shows a homodimer in the asymmetric unit, while a plausible tetramer can be generated by crystal symmetry. Each monomer is complexed to β-D-glucose and ADP. While the studied hexokinases exert a preference for α-Dglucose, enzymatic assays using different D-glucose anomers further indicate that TcGlcK uses preferentially the β anomer as substrate. Comparison of enzyme–substrate interactions between TcGlcK and other hexokinase–glucose complexes points out a mechanism acquired by hexokinases to enhance their affinity for α-D-glucose.

Results Structure solution His-tagged recombinant TcGlcK was expressed in the E. coli BL21 strain and purified to homogeneity by nickel affinity chromatography. Crystals of TcGlcK in complex with D-glucose and ADP were obtained by the hanging-drop, vapor-diffusion method, using PEG3350 as precipitant agent and di-ammonium hydrogen citrate as additive. A complete native dataset was collected to 2.1 Å maximum resolution. All molecular replacement attempts to derive initial phases failed, as was expected from the low sequence identity (below 16%) between TcGlcK and EcGlcK, the best model available. Se-Met derivative TcGlcK crystals were produced under the conditions used to grow the native crystals. A single Se-Met TcGlcK crystal was used to collect three complete data sets (peak, inflection and remote), to 2.6 Å maximum resolution, and to solve the structure by a MAD approach as further

Crystal Structure of Trypanosoma cruzi Glucokinase

1217

Table 1. Data-collection and processing and refinement statistics Dataset

Native

Space group Cell parameters a (Å) b (Å) c (Å) α = β = γ (deg.) Wavelength (Å) Resolutiona (Å) Measured reflections Unique reflections Completeness (%) Rsymb I/σ(I) Refinement R/Rfreec (%) rmsd from ideal Bond lengths (Å) Bond angles (deg.) Mean B-factor (Å2)

P21212

P212121

107.9 125.7 65.1 90 0.8140 30–2.1 (2.18–2.1) 362,838 52,608 99.8 (98.5) 0.103 (0.898) 18.1 (2.0)

108.8 126.2 132.3 90 0.9776 35–2.8 (2.95–2.8) 353,692 45,376 99.6 (99.4) 0.076 (0.246) 20 (5.2)

a b c

Peak

Inflection

0.9769 358,247 45,411 99.7 (99.6) 0.068 (0.228) 21.2 (5.7)

Remote

0.9732 313,282 45,312 99.5 (99.1) 0.071 (0.363) 17.7 (3.5)

19.8/25.6 0.011 1.359 40.8

Values in parentheses are for the outer resolution shells. Rsym = ∑|Io–bIN|/∑Io, where bIN is the average intensity for symmetry-related reflections. R = ∑‖Fo|–|Fc‖ / ∑|Fo|, where Fo and Fc are observed and calculated structure factors.

explained in Materials and Methods. An initial model built into the MAD-derived electron density map was transformed by molecular replacement into the cell of the native crystal. The final structure was further constructed and refined against the native higher resolution data set. Data processing and refinement statistics are presented in Table 1. The complete refined asymmetric unit contains two polypeptide chains, two ADPs, two β-D-glucose molecules and 483 water molecules. Clear electron density is present for all ligand molecules and for most of the polypeptide backbone. Six additional residues, from the His-tag extension, are present at the N terminus of both polypeptide chains. Residues 197–199, of both subunits, were not included in the final model due to poor electron density. After complete refinement, 89.9% of the residues were in the most favored regions of the Ramachandran plot. Overall fold and quaternary structure The TcGlcK structure contains a homodimer in the asymmetric unit. Each monomer is composed of two domains, a small α/β domain (residues 1–130 and 353–367) and a large α + β domain (residues

145–352) connected by an α-helix (α4). The active site is located in a deep cleft between these domains. The small domain consists of a central, mixed sixstranded β-sheet (β1–β5 and β9, with β3 antiparallel to the others) surrounded on one side by the C-terminal α-helix (α15) and on the other side by three α-helices (α1-α3), and a small three-stranded anti-parallel β-sheet (β6-β8). The large domain contains a six-stranded mixed β-sheet (β10–β15, with β10 and β12 anti-parallel) and a cluster of α-helices (α7–α15) with a large central α-helix (α11). Structures of several members of the hexokinase family are available for comparison with TcGlcK. Structural superposition and C α rms deviation suggest that TcGlcK is related more closely to the bacterial glucokinase than to eukaryote hexokinases (Table 2). The TcGlcK monomer structure resembles that of EcGlcK.21 The main differences between the TcGlcK and EcGlcK monomers are located in the N-terminal domain. The most notable difference is an additional strand (β1) and α-helix (α1) present in TcGlcK, without equivalents in EcGlcK (Figure 1). For EcGlcK, it has been shown that the domains undergo a large displacement with regard to each other upon substrate binding. 21 Comparison between the glucose-bound and the free EcGlcK

