The adaptability of the active site of trypanosomal triosephosphate isomerase as observed in the crystal structures of three different complexes

Crystals of triosephosphate isomerase from Trypanosoma brucei brucei have been used in binding studies with three competitive inhibitors of the enzyme's activity. Highly refined structures have been deduced for the complexes between trypanosomal triosephosphate isomerase and a substrate analogue (glycerol-3-phosphate to 2.2 A), a transition state analogue (3-phosphonopropionic acid to 2.6 A), and a compound structurally related to both (3-phosphoglycerate to 2.2 A). The active site structures of these complexes were compared with each other, and with two previously determined structures of triosephosphate isomerase either free from inhibitor or complexed with sulfate. The comparison reveals three conformations available to the "flexible loop" near the active site of triosephosphate isomerase: open (no ligand), almost closed (sulfate), and fully closed (phosphate/phosphonate complexes). Also seen to be sensitive to the nature of the active site ligand is the catalytic residue Glu-167. The side chain of this residue occupies one of two discrete conformations in each of the structures so far observed. A "swung out" conformation unsuitable for catalysis is observed when sulfate, 3-phosphoglycerate, or no ligand is bound, while a "swung in" conformation ideal for catalysis is observed in the complexes with glycerol-3-phosphate or 3-phosphonopropionate. The water structure of the active site is different in all five structures. The results are discussed with respect to the triosephosphate isomerase structure function relationship, and with respect to an on-going drug design project aimed at the selective inhibition of glycolytic enzymes of T. brucei.     

Highly specific inactivation of triosephosphate isomerase from Trypanosoma cruzi

We searched for molecules that selectively inactivate homodimeric triosephosphate isomerase from Trypanosoma cruzi (TcTIM), the parasite that causes Chagas' disease. We found that some benzothiazoles inactivate the enzyme. The most potent were 3-(2-benzothiazolylthio)-propanesulfonic acid, 2-(p-aminophenyl)-6-methylbenzothiazole-7-sulfonic acid, and 2-(2-4(4-aminophenyl)benzothiazole-6-methylbenzothiazole-7-sulfonic acid. Half-maximal inactivation by these compounds was attained with 33, 56, and 8 microM, respectively; in human TIM, half-maximal inactivation required 422 microM, 3.3 mM, and 1.6 mM. In TcTIM, the effect of the benzothiazoles decreased as the concentration of the enzyme was increased. TcTIM has a cysteine (Cys 15) at the dimer interface, whereas human TIM has methionine in that position. In M15C human TIM, the benzothiazole concentrations that caused half-maximal inactivation were much lower than in the wild type. The overall findings suggest that the benzothiazoles perturb the interactions between the two subunits of TcTIM through a process in which the interface cysteine is central in their deleterious action.     

Exploring the possible binding sites at the interface of triosephosphate isomerase dimer as a potential target for anti-tripanosomal drug design

To explore the possible binding sites at the interface of tripanosomal triosephosphate isomerase, fully flexible benzothiazoles were docked onto the dimer interface. Docking studies revealed that the most favorable interactions occur in the aromatic clusters of the dimeric form. Hence is purposed that the dimer disruption is not via Cys 15, as presented in last studies, but it could be carried out through the unstabilization of pi-pi interactions of two aromatic clusters present in the interface. These studies enable a novel alternative for rational structure-based anti-tripanosomal drug design.     

Structure determination of the glycosomal triosephosphate isomerase from Trypanosoma brucei brucei at 2.4 A resolution

