Differences in the intersubunit contacts in triosephosphate isomerase from two closely related pathogenic trypanosomes

Maldonado, Ernesto; Soriano-Garcı́a, Manuel; Moreno, A.; Cabrera, Nallely; Garza‐Ramos, Georgina; Gómez‐Puyou, Marietta Tuena de; Gómez‐Puyou, Armando; Pérez‐Montfort, Ruy

doi:10.1006/jmbi.1998.2094

Cited by 64 publications

(68 citation statements)

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“…In the case of TIMs from parasites such as Trypanosoma brucei (34), Trypanosoma cruzi (35), Plasmodium falciparum (36), and Leishmania mexicana (37), a cysteine residue is present at the dimer interface; interestingly, in the enzyme from mammals the corresponding residue is methionine, whereas in yeast it is leucine (Fig. 1).…”

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Inhibition of Plasmodium falciparum Triose-phosphate Isomerase by Chemical Modification of an Interface Cysteine

Maithal¹,

Ravindra²,

Balaram³

et al. 2002

Journal of Biological Chemistry

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Plasmodium falciparum triose-phosphate isomerase, a homodimeric enzyme, contains four cysteine residues at positions 13, 126, 196, and 217 per subunit. Among these, Cys-13 is present at the dimer interface and is replaced by methionine in the corresponding human enzyme. We have investigated the effect of sulfhydryl labeling on the parasite enzyme, with a view toward developing selective covalent inhibitors by targeting the interface cysteine residue. Differential labeling of the cysteine residues by iodoacetic acid and iodoacetamide has been followed by electrospray ionization mass spectrometry and positions of the labels determined by analysis of tryptic fragments. The rates of labeling follows the order Cys-196 > Cys-13 > > Cys-217/ Cys-126, which correlates well with surface accessibility calculations based on the enzyme crystal structure. Iodoacetic acid labeling leads to a soluble, largely inactive enzyme, whereas IAM labeling leads to precipitation. Carboxyl methylation of Cys-13 results in formation of monomeric species detectable by gel filtration. Studies with an engineered C13D mutant permitted elucidation of the effects of introducing a negative charge at the interface. The C13D mutant exhibits a reduced stability to denaturants and 7-fold reduction in the enzymatic activity even under the concentrations in which dimeric species are observed.The search for new therapeutic agents active against various pathogens has led to the development of inhibitors targeted to inactivate key parasitic enzymes (1-6). These approaches have led to targeting the enzymes present typically in the pathogen only (7) or selectively targeting the pathogen enzyme, if the homologous form exists in the host (8). Two distinct approaches to inactivate target enzymes may be considered. First, the design of inhibitors that compete for the active site, and second, the design of molecules that target subunit interfaces in multimeric proteins and interfere with protein assembly. In the case of many key enzymes the high degree of conservation of the active site in both host and parasite enzymes renders selective targeting difficult. Interfaces may show greater structural variations permitting a distinction between the host and the pathogen enzymes. Two distinct strategies usually considered for disrupting intersubunit contacts in proteins are: (i) the design of synthetic peptides and peptidomimetics of interface segments, which may interfere with the subunit association (9 -11), and (ii) covalent chemical modification of reactive residues at the interface, which may cause disruption of the oligomeric state of the protein (12)(13)(14). In this regard, glycolytic enzymes are attractive targets, primarily because of their central role in energy production in the parasites (15-18).Triose-phosphate isomerase (TIM) 1 is an important glycolytic enzyme that catalyzes the interconversion of glyceraldehyde 3-phosphate to dihydroxyacetone phosphate (19,20). From the available knowledge of the structure of TIMs it is seen that the enzyme is a homo...

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“…catalytic activity (12,15,35,38,39). Although these studies have revealed quite clearly that the interface cysteine residue is critical for the enzymatic activity, a clear correlation of the rates of cysteine labeling with enzyme inactivation and structural perturbation is not available.…”

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Inhibition of Plasmodium falciparum Triose-phosphate Isomerase by Chemical Modification of an Interface Cysteine

Maithal¹,

Ravindra²,

Balaram³

et al. 2002

Journal of Biological Chemistry

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“…Each TIM monomer consists of an eightfold repeat of a b-strand-loop-a-helix-loop motif, which folds up to form a (b/a) 8 barrel. 23 The reported catalytic triad residues, Glu/His/Lys, belong to loops connecting the b-strands to the following a-helices. We find a structural pattern of CEEHKT conserved in the members of TIM family derived from diverse biological origins such as Bacillus stearothermophilus, Archaeon, Trypanosoma cruzi, One row indicates a representative pattern from a cluster.…”

Section: Clustering Of the Non-redundant Scop Representatives And Stamentioning

confidence: 99%

“…Of this pattern, the residue positions of Glu167/His95/Lys13 residues match with the reported active site residues of TIM. 22,23 However, the residues, Cys126/Glu97/Thr75 (5tim numbering ) have not yet been implicated in catalysis.…”

Section: Clustering Of the Non-redundant Scop Representatives And Stamentioning

confidence: 99%

Parameterization and Classification of the Protein Universe via Geometric Techniques