Table 2. Structural superposition of Trypanosoma cruzi glucokinase (TcGlcK) with Escherichia coli glucokinase (EcGlcK), Arthrobacter sp. glucomannokinase (ArGMK), human hexokinase type I (HsHKI) and type IVor glucokinase (HsGlcK) and Schistosoma mansoni hexokinase (SmHK)

EcGlcK ArGMK HsHK1 HsGlcK SmHK

Superposed residues

Identity (%)

Cα rmsd (Å)

Mass (kDa)

D-Glucose

anomer

Resolution (Å)

PDB code (reference)

287 225 255 259 263

20.5 17.3 13.7 13.5 14.8

2.2 2.6 3 3 3.2

50 30 100 50 50

β β α α α

2.2 1.8 1.9 2.3 2.6

1sz2 (21) 1woq (22) 1cza (16) 1v4s (17) 1bdg (18)

1218

Crystal Structure of Trypanosoma cruzi Glucokinase

Figure 1. Structure-based sequence alignment of T. cruzi glucokinase (TcGlcK), E. coli glucokinase (EcGlcK) and Arthrobacter sp. glucomannokinase (ArGMK). Gray boxes P1 and P2 both indicate phosphate-binding sites. Residues involved in hydrogen bond contacts to ADP and β-D-glucose are marked with ● and ○, respectively. Underlined residues participate in tetrameric contacts. Secondary structure elements were assigned with the DSSP program,43 and the Figure was prepared with ESPript.44

Crystal Structure of Trypanosoma cruzi Glucokinase

showed that the small domain undergoes a ∼15° rotation with respect to the large domain upon glucose binding, resulting in an open to closed transition. Superposition of the TcGlcK onto the “open” and “closed” EcGlcK structures results in a Cα deviation of 2.6 Å and 2.2 Å, respectively. This confirms that glucose/ADP-bound TcGlcK is also in a closed conformation. Two TcGlcK monomers assemble into the homodimer found in the asymmetric unit of the native crystal. This subunit interface buries an accessible surface area of 1860 Å2 (corresponding to 12% of the total surface of the dimer). A plausible tetramer can be generated from the dimer in the asymmetric unit by applying crystal symmetry (Figure 2). The same complete tetramer was also observed in the asymmetric unit of the Se-Met derivative crystal although the refinement of this structure has not been pursued any further. The tetramer contacts are exclusively established by residues belonging to α-helix α7 of the large domain (underlined residues in Figure 1). This tetramer interface buries an accessible surface area of only 483 Å2, indicating a relatively weak interaction. Yet, 12 hydrogen bonds are concentrated in this small contact area (compared to 15 hydrogen bonds in the dimer interface). Mass spectrometry analysis performed with the Se-Met derivative and the non-derivative TcGlcK purified by nickel affinity chromatography resulted in molecular masses of 89,620.0(±3.0) Da and 88,304.0(±3.4) Da, respectively. This mass difference of 1316 Da corresponds to the TcGlcK dimer with 14 Se-Met per monomer taking a mass increase of 47.0 Da per modified Met residue. Size-exclusion chromatography through a column pre-packed with a silica-based resin and equilibrated in the presence of 50 mM D-glucose was performed to further purify the native TcGlcK. Under these conditions, the TcGlcK eluted as a monomer with a molecular mass of 44.4 kDa and specific activity of

1219 34 units/mg. This molecular mass was further confirmed by mass spectrometry with a measured mass of 43,585(±8) Da. These results confirm that TcGlcK is active as a monomer and, together with data from additional size-exclusion chromatography experiments (A.J.C. et al., unpublished results), can exist also at higher oligomeric states like dimer and tetramer, depending on the protein concentration, the ionic strength of the buffer and the presence of ligands. Ligand binding in the active site One β-D-glucose and one ADP molecule are associated with each TcGlcK monomer in the active site located between the small and large domains. The β-D-glucose interacts via hydrogen bonds with residues Asn105, Asn130, Asp131 (bidentate), Glu207 (bidentate) and Glu236 (Figures 1 and 3(a)). While Asn105, Asn130 and Asp131 are provided by the small domain, Glu207 and Glu236 are located on the large domain. Glu207 is located between β13 and η4, and Glu236 is in the small αhelix α8. An α-helix, α7 is positioned between the conserved, glucose-binding residues Glu207 and Glu236. This α-helix, which corresponds to α4 in EcGlcK but is absent from ArGMK (see Figure 1), plays an important role in the tetramer formation of TcGlcK. The adenine moiety of ADP interacts with its 6-amino group and its N1 via bidentate hydrogen bonds with residue Asn312, located in α12 (Figure 3(b)). The adenine N3 interacts via a water molecule with the main chain carbonyl group of Gly184 and the Tyr282 hydroxyl group. The ribose moiety of ADP interacts with Asn308 (ribose O4′) and Thr185 (ribose 3′OH). The β phosphoryl group makes two hydrogen bonds with Thr185, one with its main chain amide and one with its side-chain hydroxyl group.