The three-dimensional crystal structure of the enzyme triosephosphate isomerase from the unicellular tropical blood parasite Trypanosoma brucei brucei has been determined at 2.4 A resolution. This triosephosphate isomerase is sequestered in the glycosome, a unique trypanosomal microbody of vital importance for the energy-generating machinery of the trypanosome. The crystals contain one dimer per asymmetric unit. The structure could be solved by the method of molecular replacement, using the refined co-ordinates of chicken triosephosphate isomerase as a search model. The positions and individual isotropic temperature factors of the 3792 atoms of the complete dimer have been refined by the Hendrickson & Konnert restrained refinement procedure. While tight restraints have been maintained on the bonded distances, the R-factor has dropped to 23.2% for 12317 reflections between 6 A and 2.4 A. A total of 0.6 mg of enzyme was used for establishing the correct crystallization conditions and solving the three-dimensional structure. Although the sequences of trypanosomal and chicken triosephosphate isomerase are identical at only 52% of the 247 common positions, the overall folds are very similar. The architecture of the active sites is virtually the same with 85% of the side-chains being identical. On the other hand, the residues involved in the dimer contacts are the same at only 55% of the positions. Nevertheless, the position of the local 2-fold axis in the chicken and glycosomal dimers is similar. A remarkable feature of glycosomal triosephosphate isomerase is its high overall positive charge. This extra charge is concentrated in four clusters of positively charged side-chains on the surface of the dimer, quite far away from the active site. These clusters may be involved in the mechanism of import of this triosephosphate isomerase into the glycosome.     

Toward a rational design of selective multi-trypanosomatid inhibitors: a computational docking study

Compound V7, a benzothiazole which was recently found as selective inhibitor of trypanosomal TIMs, was docked into TIMs from Trypanosoma cruzi, Trypanosoma brucei, Entamoeba histolytica, Plasmodium falciparum, yeast, and human. Structural analyses revealed the importance of the accessibility to the two aromatic clusters located at the dimer’s interface for the selective inhibition of trypanosomal TIMs. Thus, it was found that different accessibilities of the protein interface of TIMs plays an important role in the inhibitory activity of benzothiazoles. These findings will contribute to the rational development and improvement of benzothiazoles to be used as multi-trypanosomatid inhibitors.     

Crystal structure of recombinant human triosephosphate isomerase at 2.8 A resolution. Triosephosphate isomerase-related human genetic disorders and comparison with the trypanosomal enzyme.

The crystal structure of recombinant human triosephosphate isomerase (hTIM) has been determined complexed with the transition-state analogue 2-phosphoglycolate at a resolution of 2.8 A. After refinement, the R-factor is 16.7% with good geometry. The asymmetric unit contains 1 complete dimer of 53,000 Da, with only 1 of the subunits binding the inhibitor. The so-called flexible loop, comprising residues 168-174, is in its "closed" conformation in the subunit that binds the inhibitor, and in the "open" conformation in the other subunit. The tips of the loop in these 2 conformations differ up to 7 A in position. The RMS difference between hTIM and the enzyme of Trypanosoma brucei, the causative agent of sleeping sickness, is 1.12 A for 487 C alpha positions with 53% sequence identity. Significant sequence differences between the human and parasite enzymes occur at about 13 A from the phosphate binding site. The chicken and human enzymes have an RMS difference of 0.69 A for 484 equivalent residues and about 90% sequence identity. Complementary mutations ensure a great similarity in the packing of side chains in the core of the beta-barrels of these 2 enzymes. Three point mutations in hTIM have been correlated with severe genetic disorders ranging from hemolytic disorder to neuromuscular impairment. Knowledge of the structure of the human enzyme provides insight into the probable effect of 2 of these mutations, Glu 104 to Asp and Phe 240 to Ile, on the enzyme. The third mutation reported to be responsible for a genetic disorder, Gly 122 to Arg, is however difficult to explain. This residue is far away from both catalytic centers in the dimer, as well as from the dimer interface, and seems unlikely to affect stability or activity. Inspection of the 3-dimensional structure of trypanosomal triosephosphate isomerase, which has a methionine at position 122, only increased the mystery of the effects of the Gly to Arg mutation in the human enzyme.     

Characterization of the functional gene and several processed pseudogenes in the human triosephosphate isomerase gene family.

The functional gene and three intronless pseudogenes for human triosephosphate isomerase were isolated from a recombinant DNA library and characterized in detail. The functional gene spans 3.5 kilobase pairs and is split into seven exons. Its promoter contains putative TATA and CCAAT boxes and is extremely rich in G and C residues (76%). The pseudogenes share a high degree of homology with the functional gene but contain mutations that preclude the synthesis of an active triosephosphate isomerase enzyme. Sequence divergence calculations indicate that these pseudogenes arose approximately 18 million years ago. We present evidence that there is a single functional gene in the human triosephosphate isomerase gene family.     

