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Eur. J. Biochem. 271, 2765–2772 (2004) Ó FEBS 2004 doi:10.1111/j.1432-1033.2004.04205.x The crystal structure of glucose-6-phosphate isomerase from Leishmania mexicana reveals novel active site features Artur T. Cordeiro1, Paul A. M. Michels2, Luiz F. Delboni3 and Otávio H. Thiemann1 1 Laboratory of Protein Crystallography and Structural Biology, Physics Institute of São Carlos, University of São Paulo, São Carlos-SP, Brazil; 2Research Unit for Tropical Diseases and Laboratory of Biochemistry, Christian de Duve Institute of Cellular Pathology, Brussels, Belgium; 3Pontificia Universidade Católica de Minas Gerais, Poços de Caldas-MG, Brazil Glucose-6-phosphate isomerase catalyzes the reversible aldose-ketose isomerization of D-glucose-6-phosphate to D-fructose-6-phosphate in glycolysis and gluconeogenesis, and in the recycling of hexose-6-phosphate in the pentose phosphate pathway. The unicellular protozoans, Trypanosoma brucei, T. cruzi and Leishmania spp., of the order Kinetoplastida are important human parasites responsible for African sleeping sickness, Chagas’ disease and leishmaniases, respectively. In these parasites, glycolysis is an important (and in some cases the only) metabolic pathway for ATP supply. The first seven of the 10 enzymes that participate in glycolysis, as well as an important fraction of the enzymes of the pentose phosphate pathway, are compartmentalized in peroxisome-like organelles called glycosomes. The dependence of the parasites on glycolysis, the importance of the pentose phosphate pathway in defense against oxidative stress, and the unique compartmentalization of these pathways, point to the enzymes contained in the glycosome as potential targets for drug design. The present report describes the first crystallographic structure of a parasite (Leishmania mexicana) glucose-6-phosphate isomerase. A comparison of the atomic structure of L. mexicana, human and other mammalian PGIs, which highlights unique features of the parasite’s enzyme, is presented. Leishmania mexicana is a human protozoan pathogen belonging to the order Kinetoplastida [1,2]. Among the kinetoplastid organisms, several human parasites are present, including Trypanosoma brucei, T. cruzi and various Leishmania species that are responsible for diseases such as African sleeping sickness, Chagas’ disease and leishmaniases, respectively, causing serious health problems in tropical and subtropical areas, which, in several cases, are fatal if left untreated. This scenario is aggravated by a lack of effective, available drugs for the treatment of infected individuals, and the reports of drug-resistant parasite strains. Leishmania infection may lead to disorders that can manifest themselves in three different clinical forms – cutaneous, visceral and mucocutaneous leishmaniasis – depending on the Leishmania species involved. The actual treatment for leishmaniasis is based mainly on antimonial compounds that are of low specificity and cause undesirable side-effects [1,2]. Glycolysis is an important, and in some cases the only, metabolic pathway for the ATP supply of these parasites. The first seven of the 10 enzymes that participate in glycolysis are compartmentalized in peroxisome-like organelles called glycosomes [3], a characteristic of all members of the Kinetoplastida order. A consequence of this organellar localization is that the kinetoplastid glycolytic enzymes differ in many kinetic and structural properties from their counterparts in other organisms, and that the flux through the pathway is regulated in a different manner [2,3]. Not only glycolysis is found in glycosomes; also found is a significant fraction of many enzymes of the pentose phosphate pathway [4,5], which uses sugars for the formation of D-ribose-5-phosphate for nucleotide synthesis and NADPH, for biosynthetic processes, and for defense against oxidant stress. This process is also very important for the trypanosomes and leishmanias, particularly to combat oxidative attack by the host. Therefore, both the glycolytic and pentose phosphate pathways have been indicated as promising drug targets [2,6,7]. Glucose-6-phosphate isomerase (often still