Figure 2. Tetrameric state of TcGlcK. The small domains of all four monomers are colored red. The large domains of subunits A and D are colored gray, and B and C are colored green. The tetramer corresponds to the asymmetric unit of the Se-Met derivative crystal, and each dimer (AB or CD) corresponds to an asymmetric unit of the crystal of the native protein. The α-helix (α7 according to Figure 1) responsible for the dimer–dimer interaction is seen in the core of the tetramer.

1220

Crystal Structure of Trypanosoma cruzi Glucokinase

After equilibration of the α- and β-D-glucose solutions by incubation at 60 °C for 10 min, the v/S curves changed to intermediate values closer to the unheated β-D-glucose curve (Figure 5).

Discussion We present the crystal structure of the TcGlcK in complex with its substrate β-D-glucose and one of its reaction products, ADP. This TcGlcK structure presents the first image of an ATP-dependent glucokinase determined in complex with an ADP molecule. A putative ATP-binding site has been suggested in the EcGlcK structure 21 based on homology comparison to hexokinase in complex with glucose and ADP.16 However, modeling the adenosine interactions in the hexokinase family is not a trivial exercise, due to the poor sequence identity among members of this protein family. Apart from the residues interacting with the hexose, only two short patterns, predicted to interact with the phosphoryl groups of the ATP molecule, are well conserved between hexokinases and glucokinases. These regions were originally named phosphate sites 1 and 2 (see Figure 1) and were identified in other proteins containing an ATPase domain.23 The availability of the TcGlcK structure complexed with ADP provides a more realistic insight into the interactions made by the α- and β-phosphoryl groups and the adenosine moiety with the protein residues. Multiple oligomeric states of TcGlcK Figure 3. 2|Fo|–|Fc| electron density maps for (a) β-D-glucose and (b) ADP found in each TcGlcK active site. β-D-Glucose and ADP maps have contour levels of 1.5 σ and 1.0 σ, respectively. Hydrogen bonds between ligands and TcGlcK residues are shown as green dashes. Residues located in small or large domains are presented with their Cα atoms colored red and gray, respectively.

Anomer preference of TcGlcK The observation of a β-D-glucose molecule in the active site of the TcGlcK crystal structure raises the question of whether this enzyme preferentially binds this glucose anomer. To determine the anomer specificity, the enzyme's catalytic constants were determined for the reaction when either the α or the β-D-glucose anomer was provided as substrate and with an equilibrated solution of substrates, using two different assay systems, as described in Materials and Methods. No significant difference in the enzymatic constants was observed when comparing the results obtained by each assay system. The data given in Figure 5 and Table 3 show that TcGlcK has a slight preference for the β anomer. The ratios Km α-D-glc/Km β-D-glc and Vmax α-D-glc/Vmax β-D-glc are 3.9 and 0.6, respectively.

The recombinant TcGlcK has been shown to exist in equilibrium between monomeric and dimeric states, dependent on the protein concentration (A.J.C. et al., unpublished results). In this work, we confirm by electrospray mass spectrometry the existence of both TcGlcK states in solution. The TcGlcK dimeric state was obtained after nickel affinity chromatography and further concentration. The monomeric TcGlcK was found after an additional size-exclusion purification step in the presence of 50 mM glucose. In the TcGlcK crystal, where the protein concentration is higher than 10 mg/ml, we can observe a tetrameric molecule with 222 symmetry as the highest oligomeric state (Figure 2). The tetrameric state observed in the crystal packing and the dimeric state obtained by mass spectrometry before size-exclusion experiments suggest that an increase in protein concentration favors higher oligomeric states. From a mechanistic point of view, however each individual TcGlcK monomer is equipped with all required ATP/Mg 2+ and glucose-interacting residues. Hence, there is no obvious need for TcGlcK to acquire higher oligomeric states in order to bind both substrates in a reactive conformation. The presumed tetramer found in the crystal is stabilized by interactions between residues localized in helix α7, in the large domain (Figure 1, underlined

Crystal Structure of Trypanosoma cruzi Glucokinase

1221

Figure 4. Detailed view of the TcGlcK tetramer-stabilizing interactions. Monomer chains are indicated inside circles. Asp220 makes hydrogen bonds to the main-chain amine group of residues Ile219 and Asp220 in α7. The Ile219 residues of all four subunits form a hydrophobic core. Bidentate hydrogen bonds between Gln223 residues of pairwise opposing subunits connect the dimers.