Derivatization of the interface cysteine of triosephosphate isomerase from Trypanosoma brucei and Trypanosoma cruzi as probe of the interrelationship between the catalytic sites and the dimer interface

In the interface of homodimeric triosephosphate isomerase from Trypanosoma brucei (TbTIM) and Trypanosoma cruzi (TcTIM), one cysteine of each monomer forms part of the intersubunit contacts. The relatively slow derivatization of these cysteines by sulfhydryl reagents induces progressive structural alterations and abolition of catalysis [Garza-Ramos et al. (1998) Eur. J. Biochem. 253, 684-691]. Derivatization of the interface cysteine by 5, 5-dithiobis(2-nitrobenzoate) (DTNB) and methylmethane thiosulfonate (MMTS) was used to probe if events at the catalytic site are transmitted to the dimer interface. It was found that enzymes in the active catalytic state are significantly less sensitive to the thiol reagents than in the resting state. Maximal protection against derivatization of the interface cysteine by thiol reagents was obtained at near-saturating substrate concentrations. Continuous recording of derivatization by DTNB showed that catalysis hinders the reaction of sulfhydryl reagents with the interface cysteine. Therefore, in addition to intrinsic structural barriers, catalysis imposes additional impediments to the action of thiol reagents on the interface cysteine. In TcTIM, the substrate analogue phosphoglycolate protected strongly against DTNB action, and to a lesser extent against MMTS action; in TbTIM, phosphoglycolate protected against the effect of DTNB, but not against the action of MMTS. This indicates that barriers of different magnitude to the reaction of thiol reagents with the interface cysteine are induced by the events at the catalytic site. Studies with a Cys14Ser mutant of TbTIM confirmed that all the described effects of sulfhydryl reagents on the trypanosomal enzymes are a consequence of derivatization of the interface cysteine.     

Comparison of the refined crystal structures of liganded and unliganded chicken, yeast and trypanosomal triosephosphate isomerase.

The refined crystal structures of chicken, yeast and trypanosomal triosephosphate isomerase (TIM) have been compared. TIM is known to exist in an "open" (unliganded) and "closed" (liganded) conformation. For chicken TIM only the refined open structure is available, whereas for yeast TIM and trypanosomal TIM refined structures of both the open and the closed structure have been used for this study. Comparison of these structures shows that the open structures of chicken TIM, yeast TIM and trypanosomal TIM are essentially identical. Also it is shown that the closed structures of yeast TIM and trypanosomal TIM are essentially identical. The conformational difference between the open and closed structures concerns a major shift (7 A) in loop-6. Minor shifts are observed in the two adjacent loops, loop-5 (1 A) and loop-7 (1 A). The pairwise comparison of the three different TIM barrels shows that the 105C alpha atoms of the core superimpose within 0.9 A. The sequences of these three TIMs have a pairwise sequence identity of approximately 50%. The residues that line the active site are 100% conserved. The residues interacting with each other across the dimer interface show extensive variability, but the direct hydrogen bonds between the two subunits are well conserved. The orientation of the two monomers with respect to each other is almost identical in the three different TIM structures. There are 56 (22%) conserved residues out of approximately 250 residues in 13 sequences. The functions of most of these conserved residues can be understood from the available open and closed structures of the three different TIMs. Some of these residues are quite far from the active site. For example, at a distance of 19 A from the active site there is a conserved saltbridge interaction between residues at the C-terminal ends of alpha-helix-6 and alpha-helix-7. This anchoring contrasts with the large conformational flexibility of loop-6 and loop-7 near the N termini of these helices. The flexibility of loop-6 is facilitated by a conserved large empty cavity near the N terminus of alpha-helix-6, which exists only in the open conformation.     