called by its old name, phosphoglucose isomerase; PGI) is the second enzyme in glycolysis and catalyzes the reversible aldoseketose isomerization of D-glucose 6-phosphate (D-Glc6P) to D-fructose 6-phosphate (D-Fru6P). It is also an enzymatic link between glycolysis and the pentose phosphate pathway. Correspondence to O. H. Thiemann, Laboratory of Protein Crystallography and Structural Biology, Department of Physics and Informatics, Physics Institute of São Carlos, University of São Paulo, Avenue Trabalhador Sãocarlense 400, PO Box 369, 13566–590, São Carlos-SP, Brazil. Fax: + 55 16 273 9881, Tel.: + 55 16 273 8089, E-mail: [email protected] Abbreviations: D-Fru6P, D-fructose-6-phosphate; D-Glc6P, D-glucose6-phosphate; dPGI-Lm, N-terminally deleted glucose-6-phosphate isomerase from Leishmania mexicana; PGI, glucose-6-phosphate isomerase; PGI-Lm, glucose-6-phosphate isomerase from Leishmania mexicana. Enzyme: glucose-6-phosphate isomerase (E.C. 5.3.1.9). Note: The PDB ID code for the solution structures of Leishmania mexicana glucose-6-phosphate isomerase full-length PGI-Lm is 1Q5O and of the form with the 48 residues deleted from its N-terminus (dPGI-Lm), 1Q1I. (Received 3 December 2003, revised 31 March 2004, accepted 6 May 2004) Keywords: Leishmania; phosphoglucose isomerase; glycolysis; substrate–enzyme; human PGI. Ó FEBS 2004 2766 A. T. Cordeiro et al. (Eur. J. Biochem. 271) In the pentose phosphate pathway, PGI recycles one of the products (D-Fru6P) back into the substrate (D-Glc6P) for glucose-6-phosphate dehydrogenase, representing the initial step of the pathway. The pentose phosphate pathway appears to have a dual localization in both Leishmania spp. and T. brucei, as several of its enzymes have been shown to be present in both the cytosol and the glycosomes [4,5]. Whereas most glycolytic enzymes are entirely or predominantly present inside the organelles, PGI, the enzyme shared with the pentose phosphate pathway, is indeed found in both compartments, although in a ratio that differs between Kinetoplastida species. In bloodstream-form T. brucei, most ( 85%) PGI resides in the glycosomes, but in cultured L. mexicana promastigotes (representative of the insect-infective stage), PGI activity was detected mainly in the cytosol, with the remainder (less than 10%) associated with the glycosome [8,9]. This is consistent with a higher pentose phosphate pathway activity and a lower glycolytic activity in promastigotes when compared to the bloodstream form of T. brucei [10–12]. The involvement of PGI in two important pathways of Leishmania metabolism may make it interesting for drug targeting. Recently, a 50% growth inhibition in bloodstream-form T. brucei was observed as a consequence of decreasing the level of PGI by RNA interference. This result is indicative of the central role of PGI in the parasite metabolism [13]. In this report we present the atomic structure of L. mexicana PGI (PGI-Lm) obtained by X-ray diffraction techniques. The comparison of this structure with the available mammalian PGI structures allowed the identification of significant differences between the enzyme of the parasite and its human homologue, which may be exploited in future drug design. Experimental procedures Crystallization data collection and structure determination Two different constructs of the PGI gene from L. mexicana have been expressed in Escherichia coli BL21(DE3) and purified to homogeneity [14]. The two forms correspond to the entire 604 amino acid PGI sequence (PGI-Lm) and a polypeptide from which the N-terminal 47 amino acid residues were deleted (dPGI-Lm), respectively. The N-terminal deletion from L. mexicana PGI does not interfere with the catalytic process. The N-terminal extension is believed to represent an unorganized structure, possibly related to the glycosomal localization of the protein [8,9,14], and could be interfering in the crystallization process. Both forms of the bacterially expressed L. mexicana PGI (PGI-Lm and dPGI-Lm) were successfully crystallized by the hanging drop vapor-diffusion technique, but the dPGI-Lm crystals presented a better intrinsic order [14]. A complete PGI-Lm dataset of 228 frames was collected at 100 K from a single