Tendulkar

Wangikar

Sohoni

et al. 2003

Journal of Molecular Biology

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We present a scheme for the classification of 3487 non-redundant protein structures into 1207 non-hierarchical clusters by using recurring structural patterns of three to six amino acids as keys of classification. This results in several signature patterns, which seem to decide membership of a protein in a functional category. The patterns provide clues to the key residues involved in functional sites as well as in protein -protein interaction. The discovered patterns include a "glutamate double bridge" of superoxide dismutase, the functional interface of the serine protease and inhibitor, interface of homo/hetero dimers, and functional sites of several enzyme families. We use geometric invariants to decide superimposability of structural patterns. This allows the parameterization of patterns and discovery of recurring patterns via clustering. The geometric invariant-based approach eliminates the computationally explosive step of pair-wise comparison of structures. The results provide a vast resource for the biologists for experimental validation of the proposed functional sites, and for the design of synthetic enzymes, inhibitors and drugs.

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“…The active site of TIM lies at the carboxyl-terminal end of the ␤-barrel, and the catalytic residues belong to loops connecting the ␤-strands to the following ␣-helices (Lys 10 , His 94 , and Glu 166 ; B. stearothermophilus TIM numbering). At present, the three-dimensional structures of 11 TIMs are known: those of chicken (12), yeast (13), Trypanosoma brucei (14), Escherichia coli (15), human (16), B. stearothermophilus (17), Plasmodium falciparum (18), Vibrio marinus (19), Trypanosoma cruzei (20), Leishmania mexicana (21), and T. maritima. 2 B. stearothermophilus TIM (bTIM) is reported to display moderately thermophilic enzymatic properties (22).…”

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Lys13 Plays a Crucial Role in the Functional Adaptation of the Thermophilic Triose-phosphate Isomerase fromBacillus stearothermophilus to High Temperatures

Alvarez

Maes

Mainfroid

et al. 1999

Journal of Biological Chemistry

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The thermophilic triose-phosphate isomerases (TIMs) of Bacillus stearothermophilus (bTIM) and Thermotoga maritima (tTIM) have been found to possess a His 12 -Lys 13 pair instead of the Asn 12 -Gly 13 pair normally present in mesophilic TIMs. His 12 in bTIM was proposed to prevent deamidation at high temperature, while the precise role of Lys 13 is unknown. To investigate the role of the His 12 and Lys 13 pair in the enzyme's thermoadaptation, we reintroduced the "mesophilic residues" Asn and Gly into both thermophilic TIMs. Neither double mutant displayed diminished structural stability, but the bTIM double mutant showed drastically reduced catalytic activity. No similar behavior was observed with the tTIM double mutant, suggesting that the presence of the His 12 and Lys 13 cannot be systematically correlated to thermoadaptation in TIMs. We determined the crystal structure of the bTIM double mutant complexed with 2-phosphoglycolate to 2.4-Å resolution. A molecular dynamics simulation showed that upon substitution of Lys 13 to Gly an increase of the flexibility of loop 1 is observed, causing an incorrect orientation of the catalytic Lys 10 . This suggests that Lys 13 in bTIM plays a crucial role in the functional adaptation of this enzyme to high temperature. Analysis of bTIM single mutants supports this assumption.Understanding the molecular adaptation of proteins to extreme temperatures remains a major challenge for protein scientists. Over the last 2 decades, several studies have attempted to establish a general rule leading to thermostability, but no overall consensus explanation could be formulated (1). For example, although the composition of thermophilic proteins shows that certain amino acids are preferred to ones present in mesophilic homologues, no general trends emerge. Studies have demonstrated the need to place the analysis of primary structure in a three-dimensional structural context (2); structural determinants such as ionic networks (3, 4), hydrophobic packing (5, 6), and cooperative associations (5, 7) seem to better reflect the general features associated with thermal adaptation. All of them, individually or in combination, enable thermophilic proteins to adapt to extreme temperatures.Thermotoga maritima is a hyperthermophilic eubacterium of the order Thermotogales, the oldest branch within the bacterial domain. Its optimal growth temperature is 80°C (8). Bacillus stearothermophilus is a moderately thermophilic endosporeforming Gram-positive rod growing optimally at about 65°C (9). Several glycolytic pathway enzymes from these strains have been produced by recombinant DNA technology, and all of them display optimal catalytic activity at temperatures above the optimal growth temperature of the source organism (1). Triose-phosphate isomerase (TIM) 1 is one of them. TIM is a homodimer catalyzing the interconversion of dihydroxyacetone phosphate and D-glyceraldehyde 3-phosphate in the glycolytic pathway (10). It is the prototype of a family comprising many different members sharing a common foldi...

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Differences in the intersubunit contacts in triosephosphate isomerase from two closely related pathogenic trypanosomes

Cited by 64 publications

References 46 publications

Inhibition of Plasmodium falciparum Triose-phosphate Isomerase by Chemical Modification of an Interface Cysteine

Inhibition of Plasmodium falciparum Triose-phosphate Isomerase by Chemical Modification of an Interface Cysteine

Parameterization and Classification of the Protein Universe via Geometric Techniques

Lys13 Plays a Crucial Role in the Functional Adaptation of the Thermophilic Triose-phosphate Isomerase fromBacillus stearothermophilus to High Temperatures

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