The crystal structure of the "open" and the "closed" conformation of the flexible loop of trypanosomal triosephosphate isomerase

Triosephosphate isomerase has an important loop near the active site which can exist in a "closed" and in an "open" conformation. Here we describe the structural properties of this "flexible" loop observed in two different structures of trypanosomal triosephosphate isomerase. Trypanosomal triosephosphate isomerase, crystallized in the presence of 2.4 M ammonium sulfate, packs as an asymmetric dimer of 54,000 Da in the crystallographic asymmetric unit. Due to different crystal contacts, peptide 167-180 (the flexible loop of subunit-1) is an open conformation, whereas in subunit-2, this peptide (residues 467-480) is in a closed conformation. In the closed conformation, a hydrogen bond exists between the tip of the loop and a well-defined sulfate ion which is bound to the active site of subunit-2. Such an active site sulfate is not present in subunit-1 due to crystal contacts. When the native (2.4 M ammonium sulfate) crystals are transferred to a sulfate-free mother liquor, the flexible loop of subunit-2 adopts the open conformation. From a closed starting model, this open conformation was discovered through molecular dynamics refinement without manual intervention, despite involving C alpha shifts of up to 7 A. The tip of the loop, residues 472, 473, 474, and 475, moves as a rigid body. Our analysis shows that in this crystal form the flexible loop of subunit-2 faces a solvent channel. Therefore the open and the closed conformations of this flexible loop are virtually unaffected by crystal contacts. The actual observed conformation depends only on the absence or presence of a suitable ligand in the active site.     

Inhibition of triosephosphate isomerase from Trypanosoma brucei with cyclic hexapeptides.

Two series of oligopeptides have been synthesized. Their effects on the activity of purified triosephosphate isomerase from Trypanosoma brucei and various other organisms have been studied. Using detailed three-dimensional structure information, the first series consisted of both cyclic and linear hydrophilic peptides that were designed to mimic the beta turns of the subunit interface loops of the trypanosome triosephosphate isomerase dimer. None of these exerted any inhibitory effect. The second series consisted of more hydrophobic cyclic peptides, originally designed to inhibit a hepatic transport system. Several of these were very effective in inhibiting the trypanosome triosephosphate isomerase, but not the homologous enzymes from rabbit, dog, yeast or Escherichia coli. The most active peptide, cyclo[-Trp-Phe-D-Pro-Phe-Phe-Lys(Z)-], exerted 50% inhibitory activity at a concentration of 3 microM. The nature of the inhibitory action of one of these compounds cyclo[-Trp-Tyr(OSO3Na)-D-Pro-Phe-Thr(OSO3Na)-Lys(Z)-] was studied in more detail. Its inhibition was noncompetitive and reversible and more than one peptide was able to bind/active site.     

The structure of the thioredoxin-triosephosphate isomerase complex provides insights into the reversible glutathione-mediated regulation of triosephosphate isomerase

Protein-protein interactions are crucial for many biological functions. The redox interactome encompasses numerous weak transient interactions in which thioredoxin plays a central role. Proteomic studies have shown that thioredoxin binds to numerous proteins belonging to various cellular processes, including energy metabolism. Thioredoxin has cross talk with other redox mechanisms involving glutathionylation and has functional overlap with glutaredoxin in deglutathionylation reactions. In this study, we have explored the structural and biochemical interactions of thioredoxin with the glycolytic enzyme, triosephosphate isomerase. Nuclear magnetic resonance chemical shift mapping methods and molecular dynamics-based docking have been applied in deriving a structural model of the thioredoxin-triosephosphate isomerase complex. The spatial proximity of active site cysteine residues of thioredoxin to reactive thiol groups on triosephosphate isomerase provides a direct link to the observed deglutathionylation of cysteine 217 in triosephosphate isomerase, thereby reversing the inhibitory effect of S-glutathionylation of triosephosphate isomerase.     

Determination of the amino acid requirements for a protein hinge in triosephosphate isomerase.