crystal grown under conditions previously reported [14], using a RIGAKU X-ray source and a MAR345 image plate detector. The crystalto-detector distance was set to 250 mm and each frame was exposed for 4 min with a phi oscillation of 0.75°. The dataset was processed using DENZO and SCALEPACK [15]. The molecular replacement solution obtained in the previous work was used as an initial model in the refinement procedure. Crystals of dPGI-Lm were soaked for 5 min in cryoprotectant solution consisting of the reservoir solution and 17.5% methane pentanediol (w/v) containing 3.0 mM D-Fru6P and then flash-frozen in liquid nitrogen. A complete dataset of 140 frames was collected at 100 K for the dPGI-Lm at the X25 beam line of the Brookhaven National Laboratory. This beam line is equipped with a Q315 ccd detector, which allowed the collection of reflections at a 2.3 Å resolution limit with the detector placed 325 mm from the crystal. Each frame was exposed for 15 s with a phi oscillation of 0.75°. The frames were integrated using MOSFLM [16] and the reflections were scaled using SCALA from the CCP4 package [17]. The refined PGI-Lm structure, collected using the RIGAKU X-ray source, was used as initial model in the refinement of the dPGI-Lm. Structure refinement All refinement procedures were performed using the CNS program [18], except for a final TLS parameters refinement performed with REFMAC5 from the CCP4 package [17]. The first structure refined was the PGI-Lm. One monomer of the previously reported solution, obtained using the AMORE program [19], was submitted to a rigid body routine with data ranging between 30 and 3 Å resolution. Several cycles of simulated annealing, using the maximum likelihood method, were performed, followed by coordinate and B-factor refinement using all data up to the 2.6 Å resolution border. Local corrections and N-terminal amino acid residue additions were performed using the O program [20] while inspecting the 2rA|Fo|-D|Fc| map and the m|Fo|-D|Fc| difference map. Water molecules were introduced running the Ôwater-pickÕ script of CNS. Finally, all model atoms were used to define a single group for refinement of TLS parameters with REFMAC5 [17]. The dPGI-Lm data were refined following the same procedures adopted for the PGI-Lm. The water molecules and additional N-terminal residues from the refined PGILm structure were removed from dPGI-Lm. The modified dPGI-Lm polypeptide chain was used as an initial model for the rigid body refinement. Prior to water addition, D-Fru6P was placed into the only large electron density cloud observed at the difference map contoured at 5 s. The correct orientation of the phosphate group, relative to the protein residues involved in its coordination, was driven by structural similarity to the previously reported rabbit PGI in complex with D-Fru6P [21]. Additional information of the D-Fru6P conformation was obtained by observing the 2rA|Fo|-D|Fc| map contoured at 1 s. Water molecules were added using the Ôwater-pickÕ script of CNS with the same parameters as applied to the PGI-Lm. A single group containing all atoms from the model was used for refinement of TLS parameters with REFMAC5 [17]. Structure comparison Superposition and root mean square (r.m.s.) calculations were performed using the Ca atoms and the SWISS PDB Ó FEBS 2004 Leishmania PGI crystal structure (Eur. J. Biochem. 271) 2767 program [22]. D-Fru6P and dPGI-Lm side-chain contacts were analyzed by using the LIGPLOT program [23]. Figures were prepared using the PYMOL program [24]. VIEWER Results and discussion Structure refinement The previously reported molecular replacement solution [14] was characterized as containing one homodimer molecule per asymmetric unit in the P61 space group. The processing of the data described in this study indicate that the noncrystallographic twofold axe, which relates the monomers in the asymmetric unit, described previously, is coincident to a real crystal axe from the higher-symmetry P6122 space group. The choice of P6122 crystal space group reduced the asymmetric unit content to a single monomer. The first Leishmania PGI structure refined, PGI-Lm (pdb code, 1Q5O), had a total of 216 water molecules