Structure of <i>Lm</i>TK.

<p>(A) The protomer in complex with dThd and phosphate, ligands as spheres coloured by atom type. (B) Superposition using SSM [<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0003781#pntd.0003781.ref055" target="_blank">55</a>] showing the loop region corresponding to <i>Lm</i>TK residues 42–59 of <i>Lm</i>TK-dThd in lemon, <i>Lm</i>TK-dTTP in green, <i>Lm</i>TK-AP₅dT in ice blue, <i>Bc</i>TK (2ja1) in orange and hTK1 (1w4r) in pale crimson. The overall fold of <i>Lm</i>TK-dThd is shown in white. (C) The dimer in the asymmetric unit in the dThd-phosphate complex with the ligands shown as spheres coloured by atom type. (D) The tetramer generated using PISA [<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0003781#pntd.0003781.ref056" target="_blank">56</a>]. Figure made using CCP4mg [<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0003781#pntd.0003781.ref057" target="_blank">57</a>].</p

Stereo views of the ligand sites after superposition with SSM [55] in CCP4mg [57].

<p>(A) The dThd binding site of <i>h</i>TK1 (lemon), <i>Lm</i>TK-dThd (light blue) and <i>Lm</i>TK-dTTP (ice blue). (B) <i>Lm</i>TK-dThd (dark cyan), <i>Lm</i>TK-dTTP (ice blue), <i>Lm</i>TK-AP₅dT (light blue) and <i>Tm</i>TK-dThd-AppNHp (tan). The P-loop of <i>Lm</i>TK-AP₅dT is shown as a ribbon. Amino acid residues and ligands are shown as cylinders coloured by atom type with C and P atoms coloured by model. Hydrogen bonds are shown as dashed lines.</p

Analytical ultracentrifugation of C-terminally His-tagged <i>Lm</i>TK.

<p>Superposition of the c(s) distributions of the unliganded <i>Lm</i>TK and <i>Lm</i>TK-dThd-AppNHp samples are shown at concentrations in the range of 1–8 S.</p

Kinetic analysis of <i>Lm</i>TK.

<p>(A) ATP substrate dependent specific activity. Experiments were performed at 25 μM dThd and kinetic constants obtained by a least-squares best fit of the experimental data to the Hill equation. (B) Inhibition of enzyme activity by dTTP. Experiments were performed at 25 μM dThd and 500 μM ATP. The IC₅₀ value was calculated by non-linear regression analysis of the data. (C) The modulating effect of ATP on the inhibition of <i>Lm</i>TK by dTTP. dTMP formation was monitored at variable ATP concentrations and 25 μM dThd; in the absence of dTTP and in the presence of 3.5 μM and 7 μM dTTP. (D) Inhibition of enzyme activity by AP₅dT. Experiments were performed at 25 μM dThd and 500 μM ATP. The IC₅₀ value was calculated by non-linear regression analysis. Each point represents the average of three determinations, and error bars represent the standard deviation.</p

Kinetic constants and substrate specificity of recombinant <i>Lm</i>TK and <i>h</i>TK1.

<p>Kinetic parameters for <i>Lm</i>TK are means of two or more independent experiments.</p><p>N.D. Not determined.</p><p>^a Activity measurements at 10 mM substrate concentration.</p><p>^b Kinetic constants obtained at 37°C by Munch-Peterson et al. [<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0003781#pntd.0003781.ref043" target="_blank">43</a>].</p><p>Kinetic constants and substrate specificity of recombinant <i>Lm</i>TK and <i>h</i>TK1.</p

Crystallographic data and statistics.

<p>¹ Ramachandran Plot generated with COOT (Emsley <i>et al</i>., 2010).</p><p>² Poor Rotamer analysis performed using <i>Molprobity</i> (Chen <i>et al</i>., 2010).</p><p>Crystallographic data and statistics.</p

Excel spreadsheet containing, in separate sheets, all numerical values for Figs 1, 2A, 2B, 3A, 3B, 4A, 4B, 5A, 6, S2B, S3A, S3B, S4A and S4B.

Excel spreadsheet containing, in separate sheets, all numerical values for Figs 1, 2A, 2B, 3A, 3B, 4A, 4B, 5A, 6, S2B, S3A, S3B, S4A and S4B.</p

Rate of action against <i>T</i>. <i>cruzi</i> amastigotes.

(A) Time-dependent correlation between compound dose and T. cruzi amastigote growth inhibition upon 24, 48 and 72 hours exposure plotted as 10-point dose-response curves. Dose-response data obtained for posaconazole after 24, 48 and 72 h exposure at higher concentrations is indicated. (B) Maximum percentage activity against intracellular forms of T. cruzi obtained after 24, 48 and 72 hours treatment with 50 μM strasseriolide C (blue bars), 50 μM benznidazole (green bars), 20 μM nifurtimox (red bars) or 100 nM posaconazole (purple bars) in rate of kill experiments with amastigote forms of the parasite.</p

Quantification of intracellular ATP levels.

(A) ATP standard curve used for interpolating RLU read outs. (B) T. cruzi epimastigotes were treated with strasseriolide C for 72 hours at increasing concentrations, and ATP content was determined using Cell-Titer Glo luminescent viability assay and normalized by the number of cells per well. Results correspond to the mean of three replicates for each condition. (TIF)</p

Rate of action against <i>T</i>. <i>cruzi</i> trypomastigotes.

(A) Effect of different concentrations of strasseriolide C, benznidazole and nifurtimox on T. cruzi trypomastigotes viability. Loss of viability upon 24, 48 and 72 hours drug treatment was measured by the luminescent method at 1/2 serial concentrations ranging from 50 μM strasseriolide and benznidazole and from 10 μM for nifurtimox. Results correspond to the mean value of 3 replicates and error bars represent the standard deviation. (B) Maximum percentage activity against extracellular forms of T. cruzi reached following 24 and 48 hours treatment with 50 μM strasseriolide C (blue bars), 50 μM benznidazole (green bars), 10 μM nifurtimox (red bars) or 2 μM posaconazole (purple bars) in rate of kill experiments with trypomastigote forms of the parasite.</p