We have determined the sequence requirements for a protein hinge in triosephosphate isomerase. The codons encoding the hinge at the C-terminus of the active-site lid of triosephosphate isomerase were replaced with a genetic library of all possible 8,000 amino acid combinations. The most active of these 8,000 mutants were selected using in vivo complementation of a triosephosphate isomerase deficient strain of E. coli, DF502. Approximately 3% of the mutants complement DF502 with an activity that is above 70% of wild-type activity. The sequences of these hinge mutants reveal that the solutions to the hinge flexibility problem are varied. Moreover, these preferences are sequence dependent; that is, certain pairs occur frequently. They fall into six families of similar sequences. In addition to the hinge sequences expected on the basis of phylogenetic analysis, we selected three new families of 3-amino-acid hinges: X(A/S)(L/K/M), X(aromatic/beta-branched)(L/K), and XP(S/N). The absence of these hinge families in the more than 60 known species of triosephosphate isomerase suggests that during evolution, not all of sequence space is sampled, perhaps because there is no neutral mutation pathway to access the other families.     

The influence of pH on the interaction of inhibitors with triosephosphate isomerase and determination of the pKa of the active-site carboxyl group.

Ionization effects on the binding of the potential transition state analogues 2-phosphoglycolate and 2-phosphoglycolohydroxamate appear to be attributable to the changing state of ionization of the ligands themselves, therefore it is unnecessary to postulate the additional involvement of an ionizing residue at the active site of triosephosphate isomerase to explain the influence of changing pH on Ki in the neutral range. The binding of the competitive inhibitor inorganic sulfate is insensitive to changing pH in the neutral range. 3-Chloroacetol sulfate, synthesized as an active-site-specific reagent for triosephosphate isomerase, is used to provide an indication of the pKa of the essential carboxyl group of this enzyme. Previously described active-site-specific reagents for the isomerase were phosphate esters, and their changing state of ionization (accompanied by possible changes in their affinity for the active site) may have complicated earlier attempts to determine the pKa of the essential carboxyl group from the pH dependence of the rate of inactivation. Being a strong monoprotic acid, chloroacetol sulfate is better suited to the determination of the pKa of the carboxyl group. Chloroacetol sulfate inactivates triosephosphate isomerase by the selective esterification of the same carboxyl group as that which is esterified by the phosphate esters described earlier. From the pH dependence of the rate of inactivation of yeast triosephosphate isomerase, the apparent pKa of the active-site carboxyl group is estimated as 3.9 +/- 0.1.     

Structure of the complex between trypanosomal triosephosphate isomerase and N-hydroxy-4-phosphono-butanamide: binding at the active site despite an "open" flexible loop conformation.

The structure of triosephosphate isomerase from Trypanosoma brucei complexed with the competitive inhibitor N-hydroxy-4-phosphono-butanamide was determined by X-ray crystallography to a resolution of 2.84 A. Full occupancy binding of the inhibitor is observed only at one of the active sites of the homodimeric enzyme where the flexible loop is locked in a completely open conformation by crystal contacts. There is evidence that the inhibitor also binds to the second active site of the enzyme, but with low occupancy. The hydroxamyl group of the inhibitor forms hydrogen bonds to the side chains of Asn 11, Lys 13, and His 95, whereas each of its three methylene units is involved in nonpolar interactions with the side chain of the flexible loop residue Ile 172. Interactions between the hydroxamyl and the catalytic base Glu 167 are absent. The binding of this phosphonate inhibitor exhibits three unusual features: (1) the flexible loop is open, in contrast with the binding mode observed in eight other complexes between triosephosphate isomerase and various phosphate and phosphonate compounds; (2) compared with these complexes the present structure reveals a 1.5-A shift of the anion-binding site; (3) this is the first phosphonate inhibitor that is not forced by the enzyme into an eclipsed conformation about the P-CH2 bond. The results are discussed with respect to an ongoing drug design project aimed at the selective inhibition of glycolytic enzymes of T. brucei.     

Structural and mutagenesis studies of leishmania triosephosphate isomerase: a point mutation can convert a mesophilic enzyme into a superstable enzyme without losing catalytic power