added, resulting in an Rfactor value of 19.5% and an Rfree value of 24.9%. The refinement of dPGILm-D-Fru6P was concluded with Rfactor and Rfree values of 22.0 and 25.9%, respectively. A total of 180 water molecules and one D-Fru6P are present in the final dPGILm/D-Fru6P model (pdb code, 1Q1I). The refinement of TLS parameters contributed to a decrease of 2% in final R and Rfree values for both molecules. Additional information of both refined structures is presented in Table 1. Description of structures The L. mexicana PGI structure is a homodimer and the monomer subunit is composed of a large and a small a/b sandwich domain and has an extended C-terminal segment (comprising two a-helices) that embraces the other monomer (Fig. 1). There are two catalytic sites per dimer molecule; the catalytic sites are located in the dimer interface formed by adjacent monomers. The PGI-Lm has an overall fold similar to those described for rabbit, human and pig PGIs [25–27]. The main difference is the presence of an N-terminal sequence in PGI-Lm, which may be involved in the functioning of the enzyme inside the glycosome, as discussed previously [8,9,14]. For the first 44 and the last residue – Leu605 – of PGI-Lm (Fig. 2), no electron density was distinguishable in the 2rA|Fo|-D|Fc| map of both chains of the homodimer. The absence of continuous electron density for residues 1–44 indicates that the N-terminal sequence is not ordered. Based on the electron density map and sequence alignment, four additional residues (Val45 to Ser48) could be added to that region of PGI-Lm (Fig. 2). The superposition of the PGI-Lm and the N-terminally deleted dPGI-Lm resulted in an r.m.s. of 0.4 Å. The main difference between the two structures is found in a 10-residue loop (of amino acids Gly433 to Ala442) in which the catalytic His441 is located. Owing to the improved electron density map and higher-resolution data obtained with dPGI-Lm crystals, it was possible to identify the correct Ca trace for this 10-residue loop. Differences vs. mammalian PGIs Table 1. Statistics for data collection and refinement. dPGI-Lm, N-terminally deleted glucose-6-phosphate isomerase from Leishmania mexicana; PGI-Lm, glucose-6-phosphate isomerase from Leishmania mexicana. Structure Data collection Space group Cell dimension a, b and c (Å) a, b and c (°) Resolution range (Å) Unique reflections Redundancy Completeness (last shell) (%) R-sym (%) Refinement Number of atoms Protein Heteroatoms Solvent Used reflections R-factor R-free Rms bond angle <B-factor> (Å2) PGI-Lm dPGI-Lm P6122 P6122 85.74, 85.74 and 350.43 90, 90 and 120 30–2.6 24333 6.7 98.5 (98.8) 5.8 85.13, 85.13 and 350.09 90, 90 and 120 74.53–2.35 32626 7.1 100 (100) 6.1 8607 0 216 23091 19.5 24.9 8649 32 180 30902 22.0 25.9 0.022 1.83 30.9 0.018 1.69 32.8 A significant difference in Ca-r.m.s. can be observed in part of the small domain of Leishmania PGI compared with its Fig. 1. Cartoon representation of Leishmania mexicana glucose-6phosphate isomerase (PGI-Lm). The native enzyme is a homodimer with the monomer subunit formed by two a/b sandwich domains (large and small domains) and a C-terminal a-helix segment that embraces the adjacent monomer. The catalytic residues are distributed among the small domain and C-terminal segment from one monomer, and at the large domain from the other monomer. 2768 A. T. Cordeiro et al. (Eur. J. Biochem. 271) Ó FEBS 2004 Fig. 2. Structure-based alignment of glucose-6-phosphate isomerase (PGI) sequences from different organisms: Leishmania mexicana, Trypanosoma brucei (Tryp; SwissProt code: P13377), human [28], rabbit [25] and pig [27]. The secondary elements of N-terminally deleted glucose-6phosphate isomerase from Leishmania mexicana (PGI-Lm) are represented as h (a-helices) and s (b-sheet strands). The small-domain helix and sheet elements are underlined. Residues in bold are involved in the coordination of the substrate’s phosphate group. Positions of the possible catalytic residues, described in previous work [21], are colored in magenta. In the Leishmania (Leish) sequence, the residues shown in italics are not seen in the electronic density maps of PGI-Lm; the PGI-Lm residues in the black box have a mean