Identification of Amino Acids that Account for Long-Range Interactions in Two Triosephosphate Isomerases from Pathogenic Trypanosomes

Abstract

For a better comprehension of the structure-function relationship in proteins it is necessary to identify the amino acids that are relevant for measurable protein functions. Because of the numerous contacts that amino acids establish within proteins and the cooperative nature of their interactions, it is difficult to achieve this goal. Thus, the study of protein-ligand interactions is usually focused on local environmental structural differences. Here, using a pair of triosephosphate isomerase enzymes with extremely high homology from two different organisms, we demonstrate that the control of a seventy-fold difference in reactivity of the interface cysteine is located in several amino acids from two structurally unrelated regions that do not contact the cysteine sensitive to the sulfhydryl reagent methylmethane sulfonate, nor the residues in its immediate vicinity. The change in reactivity is due to an increase in the apparent pKa of the interface cysteine produced by the mutated residues. Our work, which involved grafting systematically portions of one protein into the other protein, revealed unsuspected and multisite long-range interactions that modulate the properties of the interface cysteines and has general implications for future studies on protein structure-function relationships.

Identification of the critical residues responsible for differential reactivation of the triosephosphate isomerases of two trypanosomes

“…[17]. Briefly, the DNA sequences X03921 for TbTIM and U53867 for TcTIM were used to construct the chimeric proteins.…”

“…Other chimeras used in this work, which include TcTIM1–3;TbTIM4–8, TcTIM1,2;TbTIM3–8 and TcTIM1;TbTIM2–8, were made as described by García-Torres et al . [17]. The gene of chimeric protein TcTIM3–8;TbTIM1,2 was made with three PCRs using Accuzyme DNA polymerase (Bioline, Taunton, MA).…”

New chemotypes as Trypanosoma cruzi triosephosphate isomerase inhibitors: a deeper insight into the mechanism of inhibition

“…Recently, we reported 14,18 that it is possible to use chimeras of TcTIM and TbTIM in order to identify the amino acids or amino acid sequences that are involved in a given property of the enzymes. Thus, we examined the effect of compounds 2 and 3 on a TcTIM in which regions 1 (residues 1-35) and 4 (residues 92-119) were replaced by the corresponding regions of TbTIM, namely TcTIM 2,3,5-8 (Figure 5a).…”

1,2,4-thiadiazol-5(4H)-ones: a new class of selective inhibitors of Trypanosoma cruzi triosephosphate isomerase. Study of the mechanism of inhibition

“…The mutant was inactivated by compound 1 and was slightly more sensitive than the wild type. Recently, we reported 16 that it is possible to use chimeras of TcTIM and TbTIM in order to identify the amino acids or amino acid sequences that are involved in a given property of the enzymes. Thus, we examined the effect of compound 1 on a TcTIM in which regions 1 (residues 1-35) and 4 (residues 92-119) were replaced by the corresponding regions of TbTIM, namely TcTIM 2,3,5-8 ( Figure 6).…”

“…We further show that, although compound 1 binds non-covalently to the enzyme, the binding character is strong as revealed by diffusion nuclear magnetic resonance (DOSY-NMR) experiments, and it is an effective inhibitor of T. cruzi growth. 16 and TcTIMC15A (a mutant protein, that contains an Ala-residue at position 14 of the sequence instead of a Cys present in wild-type TcTIM) 17 , were expressed in Escherichia coli and purified as described in the referred literature. After purification, the enzymes, dissolved in 100 mM triethanolamine, 10 mM EDTA and 1 mM dithiothreitol (pH 8), were precipitated with ammonium sulfate (75% saturation) and stored at 4 °C.…”