residues). In the TcGlcK structure, the pair of α7 helices of the AB dimer interacts with the pair of α7 helices of the CD dimer in a head-to-tail fashion (Figure 2). Four hydrogen bonds, between Gln223 (A)-Gln223(D) and Gln223(B)-Gln223(C), connect parallel α7 helices from the opposite dimers (see Figure 4). The Asp220(A) side-chain carboxyl group makes hydrogen bonds to the main-chain amine groups of Ile219(C) and Asp220(C); the same interactions connect Asp220(C) to Ile219(A) and Asp220(A). These interactions connect monomers A and C, and a similar hydrogen-bond network is observed between subunits B and D. Moreover, the Ile219 side-chains of all subunits are symmetrically packed by hydrophobic interactions. In summary, between the monomers of each dimer, AB or CD, a

Figure 5. TcGlcK velocity (μM NADPH produced per minute) versus substrate concentration plot, using solutions of different anomeric forms of the glucose substrate: α-D-glucose (■), heated α-D-glucose (□), β-D-glucose (●) and heated β-D-glucose (○). Stock solutions of all substrates were prepared at 0 °C at a 50-fold higher concentration than that used during the measurements. To equilibrate the α and β-anomers, solutions were heated to 60 °C for 10 min and then cooled to 0 °C before being used in the measurements.

combination of hydrophobic and hydrogen bond interactions is established, while opposite dimers interact almost exclusively via hydrogen bonds. A comparison of EcGlcK versus TcGlcK dimers shows that when monomers A from the different molecules are superposed, the adjacent monomers B do not superpose as well. Upon superposition of the A subunits, a rotation of 17° of the B subunits vis-à-vis each other is observed. One consequence of this change in dimer organization is that in EcGlcK the α4 helices (equivalent to helix α7 in TcGlcK) from both subunits in the dimer lie parallel with each other. A cross-linked connection between two EcGlcK dimers as observed for the TcGlcK tetramer would therefore require major rearrangements in the α4 orientation. Moreover, Ile219 and Asn223 of TcGlcK are substituted in EcGlcK by Glu167 and Ile171. These substitutions would not allow EcGlcK to establish the same tetrameric interactions as observed in TcGlcK. Indeed, the EcGlcK was characterized by dynamic light-scattering as a dimeric molecule.10 In ArGMK, which is a monomer in solution,24 there is no α-helix segment corresponding to TcGlcK α7 (or EcGlcK α4). The segment between Glu168 and Glu181, equivalent to Glu207 and 236 from TcGlcK, is reduced to the helix turn η12 and small β-hairpin β11-β12.

Table 3. Kinetic constants of TcGlcK obtained for different anomer solutions of D-glucose D-Glucose

α αa βa β

anomer

Km (mM)

Vmax (μmol NADPH.min−1)

2.75 ± 0.1 0.85 ± 0.1 1.05 ± 0.1 0.70 ± 0.05

33.1 ± 0.5 48.4 ± 1.8 51.5 ± 1.9 55.6 ± 1.6

a These anomer solutions were equilibrated by heating at 60 °C for 10 min, followed by cooling to 0 °C before measurement.

1222 Ligand binding and implications for the catalytic mechanism Members of the hexokinase family, including hexokinases, glucokinases and Euryarchaeota ADP-dependent glucokinases, contain two highly conserved β-hairpins per subunit (called P1 and P2), one in each domain, that are responsible for coordination of the phosphoryl groups of ATP or ADP. 23 Relevant differences in the active site between ADP and ATP-dependent glucokinases are therefore restricted to the adenosine-binding interactions. In the ADP-dependent glucokinase of Thermococcus litoralis, the phosphates α and β of ADP are the positional equivalents of the phosphates β and γ of ATP in ATP-dependent glucokinases. As a consequence, the position of the adenosine moiety shows a shift by one phosphate unit.25 In the TcGlcK structure, the phosphate-binding loops P1 and P2 are located between strands β2-β3 and β11-β12, respectively (Figures 1 and 6). Thr185, located on P2, makes two hydrogen bonds to the phosphate β oxygen atoms (Figure 3(b)). The α phosphate group interacts via a water-mediated hydrogen bond with the Glu307 main-chain amide group. The nucleoside moiety of the bound ADP molecule is anchored by Asn308, which makes a hydrogen bond to the ribose ring oxygen, and by Asn312, which forms a bidentate hydrogen bond with the adenine ring N1 and adenine amine group at position 6 (Figure 3(b)). The three ADP-interacting