Ca r.m.s. deviation of 3.3 Å when superimposed to the human PGI [28]. The PGI-Lm residues in the gray box superimpose with human PGI with a mean Ca r.m.s. of 0.9 Å. Loops A and B are colored green and yellow, respectively. PGI-Lm residues, marked above the alignment using the letter ÔcÕ, are in contact with residues of the opposite monomer by a distance of less than 3.6 Å. The ÔoÕ marks residues of PGI-Lm that are in hydrophobic contact with Met337 (marked with #). human homologue [28] (pdb code, 1jlh), although both present the same overall fold. The small domain from all PGIs is connected to the large domain by two long a-helices named LH-n (long helix connected to the small domain Nterminus) and LH-c (long helix connected to the small domain C-terminus). The PGI-Lm small domain encompasses residues 197–316. The superposition of Leishmania and human PGI (pdb code, 1jlh), using the large domain and the first a-helix (h-l) of the small domain (Fig. 2), results in a Ca-r.m.s. of 0.9 Å for the considered residues. The calculated Ca-r.m.s. for the remaining residues (small domain, except for the h-l a-helix) is 3.3 Å (Fig. 3B). Superposition of just the small-domain residues, including the h-l a-helix, from each monomer results in a mean Car.m.s. of 0.7 Å. This r.m.s. analysis indicates a rigid body displacement between the large and small domains when comparing human and Leishmania PGI. The fact that dimer assembly is driven by contacts between residues located exclusively in the large domain (Fig. 1) suggests that residues connecting large and small domains from the same monomer are responsible for the differences between the PGI structures. Moreover, the high Ó FEBS 2004 structural similarity of the small domain region (r.m.s. of 0.7 Å), observed between the mammalian and parasite PGIs, point to a conserved packing of the secondary structure elements of this domain. Amino acid substitutions in the small domain do not result in significant alterations of the domain interfaces between the two PGIs. We identified, from this superposition analysis, that the presence of Met337 in PGI-Lm, and Ala284 at a structurally equivalent site in the human PGI, contribute significantly to the smalldomain position differences observed between both structures. Met337 is located in an a-helical segment connecting the C-terminal side of the small domain to the LH-c of the large domain (Fig. 2). The first a-helix of the small domain (h-l) is positioned adjacent to the large domain, establishing the main interdomain contacts (Fig. 3A). The mean Ca-r.m.s. for h-l, calculated between human and PGI-Lm, is equivalent to the Ca-r.m.s. calculated between the large domains (< 0.9 Å). The direction of the h-l a-helix can be associated with an imaginary axis describing a rigid body movement of the small domain between the superimposed structures (Fig. 3B). The substitution of Ala284 in human PGI with Met337 in PGI-Lm causes a local stress in both long ahelices (LH-n and LH-c) connecting the large and small domains. Met337 is located at a short a-helix segment (Hmet), side-by-side with h-l of the small domain, and makes hydrophobic contacts to conserved residues in LH-c and LH-n. The Ca atoms of Met337 in PGI-Lm and Ala284 in human PGI are separated by a distance of 1.6 Å, while the Ca atoms of the first residue of LH-n, Tyr342 (in PGI-Lm) and Phe289 (in human PGI) are at a distance of 2.5 Å (Fig. 3C). Finally, it is clear from the structural superposition that PGI-Lm presents its small domain in a more open conformation when compared to the human homologue, resulting in a larger active-site cavity. This may explain the difference in the affinity of the proteins for their substrate and product. A higher Km value for the substrate D-Fru6P was measured for PGI-Lm (242 lM) when compared with that of the human enzyme (99 lM) [14]. Substrate binding The position of D-Fru6P in the dPGI-Lm structure is clearly seen in the difference map calculated in the absence of the ligand (Fig. 4A). However, as it is not clear whether D-7Fru6P is in the open or closed conformation, both Fig. 3. Structural details of the Leishmania PGI. (A) Cartoon representation of large (blue) and small (green) domains