The dimeric enzyme triosephosphate isomerase (TIM) has a very tight and rigid dimer interface. At this interface a critical hydrogen bond is formed between the main chain oxygen atom of the catalytic residue Lys13 and the completely buried side chain of Gln65 (of the same subunit). The sequence of Leishmania mexicana TIM, closely related to Trypanosoma brucei TIM (68% sequence identity), shows that this highly conserved glutamine has been replaced by a glutamate. Therefore, the 1.8 A crystal structure of leishmania TIM (at pH 5.9) was determined. The comparison with the structure of trypanosomal TIM shows no rearrangements in the vicinity of Glu65, suggesting that its side chain is protonated and is hydrogen bonded to the main chain oxygen of Lys13. Ionization of this glutamic acid side chain causes a pH-dependent decrease in the thermal stability of leishmania TIM. The presence of this glutamate, also in its protonated state, disrupts to some extent the conserved hydrogen bond network, as seen in all other TIMs. Restoration of the hydrogen bonding network by its mutation to glutamine in the E65Q variant of leishmania TIM results in much higher stability; for example, at pH 7, the apparent melting temperature increases by 26 degrees C (57 degrees C for leishmania TIM to 83 degrees C for the E65Q variant). This mutation does not affect the kinetic properties, showing that even point mutations can convert a mesophilic enzyme into a superstable enzyme without losing catalytic power at the mesophilic temperature.     

Synthesis of the pentadecapeptide sequence of the active site of rabbit muscle triosephosphate isomerase.

The pentadecapeptide fragment, Trp-Val-Leu-Ala-Tyr-Glu-Pro-Val-Trp-Ala-Ile-Gly-Thr-Gly-Lys, which constitutes a part of the active site of rabbit muscle triosephosphate isomerase has been synthesized. It does not exhibit any catalytic activity typical of triosephosphate isomerase.     

Human triosephosphate isomerase cDNA and protein structure. Studies of triosephosphate isomerase deficiency in man

Nine cDNA clones of human adult liver triosephosphate (TP) isomerase have been isolated and characterized. All nine appear to be derived from a single mRNA species. DNA sequencing of one clone, designated pHTPI-5a, defined the last two nucleotides of the methionine initiation codon, the entire 744-nucleotide coding region of the mature polypeptide, and the entire 448-nucleotide 3' untranslated region. The frequency of TP isomerase clones in the cDNA library suggests that TP isomerase mRNA is present in adult liver at approximately 25 copies/cell. A single, low abundance TP isomerase mRNA species was detected in RNA isolated from normal human fibroblast cell lines. Analysis of TP isomerase mRNA levels in cultured fibroblasts of individuals that are homozygous for TP isomerase deficiency revealed normal levels in one and approximately 40% of normal levels in another. From this small patient sampling, it can be concluded that the genetic basis for TP isomerase deficiency is heterogeneous.     

Perturbation of the dimer interface of triosephosphate isomerase and its effect on Trypanosoma cruzi

Background

Chagas disease affects around 18 million people in the American continent. Unfortunately, there is no satisfactory treatment for the disease. The drugs currently used are not specific and exert serious toxic effects. Thus, there is an urgent need for drugs that are effective. Looking for molecules to eliminate the parasite, we have targeted a central enzyme of the glycolytic pathway: triosephosphate isomerase (TIM). The homodimeric enzyme is catalytically active only as a dimer. Because there are significant differences in the interface of the enzymes from the parasite and humans, we searched for small molecules that specifically disrupt contact between the two subunits of the enzyme from Trypanosoma cruzi but not those of TIM from Homo sapiens (HTIM), and tested if they kill the parasite.

Methodology/Principal Findings

Dithiodianiline (DTDA) at nanomolar concentrations completely inactivates recombinant TIM of T. cruzi (TcTIM). It also inactivated HTIM, but at concentrations around 400 times higher. DTDA was also tested on four TcTIM mutants with each of its four cysteines replaced with either valine or alanine. The sensitivity of the mutants to DTDA was markedly similar to that of the wild type. The crystal structure of the TcTIM soaked in DTDA at 2.15 Å resolution, and the data on the mutants showed that inactivation resulted from alterations of the dimer interface. DTDA also prevented the growth of Escherichia coli cells transformed with TcTIM, had no effect on normal E. coli, and also killed T. cruzi epimastigotes in culture.

Conclusions/Significance

By targeting on the dimer interface of oligomeric enzymes from parasites, it is possible to discover small molecules that selectively thwart the life of the parasite. Also, the conformational changes that DTDA induces in the dimer interface of the trypanosomal enzyme are unique and identify a region of the interface that could be targeted for drug discovery.