Crystal Structure of Trypanosoma cruzi Glucokinase

residues Glu307, Asn308 and Asn312 are all located on helix α12 of TcGlck. The adenosine group is wedged between α12 and the first turn of α9. This first turn of α9 presents a conserved sequence pattern reading S-(G/A)-X-(G/A) (Figure 1). The small sidechain residues in this pattern permit the accommodation of the ADP ribose moiety, explaining the need for their conservation. Ser240 in α9 makes a hydrogen bond to the Glu236 main chain carbonyl group, in α8. The orientation of α8 and α9 is important, since Glu236 participates in the carbohydrate coordination (Figure 6). In analogy with other glucokinases and hexokinases,16,21 an SN2 mechanism can be envisioned for TcGlcK, comprising a nucleophilic attack of the O6 of glucose on the electropositive P atom of the γ-phosphoryl of ATP. In this respect, Asp131 of TcGlcK is ideally oriented, at 2.8 Å from the O6 of glucose, to act as a catalytic base by abstracting a proton from this hydroxyl group (Figure 3(a)). Such a role of Asp131 as a general base is in agreement with a similar proposed role for the corresponding residues Asp657 and Asp100 in HsHKI 16 and EcGlcK,21 respectively. No specific residue to function as a general acid for the protonation of ADP as the leaving group, has been identified in mammalian or bacterial hexose kinase family members. However, the possibility that a Mg2+-coordinated water molecule might fulfill such a role has been suggested for HsHKI.16 Another catalytic strategy employed by hexokinases is the stabilization of the developing negative charge on the γ-phosphoryl of ATP in the transition state of the (associative) nucleophilic displacement reaction. This is accomplished by coordination to the magnesium ion and via the positioning of a positively charged residue in proximity to the γ-phosphoryl in the transition state. In HsHK1, an arginine residue, Arg539 (corresponding to Lys15 in the ATP-dependent Sulfolobus tokodaii hexokinase26) is recruited for this role. This residue corresponds to Arg36 in TcGlcK, located adjacent to the P1 loop. In our ADP/glucose-bound TcGlcK structure, the guanidinium group of Arg36 is located at a distance of about 6 Å from the β-phosphoryl of ADP. However as is often seen in kinases or ATPases, the P1 loop (or even the entire small subunit) could undergo further conformational changes upon binding of Mg2+/ATP or in the transition state, bringing Arg36 to an appropriate distance for charge stabilization. Discrimination between glucose anomers in the active site of hexo- and glucokinases

Figure 6. Detailed view of the TcGlcK complex with β-D-glucose and ADP. Residues interacting with β-Dglucose, the ADP adenosine group and the β phosphate group are represented by sticks and colored blue, cyan and magenta, respectively. Ser240, Gly241 and Gly243 in the first turn of α9 are colored orange. The hydrogen bond between Ser240 and Glu236 is represented by a broken line. Phosphate sites 1 and 2 (P1 and P2) are colored magenta. Some protein segments were excluded from the image to allow a clear view of the active site.

D-Glucose and D-glucose 6-phosphate are examples of hexose carbohydrates that, once in solution, undergo spontaneous interconversion between α and β anomeric forms. This reaction, known as mutarotation, happens at slow rates when compared to the corresponding enzyme-assisted process. Rate constants for spontaneous mutarotation of glucose and glucose 6-phosphate are 0.015 min−1 and 0.09 min−1, respectively.27 In equilibrated solu-

Crystal Structure of Trypanosoma cruzi Glucokinase

tions of D-glucose and D-glucose 6-phosphate, the ratio of the α to β anomer is 33:66 and 20:80, respectively. Several enzymes of the carbohydrate metabolism show different levels of anomeric selectivity, which can vary from a moderate preference to full specificity. Salas et al.28 demonstrated, by kinetic assays, that yeast hexokinase has a moderate preference for α-D-glucose, while muscle phosphoglucomutase and yeast glucose-6-phosphate dehydrogenase are specific for α and β-D -glucose 6-phosphate, respectively. The low spontaneous mutarotation rates and the anomer selectivity of key enzymes of sugar metabolism highlight the importance of mutarotases to support fast metabolic fluxes. Well known examples of mutarotases are aldose 1-epimerase, which shows a fourfold higher catalytic constant for the reaction with D-galactose over D-glucose anomers,29 and hexose-6-phosphate mutarotase, which catalyzes the mutarotation between D-glucose 6-phosphate anomers.30 On the basis of sequence identity (50%) with yeast hexokinase, and a complete conservation of glucoseinteracting residues, a preference for the α anomer is expected for T. cruzi hexokinase.28 In contrast to the hexokinases, our kinetic and structural data now indicate a moderate preference of the TcGlcK for the β anomer of glucose. The different anomer selectivity between hexokinase and glucokinase might explain why both enzymes were found to coexist in glycosomal fractions of T. cruzi and L. mexicana (A.J.C., unpublished results). Considering that in an equilibrated glucose solution β-Dglucose is the most abundant anomer, the hexokinases should possess a method for selection of the α anomer. The structural comparison between hexokinase and glucokinase crystallized in complex with D-glucose (Table 2) now allows us to propose the mechanism for such a discrimination. In HsHKI, the α-D-glucose molecule is coordinated by Asn656, Glu657, Glu708, Glu742, Thr620 and Lys621 (Figure 7). Similar interactions are found in SmHK and HsGlcK glucose complexes. The first four residues listed above are equivalent to Asn130, Asp131, Glu207, and Glu236 in TcGlcK. However, residues corresponding to Thr620 and Lys621 are missing from the glucokinase structures. These two additional residues are highly conserved in the hexokinase group; they form part of the protein sequence signature, [LIVM]-G-F-[TN]-F-S-[FY]-P-x(5)[LIVM]-[DNST]-x(3)-[LIVM]-x(2)-W-T-K-x-[LF] (PROSITE, PDOC00370), used to identify new members of this enzyme group. The modeling of a β-D-glucose molecule in the human hexokinase active site shows that, in the β conformation, the hydroxyl group at C1 would make unfavorable contacts with Cγ and Cε of Lys621 (Figure 7). Moreover, in the β configuration, a hydrogen bond with Glu742 would be lost. Members of the glucokinase group, including TcGlcK, do not have the WTK tri-peptide in their protein signature that is present in the hexokinase group. As a consequence, they are expected not to discriminate against the β anomer of D-glucose.