from Leishmania mexicana glucose-6-phosphate isomerase (PGI-Lm). The large and small domains are connected by two long a-helices, named LH-n and LH-c; Met337 is located at a-helix ÔH-metÕ in the large domain, which is placed side-by-side with Ôh-lÕ in the small domain (B). Superimposed monomers from human (gray) [28] and PGI-Lm (green) highlight the 2 Å side-displacement of their small domains. An imaginary rotation axis can be placed close to h-l, indicated by the vertices of the drawn lines. (C) Detailed view of Met337 neighboring residues located in both LH-n and LH-c of PGI-Lm (green). The small domain from human PGI is represented by the gray surface. The presence of Met337 at the corresponding position of Ala284 in human PGI causes the displacement of Tyr342 (Phe286 in human PGI) located in the LH-c of PGI-Lm. Leishmania PGI crystal structure (Eur. J. Biochem. 271) 2769 2770 A. T. Cordeiro et al. (Eur. J. Biochem. 271) Ó FEBS 2004 Fig. 4. Stereoview of the active site of N-terminally deleted glucose-6-phosphate isomerase from Leishmania mexicana (dPGI-Lm) with bound D-fructose-6-phosphate (D-Fru6P). (A) Electron density map 2rA|Fo|-D|Fc| (contoured at 1r), colored green, yellow and magenta for loop A, loop B and D-Fru6P, respectively; monomers A and B are represented by gray and blue sticks, respectively. (B) Hydrogen bonds between D-Fru6P and dPGI-Lm residues are colored yellow, while red dot lines represent the separation between atoms that form hydrogen bonds in the D-Fru6P/rabbit PGI complex [21]. These distances indicate that loops A and B from PGI-Lm should move 5 Å to place D-Fru6P in a similar orientation described for D-Fru6P/rabbit PGI. possibilities were explored and finally a closed conformation was chosen owing to a better fit with the difference map. The comparison with the rabbit PGI/D-Fru6P [21] revealed that the phosphate group of D-Fru6P interacts with the same residues in both structures. The only exception is Ser209 (Ser159 in rabbit PGI) which, owing to the open conformation of dPGI-Lm, is making a hydrogen bond to O4 of D-Fru6P. The other residues – Ser259, Lys260, Thr261, Thr264 and Thr267 (corresponding to Ser209, Lys210, Thr211, Thr214 and Thr217 in rabbit PGI) – maintain a similar hydrogen bond network to the three oxygen atoms of the phosphate group (Fig. 4B). The main difference observed in the D-Fru6P coordination between the mammalian and Leishmania PGIs is a result of the open conformation of the active site of the latter. In the rabbit PGI in complex with D-Fru6P [21], residues His388(b) and Glu357 are less than 3 Å from the oxygen atom and hydroxyl groups of the substrate ring. Based on this enzyme–substrate complex structure, it was proposed that His388(b) participates in the substrate ring opening step, and Glu357 acts as a proton exchanger in the isomerization step [27]. In dPGI-Lm, His441(b) makes a hydrogen bond to O2 of D-Fru6P, and Glu410 is 7.7 Å distant from the closest hydroxyl group of the D-Fru6P ring (Fig. 4B). To bring D-Fru6P to a similar orientation, as observed in the rabbit PGI complex, would require a movement of 5 Å in the direction of Glu410. Two possibilities of such dislocation were analyzed. The first would require a 2.5 Å displacement of the small domain followed by a 2.5 Å movement of loops A and B. These loops in the rabbit [29] and human [28] PGIs comprise residues 209–215 and 246–262, respectively (numbered according to the human PGI sequence). Loops A and B are known to undergo a rigid body displacement of Ó FEBS 2004 Leishmania PGI crystal structure (Eur. J. Biochem. 271) 2771 Fig. 5. Neighboring PGI molecules contact surface. (A) Ribbon representation of deleted glucose-6-phosphate isomerase from Leishmania mexicana (dPGI-Lm), showing the surface of a neighboring molecule present in the crystal packing. Loop A, loop B and D-fructose-6phosphate (D-Fru6P), are colored green, yellow and magenta, respectively. Each monomer forming the dimer unit is colored in gray or blue. (B) Detailed view of hydrogen bond interactions in the crystal interface and hydrophobic interaction between Phe260 (in loop A) and Phe308 (in loop B). The close packing of dPGI-Lm symmetry-related molecules restricts the movement of loops A and B. The flexibility of these loops is necessary for the effective approximation of substrate to catalytic residues His441 and Glu410 in the active site. The colored patches in the symmetry-related molecule surface are related to the presence of oxygen (red) or nitrogen (blue) exposed atoms. 