Three unrelated and unexpected amino acids determine the susceptibility of the interface cysteine to a sulfhydryl reagent in the triosephosphate isomerases of two trypanosomes.

Proteins with great sequence similarity usually have similar structure, function and other physicochemical properties. But in many cases, one or more of the physicochemical or functional characteristics differ, sometimes very considerably, among these homologous proteins. To better understand how critical amino acids determine quantitative properties of function in proteins, the responsible residues must be located and identified. This can be difficult to achieve, particularly in cases where multiple amino acids are involved. In this work, two triosephosphate isomerases with very high similarity from two related human parasites were used to address one such problem. We demonstrate that a seventy-fold difference in the reactivity of an interface cysteine to the sulfhydryl reagent methylmethane sulfonate in these two enzymes depends on three amino acids located far away from this critical residue and which could not have been predicted using other current methods. Starting from previous observations with chimeric proteins involving these two triosephosphate isomerases, we developed a strategy involving additive mutant enzymes and selected site directed mutants to locate and identify the three amino acids. These three residues seem to induce changes in the interface cysteine in reactivity by increasing (or decreasing) its apparent pKa. Some enzymes with four to seven mutations also exhibited altered reactivity. This study completes a strategy for identifying key residues in the sequences of proteins that can have applications in future protein structure-function studies

Effect of MMTS on WT TbTIM, WT TcTIM and on different site directed mutants of selected amino acids of regions 1 and 4 on the sequence of WT TbTIM.

<p>Enzymes were incubated at a concentration of 250 μg/mL in 100 mM TEA, 10 mM EDTA pH 7.4 and 5–100 μM MMTS for 2 h at 25°C. At that time, the activity of the samples was determined, including a sample without MMTS to calculate the percentage of remaining activity. Panel a) effect of MMTS on WT TcTIM, WT TbTIM and TbTIM: F28L, A100L, A115Q. Panel b) effect of MMTS on WT TcTIM, WT TbTIM and TbTIM: A100L, A115Q, TbTIM: F28L, A100L and TbTIM: F28L, A115Q. Panel c) effect of MMTS on WT TcTIM, WT TbTIM and TbTIM: F28L, TbTIM: A100L and TbTIM: A115Q. Assays were performed independently three times.</p

Effect of MMTS on WT TbTIM, WT TcTIM and on different site directed mutants of selected amino acids of regions 1 and 4 of the sequence of WT TbTIM onto WT TcTIM.

<p>Enzymes were incubated at a concentration of 250 μg/mL in 100 mM TEA, 10 mM EDTA pH 7.4 and 5–100 μM MMTS for 2 h at 25°C. At that time, the activity of the samples was determined, including a sample without MMTS to calculate the percentage of remaining activity. Panel a) effect of MMTS on WT TcTIM, WT TbTIM and TcTIM: E26D, T27L, L28F, A30S, T32S, L100A, Q115A, TcTIM: E26D, T27L, L28F, A30S, L100A, Q115A and TcTIM: E26D, T27L, L28F, L100A, Q115A. Panel b) effect of MMTS on WT TcTIM, WT TbTIM and TcTIM: E26D, T27L, L28F, A30S, T32S, L100A; TcTIM: E26D, T27L, L28F, A30S, L100A and TcTIM: E26D, T27L, L28F, L100A. Assays were performed independently three times.</p

pKa values of Cys 14/15 in WT TbTIM and WT TcTIM and some mutant enzymes.

<p>pKa values of Cys 14/15 in WT TbTIM and WT TcTIM and some mutant enzymes.</p

Location of regions 1 and 4 in a ribbon diagram of the structure of WT TbTIM and aligned sequences of regions 1 and 4 of WT TbTIM and WT TcTIM.

<p>The ribbon diagram of the structure of 5TIM in the PDB database, corresponding to a dimer of WT TbTIM, is shown in red. Regions 1 and 4 are shown in blue and marine blue for monomers A and B, respectively. The n-terminus of each monomer and the position of amino acids 35, 92 and 119 are indicated by the corresponding text or number, sometimes using an arrow, on the diagram. The interface cysteines of both monomers are shown as yellow spheres. In the alignments, the differences in the amino acids are highlighted as conservative (similar size and polarity) in grey, semiconservative (similar polarity) in green, and without similarity in cyan. Secondary structure elements are shown below as dark green lines (loops), arrows (beta sheets) and barrels (alpha helixes).</p

Effect of MMTS on WT TbTIM, WT TcTIM and on different additive mutants of region 4.

<p>Enzymes were incubated at a concentration of 250 μg/mL in 100 mM TEA, 10 mM EDTA pH 7.4 and 5–100 μM MMTS for 2 h at 25°C. At that time, the activity of the samples was determined, including a sample without MMTS to calculate the percentage of remaining activity. Panel a) effect of MMTS on WT TcTIM, WT TbTIM, R4M1 and TbTIM 1–3, 5–8; TcTIM 4. Panel b) effect of MMTS on WT TcTIM, WT TbTIM and R4M2, R4M3, R4M4 and R4M5. Assays were performed independently three times.</p

Identification of amino acids that account for long-range interactions in two triosephosphate isomerases from pathogenic trypanosomes.

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