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Figure 7. Superposition of β-D-glucose (magenta) with α-D-glucose (green) present or fitted in the active site of human hexokinase I.16 Hydrogen bonds that are identical for both anomers are in gray, those bonds exclusive for either α- or β-D-glucose are given in green and magenta, respectively. Unfavorable contacts between the Lys621 side-chain carbon atoms and the β-D-glucose hydroxyl group at C1 are indicated by red dashes.

Our current TcGlcK structure and the catalytic constants obtained for α- and β-D-glucose confirm the lack of α-anomer specificity in this member of the glucokinase group. In contrast, TcGlcK has a slight preference for β-D-glucose, as indicated by its approximately fourfold lower Km and twofold higher Vmax with β-D-glucose as substrate compared to the values obtained for α-D-glucose. This preference for the β anomer in TcGlcK could be achieved by a weak hydrogen bond with Asn105, which would not be possible with the α anomer (Figure 3(a)). These ratios are probably underestimates, since spontaneous mutarotation should have happened within the time-scale of the measurement.

Concluding Remarks This work describes the structure of TcGlcK in complex with β-D-glucose and ADP. The structure was used to assign the interactions that stabilize the tetrameric and dimeric states. The well-defined electron density associated with β-D-glucose raised the hypothesis that TcGlcK could be selective for the β anomer of D-glucose. That would differentiate the substrate specificity between hexokinases and glucokinases, and provide an explanation for the coexistence of both enzymes within the glycosomes of T. cruzi and different Leishmania species. Kinetic assays with both glucose anomers confirm the TcGlcK preference for β-D-glucose. A structural comparison between hexokinases and glucokinases resulted in the identification of a highly conserved motif, comprising the residues WTK of the hexokinase active site, which is involved in the discrimination of hexokinases in favor of the α-D-glucose anomer.

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Materials and Methods Expression and purification His-tagged recombinant TcGlcK was expressed and purified to homogeneity by nickel-affinity chromatography (A.J.C. et al., unpublished results). Briefly, E. coli strain BL21(DE3) harboring the recombinant plasmid TcGlcK/pET28a was grown in ZYM-5052 auto-induction medium for 24 h at 22 °C.31 Cells were centrifuged, resuspended in 0.1 M Hepes (pH 7.6), 10 mM imidazole, 2 mM MgCl2, and disrupted in a French press at 6.9 MPa. The soluble protein extract was loaded onto a column containing 1 ml of HisLink resin (Promega), and purified as recommended by the resin manufacturer. TcGlcK eluted from the column in 0.1 M Hepes (pH 7.6), 350 mM imidazole, 2 mM MgCl2. In order to obtain the Se-Met derivative TcGlcK, the recombinant E. coli cells were grown in 100 ml of Overnight Express Autoinduction System 2 medium (Novagen). The expression and purification procedures for the Se-Met derivative TcGlcK were identical with those used for the native protein. Size-exclusion chromatography and mass spectrometry Mass spectrometry analysis was performed with two different TcGlcK preparations. The first set of samples was prepared with Se-Met derivative (9 mg/ml) and the nonderivative TcGlcK (2.7 mg/ml) eluted from the HisLink column. In order to reduce the salt content from the elution buffer, 20 μg of protein was diluted in 200 μl of 5% (v/v) acetonitrile in 0.1% (v/v) trifluoroacetic acid and loaded onto a C18 reverse phase spin column (Agilent). After extensive washing with 5% acetonitrile in 0.1% trifluoroacetic acid, the protein was eluted with 100 μl of 70% acetonitrile in 0.1% (v/v) formic acid and submitted directly to mass analysis. The second sample was prepared with native TcGlcK purified via the HisLink column, concentrated to 5 mg/ml in an Amicon 30 k centrifugal filter device (Millipore) and loaded onto a sizeexclusion chromatography column Biosep-SEC-S3000 (Phenomenex®) pre-equilibrated with 0.05 M triethanolamine (pH 7.5), 0.15 M NaCl, 2 mM MgCl2, and 50 mM glucose. Before loading the TcGlcK onto the column, glucose was added to the protein solution to a final concentration of 50 mM. After elution, fractions containing TcGlcK were identified by enzymatic activity and SDSPAGE. The salt content of the eluate was reduced tenfold in an Amicon 30 k centrifugal filter device. The sample was diluted to 5 pmol/μl in 70% acetonitrile in 0.1% formic acid and submitted to mass analysis. Mass spectra were acquired with a LCQ DECA XP Plus ion trap mass spectrometer (ThermoFinnigan, San José, CA, USA) equipped with a microflow electrospray ionization probe. Full MS spectra were taken in two mass ranges (500–2000 m/z) or (500– 4000 m/z) depending on the oligomeric state. In-source collision energy was set at 65% to reduce formation of adducts. Spectra were averaged and deconvoluted with the BIOMASS software within the BIOWORKS 3.2 software package (ThermoFinnigan). Crystallization, data collection and phasing His-tagged TcGlcK was concentrated to 10 mg/ml in 50 mM Hepes (pH 7.5), 0.2 M imidazole, 2 mM MgCl2