2.5 Å in order to coordinate the phosphate group of the substrate. Loops A (residues 256–262) and B (residues 298– 314) of PGI-Lm (Fig. 2) are structurally conserved and capable of undergoing a similar displacement, as observed for the mammalians PGIs. The movement, of 2.5 Å, of the small domain, however, would result in improper contacts with the surrounding residues, specifically with the Met337 described above. Another explanation for making the required contact would involve the movement of loops A and B by the whole distance of 5 Å that separates the substrate from the correct orientation with the catalytic residues (His441b and Glu410a, according to the PGI-Lm sequence). The simultaneous movement of these loops is mediated by Phe260 located in loop A that makes contact with loop B, forcing it to dislocate together with loop A. Residues of loop B are accessible to solvent and would not be restricted in such dislocation by interactions with other parts of the small domain. The residues from loop B which are not accessible to solvent – Val304, Phe307 and Ile309 – are in hydrophobic contact with Phe261 of loop A. Such orchestrated movement of both loops would bring D-Fru6P closer to the proposed catalytic residues, allowing the isomerization reaction. Crystal contacts One area of crystal contact of symmetry-related dPGI-Lm molecules occurs between Gln263 (loop A) and Lys304 (loop B) of one molecule and residues His80, Gln81 and Gln85 of its symmetry-related molecule (Fig. 5). These contacts represent the main hydrogen bonds between the related molecules and, together with weaker interactions, are believed to hold loops A and B in the open conformation observed in the crystal packing. These interactions have allowed D-Fru6P to reach the correct phosphate coordination residues (Fig. 4A), but blocked the movement of the flexible loops, obstructing the enzyme to close the catalytic cavity, as seen in the rabbit-PGI/D-Fru6P complex [21]. The crystalline contacts do not contribute to the orientation of the small domain. The large and small domains from Leishmania PGI form the unique and rigid body that is different from the mammalians PGIs. The conserved loops A and B are believed to be the only flexible region in PGI structures. Conclusions Both a full-length and an N-terminally deleted form of L. mexicana PGI were crystallized, the latter in the presence of D-Fru6P, and their structures refined to 2.6 Å and 2.35 Å, respectively. A comparison of these structures with those of human and other mammalian PGIs highlights unique features of the parasite enzyme. Although the overall fold of human and L. mexicana PGI appeared to be similar, and the substrate-binding residues to be conserved, a significant difference was detected in the position of the 2772 A. T. Cordeiro et al. (Eur. J. Biochem. 271) small domain that presents a more open conformation in the enzyme of the parasite. As a result, a larger active-site cavity is created, providing a possible explanation for the different values of kinetic parameters, as measured for the human and parasite enzymes. This larger cavity may be used in future rational drug design strategies, to develop selective inhibitors of the parasite enzyme which are too large to be accommodated by the catalytic site of the mammalian PGI. Acknowledgements This work was supported, in part, by research grants 98/14979-7 (FAPESP) and 478127/01-4 (CNPq) to O. H. Thiemann. A. T. Cordeiro is a recipient of a FAPESP student fellowship (project number 00/14960-6). We would like to thank the members of the Protein Crystallography and Structural Biology Group (IFSC-USP) for helpful discussions in the course of this work. We thank, in particular, Drs Robert Sweet, Denize Kranz and Michael Becker, and all RapiData 2003 course staff for their assistance in data collection. 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