Crystal Structure of Trypanosoma cruzi Glucokinase using an Amicon 30 k centrifugal filter device. Before crystallization, ADP (Roche Applied Science) and D-glucose (Acros) were added to the protein solution to a final concentration of 5.0 mM and 12.5 mM, respectively. Initial crystallization conditions were screened by the sparsematrix method with solutions from Nextal Screening Suites (Qiagen). Crystals were grown by the hangingdrop, vapor-diffusion method using mixed equal volumes (2 μl) of protein and reservoir solution, with equilibration against 0.5 ml of reservoir solution, at 16 °C. Native TcGlcK crystals appeared within one day in 20% (w/v) PEG3350 and 0.2 M di-ammonium hydrogen citrate (PEG's Suite, formulation 96; Qiagen). Additional efforts were made to optimize the crystal quality by varying the original concentration of precipitant agent and additive. Crystals suitable for X-ray diffraction measurements were obtained with PEG3350 in the range of 12–20% in combination with di-ammonium hydrogen citrate concentrations varying from 0.1–0.4 M. A crystal of native TcGlcK grown in 20% PEG3350 and 0.4 M di-ammonium hydrogen citrate was transferred to a cryoprotectant solution containing 20% (v/v) glycerol, 24% PEG3350, 0.3 M di-ammonium hydrogen citrate, 8.5 mM D-glucose and 12.5 mM ADP before being flash-frozen in a nitrogen stream at 100 K at the X11 beam line at EMBL/DESY, Hamburg (Germany). The native data set was processed with DENZO and SCALEPACK.32 The Se-Met derivative TcGlcK protein solution was concentrated to 12 mg/ml in 50 mM Hepes (pH 7.5), 0.2 M imidazole, 2 mM MgCl2. Before crystallization, ADP and D-glucose were added to final concentrations of 5.0 mM and 12.5 mM, respectively. From 12–16% (w/v) PEG3350 and 0.1–0.2 M di-ammonium hydrogen citrate combinations were used in attempts to obtain crystals. Hanging drops containing 2 μl of protein solution mixed with 2 μl of reservoir solution were equilibrated with 0.5 ml of reservoir solution at 16 °C. One crystal grown in 16% (w/v) PEG3350 and 0.15 M di-ammonium citrate was transferred through solutions containing increasing concentrations of glycerol, while preserving the precipitant and additives as originally in the drop. The glycerol content was increased in steps of 5% (v/v), each of 2 min duration, to a final cryoprotectant concentration of 25% (v/v). After passing through the 25% (v/v) glycerol solution, the crystal was flash-frozen in a nitrogen stream at 100 K. Three datasets suitable for multiple wavelength anomalous dispersion phasing methods were collected at the BW7A beam line at EMBL/DESY, Hamburg (Germany). Each data set was processed with MOSFLM33 and SCALA34 (see Table 1 for data processing statistics). Initial phases were estimated with the autoSHARP35 script in the CCP4i interface.36 The three Se-Met derivative datasets, with 14 heavy-atom sites per subunit, four subunits in the asymmetric unit and a subunit molecular mass of 42 kDa, were used as input data. The AutoSHARP parameter was set to the highest level of accuracy; SHELXD37 and ARPwARP38 subroutines were selected for heavy-atom detection and automated model building steps, respectively. Model inspection and extension were performed with Coot.39 The MOLREP program40 was used to find the orientation of the partially built model in the native TcGlcK cell. Additional model extension was alternated with REFMAC41 restrained refinement cycles using the native reflections amplitudes. The final model geometric parameters were verified with PROCHECK.42 All structural representations were prepared with Pymol†. † http://pymol.sourceforge.net/

Crystal Structure of Trypanosoma cruzi Glucokinase Anomer specificity assays Glucokinase activity of TcGlK was assayed using two alternative coupled systems, one with yeast glucose-6phosphate dehydrogenase (yG6PDH, Roche Applied Science) and the other with pyruvate kinase (PyK, Roche Applied Science) and lactate dehydrogenase (LDH, Sigma). In the first system, yG6PDH converts β-D-glucose 6-phosphate into D-glucono-1,5-lactone 6-phosphate with the concomitant reduction of NADP+ to NADPH. The formation of NADPH was monitored at 340 nm in a Beckman-Coulter DU-800 spectrophotometer. Measurements were performed at 20 °C in 0.1 M triethanolamine (pH 7.6), 2.5 mM MgCl2, 5 mM ATP, 0.64 mM NADP+ (Roche Applied Science), 1.4 U of yG6PDH and 0.02 U of TcGlcK. In the second system, PyK converts phosphoenolpyruvate into pyruvate using the ADP produced by TcGlcK as phosphoryl acceptor; subsequently, LDH converts the pyruvate formed into lactate with the concomitant oxidation of NADH to NAD+. The disappearance of NADH was monitored spectrophotometrically at 340 nm and 20 °C in 0.1 M triethanolamine (pH 7.6), 2.5 mM MgCl2, 5 mM ATP, 1 mM phosphoenolpyruvate, 0.3 mM NADH, 4 U of PyK, 5 U of LDH and 0.02 U of TcGlcK. In order to reduce the spontaneous mutarotation rate, α-D-glucose (Acros) and β-D-glucose (MP Biomedicals) were dissolved in cold water (4 °C) at a 50-fold higher concentration than that used in the assay and kept in an ice-bath until the kinetic measurements. For each measurement, 10 μl of substrate was pipetted into a quartz cuvette and the reaction was started by the addition of 490 μl of a mixture containing the buffer, cofactors and enzymes in the concentrations described above. The assay concentrations for α-D-glucose were 0.2 mM, 0.4 mM, 0.8 mM, 1.6 mM, 3.2 mM and 6.4 mM; for β-D-glucose the values were 0.05 mM, 0.1 mM, 0.2 mM, 0.4 mM, 0.8 mM and 1.6 mM. For assays with an equilibrated mixture of anomers, α- and β-D-glucose solutions were heated for 10 min at 60 °C, cooled in an ice-bath and used for TcGlcK enzyme activity measurements at concentrations of 0.1 mM, 0.2 mM, 0.4 mM, 0.8 mM, 1.6 mM and 3.2 mM. Values of the Km and Vmax of TcGlcK for glucose were obtained by fitting the reaction velocity versus substrate concentration plots to the equation: V ¼ Vmax S=ðS þ Km Þ GlcK enzymatic units (U) are defined as the amount of protein necessary to produce 1 μM NADPH per min. For glucose-6-phosphate dehydrogenase, PyK and LDH, units were used as defined by the manufacturers. Data Bank accession code Coordinates and structure factors for the TcGlcK complexed with β-D-glucose and ADP have been deposited in the RCSB Protein Data Bank under the accession code 2q2r.

Acknowledgements A.T. acknowledges a ‘Unicore’ post-doctoral fellowship from the Christian de Duve Institute of Cellular Pathology (ICP, Brussels), and A.C.

1225 acknowledges financial support from FONACIT (Venezuela) to spend one year in the laboratory (ICP) in Brussels. W.V. acknowledges the FWOVlaanderen for a post-doctoral grant. This research was supported by grants from the ‘Fonds de la Recherche Scientifique Médicale’ (FRSM) of the ‘Communité française de Belgique’ (contract no. 2.4653.06, to P.M.), the Interuniversity Attraction Poles-Belgian Federal Office for Scientific, Technical and Cultural Affairs (contract no. P5-29 to P.M. and contract P5-05 to D.V.), and the European Commission (INCO-DEV programme, contract no. ICA4-CT2001-10075, to P.M. and J.C.). We are grateful to Dr Fred Opperdoes (ICP) for helpful discussions. The authors acknowledge the EMBL and EMBL staff for beamtime and assistance at beamlines X11 and BW7A (DESY, Hamburg).

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Edited by M. Guss (Received 30 May 2007; received in revised form 6 July 2007; accepted 11 July 2007) Available online 26 July 2007